scholarly journals A study of rapid mitochondrial structural changes in vitro by spray-freeze-etching

1978 ◽  
Vol 77 (1) ◽  
pp. 134-147 ◽  
Author(s):  
RD Lang ◽  
JR Bronk
Keyword(s):  
Time Course ◽  
Structural Changes ◽  
Rat Liver Mitochondria ◽  
Sampling Device ◽  
Rapid Sampling ◽  
Outer Compartment ◽  
Matrix Volume ◽  
The Matrix ◽  
Freeze Etching

The spray-freeze-etching technique has been used to study energy-linked mitochondrial structural changes in rat liver mitochondria incubated in vitro. The technique involves spraying the suspension of mitochondria into liquid propane at -190 degrees C, and does not require the use of cryoprotectants or chemical fixatives. The results confirmed that freshly isolated mitochondria have a condensed matrix and that this expands at the expense of the outer compartment to give the orthodox configuration when the mitochondria are incubated in a K+ medium in the presence of substrate and phosphate. Addition of adenosine diphosphate (ADP) caused a rapid shrinkage of the matrix compartment, and the time-course and extent of this shrinkage has been measured quantitatively by coupling a rapid sampling device to the spray-freezing apparatus. These data show that for orthodox mitochondria the onset of phosphorylation is accompanied by a reduction of 30% in the matrix volume in 20's, and there is no evidence that the decrease in matrical volume affects the phosphorylation efficiency. These results suggest that natural ionophores in the mitochondrial inner membrane make it permeable enough to permit a rapid readjustment of matrix volume after the addition of ADP, and that the associated ion movement does not cause uncoupling of oxidative phosphorylation.

Effect of carbon tetrachloride poisoning on potassium, sodium and water content of liver mitochondria

1959 ◽  
Vol 197 (1) ◽  
pp. 121-125 ◽  
Author(s):  
Leonard Share ◽  
Richard O. Recknagel
Keyword(s):  
Carbon Tetrachloride ◽  
Water Content ◽  
Time Course ◽  
Liver Mitochondria ◽  
Liver Fat ◽  
Direct Result ◽  
Rat Liver Mitochondria ◽  
Water Metabolism ◽  
Force Feeding

A physiological test of mitochondrial function, which measures the ability of the mitochondria to concentrate potassium in vitro, was employed in a study of carbon tetrachloride-induced hepatotoxicity. The force feeding of carbon tetrachloride resulted in a) a reduction in the potassium content of rat liver mitochondria, b) an impairment in the ability of potassium depleted mitochondria to reaccumulate potassium and c) an increased water content of the mitochondria. The changes could be demonstrated unequivocally at 10.5 hr. and became extensive at 20 hr. The results do not support the view that the increase in liver fat is the direct result of a lesion in the mitochondria since the time course of impairment in mitochondrial potassium and water metabolism lags behind the fatty changes of the whole liver.

The Effect of Insulin on Mitochondrial Particles

1967 ◽  
Vol 25 ◽  
pp. 160-161 ◽  
Author(s):  
Burton B. Silver ◽  
James C. Hall
Keyword(s):  
Bovine Serum Albumin ◽  
Structural Changes ◽  
Normal Values ◽  
Liver Mitochondria ◽  
Synergistic Action ◽  
Diabetic Rat ◽  
Rat Liver Mitochondria ◽  
Structural Studies

Correlative biochemical and structural studies have shown that insulin and Mg++ may act to alter the configuration and also enhance the efficiency of coupled phosphorylation in sonicated fragments of diabetic rat liver mitochondria. The diabetic preparations had consistently lowered P:O ratios which returned to normal values with addition of insulin in vivo or in vitro. Optimum coupling and structural changes with insulin required a Mg++ concentration of 5 × 10−5 M. Insulin remained effective diluted to a concentration of 2 × 10−4 I.U. per ml. Glutathione, bovine serum albumin, and Zn++ were ineffective in producing either coupling or structural changes. There seems to be a synergistic action of insulin and Mg++ in restoring P:O ratios in diabetic particles while simultaneously altering the structure toward normal control appearances. Fragments negatively stained with phosphotungstate indicated that the normal particles had well defined cristae with numerous evenly distributed, stalked subunits, 90 Å in diameter.

scholarly journals d-Lactate transport and metabolism in rat liver mitochondria

2002 ◽  
Vol 365 (2) ◽  
pp. 391-403 ◽  
Author(s):  
Lidia de BARI ◽  
Anna ATLANTE ◽  
Nicoletta GUARAGNELLA ◽  
Giovanni PRINCIPATO ◽  
Salvatore PASSARELLA
Keyword(s):  
Lactate Dehydrogenase ◽  
Rat Liver ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Lactate Metabolism ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
The Matrix ◽  
Inside Out

In the present study we investigated whether isolated rat liver mitochondria can take up and metabolize d-lactate. We found the following: (1) externally added d-lactate causes oxygen uptake by mitochondria [P/O ratio (the ratio of mol of ATP synthesized to mol of oxygen atoms reduced to water during oxidative phosphorylation) = 2] and membrane potential (Δψ) generation in processes that are rotenone-insensitive, but inhibited by antimycin A and cyanide, and proton release from coupled mitochondria inhibited by α-cyanocinnamate, but not by phenylsuccinate; (2) the activity of the putative flavoprotein (d-lactate dehydrogenase) was detected in inside-out submitochondrial particles, but not in mitochondria and mitoplasts, as it is localized in the matrix phase of the mitochondrial inner membrane; (3) three novel separate translocators exist to mediate d-lactate traffic across the mitochondrial inner membrane: the d-lactate/H+ symporter, which was investigated by measuring fluorimetrically the rate of endogenous flavin reduction, the d-lactate/oxoacid antiporter (which mediates both the d-lactate/pyruvate and d-lactate/oxaloacetate exchanges) and d-lactate/malate antiporter studied by monitoring photometrically the appearance of the d-lactate counteranions outside mitochondria. The d-lactate translocators, in the light of their different inhibition profiles separate from the monocarboxylate carrier, were found to differ from each other in the Vmax values and in the inhibition and pH profiles and were shown to regulate mitochondrial d-lactate metabolism in vitro. The d-lactate translocators and the d-lactate dehydrogenase could account for the removal of the toxic methylglyoxal from cytosol, as well as for d-lactate-dependent gluconeogenesis.

scholarly journals Selective permeability of rat liver mitochondria to purified aspartate aminotransferases in vitro

1977 ◽  
Vol 164 (3) ◽  
pp. 685-691 ◽  
Author(s):  
E Marra ◽  
S Doonan ◽  
C Saccone ◽  
E Quagliariello
Keyword(s):  
Rat Liver ◽  
Aspartate Aminotransferase ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Transferase Activity ◽  
Selective Permeability ◽  
The Matrix ◽  
Mitochondrial Aspartate Aminotransferase

1. A method was devised to allow determination of intramitochondrial aspartate amino-transferase activity in suspensions of intact mitochondria. 2. Addition of purified rat liver mitochondrial aspartate aminotransferase to suspensions of rat liver mitochondria caused an apparent increase in the intramitochondrial enzyme activity. No increase was observed when the mitochondria were preincubated with the purified cytoplasmic isoenzyme. 3. These results suggest that mitochondrial aspartate aminotransferase, but not the cytoplasmic isoenzyme, is able to pass from solution into the matrix of intact rat liver mitochondria in vitro. 4. This system may provide a model for studies of the little-understood processes by which cytoplasmically synthesized components are incorporated into mitochondria in vivo.

scholarly journals Liver mitochondrial pyrophosphate concentration is increased by Ca2+ and regulates the intramitochondrial volume and adenine nucleotide content

1987 ◽  
Vol 246 (3) ◽  
pp. 715-723 ◽  
Author(s):  
A M Davidson ◽  
A P Halestrap
Keyword(s):  
Adenine Nucleotide ◽  
Hormone Treatment ◽  
Liver Mitochondria ◽  
Mitochondrial Swelling ◽  
Rat Liver Mitochondria ◽  
Nucleotide Content ◽  
Mitochondrial Inner Membrane ◽  
Matrix Volume ◽  
The Matrix ◽  
K Permeability

1. The matrix pyrophosphate (PPi) content of isolated energized rat liver mitochondria incubated in the presence of ATP, Mg2+, Pi and respiratory substrate was about 100 pmol/mg of protein. 2. After incubation with sub-micromolar [Ca2+], this was increased by as much as 300%. There was a correlation between the effects of Ca2+ on PPi and on the increase in matrix volume reported previously [Halestrap, Quinlan, Whipps & Armston (1986) Biochem. J. 236, 779-787]. Half-maximal effects were seen at 0.3 microM-Ca2+. 3. Coincident with these effects, the total adenine nucleotide content increased in a carboxyatractyloside-sensitive manner. 4. Incubation with 0.2-0.5 mM-butyrate induced similar but smaller effects on mitochondrial swelling and matrix PPi and total adenine nucleotide content. Addition of butyrate after Ca2+, or vice versa, caused Ca2+-induced mitochondrial swelling to stop or reverse, while matrix PPi increased 30-fold. 5. Addition of atractyloside or the omission of ATP from incubations greatly enhanced swelling induced by Ca2+ without increasing matrix PPi. 6. Swelling of mitochondria incubated under de-energized conditions in iso-osmotic KSCN was progressively enhanced by the addition of increasing concentrations of PPi (1-20 mM) or valinomycin. 7. In iso-osmotic potassium pyrophosphate swelling was slow initially, but accelerated with time. This acceleration was inhibited by ADP, whereas carboxyatractyloside induced rapid swelling. Swelling in other iso-osmotic PPi salts showed that the rate of entry decreased in the order NH4+ greater than K+ greater than Na+ greater than Li+, whereas choline, tetramethylammonium and Tris did not enter. It is suggested that the adenine nucleotide translocase transports small univalent cations when PPi is bound and that PPi can also be transported when the transporter is in the conformation induced by carboxyatractyloside. 8. It is concluded that Ca2+ and butyrate cause swelling of energized mitochondria through this effect of PPi on K+ permeability of the mitochondrial inner membrane. 9. Freeze-clamped livers from rats treated with glucagon or phenylephrine show 30-50% increases in tissue PPi. It is proposed that Ca2+-mediated increases in mitochondrial PPi are responsible for the increase in matrix volume and total adenine nucleotide content observed after hormone treatment.

scholarly journals The Role of Interleukin-6 (IL-6) in Human Sulfur Mustard (HD) Toxicology

2001 ◽  
Vol 20 (5) ◽  
pp. 281-296 ◽  
Author(s):  
Carmen M. Arroyo ◽  
Clarence A. Broomfield ◽  
Brennie E. Hackley
Keyword(s):  
Interleukin 6 ◽  
Time Course ◽  
Structural Changes ◽  
Sulfur Mustard ◽  
Skin Irritation ◽  
Epidermal Keratinocytes ◽  
Early Event ◽  
Mrna Levels ◽  
Human Epidermal Keratinocytes

The authors applied in vitro models of controlled damage to human epidermal keratinocytes (HEKs), human skin fibroblasts (HSFs), and human breast skin tissue (HBST) to examine the mechanism responsible for sulfur mustard (HD)-induced interleukin-6 (IL-6) alterations. Treatment with 100 μM HD for 24 hours resulted in a significant increased amount of IL-6 being secreted by HEKs (HD-exposed to control ratio [E/C] = 4.15 ± 0.07) and by HSFs (E/C = 7.66 ± 0.04). Furthermore, the HD-induced secretion of IL-6 in HEKs was neutralized with monoclonal human IL-6 antibodies. The secretion of IL-6 in HBST supernatant exposed to HD produced conflicting results. Although an increase of IL-6 was observed in control superfusion media from HBST, IL-6 levels were observed to decrease as the concentration of HD increased. Time course of IL-6 mRNA levels were performed using a competitive polymerase chain reaction (PCR) and human IL-6 mRNA assay detection kit in control and HD (100 μM)-treated HEKs cells. IL-6 mRNA transcripts in HD-exposed HEKs were first observed within 2 hours, dropped at 5 to 6 hours, and increased by ® 2.2-fold and 8.5-fold at 24 to 48 hours after HD exposure, respectively, as detected by the Xplore mRNA Quantification System. Surface-enhanced laser desorption ionization (SELDI) mass spectrometry was also applied to study the secretion pattern of IL-6 on lysate preparations of HBST. A peak in the area of 23,194 to 23,226 Da was detected using antibody coupled to the chip. This peak was assigned to correspond to the mass of the IL-6 glycoprotein. Recombinant human IL-6 (rhIL-6) exposed to HD lacked the second disulfide bridge and was partially unfolded, as determined by nuclear magnetic resonance-nuclear Overhauser enhancement and exchange spectroscopy (NMR-NOESY). The disappearance of the resonance peak at 3.54 ppm and the appearance of a new chemical shift at 1.85 ppm suggested that a change in structure had occurred in the presence of HD. From the data, the possibility cannot be excluded that IL-6 might be involved in the early event of structural changes of the signal transducer glycoprotein that indirectly initiates the cascade of events such as skin irritation and blister formation observed in the pathophysiology of HD injury.

THE ACTION OF OXYTOCIN AND SYNTHETIC ANALOGUES ON THE ISOLATED MAMMARY-GLAND MYOEPITHELIUM OF THE LACTATING RAT; EFFECT OF SOME IONS

1967 ◽  
Vol 38 (1) ◽  
pp. 13-24 ◽  
Author(s):  
I. POLÁČEK ◽  
I. KREJČÍ ◽  
J. RUDINGER
Keyword(s):  
Mammary Gland ◽  
Time Course ◽  
Structural Changes ◽  
Contractile Response ◽  
Organ Bath ◽  
Synthetic Analogues ◽  
Uterine Smooth Muscle ◽  
Rat Mammary Gland ◽  
High Concentrations

SUMMARY The contractile response of mammary-gland strips from lactating rats to oxytocin varied with the concentration of calcium ions in the organ bath; calcium could be partially substituted by strontium but not by magnesium. High concentrations of potassium did not substantially affect the response to oxytocin. In these and several other respects the mammary gland myoepithelium resembles uterine smooth muscle, notwithstanding the differences in morphology and embryonal origin. The effects of 12 synthetic analogues of oxytocin modified in sequence positions 1, 2, and 3 of the peptide chain on the rat mammary-gland strip were examined. None of the analogues showed a higher potency than oxytocin. However, the mammary-gland strip was less sensitive to the structural changes represented in this series of analogues than the rat uterus in vitro. The action of some of the analogues on the mammary-gland strip differs from that of oxytocin in the time-course of the response.

scholarly journals Intramitochondrial regulation of fatty acid β-oxidation occurs between flavoprotein and ubiquinone A role for changes in the matrix volume

1986 ◽  
Vol 239 (3) ◽  
pp. 559-565 ◽  
Author(s):  
A P Halestrap ◽  
J L Dunlop
Keyword(s):  
Fatty Acid ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Hormonal Stimulation ◽  
Terminal Electron Acceptor ◽  
Matrix Volume ◽  
The Matrix ◽  
Range Of Values ◽  
Stimulation Of

Rat liver mitochondria were incubated in media of different osmolarities and in the presence of various substrates. Rates of oxygen consumption and mitochondrial matrix volumes were measured in the presence and absence of ADP and uncoupler. Duroquinol oxidation was insensitive to matrix volume, whereas other substrates tested showed increased rates of oxidation when the matrix volume increased from 1.0 to 1.5 microliter/mg of protein; this is the range of values measured in situ [Quinlan, Thomas, Armston & Halestrap (1983) Biochem. J. 214, 395-404]. Palmitoylcarnitine, octanoate and butyrate oxidations were particularly sensitive to the matrix volume, increasing from negligible rates to maximal rates within this range. Swelling induced by K+ uptake also stimulated palmitoylcarnitine oxidation. A similar effect of volume on substrate oxidation was seen when ferricyanide in the presence or absence of ubiquinone-1 replaced oxygen as terminal electron acceptor. Measurement of flavoprotein reduction (A 460-480) demonstrated that the locus of the effect of matrix volume is between the electron-transfer flavoprotein and ubiquinone. It is suggested that volume-mediated regulation of fatty acid and proline oxidation may be an important component of the hormonal stimulation of their oxidation.

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