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1.
Mitochondria from liver, kidney, brain, and skeletal muscle metabolized acetaldehyde. Acetaldehyde oxidation by liver and kidney mitochondria was maximal at low levels of acetaldehyde and was sensitive to rotenone, suggesting the involvement of a NAD+-dependent aldehyde dehydrogenase with a high affinity for acetaldehyde. Acetaldehyde oxidation was stimulated 50% by ADP, suggesting that, in state 4, reoxidation of NADH is rate limiting for acetaldehyde oxidation. In state 4, acetaldehyde oxidation was decreased by NAD+-dependent substrates, as well as by succinate and ascorbate. The inhibition by the latter two substrates was prevented by ADP, dinitrophenol, valinomycin, and gramicidin, but not by oligomycin. Since these compounds are linked to energy transduction and utilization, the data suggest that the inhibition is mediated via energy-dependent reversed electron transport. In state 3, all of these substrates caused considerably less inhibition of acetaldehyde oxidation, suggesting that the activity of aldehyde dehydrogenase, and not of NADH reoxidation, is probably rate limiting for acetaldehyde oxidation. The ionophores valinomycin and gramicidin stimulated acetaldehyde oxidation to a greater extent than ADP. These ionophores also stimulated acetaldehyde oxidation in the presence of ADP. Stimulation by valinomycin occurred in the presence of monovalent cations transported by this ionophore, e.g., K+, Rb+, Cs+. Stimulation by gramicidin also occurred in the presence of these cations, but did not occur with Na+ or Li+. Na+ prevents the stimulation of acetaldehyde oxidation, which occurs in the presence of gramicidin and K+. The stimulation by valinomycin and gramicidin was energy dependent and required the presence of a permeant anion. In the absence of an ionophore, potassium phosphate had no effect on acetaldehyde oxidation. These data suggest that the oxidation of acetaldehyde by rat liver and kidney mitochondria is influenced by the oxidation-reduction state of the mitochondria and by the cationic environment. With brain and muscle mitochondria, the rate of acetaldehyde oxidation increased two- to threefold as the concentration of acetaldehyde was raised from 0.167 to 0.50 mm. Acetaldehyde oxidation in these mitochondria was also sensitive; to rotenone, indicating dependence on NAD+. ADP, valinomycin, gramicidin, and succinate, compounds which either increased or decreased the rate of acetaldehyde oxidation by liver and kidney mitochondria, had no effect on acetaldehyde oxidation by muscle or brain mitochondria. In state 4, mitochondria from Becker-transplantable hepatocellular carcinoma HC-252 oxidized acetaldehyde at the same rate as liver mitochondria. However, in the presence of ADP, dinitrophenol, valinomycin and gramicidin, the rate of acetaldehyde oxidation by the tumor mitochondria was two to three times greater than that of liver mitochondria, suggesting the presence of a more active; acetaldehyde-oxidizing system in tumor than in liver mitochondria.  相似文献   

2.
The oxidation of ethanol by the liver produces acetaldehyde, which is a highly reactive compound. Low concentrations of acetaldehyde inhibited mitochondrial respiration with glutamate, β-hydroxybutyrate, or α-ketoglutarate as substrates, but not with succinate or ascorbate. High concentrations led to respiratory inhibition with all substrates. Inhibition of succinate- and ascorbate-linked oxidation by acetaldehyde correlates with the inhibition of the activities of succinic dehydrogenase and cytochrome oxidase. A site more sensitive to acetaldehyde appears to be localized prior to the NADH-ubiquinone oxidoreductase segment of the respiratory chain. Acetaldehyde inhibits energy production by the mitochondria, as evidenced by its inhibition of respiratory control, oxidative phosphorylation, the rate of phosphorylation, and the ATP-32P exchange reaction. Energy utilization is also inhibited, in view of the decrease in both substrate- and ATP-supported Ca2+ uptake, and the reduction in Ca2+-stimulated oxygen uptake and ATPase activity. The malate-aspartate, α-glycerophosphate, and fatty acid shuttles for the transfer of reducing equivalents, and oxidation by mitochondria, were highly sensitive to acetaldehyde. Acetaldehyde also inhibited the uptake of anions which participate in the shuttles. The inhibition of the shuttles is apparently caused by interference with NAD+-dependent state 3 respiration and anion entry and efflux. Ethanol (6–80 mm) had no significant effect on oxygen consumption, anion uptake, or mitochondrial energy production and utilization. The data suggest that acetaldehyde may be implicated in some of the toxic effects caused by chronic ethanol consumption.  相似文献   

3.
Isolated, intact rat liver mitochondria, without extraneous substrates but loaded with Ca2+ (20 nmol/mg), can be observed to release Ca2+ when treated with ruthenium red. Such release can be inhibited by 0.33 mM dlisocitrate but not by 10 mM dl-β-hydroxybutyrate. Assays of NADP+, NADPH, NAD+, and NADH revealed that only the reduction of NADP+ can be linked with such inhibition of Ca2+ release, not that of NAD+. Since ruthenium redinsensitive Ca2+ release is a physiological (but normally masked) process, this experimental approach avoids some potential problems ascribed to strong pyridine nucleotide oxidation. It is suggested that specific NADP+:NADPH dependent reactions are part of a physiological mechanism regulating Ca2+ release/retention.  相似文献   

4.
Summary

Rat liver mitochondria have a specific Ca2+ release pathway which operates when NAD+ is hydrolysed to nicotinamide and ADPribose. NAD+ hydrolysis is Ca2+-dependent and inhibited by cyclosporine A (CSA). Mitochondrial Ca2+ release can be activated by the prooxidant t-butylhydroperoxide (tbh) or by gliotoxin (GT), a fungal metabolite of the epipolythiodioxopiperazine group. Tbh oxidizes NADH to NAD+ through an enzyme cascade consisting of glutathione peroxidase, glutathione reductase, and the energy linked transhydrogenase, whereas GT oxidizes some vicinal thiols to the disulfide form, a prerequisite for NAD+ hydrolysis. We report now that rat skeletal muscle mitochondria also contain a specific Ca2+ release pathway activated by both tbh and GT. Ca2+ release increases with the mitochondrial Ca2+ load, is completely inhibited in the presence of CSA, and is paralleled by pyridine nucleotide oxidation. In the presence of tbh and GT, mitochondria do not lose their membrane potential and do not swell, provided continuous release and re-uptake of Ca2+ (‘Ca2+ cycling’) is prevented. These data support the notion that both tbh- and GT-induced Ca2+ release are not the consequence of an unspecific increase of the inner membrane permeability (‘pore’ formation). Tbh induces Ca2+ release from rat skeletal muscle less efficiently than from liver mitochondria indicating that the coupling between tbh and NADH oxidation is much weaker in skeletal muscle mitochondria. This conclusion is corroborated by a much lower glutathione peroxidase activity in skeletal muscle than in liver mitochondria. The prooxidant-dependent pathway promotes, under drastic conditions (high mitochondrial Ca2+ loads and high tbh concentrations), Ca2+ release to about the same extent and rate as the Na+/Ca2+ exchanger. This renders the prooxidant-dependent pathway relevant in the pathophysiology of mitochondrial myopathies where its activation by an increased generation of reactive oxygen species probably results in excessive Ca2+ cycling and damage to mitochondria.  相似文献   

5.
The respiratory rate of rat liver mitochondria in the presence of NADH as exogenous substrate is enhanced by the addition of CaCl2 (> 50 μM) when inorganic phosphate is present in the medium. The Ca-induced oxidation of NADH is inhibited by rotenone but is not affected by uncoupling agents. EDTA, which does not reverse the swelling of mitochondria which occurs in the presence of Ca2+ and phosphate, is able to inhibit reversibly the Ca-stimulated NADH oxidation. A stimulation of the rate of oxidation of NADH by Ca2+ is also observed in mitochondria partially swollen in a hypotonic medium.  相似文献   

6.
EGTA (ethanedioxybis(ethylamine)tetra-acetic acid) induced a release of Ca2+ from mitochondria isolated from both rat liver and rat heart that was inhibited by Ruthenium Red. The concentration of Ruthenium Red giving half-maximal inhibition was about 350 pmol/mg of protein, a value approximately 7 times greater than that giving half-maximal inhibition of the initial rate of Ca2+ transport. The EGTA-induced release of Ca2+ was temperature-dependent and was inhibited by the local anaesthetic, nupercaine.Pi, acetate, and tributyltin in the presence of Cl?, inhibited the Ruthenium Red-sensitive Ca2+ release induced by EGTA, whereas these agents enhanced the Ruthenium Red-insensitive release of Ca2+ induced by acetoacetate in liver and heart mitochondria and by Na+ in heart mitochondria.  相似文献   

7.
The adsorption of Ca2+ to the mitochondria ofSaccharomyces cerevisiae was investigated and it was found that, in contrast with animal mitochondria, Ca2+ is not accumulated through an energydependent process but is more probably adsorbed to mitochondrial membranes. The adsorption magnitude depends both on the amount of added calcium and on the ionic composition of the medium. It was found by study of the effect of divalent cations on the respiratory activity of yeast mitochondria that (a) Ca2+ and Mg2+ inhibit their oxidation competitively with succinate or citrate, the oxidation of NADH not being affected; (b) stimulation of oxidation of NADH and inhibition of oxidation of citrate and succinate may be observed with Ca2+ in the mitochondria ofTorulopsis utilis and with Co2+ in the mitochondria ofSaccharomyces cerevisiae; (c) Zn2+ inhibits the oxidation of NADH and of citrate; (d) the rate of oxidation of NADH in the presence of Cd2+ is several-fold greater than State 3 activity—on the other hand, oxidation of suceinate and citrate is inhibited by cadmium. In comparison with animal mitochondria, the fate of Ca2+ as well as the effects of other divalent cations on the respiratory activity of yeast mitochondria are different.  相似文献   

8.
Inside-out submitochondrial particles from both potato (Solanum tuberosum L. cv. Bintje) tubers and pea (Pisum sativum L. cv. Oregon) leaves possess three distinct dehydrogenase activities: Complex I catalyzes the rotenone-sensitive oxidation of deamino-NADH, NDin(NADPH) catalyzes the rotenone-insensitive and Ca2+-dependent oxidation of NADPH and NDin(NADH) catalyzes the rotenone-insensitive and Ca2+-independent oxidation of NADH. Diphenylene iodonium (DPI) inhibits complex I, NDin(NADPH) and NDin (NADH) activity with a Ki of 3.7, 0.17 and 63 µM, respectively, and the 400-fold difference in Ki between the two NDin made possible the use of DPI inhibition to estimate NDin (NADPH) contribution to malate oxidation by intact mitochondria. The oxidation of malate in the presence of rotenone by intact mitochondria from both species was inhibited by 5 µM DPI. The maximum decrease in rate was 10–20 nmol O2 mg?1 min?1. The reduction level of NAD(P) was manipulated by measuring malate oxidation in state 3 at pH 7.2 and 6.8 and in the presence and absence of an oxaloacetate-removing system. The inhibition by DPI was largest under conditions of high NAD(P) reduction. Control experiments showed that 125 µM DPI had no effect on the activities of malate dehydrogenase (with NADH or NADPH) or malic enzyme (with NAD+ or NADP+) in a matrix extract from either species. Malate dehydrogenase was unable to use NADP+ in the forward reaction. DPI at 125 µM did not have any effect on succinate oxidation by intact mitochondria of either species. We conclude that the inhibition caused by DPI in the presence of rotenone in plant mitochondria oxidizing malate is due to inhibition of NDin(NADPH) oxidizing NADPH. Thus, NADP turnover contributes to malate oxidation by plant mitochondria.  相似文献   

9.
Chronic ethanol feeding to rats produces changes in hepatic mitochondria which persist in the absence of ethanol metabolism. The integrity of isolated mitochondria is well preserved, as evidenced by unchanged activities of latent, Mg2+- and dinitrophenol-stimulated ATPase activity, and unaltered permeability to NADH. With succinate or ascorbate as substrates, oxygen uptake by mitochondria from ethanol-fed rats was decreased compared to pair-fed controls. The decrease was comparable under state 4 or state 3 conditions, or in the presence of an uncoupler. However, with the NAD+-dependent substrates, ADP-stimulated oxygen consumption (state 3) was decreased to a greater extent than state 4 or uncoupler-stimulated oxygen consumption in mitochondria from ethanol-fed rats. This suggests that the decrease in energy-dependent oxygen consumption at site I may be superimposed upon damage to the respiratory chain. Using NAD+-dependent substrates (glutamate, α-ketoglutarate or β-hydroxybutyrate) the respiratory control ratio and the PO ratio of oxidative phosphorylation were significantly decreased in mitochondria isolated from the livers of rats fed ethanol. By contrast, when succinate or ascorbate served as the electron donor these functions were unchanged. The rate of phosphorylation is decreased 70% with the NAD+-dependent substrates because of a decreased flux of electrons, as well as a lower efficiency of oxidative phosphorylation. With succinate and ascorbate as substrates, the rate of phosphorylation is decreased 20–30%, owing to a decreased flux of electrons. These data suggest the possibility that, in addition to effects on the respiratory chain, energy-coupling site I may be damaged by ethanol feeding. Energy-dependent Ca2+ uptake, supported by either substrate oxidation or ATP hydrolysis, was inhibited by chronic ethanol feeding.Concentrations of acetaldehyde (1–3 mm) which inhibited phosphorylation associated with the oxidation of NAD+-dependent substrates had no effect on that of succinate or ascorbate. Many of the effects of chronic ethanol feeding on mitochondrial functions are similar to those produced by acetaldehyde in vitro.  相似文献   

10.
Some features of the Ca2+-transport system in mitochondria of the yeast Endomyces magnusii are considered. The Ca2+ uniporter was shown to be specifically activated by low concentrations of physiological modulators such as ADP, NADH, spermine, and Ca2+ itself. The Na+-independent system responsible for Ca2+ release from Ca2+-preloaded yeast mitochondria was characterized. The rate of the Ca2+ release was proportional to the Ca2+ load, insensitive to cyclosporin A and to Na+, inhibited by La3+, TPP+, Pi, and nigericin, while being activated by spermine. We conclude that Ca2+ release from preloaded E. magnusii yeast mitochondria is mediated by a Na+-independent pathway, very similar to that in mitochondria from nonexcitable mammalian tissues. A scheme describing an arrangement of the Ca2+ transport system of yeast mitochondria is proposed.  相似文献   

11.
Mitochondria from skeletal muscle, heart and liver of strain 129/ReJ-dy dystrophic mice and their littermate controls were characterized with respect to their respiratory and phosphorylating activities. Skeletal muscle mitochondria from dystrophic mice showed significantly lower state 3 respiratory rates than controls with both pyruvate + malate and succinate as substrates (P < 0.01). ADP/O and Ca2+/O ratios were found to be normal. A decreased rate of NADH oxidation (0.01 <P < 0.05) by sonicated mitochondrial suspensions from dystrophic mice was also seen. High respiratory rates with ascorbate + phenazine methosulfate as substrates indicated that cytochrome oxidase was not rate limiting in the oxidation of either pyruvate + malate or succinate. Skeletal muscle mitochondria from dystrophic mice showed no deficiency in any of the cytochromes or coenzyme Q. Mg2+-stimulated ATPase activity was higher in dystrophic muscle mitochondria than in controls, but basal and oligomycin-insensitive activities were virtually identical to those of controls. A significant reduction in the intramitochondrial NAD+ content (0.01 <P < 0.02) was seen in dystrophic skeletal muscle as compared to controls. Heart mitochondria from dystrophic mice showed similar, though less extensive abnormalities while liver mitochondria were essentially normal. We concluded from these results that skeletal muscle mitochondria from strain 129 dystrophic mice possess impairments in substrate utilization which may result from (1) an abnormality in the transfer of electrons on the substrate side of coenzyme Q in the case of succinate oxidation; (2) a defect on the path of electron flow from NADH to cytochrome c, and (3) a deficiency of NAD+ in the case of NAD+-linked substrates.  相似文献   

12.
The addition of calcium ions (Ca2+) to rat liver mitochondria, under conditions of rapid accumulation of 10–40 nmol Ca2+/mg protein, inhibited the oxidation of long and medium chain fatty acids to ketone bodies, whereas higher quantities of Ca2+ activated the process. The mitochondrial NADH:NAD ratio exhibited corresponding depression and elevation. Both inhibitory and stimulatory actions of Ca2+ were operative in liver mitochondria from fed and fasted rats and appear to be localized in the mitochondrial inner membranematrix region. These observations may signify involvement of Ca2+ in the regulation of fatty acid oxidation and ketogenesis.  相似文献   

13.
This study aims at characterizing NAD(P)H dehydrogenases on the inside and outside of the inner membrane of mitochondria of one phosphoenolpyruvate carboxykinase??crassulacean acid metabolism plant, Hoya carnosa. In crassulacean acid metabolism plants, NADH is produced by malate decarboxylation inside and outside mitochondria. The relative importance of mitochondrial alternative NADH dehydrogenases and their association was determined in intact??and alamethicin??permeabilized mitochondria of H. carnosa to discriminate between internal and external activities. The major findings in H. carnosa mitochondria are: (i) external NADPH oxidation is totally inhibited by DPI and totally dependent on Ca2+, (ii) external NADH oxidation is partially inhibited by DPI and mainly dependent on Ca2+, (iii) total NADH oxidation measured in permeabilized mitochondria is partially inhibited by rotenone and also by DPI, (iv) total NADPH oxidation measured in permeabilized mitochondria is partially dependent on Ca2+ and totally inhibited by DPI. The results suggest that complex I, external NAD(P)H dehydrogenases, and internal NAD(P)H dehydrogenases are all linked to the electron transport chain. Also, the total measurable NAD(P)H dehydrogenases activity was less than the total measurable complex I activity, and both of these enzymes could donate their electrons not only to the cytochrome pathway but also to the alternative pathway. The finding indicated that the H. carnosa mitochondrial electron transport chain is operating in a classical way, partitioning to both Complex I and alternative Alt. NAD(P)H dehydrogenases.  相似文献   

14.
Low concentrations of HPE and MLA inhibited state 3 respiration of rat liver mitochondria in the presence of different NAD+-dependent substrates. MLA appeared to be more active than HPE. High aldehyde concentrations inhibited the state 3 respiration with succinate. The restraint of succinate oxidation by HPE and MLA and of glutamate plus malate oxidation by MLA correlated with the inhibition of succinate and glutamate dehydrogenase activites, respectively. HPE inhibited glutamate dehydrogenase at concentrations higher than those affecting glutamate oxidation. Malate dehydrogenase activity was slightly sensitive to HPE and MLA. Both aldehydes inhibited NADH oxidation by freeze-thawed mitochondria. These results suggest the existence of a site particularly sensitive to aldehydes in the electron transport chain between the specific NAD+-linked dehydrogenases and ubiquinone.  相似文献   

15.
Chlorotetracycline inhibits the uncoupled oxidation of exogenous NADH by Jerusalem artichoke (Helianthus tuberosus L.) mitochondria extensively (over 80%) and rapidly (inhibition complete in 10 s) in the presence of added Ca2+. Half-maximal inhibition is observed at 15 μM chlorotetracycline in the presence of 2 mM Ca2+. The oxidation of succinate is only affected marginally by chlorotetracycline plus Ca2+. The inhibition of NADH oxidation and the fluorescence of CTC are well correlated. Mn2+ is the only other cation which shows an (increased) inhibition in the presence of chlorotetracycline. The inhibition by Ca2+ and chlorotetracycline disappears at acid pH, and the pH optimum in their presence is 6.4. The inhibition caused by other lipid-soluble Ca2+-chelators is not reversible or is enhanced by the addition of excess Ca2+. In contrast, inhibition caused by relatively water-soluble chelators is completely reversed by added Ca2+. It is suggested that a neutral 1:2 complex is formed between Ca2+ and chlorotetracycline which can substitute for Ca2+ bound at sites in the lipophilic phase of the inner mitochondrial membrane, which are essential for the activity of the external NADH dehydrogenase.  相似文献   

16.
N.-E.L. Saris  P. Bernardi 《BBA》1983,725(1):19-24
The effect of Sr2+ on the set point for external Ca2+ was studied in rat heart and liver mitochondria with the aid of a Ca2+-sensitive electrode. In respiring mitochondria the set point is determined by the rates of Ca2+ influx on the Ca2+ uniporter and efflux by various mechanisms. We studied the Ca2+-Na+ exchange pathway in heart mitochondria and the Δψ-modulated efflux pathway in liver mitochondria. Prior accumulation of Sr2+ was found to shift the set points towards lower external Ca2+ both in heart mitochondria under conditions of Ca2+-Na+ exchange and in liver mitochondria under conditions that should promote opening of the Δψ-modulated pathway. The effect on the set point was found to be due to inhibition of Ca2+ efflux by Sr2+ taken up by the mitochondria, while Sr2+ efflux was too slow to be measurable.  相似文献   

17.
Ca2+ plays a central role in energy supply and demand matching in cardiomyocytes by transmitting changes in excitation-contraction coupling to mitochondrial oxidative phosphorylation. Matrix Ca2+ is controlled primarily by the mitochondrial Ca2+ uniporter and the mitochondrial Na+/Ca2+ exchanger, influencing NADH production through Ca2+-sensitive dehydrogenases in the Krebs cycle. In addition to the well-accepted role of the Ca2+-triggered mitochondrial permeability transition pore in cell death, it has been proposed that the permeability transition pore might also contribute to physiological mitochondrial Ca2+ release. Here we selectively measure Ca2+ influx rate through the mitochondrial Ca2+ uniporter and Ca2+ efflux rates through Na+-dependent and Na+-independent pathways in isolated guinea pig heart mitochondria in the presence or absence of inhibitors of mitochondrial Na+/Ca2+ exchanger (CGP 37157) or the permeability transition pore (cyclosporine A). cyclosporine A suppressed the negative bioenergetic consequences (ΔΨm loss, Ca2+ release, NADH oxidation, swelling) of high extramitochondrial Ca2+ additions, allowing mitochondria to tolerate total mitochondrial Ca2+ loads of > 400 nmol/mg protein. For Ca2+ pulses up to 15 μM, Na+-independent Ca2+ efflux through the permeability transition pore accounted for ~ 5% of the total Ca2+ efflux rate compared to that mediated by the mitochondrial Na+/Ca2+ exchanger (in 5 mM Na+). Unexpectedly, we also observed that cyclosporine A inhibited mitochondrial Na+/Ca2+ exchanger-mediated Ca2+ efflux at higher concentrations (IC50 = 2 μM) than those required to inhibit the permeability transition pore, with a maximal inhibition of ~ 40% at 10 μM cyclosporine A, while having no effect on the mitochondrial Ca2+ uniporter. The results suggest a possible alternative mechanism by which cyclosporine A could affect mitochondrial Ca2+ load in cardiomyocytes, potentially explaining the paradoxical toxic effects of cyclosporine A at high concentrations. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.  相似文献   

18.
The NADH and NAD+ dependent reactions catalyzed by glutamate dehydrogenase (GDH) from sterile cultures of Lemna minor are completely inactivated by EDTA. The activities of both reactions can be fully restored by addition of Ca2+ and to a lesser extent Mn2+, Zn2+, Sr2+ or La3+, whereas Mg2+ reactivates only the NAD+ dependent reaction. Activation of the NADH reaction by Ca2+ has been studied by using partially purified, EDTA pretreated, and Mg2+ saturated GDH preparations. Saturation kinetic curves with Ca2+ were always sigmoidal, whereas saturation plots for the 3 substrates of the aminating reaction at various fixed Ca2+ concentrations showed normal Michaelis kinetics. However, a pronounced substrate inhibition at low Ca2+ levels was found, particularly with NH4+ and NADH. Product inhibition studies revealed unchanged enzyme substrate binding characteristics for NADH and 2-oxoglutarate in the Ca2+ free enzyme. A drastic alteration was established for the third substrate NH4+. The kinetic data suggest that Ca2+ governs an equilibrium between a catalytically inactive (Ca2+ free) and an active (Ca2+ saturated) enzyme form. Inactivation by removal of Ca2+ is related to an alteration in the binding characteristics or binding sequence of the substrate NH4+.  相似文献   

19.
The cardiac Na+/Ca2+ exchanger (NCX) is the major Ca2+ efflux pathway on the sarcolemma, counterbalancing Ca2+ influx via L-type Ca2+ current during excitation-contraction coupling. Altered NCX activity modulates the sarcoplastic reticulum Ca2+ load and can contribute to abnormal Ca2+ handling and arrhythmias. NADH/NAD+ is the main redox couple controlling mitochondrial energy production, glycolysis, and other redox reactions. Here, we tested whether cytosolic NADH/NAD+ redox potential regulates NCX activity in adult cardiomyocytes. NCX current (INCX), measured with whole cell patch clamp, was inhibited in response to cytosolic NADH loaded directly via pipette or increased by extracellular lactate perfusion, whereas an increase of mitochondrial NADH had no effect. Reactive oxygen species (ROS) accumulation was enhanced by increasing cytosolic NADH, and NADH-induced INCX inhibition was abolished by the H2O2 scavenger catalase. NADH-induced ROS accumulation was independent of mitochondrial respiration (rotenone-insensitive) but was inhibited by the flavoenzyme blocker diphenylene iodonium. NADPH oxidase was ruled out as the effector because INCX was insensitive to cytosolic NADPH, and NADH-induced ROS and INCX inhibition were not abrogated by the specific NADPH oxidase inhibitor gp91ds-tat. This study reveals a novel mechanism of NCX regulation by cytosolic NADH/NAD+ redox potential through a ROS-generating NADH-driven flavoprotein oxidase. The mechanism is likely to play a key role in Ca2+ homeostasis and the response to alterations in the cytosolic pyridine nucleotide redox state during ischemia-reperfusion or other cardiovascular diseases.  相似文献   

20.
Summary Rapid uptake of Ba2+ by respiring rat liver mitochondria is accompanied by a transient stimulation of respiration. Following accumulation of Ba2+, e.g. at a concentration of 120 nmol per mg protein, the mitochondria exhibit reduced rates of state 3 and uncoupler-stimulated respiration. ADP-stimulated respiration is inhibited at a lower concentration of Ba2+ than is required to affect uncoupler-stimulated respiration, suggesting a distinct effect of Ba2+ on mechanisms involved in synthesis of ATP. Ba2+, which has an ionic radius similar to that of K+, inhibits unidirectional K+ flux into respiring rat liver mitochondria. This effect on K+ influx is observable at concentrations of Ba2+, e.g. 23 to 37 nmol per mg protein, which cause no significant change in state 4 or uncoupler-stimulated respiration. The accumulated Ba2+ decreases the measuredV max of K+ influx, while having little effect on the apparentK m for K+. The inhibition of K+ influx by Ba2+ is seen in the presence and absence of mersalyl, an activator of K+ influx. In contrast, under the conditions studied, Ba2+ has no apparent effet on the rate of unidirectional K+ efflux. These data are consistent with the idea that K+ may enter and leave mitochondria via spearate mechanisms.  相似文献   

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