常氧及低氧下孵育液中钙浓度改变对心肌线粒体钙含量及其呼吸功能损伤的影响

在常氧孵育中,当孵育介质自由钙离子浓度升高时,离体心肌线粒体钙含量显著增加。同时,线粒体状态4呼吸速率也明显加快并与其钙含量的增加呈正相关关系。在低氧孵育中,当孵育介质自由钙离子浓度升高时,离体心肌线粒体钙含量没有明显的增加,其状态4呼吸速率虽有加快但程度明显较常氧孵育时低。另外,在低孵育介质自由钙离子浓度(pCa8.0)的条件下,低氧可引起轻微的线粒体状态4呼吸速率加快。从以上结果作者推测,低氧引起心肌细胞的线粒体损伤可能主要不是低氧直接对线粒体作用所造成的,而是由低氧引起的心肌细胞胞浆环境变化对线粒体破坏的结果。其中胞浆自由钙离子的升高可能是一个的原因。     

Ultrastructural characteristics of mitochondria during cell adaptation to rotenone

A study was made of respiration, heat production, K+ output and ultrastructure of wheat root cells treated for 6 h with rotenone (10 microM), an inhibitor of HADH-ubiquinone oxidoreductase (Complex I). Besides, the involvement of alternative pathways for adaptation to this inhibitor was studied. After 20 min of treatment, a brightened mitochondrial matrix and mitochondria with torus shapes were observed. We propose that the outer area of mitochondria increases due to their torus shapes, and this can point to the activating of extremal NAD(P)H-dehydrogenase, which uses enternal NAD(P)H. Further on the normal ultrastructure of mitochondria was observed, which may result from activation of succinate dehydrogenase and rotenone resistant NAD(P)H-dehydrogenase. After 1 h of treatment, a decrease in respiration, heat production, K+ output and pH increase of incubation medium were observed. Starting from 2 h of incubation and up to the end of the experiment, an increase of respiration and heat production was observed, pointing to the activation of oxidative phosphorilation. Besides, re-entry of K+ and pH decrease in the incubation medium were observed. We conclude that these findings may indicate to a possible adaptation of root cells to this inhibitor. We propose that the torus shape of mitochondria may be associated with function of external NAD(P)H-dehydrogenase.     

Respiratory behaviour of sea-urchin spermatozoa. I. Effect of pH and egg water on the respiratory rate.

Effects of pH and egg water on the respiration of sea-urchin spermatozoa were polarographically studied in three sea-urchins and one starfish species. Sea-urchin sperm respiration is extremely sensitive to change in the pH of the suspending medium over a wide range. In normal-sea water, the pH of the sperm suspension decreased from 8.02 to 7.62, after four to five minutes' incubation at 18 degrees C. The Respiratory Dilution Effect could be recognized in the same medium. However, when sea water was buffered with HEPES at pH 8.2, the Effect was no longer observed. The diffusate from egg water (jelly coat solution) brought about a striking increase in the respiration when added to moderately respiring spermatozoa in HEPES-sea water of pH values lower than 7.9. No inccrease in the respiration was observed when the diffusate was added to vigorously respiring spermatozoa in HEPES-sea water of pH values higher than 8.2. Sperm motility was also inhibited by acid pH, and this inhibition was reversed by the addition of the diffusate. It does not seem that there is any species-specificity among three sea-urchins and one starfish used. The role of the diffusate is discussed in relation to the penetration of spermatozoa through the jelly coat to the egg surface.     

Modulation of mammalian sperm motility by quercetin

The flavonoid quercetin inhibits collective motility of ejaculated ram spermatozoa in the first 2 hr of incubation; during the next 3-4 hr motility is stimulated. To explain this interesting effect, we followed the influence of quercetin on sperm glycolysis, extracellular pH, ATP content, mitochondrial respiration, and lipid peroxidation. The collective motility of untreated cells is decreased to about 40% of the original motility during two hours of incubation. During this time, the rate of glycolysis is constant, respiration rate is increasing, there is no change in ATP content, the rate of lipid peroxidation is very slow, and the extracellular pH became very acidic (pH 5.5). It is concluded that motility is decreased due to this acidification. This acidification is prevented to some extent by quercetin, which indirectly inhibits glycolysis. Quercetin inhibits motility due to the inhibition of the plasma membrane calcium pump, as we showed previously (Breitbart et al., J Biol Chem 260:11548-11553, 1985). The motility of untreated cells is arrested after 3.5 hr of incubation, whereas quercetin-treated cells show high motility, which continues for additional 2-3 hr. After 3.5 hr, the control cells show no glycolytic activity, ATP content and respiration rates are decreased, and rate of lipid peroxidation is highly increased. At this time, quercetin-treated cells show no glycolytic activity, only a small decrease in ATP content and respiratory rate, and a very low rate of lipid peroxidation. Based on these data it is concluded that sperm motility after 3.5 hr of incubation is dependent mainly on mitochondrial respiration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Urea-induced hypometabolism in the hibernating wood frog (Rana sylvatica) is not reflected in isolated mitochondria

It has long been speculated that urea accumulated during seasonal dormancy contributes to metabolic depression. Recent work suggests urea can indeed act as a metabolic depressant during dormancy in a number of taxonomically diverse species of ectotherms. The mechanisms by which urea exerts its hypometabolic effect are unknown, but potentially stem from inhibition of mitochondrial respiration. We isolated mitochondria from Rana sylvatica skeletal muscle, an organ that is metabolically responsive to urea, and measured respiration rates in the absence or presence of 80 mmol l⁻¹ urea in the respiration medium. Because the effect of urea may be influenced by the intracellular milieu, in these experiments we varied substrate (pyruvate or palmitoylcarnitine), temperature (4, 10, or 15°C), and pH (6.8 or 7.4). Oxygen consumption of control and urea-treated mitochondria was sensitive to each of these variables, but neither state 3 nor state 4 respiration was reduced by urea treatment and, to the contrary, urea treatment slightly increased state 4 respiration at higher test temperatures. Although we did not test the efficacy of other incubation times or urea concentrations, the outcome of our experiment intimates that the urea-induced hypometabolism observed in hibernating R. sylvatica results from inhibition of energy-utilizing processes elsewhere in the cell, rather than a direct inhibition of mitochondrial respiration. Future investigation into urea’s effects on non-mitochondrial metabolic pathways is necessary to uncover the mechanisms by which urea depresses metabolic rate.     

A study of the effects of flocalin on respiration and potassium transport of rat-heart and liver mitochondria

The effects of the drug flocalin, which possesses cardioprotective properties, on the respiration rates of rat-heart and liver mitochondria in different functional states, the efficiency of oxidative phosphorylation, as well as the transport of potassium ions in these organelles, were studied. It was found that flocalin at concentrations of 7–30 μm stimulated respiration of rat-heart and liver mitochondria in V ₂ and V ₄ states in the presence of succinic add as a respiration substrate in a potassium-containing medium. In the absence of potassium ions in the incubation medium, flocalin had no effect on mitochondrial respiration in these states. Studying the functioning of the potassium transport system revealed that flocalin at these concentrations dose-dependently activated the ATP-dependent transport of potassium ions in rat-heart and liver mitochondria. The data we obtained indicate that the cardioprotective effect of flocalin can be associated with activation of the ATP-dependent potassium channel of the inner mitochondrial membrane.     

The role of lipolysis and lipid peroxidation in the induction of ion transport in mitochondria after freezing and thawing

The reasons of ion transport induction observed after freezing-thawing of the rat liver mitochondria were investigated. It is found that a fall in the membrane potential value, an increase in the respiration rate and K+ ion release from mitochondria to the incubation medium with phosphate are averted when succinate is replaced by the Kreb's cycle substrates and when ionol (an antioxidant) or nupercain (an inhibitor of mitochondrial phospholipase A2) are incorporated into the incubation medium. The induction of ion transport is caused by the activation of peroxide processes and of lipolysis associated with them. It is supposed that under experimental conditions after freezing-thawing a degree of pyridine nucleotides and glutathione reduction lowers, that in its turn leads to the inhibition of the glutathione-peroxidase activity and development of peroxide processes.     

The effect of substrate, ADP and uncoupler on the respiration of tomato pollen during incubation in vitro at moderately high temperature

Pollen of tomato cv. Supermarmande was collected from greenhouse-grown plants at various intervals throughout the year and arbitrarily classified as of high, medium or low respiratory activity on the basis of CO₂ production during 8 h incubation in vitro at 30°C, a temperature that is considered to be moderately high for tomato fruit set. After an initial burst of respiration during the first stage of hydration at 30°C (>1 h), the respiration rate of pollen of all three categories declined, the decrease being greater in the lots with a low or medium respiratory activity than in the high category. During hydration (10 min after the start of incubation), the addition of succinate or reduced β-nicotinamide adenine dinucleotide (NADH) to the substrate increased the respiratory rate of slowly-respiring pollen more than that of fast-respiring pollen, but carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and adenosine 5′-diphosphate (ADP) had less effect. After 1–4 h incubation, the respiration rate of the slow- or medium-respiring pollen lots had decreased, but was stimulated by succinate or NADH, and to a lesser degree by ADP. By 7 h, the respiration rate of all pollen lots had declined and was stimulated less by substrate, ADP or CCCP. The oxidation of NADH by tomato pollen contrasts with the failure of other pollen species to utilize this substrate; moreover, a synergistic effect of NADH and succinate was consistently observed. We conclude that the decline in respiration during incubation for up to 4 h at 30°C may reflect a lack of respiratory substrate. After 7 h, however, the decreased response to substrate indicates a loss of mitochondrial integrity or an accumulation of metabolic inhibitors. It is concluded that at 30°C (a moderately high temperature for tomato pollen), the initially high rate of respiration leads to exhaustion of the endogenous respiratory substrates (particularly in pollen with low to medium respiratory activity), but subsequently to ageing and a loss of mitochondrial activity.     

Detection and functional role of local gradients of H+-ions on the intracellular mitochondrial membrane with covalently linked pH-probe

The study is devoted to the registration of local H+ gradients on the inner membrane of mitochondria under conditions of H+ pump functioning were recorded. By using a covalently linked pH probe (fluorescein isothiocyanate), a local increase in the activity of hydrogen ions on the outer face of the inner mitochondrial membrane in the presence of the respiration substrate at increased permeability of the membrane for K+ was registered. It was also found that the buffer capacity of medium affects the respiration rate of completely uncoupled mitochondria; a change in respiration rate strictly correlates with changes in local H+ gradients on the mitochondrial membrane. It was concluded that local gradients of H+ activity can control the rate of functioning of H+ pumps. It was shown that, under certain conditions, the system of H+ pumps incorporated into succinate oxidase of mitochondria functions as a nonliner system.     

Changes in the quantitative composition of cytochromes and the functional activity of heart mitochondria during ischemia

Differential cytochrome spectra and their fourth degree derivatives were recorded at 77 degrees K temperature. During myocardial ischemia (2-h autolysis), only cytochrome c content was found to be decreased in isolated mitochondria. According to these data mitochondrial state 3 respiration with succinate decreased only in a medium without cytochrome c. Before ADP addition mitochondrial respiration increased but in a medium with cytochrome c. This was followed by an increase in the respiration rate minimized by bromthymole blue, an inhibitor of dicarboxylate transport. It is inferred that these alterations seen in ischemia are linked with increased permeability of mitochondrial membranes: external for cytochrome c, and internal for inorganic ions and low-molecular compounds.     

Influence of ethanol on alternative oxidase in mitochondria from callus-forming potato tuber discs

Ethanol, when added to the incubation medium of callus-forming potato tuber discs, inhibits callus growth and causes an increase of the mitochondrial antimycin-A resistant respiration, expressed as a percentage of state III-respiration. This increase in resistance to antimycin-A is the result of a poor development of the cytochrome pathway in tissue discs treated with ethanol. The development of the antimycin-A resistant alternative oxidase sensitive to chelator is about the same for treated and untreated discs. The respiratory control (RC) ratio of the mitochondrial respiration increases after addition of a chelator, which inhibits the alternative pathway. The RC ratio of the uninhibited mitochondrial respiration appears to be inversely related to the capacity of the alternative pathway, when mitochondrial preparations with different capacities to transfer electrons via the alternative path are compared. From the experimentally observed relation between RC-ratio and alternative oxidase capacity, it was concluded that at least half of the capacity of the alternative path is used in uninhibited state IV respiration.     

A method for isolating lung mitochondria from rabbits, rats, and mice with improved respiratory characteristics.

Previous methods for isolating lung mitochondria, particularly from rabbits, have yielded preparations which exhibit low respiratory control ratios (RCRs). We now report a method for the isolation of lung mitochondria from rabbit, rat, and mouse with RCRs, ADP/O ratios, and rates of substrate oxidation comparable to those for liver mitochondria. These mitochondrial preparations fail to oxidize exogenously added NADH and exhibit RCRs, during succinate oxidation, which closely approximate those obtained with NADH-linked substrates. However, an otherwise latent Mg²⁺-stimulated ATPase activity can still be elicited when Mg²⁺ is added to the mitochondrial incubation medium. This ATPase activity is insensitive to oligomycin and atractyloside, indicating that the source is from contaminating endoplasmic reticulum. The pH and EDTA concentration for maximum substrate oxidation and RCR were found to be 7.2 and 0.1 mm, respectively. State 4 respiration was affected by pH and EDTA concentration while state 3 respiration appeared to be independent of these two factors over the ranges studied.     

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.     

The decrease of mitochondrial substrate uptake caused by trialkyltin and trialkyl-lead compounds in chloride media and its relevance to inhibition of oxidative phosphorylation.

1. In a 100 mM-KCl medium (pH 6.8) containing ATP, triethyltin (1 muM) causes a decrease in the uptake of pyruvate, malate, citrate or beta-hydroxybutyrate by rat liver mitochondria, but no decrease is observed in a 100 mM-KNO3 medium. This response is not modified by the presence of rotenone in the incubation medium. 2. In the KCl medium at least 1 muM-triethyltin is required to cause maximum inhibition of pyruvate uptake. 3. Trimethyltin, tributyltin and the trialkyl-lead analogues at 1 muM, to varying degrees, also cause a decrease in pyruvate uptake by mitochondria only in the KCl medium. 4. Triethyltin stimulates resting respiration of mitochondria with all the substrates tested in the KCl medium but not in the KNO3 medium, yet this stimulation of O2 uptake occurs under conditions when substrate uptake is decreased. 5. In contrast, both O2 uptake during state 3 respiration and ATP synthesis when linked to the oxidation of pyruvate, malate or citrate are strongly inhibited by 1 muM-triethyltin in a KCl medium, but O2 uptake and ATP synthesis during the oxidation of beta-hydroxybutyrate are only slightly affected. In a KNO3 medium O2 uptake and ATP synthesis linked to the oxidation of all substrates are only slightly affected. 6. The relevance of the decrease in substrate uptake by mitochondria caused by triethyltin in a KCl medium to the greater sensitivity of various mitochondrial functions observed in vitro is discussed. It is concluded that decrease of matrix substrate content is probably not the major cause of the greater sensitivity of oxidative phosphorylation to triethyltin in a KCl medium observed previously.     

The effects of diphenyleneiodonium and of 2,4-dichlorodiphenyleneiodonium on mitochondrial reactions. Mechanism of the inhibition of oxygen uptake as a consequence of the catalysis of the chloride/hydroxyl-ion exchange.

1. Increasing the substrate concentration only decreased the inhibition of mitochondrial oxidations by diphenyleneiodonium or by 2,4-dichlorophenyleneiodonium by a small amount. 2. Diphenyleneiodonium and 2,4-dichlorodiphenyleneiodonium lowered the amounts of succinate, citrate and glutamate accumulated in the matrix of mitochondria in the presence of Cl-, but not in its absences. 2,4-Dichlorodiphenyleneiodonium decreased the accumulation of substrates by mitochondria oxidizing glycerol 3-phosphate. 3. Diphenyleneiodonium caused an alkalinization of the medium with an anaerobic suspension of mitochondria, which was only partly reversed by Triton X-100. 4. The rate of proton extrusion by mitochondria oxidizing succinate was not altered by diphenyleneiodonium or by 2,4-dichlorodiphenyleneiodium, although the rate of decay of proton pulses was increased. 5. 2,4-Dichlorodiphenyleneiodonium shifted the pH optimum for succinate oxidation by intact mitochondria from pH 7.2 to 8.0, whereas there was no effect on that of freeze-thawed mitochondria, which was pH 8.0. 6. The concentration of 2,4-dichlorophenyleneiodonium required to inhibit respiration by 50% is less the higher the absolute rate of oxygen uptake. 7. EDTA, but not EGTA [ethanedioxybis(ethylamine)-tetra-acetic acid] increased the inhibition of respiration by diphenyleneiodonium, 2,4-dichlorodiphenyleneiodonium and by tri-n-propyltin. 8. It is concluded that diphenyleneiodonium and 2,4-dichlorodiphenyleneiodonium limit respiration in Cl--containing medium by causing an acidification of the matrix, and that there are pH-sensitive sites in the respiratory chain between NADH and succinate, and between succinate and cytochrome c.     

Effect of the outer mitochondrial membrane fraction on mitoplast respiration

Additions of the fraction of outer mitochondrial membranes to the mitoplast suspension is shown to bring about an increase of the ADP-stimulated respiration rate, indices of respiration control and uncoupled respiration. This effect is not a result of the cytochrome c presence in the fraction of outer membranes. In the glycerol-containing medium which causes dissociation of intermembrane contacts the coupling effect of outer membranes on mitoplast respiration is not revealed. It is concluded that the outer membrane in contact with the inner one takes part in realization of the mitochondrial coupling.     

Microphotometric determination of enzymes in brain sections. V. Glycerophosphate dehydrogenases

An incubation medium was adapted for the microphotometric determination (kinetic and end-point measurements) of the activities of mitochondrial alpha-glycerophosphate dehydrogenase (GPDH) in the rat hippocampus. For comparison, the activities of the cytoplasmic NAD-linked alpha-glycerophosphate dehydrogenase were also measured. The study showed that in the demonstration of both enzymes the use of an exogenous electron carrier is necessary. Both enzymes react to phenazine methosulfate (PMS) which transfers reduction equivalents to the electron acceptor nitroblue tetrazolium chloride (NBT), thus causing a coreaction of GPDH in the demonstration of NAD-GPDH. Therefore, only the NAD-independent GPDH which is stimulated by menadione, can be selectively demonstrated in the histochemical procedure applied. The final incubation medium of GPDH consisted of 15 mM L-glycerol 3-phosphate, 5 mM NBT, 0.4 mM menadione, 7.5% polyvinyl alcohol in 0.5 M Hepes buffer, pH 8; the final pH of the incubation medium was 7.5. A linear response of the reaction lasted about 5 min. There was a linear relationship between section thickness and the formation of reaction product up to a section thickness of 14 microns. The apparent Km value at 25 degrees C was 0.6 mM. It is concluded that using menadione histochemical methods are suited to determine the mitochondrial GPDH activities in brain sections whereas using PMS a coreaction of GPDH takes place in the demonstration of NAD-GPDH, so that a histochemical quantification of NAD-GPDH cannot be recommended.     

Inhibition of Mitochondrial Permeability Transition by Low pH is Associated with Less Extensive Membrane Protein Thiol Oxidation

Ca²⁺ and inorganic phosphate-induced mitochondrial swelling and membrane protein thiol oxidation, which are associated with mitochondrial permeability transition, are inhibited by progressively decreasing the incubation medium pH between 7.2 and 6.0. Nevertheless, the detection of mitochondrial H₂O₂ production under these conditions is increased. Permeability transition induced by phenylarsine oxide, which promotes membrane protein thiol cross-linkage in a process independent of Ca²⁺ or reactive oxygen species, is also strongly inhibited in acidic incubation media. In addition, we observed that the decreased protein thiol reactivity with phenylarsine oxide or phenylarsine oxide-induced swelling at pH 6.0 is reversed by diethyl pyrocarbonate, in a hydroxylamine-sensitive manner. These results provide evidence that the inhibition of mitrochondrial permeability transition observed at lower incubation medium pH is mediated by a decrease in membrane protein thiol reactivity, related to the protonation of protein histidyl residues.     

Effect of Dequalinium on Respiration and the Inner Membrane Permeability of Rat Liver Mitochondria

The effect of the lipophilic penetrating cation dequalinium on rat liver mitochondria was studied. It was found that dequalinium dose-dependently inhibits the respiration rate of rat liver mitochondria in ADP-stimulated (V₃) and DNP-stimulated (uncoupled) states. This can be due to the fact that dequalinium is a potent inhibitor of complex III of the mitochondrial respiratory chain. It was shown that dequalinium induces a high-amplitude swelling of rat liver mitochondria. The dequalinium-induced swelling of the organelles depends on the presence of inorganic phosphate in the incubation medium: in the absence of phosphate or in the presence of the phosphate carrier inhibitor mersalyl in the phosphate-containing medium, no swelling of the mitochondria was observed. At low concentrations of dequalinium (≤10 μM), this swelling is inhibited by cyclosporin A, an inhibitor of the mitochondrial permeability transition pore. At the same time, at high concentrations of dequalinium (>10 μM), cyclosporin A becomes ineffective. It was found that in the presence of dequalinium the rate of the H₂O₂ production increased in rat liver mitochondria. Possible mechanisms of toxic effect of dequalinium chloride are discussed.     

Changes in the level of tightly-bound mitochondrial phospholipids induced by exogenous factors

The effects of some parameters of the incubation medium (tonicity, H+ and Pi concentration) on the phospholipid content in rat mitochondrial extracts prepared by normal extraction and by extraction with strongly acidified solvent mixtures, were studied. It was found that after normal extraction with a chloroform-methanol mixture, 8% to 25% of mitochondrial phospholipids can be additionally extracted by acidified solvent mixtures. The concentration of weakly extractable lipids depends on the tonicity and ionic composition of the incubation medium. It was suggested that there exist in mitochondria two lipid pools, a loosely and a tightly-bound ones; the equilibrium between these pools is effectively regulated by medium tonicity and other external factors.