digitonin and Hypoxia

Glutamate is the only amino acid extracted by healthy myocardium in net amounts, with uptake further increased during hypoxic or ischemic conditions. Glutamate supplementation provides cardioprotection from hypoxic and reperfusion injury through several metabolic pathways that depend upon adequate transport of glutamate into the mitochondria. Glutamate transport across the inner mitochondrial membrane is a key component of the malate/aspartate shuttle. Glutamate transport in the brain has been well characterized since the discovery of the excitatory amino acid transporter (EAAT) family. We hypothesize that a protein similar to EAAT1 found in brain may function as a glutamate transporter in cardiac mitochondria. Rat heart total RNA was screened by reverse transcriptase-polymerase chain reaction with an array of primer pairs derived from the rat brain EAAT1 cDNA sequence, yielding a 3786-bp cDNA comprising a 1638-bp open reading frame identical to rat brain EAAT1 with flanking 5'- and 3'-untranslated regions. Northern blot analysis confirmed a 4-kb mRNA product in rat heart and brain, with greater abundance in brain. A protein of the predicted approximate 60-kD size was recognized in myocardial lysates by an anti-EAAT1 polyclonal antibody produced against an amino-terminal peptide from human EAAT1. The protein enriched in rat heart mitochondria by immunoblot, co-localized with the mitochondrial protein cytochrome c by immunohistochemistry, and further localized to the inner mitochondrial membrane upon digitonin fractionation of the mitochondria. In myocytes overexpressing EAAT1, activity of the malate/aspartate shuttle increased by 33% compared to non-transfected cells (P = 0.004). These data indicate that EAAT1 is expressed in myocardial mitochondria, and functions in the malate/aspartate shuttle, suggesting a role for EAAT1 in myocardial glutamate metabolism.

Topics: Adenoviridae; Animals; Aspartic Acid; Blotting, Northern; Brain; Cells, Cultured; Coloring Agents; Cytochromes c; Digitonin; DNA, Complementary; Excitatory Amino Acid Transporter 1; Genetic Vectors; Glutamic Acid; Hypoxia; Immunoblotting; Immunohistochemistry; Malates; Microscopy, Fluorescence; Mitochondria; Mitochondria, Heart; Myocardium; Open Reading Frames; Rats; Rats, Inbred WKY; Rats, Sprague-Dawley; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Messenger; Subcellular Fractions; Tetrazolium Salts; Thiazoles; Transfection

The aim was to clarify the factors that induce enzyme release from mitochondria during anoxia and reoxygenation.. Isolated perfused hearts or isolated mitochondria were prepared from hearts excised from rats. The amounts of lactate dehydrogenase, cytoplasmic aspartate aminotransferase, and mitochondrial aspartate aminotransferase released into the coronary effluent from perfused heart preparations were measured. To distinguish the effect of mechanical stress from that of reoxygenation, a latex balloon was placed in the left ventricular cavity to impose mechanical stress and the heartbeat was controlled with a high K+ medium. A digitonin infusion technique was used to obtain only the cytosolic compartment of the cells for analysis of the amounts of mitochondrial enzymes released into the cytosol. The effect of anoxia followed by reoxygenation on enzyme release from isolated mitochondria was studied.. On reoxygenation, mitochondrial aspartate aminotransferase was released as well as cytoplasmic enzymes, but, unlike cytoplasmic enzymes, the release was not influenced by mechanical stress. Mitochondrial injury by reoxygenation depended on the duration of the preceding anoxia. Reoxygenation of isolated mitochondria also induced enzyme release and the presence of ATP in the extramitochondrial space reduced the release of this enzyme.. Enzyme leakage from mitochondria of myocardial cells occurs during reoxygenation, irrespective of mechanical stress, and this vulnerability to oxidative stress depends on the duration of the preceding anoxic period or the concentration of cytosolic ATP.

Topics: Adenine Nucleotides; Animals; Aspartate Aminotransferases; Digitonin; Hypoxia; L-Lactate Dehydrogenase; Male; Mitochondria, Heart; Myocardium; Oxygen; Rats; Rats, Sprague-Dawley; Stress, Mechanical

The subcellular distribution of newly absorbed iron in isolated mouse duodenal enterocytes was investigated by analytical subcellular fractionation using sucrose density gradient centifugation. Two major peaks of mucosal 59Fe activity were observed: one soluble and one particulate (density 1.18-1.20 g ml-1). The latter was increased following prior exposure of animals to chronic hypoxia. The particulate 59Fe was localized to the basolateral membranes using the marker enzyme Na+, K+ activated, Mg2+ dependent, ATPase and by washing intact enterocytes with the selective plasma membrane perturbant digitonin. The basolateral membrane can be selectively labelled by in vitro incubation of intact enterocytes at 0 degrees C with 59Fe(III)-nitrilotriacetate complex, confirming the presence of a 59Fe binding site on this membrane. No significant difference in in vitro iron binding to this site was observed between normal and chronically hypoxic animals. Iron binding to the basolateral membrane was significantly higher in disrupted, compared to intact enterocytes, indicating that this site is present on both sides of the basolateral membrane. It is therefore suggested that the increased labelling of this site in hypoxia, in vivo, is a consequence of an increase in a mucosal Fe pool which is available for binding to a membrane receptor.

Topics: Adenosine Triphosphatases; Animals; Binding Sites; Cell Fractionation; Cell Membrane; Centrifugation, Density Gradient; Cytosol; Digitonin; Duodenum; Hypoxia; Intestinal Absorption; Intestinal Mucosa; Iron; Male; Mice; Precipitin Tests

The purpose of the present study is to clarify the effects of hypoxia on catecholamine release and its mechanism of action. For this purpose, using cultured bovine adrenal chromaffin cells, we examined the effects of hypoxia on high (55 mM) K(+)-induced increases in catecholamine release, in cytosolic free Ca2+ concentration ([Ca2+]i), and in 45Ca2+ uptake. Experiments were carried out in media preequilibrated with a gas mixture of either 21% O2/79% N2 (control) or 100% N2 (hypoxia). High K(+)-induced catecholamine release was inhibited by hypoxia to approximately 40% of the control value, but on reoxygenation the release returned to control levels. Hypoxia had little effect on ATP concentrations in the cells. In the hypoxic medium, [Ca2+]i (measured using fura-2) gradually increased and reached a plateau of approximately 1.0 microM at 30 min, whereas the level was constant in the control medium (approximately 200 nM). High K(+)-induced increases in [Ca2+]i were inhibited by hypoxia to approximately 30% of the control value. In the cells permeabilized by digitonin, catecholamine release induced by Ca2+ was unaffected by hypoxia. Hypoxia had little effect on basal 45Ca2+ uptake into the cells, but high K(+)-induced 45Ca2+ uptake was inhibited by hypoxia. These results suggest that hypoxia inhibits high K(+)-induced catecholamine release and that this inhibition is mainly the result of the inhibition of high K(+)-induced increases in [Ca2+]i subsequent to the inhibition of Ca2+ influx through voltage-dependent Ca2+ channels.

Topics: Adenosine Triphosphate; Adrenal Medulla; Anaerobiosis; Animals; Biological Transport, Active; Calcium; Calcium Radioisotopes; Catecholamines; Cattle; Cells, Cultured; Cytosol; Digitonin; Hypoxia; Kinetics; Potassium Chloride

Myocyte hypercontracture can be produced by adding Ca2+ to calcium-intolerant myocytes. A similar morphologic change occurs in Ca2+-free media when anoxic, ATP-depleted myocytes are reoxygenated or when respiring myocytes are lysed with digitonin. Hypercontracture in Ca2+-free media is abolished by rotenone, an inhibitor of NADH-linked respiration. Rotenone-treated, digitonin-permeabilized myocytes were used to examine the effects of MgATP, pCa, and respiration on hypercontracture. In the absence of Ca2+ (pCa 8.5), hypercontracture occurred at low MgATP but not when ATP was increased above 1 mM. At high MgATP (1-10 mM), hypercontracture was Ca2+-dependent. Succinate did not cause hypercontracture in the absence of added MgATP, but it shifted the concentration dependence for Ca2+-independent hypercontracture to lower values by regenerating ATP.

Topics: Animals; Calcium; Cell Membrane Permeability; Cell Separation; Digitonin; Hypoxia; Ion Channels; Myocardial Contraction; Myocardium; Oxygen Consumption; Rats

A method to determine intracellular distribution of ions and metabolites under conditions of low oxygen concentration was developed. The technique involves rapid digitonin fractionation and addition of cyanide to inhibit reoxygenation. Applicability of the procedure was examined by studying the distribution of the lipophilic ion triphenylmethylphosphonium, the weak acid 5,5-dimethyloxazolidine-2,4-dione, and adenine nucleotides.

Topics: Adenine Nucleotides; Animals; Cell Fractionation; Digitonin; Dimethadione; Hydrogen-Ion Concentration; Hypoxia; Ions; Male; Membrane Potentials; Mitochondria, Liver; Onium Compounds; Oxygen Consumption; Rats; Trityl Compounds

A one hour hypoxic incubation causes the release of a small but significant amount of cytosolic lactic dehydrogenase from glucose-deprived isolated adult rat heart myocytes. However, enzymes associated with the mitochondria are not liberated, and there is no increase in the number of hypercontracted cells. These observations led Piper et al. (Life Sciences 35, 127-134 [1984]) to conclude that reversibly injured myocytes can release cytosolic proteins. This conclusion was based on the supposition that irreversibly hypoxic injury must cause mitochondrial enzyme efflux and hypercontracture. The present study establishes that this supposition is invalid.

Topics: Adenosine Triphosphate; Animals; Aspartate Aminotransferases; Creatine Kinase; Cytosol; Digitonin; Glutamate Dehydrogenase; Hypoxia; Isoenzymes; L-Lactate Dehydrogenase; Malate Dehydrogenase; Microscopy, Electron; Mitochondria, Heart; Myocardium; Oxygen Consumption; Rats; Time Factors

Several studies have reported a linear correlation between ATP levels, measured on isolated adult heart cells exposed to anoxia, and cellular configuration. The case is presented here for the viewpoint that these results can best be explained by an asynchronous decline in cellular ATP levels. By asynchrony we mean that an individual cell maintains a constant high level of ATP for a lag period until some point when, over a short period of time, the ATP level suddenly drops to near zero; the time spread of the decline in total measured ATP then arises from a spread in lag periods amongst the cells. By contrast, the synchronous view assumes that the time spread of the decline in total measured ATP reflects the simultaneous slow decline of ATP levels within each cell. These viewpoints have important consequences for elucidating the etiology of cell death.

Topics: Adenosine Triphosphate; Animals; Cell Separation; Coronary Disease; Digitonin; Hypoxia; Myocardial Contraction; Myocardium; Oxygen; Rats; Sarcolemma; Time Factors

The release of lactic dehydrogenase, creatine phosphokinase, and aspartate aminotransferase from initially viable, metabolically competent adult rat heart myocytes has been examined. Freshly isolated cells contain levels of total lactic dehydrogenase, creatine phosphokinase, and aspartate aminotransferase, as well as lactic dehydrogenase and creatine phosphokinase isoenzyme profiles that are quite comparable to those of intact heart tissue. When the cells are lysed with digitonin, 89% of total lactic dehydrogenase, but only 58% of creatine phosphokinase and 27% of aspartate aminotransferase are released. The retention of creatine phosphokinase by the digitonin-lysed cells is accounted for by complete retention of mitochondrial creatine phosphokinase and 20% of MM-creatine phosphokinase. When intact myocytes are incubated under anoxic, substrate-deprived conditions, there is a gradual loss of the three enzymes to the suspending medium and a parallel increase in the fraction of the cells permeable to trypan blue. The fraction of freely soluble cytoplasmic enzymes lost was equivalent to the fraction of the cells permeable to the dye over a wide range of viability (17-95% viable by dye exclusion criteria), but permeable cells retained mitochondrial creatine phosphokinase and particulate aspartate aminotransferase. These results suggest that simultaneous and complete release of soluble cytoplasmic enzymes occurs as each individual cell sustains sarcolemmal damage.

Topics: Animals; Aspartate Aminotransferases; Cell Survival; Creatine Kinase; Digitonin; Hypoxia; Isoenzymes; L-Lactate Dehydrogenase; Muscles; Myocardium; Rats