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1.
Accumulation of Ca2+ ions by rat liver mitochondria increases the fluorescence of the reduced pyridine nucleotides in the presence of rotenone. The fluorescence increase is sensitive to the uncouplers of the oxidative phosphorylation and to the inhibitors of the electron transfer, providing the release of the accumulated Ca2+ from mitochondria. The substantial change of the fluorescence occurs in the presence of acetate, when the accumulated Ca2+ is present in the intramitochondrial space as soluble salt. Addition of Ca2+ to water solution of NADH and NADPH is followed by the slight increase of the fluorescence, while the same nucleotides dissolved in the more hydrophobic medium (methanol) considerably increase the fluorescence level by addition of Ca2+. The increase of the fluorescence of NAD(P)H in the mitochondria due to the accumulation of Ca2+ is considered not to be caused by the alkalinization of the intramitochondrial space but by the formation of the nucleotide-metal complex, localized at least partly at a hydrophobic phase.Abbreviations used FCCP p-trifluoromethoxycarbonylcyanidephenylhydrazone - TMPD tetramethyl-p-phenylenediamine  相似文献   

2.
Summary

Rat liver mitochondria contain a specific Ca2+ release pathway which operates when intramitochondrial NAD+ is hydrolyzed to ADPribose and nicotinamide. The molecular details of this pathway are incompletely understood. It has been reported that NAD+ hydrolysis and therefore Ca2+ release stimulated by t-butylhydroperoxide is prevented by 4-hydroxynonenal (HNE). The reason underlying inhibition by HNE, however, remained unclear. It has also been reported that NAD+ hydrolysis and Ca2+ release are stimulated when some vicinal thiols are cross-linked, as shown with phenylarsine oxide or gliotoxin (GT). We now show that HNE also prevents the GT-induced Ca2+ release, but only when given before GT. Conversely, GT stimulates Ca2+ release only when given before HNE. Inhibition of Ca2+ release by HNE is reduced by its preincubation with thiol compounds, the effectiveness of which increases with decreasing pKa of their sulfhydryl group. Preincubation of HNE with glutathione at high, but not at low, pH similarly reduces inhibition of Ca2+ release by HNE. These findings provide evidence that HNE inhibition of Ca2+ release is due to a modification of mitochondrial thiolates in a way that their cross-linking is prevented, and give further insight into the regulation of Ca2+ release from intact mitochondria.  相似文献   

3.
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   

4.
Ca2+ efflux from rat liver mitochondria in the presence of glutamate is stimulated by a decrease in pH from 7.3 to 6.8 and the rate is dependent on the phosphate concentration. During Ca2+ (13 μm) uptake and release at low pH (+ phosphate), swelling is minimal, but a large oxidation of pyridine nucleotides and sustained membrane depolarization occurs. The depolarization (but not Ca2+ efflux) is reversed by ruthenium red. An absolute requirement for phosphate to support Ca2+ efflux is demonstrated by using acetate or lactate to support Ca2+ uptake (efflux is depressed at pH 6.8). Preincubation with mersalyl, to block phosphate movements, with subsequent phosphate addition preceeding Ca2+ uptake also inhibits efflux. β-Mercaptoethanol then stimulates efflux concomittent with membrane repolarization. Ca2+ efflux is not a simple result of collapse of ΔpH since nigericin inhibits phosphate transport and Ca2+ release. Following Ca2+ uptake at pH 6.8, respiratory inhibition occurs, but oxygen consumption coupled to ATP synthesis can be stimulated by succinate (+ rotenone). Addition of succinate allows reuptake of Ca2+, reduction of pyridine nucleotides, and repolarization of the membrane potential. Respiratory inhibition is also seen with nigericin, but no Ca2+ efflux is observed. Coupled respiration with glutamate is seen at pH 6.8 following Ca2+ uptake in the presence of lactate with subsequent addition of phosphate to promote Ca2+ efflux. We conclude that Ca2+ efflux is not a consequence of respiratory inhibition, but is mediated solely by phosphate movements. The inhibitory effect of Mg2+ on Ca2+ efflux is probably due to Mg2+-dependent inhibition of the Ca2+ diffusion potential so that the compensatory increase in ΔpH due to membrane depolarization does not occur and phosphate entry is slowed.  相似文献   

5.
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   

6.
Abstract

The seleno-organic compound ebselen mimics the glutathione-dependent, hydroperoxide reducing activity of glutathione peroxidase. The activity of glutathione peroxidase determines the rate of hydroperoxide-induced Ca2+ release from mitochondria. Ebselen stimulates Ca2+ release from mitochondria, accelerates mitochondrial respiration and uncoupling, and induces mitochondrial swelling, indicating a deterioration of mitochondrial function. These manifestations are abolished by cyclo-sporine A, a potent inhibitor of the mitochondrial permeability transition. However, when ebselen-induced Ca2+ cycling is prevented with ruthenium red, an inhibitor of the Ca2+ uniporter, or by chelation of extramitochondrial Ca2+ by EGTA, no detectable elevation of swelling or uncoupling is observed. The release of Ca2+ from mitochondria is delayed in the absence of rotenone, i.e. when pyridine nucleotides are maintained in the reduced state due to succinate-driven reversed electron flow. We suggest that ebselen induces Ca2+ release from intact mitochondria via an NAD+ hydrolysis-dependent mechanism.  相似文献   

7.
Summary

The reduction of molecular oxygen to water provides most of the energy that enables higher organisms to exist. Oxygen reduction is a mixed blessing because incompletely reduced oxygen species are more reactive than molecular oxygen in the ground state and can, when out of control, damage biological molecules. However, incompletely reduced oxygen species may also serve useful functions, as exemplified by their control of mitochondrial Ca2+ homeostasis, the understanding of which has improved greatly during the last few years. Hydrogen peroxide can stimulate a specific Ca2+ release pathway from intact mitochondria by oxidizing mitochondrial pyridine nucleotides through the activities of glutathione peroxidase, glutathione reductase, and the energy-linked transhydrogenase. Other pro-oxidants such as menadione, alloxan, or divicine also stimulate the specific Ca2+ release, because they furnish NAD+. The specific Ca2+ release requires for its activation the hydrolysis of intramitochondrial NAD+ to ADPribose and nicotinamide, and is prevented by inhibitors of NAD+ hydrolysis and protein monoADPribosylation. Recent experiments reveal that NAD+ hydrolysis and therefore Ca2+ release is regulated by vicinal thiols in mitochondria. When reduced or alkylated, the thiols prevent hydrolysis, but when they are cross-linked hydrolysis takes place. Cyclosporine A, which also prevents NAD+ hydrolysis, acts distal of these vicinal thiols. Since mitochondrial Ca2+ handling is physiologically relevant, its control by pro-oxidants must be added to the growing list of their useful functions.  相似文献   

8.
Intra- and extra-mitochondrial Ca2+ participates in vital cellular processes. This work investigates the influence of 4-hydroxynonenal (HNE) on pro-oxidant-induced Ca2+ release from rat liver mitochondria. Ca2+ movements across the mitochondrial inner membrane, the pyridine nucleotide redox state and pyridine (nicotinamide) nucleotide hydrolysis were analysed. HNE did not influence Ca2+ uptake by mitochondria, but inhibited in a concentration-dependent manner Ca2+ release induced by t-butylhydroperoxide (tbh). Total inhibition was achieved with about 50 microM-HNE. Ca2+ release induced by the pro-oxidant alloxan was also inhibited by HNE. Oxidation of pyridine nucleotides, induced by tbh through the concerted action of glutathione peroxidase, glutathione reductase and the energy-linked transhydrogenase, was not affected by up to 50 microM-HNE. In contrast, HNE inhibited pyridine nucleotide hydrolysis in a concentration-dependent manner. The data suggest that HNE toxicity may be in part attributed to an impaired intramitochondrial Ca2+ homeostasis.  相似文献   

9.
In this study we used tightly-coupled mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts, possessing a respiratory chain with the usual three points of energy conservation. High-amplitude swelling and collapse of the membrane potential were used as parameters for demonstrating induction of the mitochondrial permeability transition due to opening of a pore (mPTP). Mitochondria from Y. lipolytica, lacking a natural mitochondrial Ca2+ uptake pathway, and from D. magnusii, harboring a high-capacitive, regulated mitochondrial Ca2+ transport system (Bazhenova et al. J Biol Chem 273:4372–4377, 1998a; Bazhenova et al. Biochim Biophys Acta 1371:96–100, 1998b; Deryabina and Zvyagilskaya Biochemistry (Moscow) 65:1352–1356, 2000; Deryabina et al. J Biol Chem 276:47801–47806, 2001) were very resistant to Ca2+ overload. However, exposure of yeast mitochondria to 50–100 μM Ca2+ in the presence of the Ca2+ ionophore ETH129 induced collapse of the membrane potential, possibly due to activation of the fatty acid-dependent Ca2+/nH+-antiporter, with no classical mPTP induction. The absence of response in yeast mitochondria was not simply due to structural limitations, since large-amplitude swelling occurred in the presence of alamethicin, a hydrophobic, helical peptide, forming voltage-sensitive ion channels in lipid membranes. Ca2+- ETH129-induced activation of the Ca2+/H+-antiport system was inhibited and prevented by bovine serum albumin, and partially by inorganic phosphate and ATP. We subjected yeast mitochondria to other conditions known to induce the permeability transition in animal mitochondria, i.e., Ca2+ overload (in the presence of ETH129) combined with palmitic acid (Mironova et al. J Bioenerg Biomembr 33:319–331, 2001; Sultan and Sokolove Arch Biochem Biophys 386:37–51, 2001), SH-reagents, carboxyatractyloside (an inhibitor of the ADP/ATP translocator), depletion of intramitochondrial adenine nucleotide pools, deenergization of mitochondria, and shifting to acidic pH values in the presence of high phosphate concentrations. None of the above-mentioned substances or conditions induced a mPTP-like pore. It is thus evident that the permeability transition in yeast mitochondria is not coupled with Ca2+ uptake and is differently regulated compared to the mPTP of animal mitochondria.  相似文献   

10.
Ca2+ uptake and the effect of the uptake inhibitors palmitoyl-CoA and palmitoylcarnitine were examined in two preparations of dog cardiac mitochondria. Mitochondria prepared by using the Nagarse technique was 2.5-fold more active in respiration-dependent Ca2+ uptake than were mitochondria isolated by using the Polytron procedure. Palmitoyl-CoA and palmitoylcarnitine inhibited Ca2+ uptake in both preparations uncompetitively, with Ki,app 0.4 and 20μm. Ca2+-uptake rates were related to, or influenced by, the concentration of mitochondrial reduced nicotinamide nucleotides, with uptake slowing as this concentration decreased. When most of the nicotinamide nucleotides was oxidized, Ca2+ release and respiratory stimulation were observed. In the presence of Ruthenium Red and palmitoyl-CoA, oxidation of nicotinamide nucleotides was abolished and the time to Ca2+ release was shortened corresponding to the time of onset of nicotinamide nucleotide oxidation in the absence of Ruthenium Red. The results suggest that NAD(P)H oxidation in the presence of rotenone was a consequence of Ca2+ re-uptake and that net Ca2+ release could be observed as reduced nicotinamide nucleotide concentrations declined. Although nicotinamide nucleotide oxidation occurred in the presence of rotenone, it was not linked in an apparent manner to acyl-group metabolism (palmitoylcarnitine was less effective than palmitoyl-CoA). Therefore either a by-pass of the rotenone block or a direct interaction of NAD(P)H with the Ca2+-uptake process was possible. Loss of NADH occurred before respiratory stimulation, and this loss may relate to decreased coupling efficiency at sites 2 and 3 of the respiratory chain, as suggested by others [Bhuvaneswaran & Wadkins (1978) Biochem. Biophys. Res. Commun. 82, 648–654].  相似文献   

11.
Parallel measurements of Ca2+ uptake, oxygen consumption, endogenous Mg2+ efflux, and swelling in rotenone-poisoned rat liver and rat heart mitochondria showed that heart mitochondria is much more resistant to uncoupling by Ca2+ in the presence of phosphate than rat liver mitochondria. The extent of Mg2+ efflux and swelling induced by Ca2+ accumulation are much less pronounced in heart mitochondria. Uncoupling and swelling in liver mitochondria seem to result from the loss of membrane-bound Mg2+ as a consequence of Ca2+ recycling across the membrane as induced by phosphate. Exogenous Mg2+ protects liver mitochondria against the deleterious effects of Ca2+ by inhibiting a ruthenium red-insensitive Ca2+ efflux induced by phosphate. Phosphate does not induce recycling of Ca2+ in heart mitochondria. On the other hand, heart mitochondria respiring on NAD-linked substrates or with succinate in the absence of rotenone behave like liver mitochondria with respect to the alterations caused by Ca2+ recycling. In heart mitochondria the recycling of Ca2+ is related to the redox state of pyridine nucleotides, which suggests that the ruthenium red-insensitive efflux of Ca2+ is subject to metabolic control. In addition it has been observed that Sr2+does not undergo cyclic movements across the membrane. The data indicate that the efflux pathway is more specific for Ca2+ than the ruthenium red-sensitive influx transporter.  相似文献   

12.
R.P. Holmes  P.R. Stewart 《BBA》1979,545(1):94-105
A method for the isolation of coupled mitochondria from the acellular slime mould Physarum polycephalum is described. The mitochondria oxidize respiratory substrates at rates comparable to those of mitochondria from other micro-organisms and show similar responses to respiratory inhibitors. ADP/O values approach similar values to those obtained with mitochondria from higher organisms: 3 with NAD-linked substrates, 2 with succinate, and 1 with ascorbate-TMPD.Mitochondria actively take up low concentrations of Ca2+ with stimulation of their respiration. With succinate or pyruvate-malate as substrates respiratory responses are depressed by Ca2+ concentrations in excess of 200 μM in the presence or absence of phosphate.Exogenous NADH is unique in supporting the uptake of large amounts of Ca2+ in the presence of phosphate and in showing an unusual ‘uncoupled’ response in the absence of phosphate.A sigmoidal relationship occurs between initial velocity of Ca2+ uptake and Ca2+ concentration with a maximum velocity of approx. 15 nmol/s per mg protein and half maximum velocity occurring at approx. 50 μM Ca2+.  相似文献   

13.
Octanoate applied to rat liver mitochondria respiring with glutamate plus malate or succinate (plus rotenone) under resting-state (State 4) conditions stimulates oxygen uptake and decreases the membrane potential, both effects being sensitive to oligomycin but not to carboxyatractyloside. Octanoate also decreases the rate of pyruvate carboxylation under the same conditions, this effect being correlated with the decrease of intramitochondrial content of ATP and increase of AMP. The decrease of pyruvate carboxylation and the change of mitochondrial adenine nucleotides are both reversed by 2-oxoglutarate. Fatty acids of shorter chain length have similar effects, though at higher concentrations. Addition of octanoate in the presence of fluoride (inhibitor of pyrophosphatase) produces intramitochondrial accumulation of pyrophosphate, even under conditions when oxidation of octanoate is prevented by rotenone. In isolated hepatocytes incubated with lactate plus pyruvate, octanoate also increases oxygen uptake and produces a shift in the profile of adenine nucleotides similar to that observed in isolated mitochondria. It decreases the ‘efficiency’ of gluconeogenesis, as expressed by the ratio between an increase of glucose production and an increase of oxygen uptake upon addition of gluconeogenic substrates (lactate plus pyruvate), and increases the reduction state of mitochondrial NAD. These effects taken together are not compatible with uncoupling, but point to intramitochondrial hydrolysis of octanoyl-CoA and probably also shorter chain-length acyl-CoAs. This mechanism probably functions as a ‘safety valve’ preventing a drastic decrease of intramitochondrial free CoA under a large supply of medium- and short-chain fatty acids.  相似文献   

14.
Addition of Pb2+ to rat kidney mitochondria is followed by induction of several reactions: inhibition of Ca2+ uptake, collapse of the transmembrane potential, oxidation of pyridine nucleotides, and a fast release of accumulated Ca2+. When the incubation media are supplemented with ruthenium red, the effect of Pb2+ on NAD(P)H oxidation, membrane , and Ca2+ release are not prevented if malate-glutamate are the oxidizing substrates; however, the latter two lead-induced reactions are prevented by ruthenium red if succinate is the electron donor. It is proposed that in mitochondria oxidizing NAD-dependent substrates, Pb2+ induces Ca2+ release by promoting NAD(P)H oxidation and a parallel drop in due to its binding to thiol groups, located in the cytosol side of the inner membrane. In addition, it is proposed that with succinate as substrate, the Ca2+-releasing effect of lead is due to the collapse of the transmembrane potential as a consequence of the uptake of Pb2+ through the calcium uniporter, since such effect is ruthenium red sensitive.  相似文献   

15.
Phosphoenolpyruvate was found to inhibit net uptake of Ca2+ by rat heart and liver mitochondria. The main action of phosphoenolpyruvate is to increase the rate of efflux of mitochondrial Ca2+. The effect of phosphoenolpyruvate on mitochondrial Ca2+ transport is antagonized by ATP and by atractylate and is observed when mitochondria are respiring in the presence of NAD-linked subtrates such as glutamate and pyruvate plus malate. In liver mitochondria phosphoenolpyruvate is also effective in the presence of succinate but not when rotenone is added. Glycolytic intermdiates other than phosphoenolpyruvate had little effect on mitochondrial Ca2+ transport.  相似文献   

16.
Rafael Moreno-Sánchez 《BBA》1983,724(2):278-285
The mechanism through which internal Ca2+ inhibits oxidative phosphorylation of rat heart mitochondria has been explored. In parallel to a Ca2+-induced diminution of the activity of the adenine nucleotide translocator, an efflux of internal adenine nucleotides is observed. The efflux of adenine nucleotides depends on the amount of Ca2+ accumulated by the mitochondria and on the time that Ca2+ remains in the mitochondria; this efflux is atractyloside insensitive. These results suggest that internal Ca2+, by inducing a lowering of the internal concentration of adenine nucleotides, diminishes the rate of exchange of adenine nucleotides via the translocase, and in consequence of oxidative phosphorylation. Under conditions in which the Ca2+-induced release of adenine nucleotides takes place, no gross changes of the permeability properties of the membrane are observed. As revealed by studies with arsenate, respiratory activity and the function of the ATPase in the direction of ATP synthesis are not affected by internal Ca2+.  相似文献   

17.
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   

18.
The ATP-supported Ca2+ uptake of heart and liver mitochondria preincubated in conditions in which electron transport had either been prevented by rotenone or antimycin, or induced by oxidizable substrates, has been studied. Mitochondria preincubated with respiratory inhibitors accumulate Ca2+ less efficiently than mitochondria preincubated with oxidizable substrates. The difference correlates with the degree of activation of the oligomycin-sensitive ATPase. The results indicate that the rate at which mitochondria take up Ca2+ in the ATP-supported system may be controlled by the reversible asociation of the inhibiting peptide (Pullman,. and Monroy, J. Biol. Chem., 238, 3762–3769) with the ATPase complex. Since this process appears to be modulated by the transmembrane electrochemical gradient, the latter may regulate the uptake of Ca2+ in a hitherto undescribed way.  相似文献   

19.
Heart mitochondria respiring in the presence of Pi release endogenous K+ to a sucrose medium when an uncoupler is added. The uncoupled mitochondria retain K+, however, if the oxidation of NAD(P)H is prevented by the addition of rotenone or antimycin. Addition of rotenone, once the uncoupler-dependent K+-efflux has been initiated, results in a rapid reduction of NAD(P) and a simultaneous decrease in permeability to K+. These changes are independent of respiration. The results suggest that a latent pathway for K+-permeability is present in the membrane, that it can be opened and closed reversibly, and that it reflects, either directly or indirectly, the redox status of mitochondrial pyridine nucleotides. The possible relationship of this putative pathway to those available for Ca2+ uptake and release is considered.  相似文献   

20.
Olga Vergun 《BBA》2005,1709(2):127-137
Ca2+-induced mitochondrial depolarization was studied in single isolated rat brain and liver mitochondria. Digital imaging techniques and rhodamine 123 were used for mitochondrial membrane potential measurements. Low Ca2+ concentrations (about 30-100 nM) initiated oscillations of the membrane potential followed by complete depolarization in brain mitochondria. In contrast, liver mitochondria were less sensitive to Ca2+; 20 μM Ca2+ was required to depolarize liver mitochondria. Ca2+ did not initiate oscillatory depolarizations in liver mitochondria, where each individual mitochondrion depolarized abruptly and irreversibly. Adenine nucleotides dramatically reduced the oscillatory depolarization in brain mitochondria and delayed the onset of the depolarization in liver mitochondria. In both type of mitochondria, the stabilizing effect of adenine nucleotides completely abolished by an inhibition of adenine nucleotide translocator function with carboxyatractyloside, but was not sensitive to bongkrekic acid. Inhibitors of mitochondrial permeability transition cyclosporine A and bongkrekic acid also delayed Ca2+-depolarization. We hypothesize that the oscillatory depolarization in brain mitochondria is associated with the transient conformational change of the adenine nucleotide translocator from a specific transporter to a non-specific pore, whereas the non-oscillatory depolarization in liver mitochondria is caused by the irreversible opening of the pore.  相似文献   

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