digitonin and malic-acid

The influence ofadenosine diphosphate (ADP) on respiration of pancreatic acinar cell mitochondria in situ was studied. The model of digitonin-treated pancreatic acini was used. It was found that succinate or a mixture of pyruvate, glutamate and malate intensified respiration ofpermeabilized cells. Low ADP concentration (100 microM) did not influence the rate of oxygen uptake, whereas at higher concentration (750 microM) brief intensification of respiration was observed when using nominally Ca(2+)-free medium. When the medium with 100 nM Ca2+ was used, ADP had no effect on oxygen uptake, while the rate of respiration stimulated by a mixture of pyruvate, glutamate and malate increased. Rate of succinate-stimulated respiration did not depend on Ca2+ content in medium. The presence of ATP in the medium reduced the stimulatory effect of ADP, but increased its duration. Intensification of respiration by ADP, occurred only at elevated Ca2+ content, was not associated with oxidative phosphorylation because oligomycin did not inhibit it. The effect ofADP might be a novel "functional marker" of development of pathological processes in the mitochondria of acinar pancreacytes.

Topics: Acinar Cells; Adenosine Diphosphate; Animals; Calcium; Cell Fractionation; Digitonin; Glutamic Acid; Malates; Male; Mitochondria; Oligomycins; Oxidative Phosphorylation; Oxygen; Pancreas; Permeability; Pyruvic Acid; Rats

Muscle biopsy analysis is regarded as the gold standard in diagnostic workups of patients with suspected mitochondrial disorders. Analysis of cultured fibroblasts can provide important additional diagnostic information. The measurement of individual OXPHOS complexes does not always provide sufficient information about the functional state of the complete mitochondrial energy-generating system. Thus, we optimized a high-throughput fluorescence-based methodology for oxygen consumption analysis in patient-derived cells.. We analyzed mitochondrial respiration in digitonin-permeabilized cells in the presence of a substrate mix containing pyruvate and malate, using a phosphorescent probe, 96-well plates, and a fluorescence plate reader.. In control fibroblasts, we observed clear stimulation by ADP of the pyruvate + malate-driven respiration. Known inhibitors of the OXPHOS system and the Krebs cycle significantly reduced respiration. In patient fibroblasts with different OXPHOS deficiencies, ADP-stimulated respiratory activity was decreased in comparison to control cells. In several patients with reduced ATP production rate in muscle tissue but with normal OXPHOS enzyme activities, the fibroblasts displayed reduced respiratory activity. Finally, we observed a clear difference between control and complex I-deficient transmitochondrial cybrid cells.. These results confirm the validity of the assay as a high-throughput screening method for mitochondrial function in digitonin-permeabilized cells. The assay allows primary and secondary mitochondrial abnormalities in muscle to be differentiated, which is of great importance with respect to counseling, and also will facilitate the search for new genetic defects that lead to mitochondrial disease.

Topics: Cell Membrane Permeability; Cell Respiration; Cells, Cultured; Digitonin; Fibroblasts; Fluorescence; High-Throughput Screening Assays; Humans; Malates; Mitochondria, Muscle; Mitochondrial Diseases; Oxygen Consumption; Pyruvic Acid

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

To evaluate the results of oxygen consumption measurement in lymphocytes for the diagnosis and treatment monitoring of pediatric patients with oxidative phosphorylation diseases.. Twenty-four children with an oxidative phosphorylation disease were studied. Results were compared with those of 87 healthy children. Oxygen consumption measurements in digitonine-permeabilized lymphocytes incubated with pyruvate plus malate and succinate were performed in a Clark-type oxygen electrode.. A total of 58% of patients showed a decreased oxygen consumption in lymphocytes incubated with pyruvate. In 4 patients, this analysis was the unique initial biochemical test, which revealed an impaired mitochondrial energy metabolism. Significant differences were observed in lymphocytes incubated with pyruvate between patients and reference values (p<0.00005), and in lymphocytes incubated with pyruvate before and after treatment (p<0.05).. This test is useful for diagnosing oxidative phosphorylation diseases in patients who did not have other biochemical alterations, although false-negative results can be found. It is not useful for treatment monitoring.

Topics: Adolescent; Child; Child, Preschool; Digitonin; DNA, Mitochondrial; Electron Transport; Energy Metabolism; Female; Humans; Indicators and Reagents; Infant; Lymphocytes; Malates; Male; Metabolic Diseases; Mitochondria; Oxidative Phosphorylation; Oxygen Consumption; Polarography; Pyruvic Acid; Reference Values; Succinic Acid

Increased mitochondrial generation of reactive oxygen intermediates (ROI) due to defective respiratory chain activity has been implicated in physiological processes such as apoptosis, in the pathogenesis of mitochondrial diseases, and as part of the normal aging process. Established methods addressing activity of the respiratory chain complexes have been limited to bulk assays for single parameters. This study describes a flow cytometry-based method and its validation for the detection of respiratory chain function in single cells permeabilized by digitonin.. Flow cytometry was used to measure mitochondrial membrane potential (DeltaPsi(m)) and reactive oxygen generation under differing conditions of respiration. This was brought about by the addition of substrates and inhibitors to digitonin-permeabilized cells. This method was validated by measurement of oxygen consumption and ATP production and by confocal microscopy.. Activity of the respiratory chain complexes assessed by DeltaPsi(m) responded to substrates and inhibitors as predicted from assessment by oxygen consumption and ATP synthesis. In addition, the flow cytometry method allows the simultaneous assessment of mitochondrial ROI generation. This was confirmed by the localization of the ROI probe, carboxy-DCF, to the same site as the mitochondrial probe observed by confocal microscopy.. This method allows the functional integrity of the respiratory chain complexes to be studied at the single-cell level, thus addressing the relationship between disordered function of respiratory chain complexes and mitochondrial ROI generation.

Topics: Adenosine Triphosphate; Apoptosis; Cell Membrane Permeability; Cell Respiration; Digitonin; Electron Transport; Flow Cytometry; Fluoresceins; Humans; Intracellular Membranes; Malates; Membrane Potentials; Microscopy, Confocal; Mitochondria; Reactive Oxygen Species; Reproducibility of Results; Succinic Acid; Tumor Cells, Cultured

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.

Topics: Adolescent; Adult; Cells, Cultured; Digitonin; Female; Fibroblasts; Glutamates; Humans; Leukocytes, Mononuclear; Malates; Middle Aged; Mitochondria; Muscle, Skeletal; Ophthalmoplegia, Chronic Progressive External; Oxygen; Permeability; Skin; Succinates

Mitochondrial dysfunction has been implicated as the cause of irreversible injury in the ischemic heart. To circumvent artifacts associated with organelle isolation, mitochondrial function was studied in intact isolated, Ca2+-tolerant rat ventricular myocytes. After 30 min of anaerobic incubation, myocyte viability decreased from 76 +/- 1 to 33 +/- 4%. Basal O2 consumption rates (nanoatoms . mg cell protein-1 . min-1) were 17.1 +/- 1.3 in aerobic cells and 51.0 +/- 9.8 in anoxic cells. Carbonylcyanide-p-trifluoromethoxyphenyl hydrazone (FCCP)-stimulated rates were 65.5 +/- 9.2 and 84.5 +/- 15.3 in aerobic and anoxic cells, respectively. Respiratory control ratio was lower in anoxic cells: 2.3 +/- 0.3 versus 4.2 +/- 0.4 in aerobic cells. These data suggest that early anoxic mitochondrial injury is due to increased permeability of the inner membrane. Addition of pyruvate, malate, and FCCP to cells made permeable by digitonin resulted in similar maximal O2 consumption rates: 276.5 +/- 31.8 in aerobic and 299.3 +/- 31.9 in anoxic cells, suggesting the electron transport chain is intact in anoxic cells. For purposes of investigating whether anoxic mitochondrial dysfunction is secondary to cellular or mitochondrial Ca2+ overload, total cell Ca2+, cytosolic free Ca2+ levels (measured by null-point titration), and mitochondrial Ca2+ contents (measured as FCCP-releasable Ca2+) were measured. There were no differences in these three parameters between aerobic and anoxic cells, suggesting that mitochondrial dysfunction and irreversible hypercontraction of isolated cardiac myocytes exposed to 30 min of anoxia are not related to Ca2+ overload.

Topics: Animals; Calcimycin; Calcium; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Membrane Permeability; Digitonin; Malates; Mitochondria, Heart; Myocardial Contraction; Oligomycins; Oxygen; Oxygen Consumption; Pyruvates; Pyruvic Acid; Rats; Ruthenium Red; Time Factors

Respiratory studies of brain mitochondria have, in general, been limited to purified preparations. Conventional procedures for mitochondrial isolation yield relatively small and potentially selected subfractions of mitochondria. Examination of respiratory responses of homogenates of rat forebrain indicated that key respiratory properties of mitochondria are fully expressed in these preparations. In a high K+ buffer, comparable to those commonly used for purified mitochondria, forebrain homogenates exhibited many of the characteristics of oxygen uptake by "free" mitochondria: requirement for both pyruvate and malate for maximal respiration, stimulation (over threefold) by ADP, stimulation by uncoupling agent [carbonyl cyanide m-chlorophenylhydrazone (CCCP)], but little effect of digitonin. In a modified Krebs-Ringer phosphate buffer (a physiological buffer), respiratory responses were primarily due to mitochondria enclosed in synaptosomes: respiration with glucose was markedly stimulated by CCCP, further stimulated by pyruvate, and extensively inhibited by digitonin (which disrupts the cholesterol-rich synaptosomal membranes). Studies with purified mitochondria and synaptosomes supported the specificity of these responses. These data indicate that classical mitochondrial responses are expressed in whole brain homogenates and, under appropriate conditions, provide functional measures of the total pools of free and synaptosomal mitochondria.

Topics: Adenosine Diphosphate; Animals; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Diencephalon; Digitonin; Malates; Male; Mitochondria; Oxygen Consumption; Potassium; Pyruvates; Pyruvic Acid; Rats; Rats, Inbred Strains; Synaptosomes; Telencephalon