Comparative study of the active cadmium efflux systems operating at the plasma membrane and tonoplast of cucumber root cells

The strategies developed by plants to avoid the toxicity of cadmium (Cd) and other heavy metals involve active sequestration of metals into the apoplast and vacuoles. The protein systems excluding heavy metals from the cell cytosol localize to the plasma membrane and tonoplast and are energized either by ATP or by the electrochemical gradient generated by H(+)-ATPase or by V-ATPase and pyrophosphatase (PPase), respectively. In this work, a comparative study on the contribution of both the plasma membrane and tonoplast in the active detoxification of plant cells after treatment with Cd was performed. The studies using plants treated and untreated with Cd reveal that both, H(+)-coupled and MgATP-driven efflux of Cd across plasma membranes and tonoplast is markedly stimulated in the presence of Cd in the environment. Previous studies on plasma-membrane localized H(+)-coupled Cd efflux together with the present data demonstrating tonoplast H(+)/Cd(2+) antiport activity suggest that H(+)-coupled secondary transport of Cd displays a lower affinity for Cd when compared with Cd primary pumps driven by MgATP. In addition, it is shown that MgATP-energized Cd efflux across both membranes is significantly enhanced by cysteine, dithiothreitol, and glutathione. These results suggest that Cd is excluded from the cytosol through an energy-dependent system as a free ion as well as a complexed form. Although both membranes contribute in the active exclusion of ionized and complexed Cd from the cytosol, the overall calculation of Cd accumulation in the everted plasma membranes and vacuolar vesicles suggests that the tonoplast and vacuole have a major function in Cd efflux from the cytosol in the roots of cucumber subjected to Cd stress.     

Iron-induced oxidative damage of corn root plasma membrane H(+)-ATPase

The effect of iron on the activity of the plasma membrane H(+)-ATPase (PMA) from corn root microsomal fraction (CRMF) was investigated. In the presence of either Fe(2+) or Fe(3+) (100-200 microM of FeSO(4) or FeCl(3), respectively), 80-90% inhibition of ATP hydrolysis by PMA was observed. Half-maximal inhibition was attained at 25 microM and 50 microM for Fe(2+) and Fe(3+), respectively. Inhibition of the ATPase activity was prevented in the presence of metal ion chelators such as EDTA, deferoxamine or o-phenanthroline in the incubation medium. However, preincubation of CRMF in the presence of 100 microM Fe(2+), but not with 100 microM Fe(3+), rendered the ATPase activity (measured in the presence of excess EDTA) irreversibly inhibited. Inhibition was also observed using a preparation further enriched in plasma membranes by gradient centrifugation. Addition of 0.5 mM ATP to the preincubation medium, either in the presence or in the absence of 5 mM MgCl(2), reduced the extent of irreversible inhibition of the H(+)-ATPase. Addition of 40 microM butylated hydroxytoluene and/or 5 mM dithiothreitol, or deoxygenation of the incubation medium by bubbling a stream of argon in the solution, also caused significant protection of the ATPase activity against irreversible inhibition by iron. Western blots of CRMF probed with a polyclonal antiserum against the yeast plasma membrane H(+)-ATPase showed a 100 kDa cross-reactive band, which disappeared in samples previously exposed to 500 microM Fe(2+). Interestingly, preservation of the 100 kDa band was observed when CRMF were exposed to Fe(2+) in the presence of either 5 mM dithiothreitol or 40 microM butylated hydroxytoluene. These results indicate that iron causes irreversible inhibition of the corn root plasma membrane H(+)-ATPase by oxidation of sulfhydryl groups of the enzyme following lipid peroxidation.     

镉胁迫对芥蓝根系质膜过氧化及ATPase活性的影响

水培条件下,以“香港白花”芥蓝品种为供试材料,研究4种不同浓度镉（0、1.0、2.0、4.0、8.0 mg/L Cd）处理对芥蓝幼苗根系质膜过氧化及ATPase活性的影响。结果表明,与对照相比,随着Cd处理浓度的增加,芥蓝根系活力呈现降低的变化趋势,而根系超氧化物歧化酶（SOD）、过氧化物酶（POD）和过氧化氢酶（CAT）活性以及丙二醛（MDA）、H2O2含量和O2-产生速率表现出升高的趋势,表明芥蓝受到活性氧物质的胁迫。1.0、2.0 mg/L Cd浓度处理下的H2O2含量与对照差异不显著,而O2-产生速率则在1.0 mg/L浓度处理下与对照差异不显著。随着Cd处理浓度的增加,芥蓝根系质膜H -ATPase和Ca2 -ATPase活性呈现出先增后减的变化趋势。1.0 mg/LCd浓度处理时,H -ATPase和Ca2 -ATPase活性与对照差异不显著（P > 0.05）,而在2.0、4.0和8.0 mg/LCd处理时,两种ATPase活性显著降低（P < 0.05）,并且与膜脂过氧化水平呈极显著的负相关（R2 > 0.969）。因此,低浓度Cd处理对芥蓝根系质膜两种ATPase活性影响较小,较高浓度Cd处理使芥蓝根系活力和质膜ATPase的损伤加重。     

Effects of heavy metal on rat liver microsomal Ca2(+)-ATPase and Ca2+ sequestering. Relation to SH groups.

In isolated hepatic microsomal vesicles the heavy metals Cd2+, Cu2+, and Zn2+ inhibit Ca2+ uptake and evoke a prompt efflux of Ca2+ from preloaded vesicles in a dose-dependent manner. N-Ethylmaleimide also inhibits Ca2+ uptake and causes Ca2+ release, but it is less effective in these respects than the heavy metals. Measurement of mannose-6-phosphatase activity indicate that the heavy metal-induced Ca2+ efflux is not caused by a general increase in membrane permeability. Heavy metals also inhibit the Ca2(+)-ATPase activity and the formation of the phosphorylated intermediate of the enzyme. In contrast, the sulfhydryl modifying reagent, N-ethylmaleimide inhibits the Ca2(+)-ATPase activity while it has a relatively small effect on Ca2+ release. Thus, the effects of these agents on Ca2+ sequestering and Ca2(+)-ATPase activity are not strictly proportional. The sulfhydryl group reducing agent dithiothreitol protects the microsomes from the effects of heavy metals, while glutathione is less protective. Addition of vanadate to vesicles, at a concentration which completely blocked the activity of the Ca2(+)-ATPase, resulted in a small and slow release of the accumulated Ca2+. Subsequent additions of heavy metals evoked a massive Ca2+ release. Thus, the effects of heavy metals on Ca2+ efflux cannot be due entirely to their inhibition of the Ca2+ pump. The heavy metal-induced Ca2+ efflux is not inhibited either by ruthenium red or tetracaine.     

Seed ageing-induced inhibition of germination and post-germination root growth is related to lower activity of plasma membrane H(+)-ATPase in maize roots

Seeds of most crops can be severely damaged and lose vigor when stored under conditions of high humidity and temperature. The aged seeds are characterized by delayed germination and slow post-germination growth. To date, little is known about the physiological mechanisms responsible for slow root growth of seedlings derived from aged seeds. Plasma membrane H(+)-ATPase is a universal H(+) pump in plant cells and is involved in various physiological processes including the elongation growth of plant cells. In the present study, we investigated the effect of a mild seed ageing treatment on plasma membrane H(+)-ATPase activity of seedling roots. Maize (Zea mays L.) seeds with 17% water content were aged at 45 degrees C for 30h. The aged seeds showed a 20% reduction in germination. Seedlings from aged seeds grew slowly during an experimental period of 120h after imbibition. Plasma membranes of maize seedling roots were isolated for investigation in vitro. Plasma membrane H(+)-ATPase (EC 3.6.3.6) activity was 14% lower for seedling roots developed from aged seeds as compared to control seeds. Protein gel immunoblotting analysis demonstrated that the reduced activity of plasma membrane H(+)-ATPase was attributed to a decrease in steady-state protein concentration of this enzyme. In conclusion, seed ageing causes a lower steady-state enzyme concentration of the H(+)-ATPase in the plasma membrane, which is related to slow germination and post-germination growth of seedling roots.     

Development of Fe-deficiency responses in cucumber (Cucumis sativus L.) roots: involvement of plasma membrane H(+)-ATPase activity

One of the mechanisms through which some strategy I plants respond to Fe-deficiency is an enhanced acidification of the rhizosphere due to proton extrusion. It was previously demonstrated that under Fe-deficiency, a strong increase in the H(+)-ATPase activity of plasma membrane (PM) vesicles isolated from cucumber roots occurred. This result was confirmed in the present work and supported by measurement of ATP-dependent proton pumping in inside-out plasma membrane vesicles. There was also an attempt to clarify the regulatory mechanism(s) which lead to the activation of the H(+)-ATPase under Fe-deficiency conditions. Plasma membrane proteins from Fe-deficient roots submitted to immunoblotting using polyclonal antibodies showed an increased level in the 100 kDa polypeptide. When the plasma membrane proteins were treated with trypsin a 90 kDa band appeared. This effect was accompanied by an increase in the enzyme activity, both in the Fe-deficient and in the Fe-sufficient extracts. These results suggest that the increase in the plasma membrane H(+)-ATPase activity seen under Fe-deficiency is due, at least in part, to an increased steady-state level of the 100 kDa polypeptide.     

Organic acid complexation,heavy metal distribution and the effect of ATPase inhibition in hairy roots of hyperaccumulator plant species

Heavy metal uptake and distribution were investigated in hairy roots of the Cd hyperaccumulator, Thlaspi caerulescens, and the Ni hyperaccumulator, Alyssum bertolonii. Hairy roots of both species contained high constitutive levels of citric, malic and malonic acids. After treatment with 20 ppm Cd or 25 ppm Ni, about 13% of the total Cd in T. caerulescens roots and 28% of the total Ni in A. bertolonii were associated with organic acids. T. caerulescens and A. bertolonii hairy roots remained healthy and grew well at high concentrations of Cd and Ni, respectively, whereas hairy roots of the non-hyperaccumulator, Nicotiana tabacum, did not. Most of the Cd in T. caerulescens and N. tabacum roots was localised in the cell walls. In contrast, 85-95% of the Ni in A. bertolonii and N. tabacum was associated with the symplasm. Growth of T. caerulescens and A. bertolonii hairy roots was severely reduced in the presence of diethylstilbestrol (DES), an inhibitor of plasma membrane H(+)-ATPase. Treatment with DES increased the concentration of Cd in the symplasm of T. caerulescens about 6-fold with retention of root viability, whereas viability and Ni transport across the plasma membrane were both reduced in A. bertolonii. These results suggest that the mechanisms of Cd tolerance and hyperaccumulation in T. caerulescens hairy roots are capable of withstanding the effects of plasma membrane depolarisation, whereas Ni tolerance and hyperaccumulation in A. bertolonii hairy roots are not.     

Inhibition of carbonic anhydrase in the gills of two euryhaline crabs,Callinectes sapidus and Carcinus maenas,by heavy metals

Two subcellular fractions of gill tissue, cytoplasm and basolateral membranes, from two species of euryhaline decapod crustaceans, Callinectes sapidus and Carcinus maenas, acclimated to low salinity, were isolated via differential centrifugation. Carbonic anhydrase activity from both fractions was titrated against a variety of heavy metals in vitro. The metals Ag(+), Cd(2+), Cu(2+) and Zn(+) showed inhibitory action against the enzyme. Ki values for these metals against cytoplasmic CA from C. sapidus were in the range of 0.05-0.5 microM (for Ag(+), Cd(2+) and Cu(2+)) and 2-6 microM for Zn(+), some of the highest sensitivities reported for CA from an aquatic organism. The Ki values for these same metals were approximately 2-3 orders of magnitude higher for cytoplasmic CA from C. maenas, indicating that there are significant differences in heavy metal sensitivity in branchial CA from the two species, and that C. maenas possesses a metal-resistant CA isoform. It required concentrations of metals in the millimolar range, however, to inhibit CA activity from the membrane fraction of the gill of both species. There were no effects on either mortality or on hemolymph osmotic and ionic concentrations in C. maenas that were exposed to 10 microM Cd or Zn(+) at 32 per thousand salinity and subsequently transferred to 10 per thousand. The presence of a metal-resistant CA isoform in the gills of C. maenas suggests that this species would not be restricted from its normal estuarine environment by heavy metal pollution.     

Inhibition of Na+/K(+)-ATPase and Mg(2+)-ATPase by metal ions and prevention and recovery of inhibited activities by chelators

Kinetics and inhibition of Na(+)/K(+)-ATPase and Mg(2+)-ATPase activity from rat synaptic plasma membrane (SPM), by separate and simultaneous exposure to transition (Cu(2+), Zn(2+), Fe(2+) and Co(2+)) and heavy metals (Hg(2+) and Pb(2+)) ions were studied. All investigated metals produced a larger maximum inhibition of Na(+)/K(+)-ATPase than Mg(2+)-ATPase activity. The free concentrations of the key species (inhibitor, MgATP(2-), MeATP(2-)) in the medium assay were calculated and discussed. Simultaneous exposure to the combinations Cu(2+)/Fe(2+) or Hg(2+)/Pb(2+) caused additive inhibition, while Cu(2+)/Zn(2+) or Fe(2+)/Zn(2+) inhibited Na(+)/K(+)-ATPase activity synergistically (i.e., greater than the sum metal-induced inhibition assayed separately). Simultaneous exposure to Cu(2+)/Fe(2+) or Cu(2+)/Zn(2+) inhibited Mg(2+)-ATPase activity synergistically, while Hg(2+)/Pb(2+) or Fe(2+)/Zn(2+) induced antagonistic inhibition of this enzyme. Kinetic analysis showed that all investigated metals inhibited Na(+)/K(+)-ATPase activity by reducing the maximum velocities (V(max)) rather than the apparent affinity (Km) for substrate MgATP(2-), implying the noncompetitive nature of the inhibition. The incomplete inhibition of Mg(2+)-ATPase activity by Zn(2+), Fe(2+) and Co(2+) as well as kinetic analysis indicated two distinct Mg(2+)-ATPase subtypes activated in the presence of low and high MgATP(2-) concentration. EDTA, L-cysteine and gluthathione (GSH) prevented metal ion-induced inhibition of Na(+)/K(+)-ATPase with various potencies. Furthermore, these ligands also reversed Na(+)/K(+)-ATPase activity inhibited by transition metals in a concentration-dependent manner, but a recovery effect by any ligand on Hg(2+)-induced inhibition was not obtained.     

Regulation of the plasma membrane potential in Pneumocystis carinii

Many protists use a H(+) gradient across the plasma membrane, the proton motive force, to drive nutrient uptake. This force is generated in part by the plasma membrane potential (DeltaPsi). We investigated the regulation of the DeltaPsi in Pneumocystis carinii using the potentiometric fluorescent dye bisoxonol. The steady state DeltaPsi in a buffer containing Na(+) and K(+) (standard buffer) was found to be -78+/-8 mV. In the absence of Na(+) and K(+) (NMG buffer) or Cl(-) (gluconate buffer), DeltaPsi was not significantly changed suggesting that cation and anion conductances do not play a significant role in the regulation of DeltaPsi in P. carinii. The DeltaPsi was also not affected by inhibitors of the Na(+)/K(+)-ATPase, ouabain (1 mM), and the K(+)/H(+)-ATPase, omeprazole (1 mM). In contrast, inhibitors of the plasma membrane H(+)-ATPase, dicyclohexylcarbodiimide (100 microM), N-ethylmaleimide (100 microM) and diethylstilbestrol (25 microM), significantly depolarized the DeltaPsi to -43+/-7, -56+/-5 and -40+/-12 mV, respectively. The data support that the plasma membrane H(+)-ATPase plays a significant role in the regulation of DeltaPsi in P. carinii.     

Differential response of wheat roots to Cu, Ni and Cd treatment: oxidative stress and defense reactions

Wheat seedlings cv. Zyta were treated with Cu, Ni and Cd at the concentrations causing approximately 50 % root growth inhibition, i.e. 12.5, 50 and 60 μM, respectively. Tissue metal accumulation, membrane permeability, lipid peroxidation, protein oxidation, concentration of thiol compounds as well as protease, glutathione S-transferase (GST) and peroxidase (POD) activities were studied in roots after 7 days of metal exposure. The metals showed different concentrations in root tissues with Cu and Cd being accumulated to the smallest and to the greatest extent, respectively. Membrane permeability was significantly enhanced by Cu and Ni but not by Cd treatment. All metals induced similar increase in protein oxidation, while significant enhancement of lipid peroxidation was observed only in the case of Cu treatment. The detected thiol compounds: cysteine (Cys), homocysteine (Hcy), γ-glutamylcysteine (γ-GluCys) and glutathione (GSH) were differently influenced by the metal treatment. Ni appeared to be the most effective inductor of GSH accumulation while both Cu and Ni similarly increased Cys content in the roots. Accumulation of γ-GluCys was found in response to Cu and Cd applications. Concentration of Hcy was enhanced by Cd treatment but exposure to Ni decreased its content below the level of detection. The activity of GST was considerably elevated by Cd and Ni treatments, while POD activity was increased only in response to Cu application. Our study showed that wheat roots differently responded to treatment with metals used at the concentrations having similar impact on growth.     

Cadmium induces changes in sucrose partitioning, invertase activities, and membrane functionality in roots of Rangpur lime (Citrus limonia L. Osbeck)

Cadmium (Cd) uptake effects on sucrose content, invertase activities, and plasma membrane functionality were investigated in Rangpur lime roots ( CITRUS LIMONIA L. Osbeck). Cadmium accumulation was significant in roots but not in shoots and leaves. Cadmium produced significant reduction in roots DW and increment in WC. Leaves and shoots did not show significant differences on both parameters. Sucrose content was higher in control roots than in Cd-exposed ones. Apoplastic sucrose content was much higher in Cd-exposed roots than in control ones. Cd-exposed roots showed a significant decrease in both cell wall-bound and cytoplasmic (neutral) invertase activities; while the vacuolar isoform did not show any change. Alterations in lipid composition and membrane fluidity of Cd-exposed roots were also observed. In Cd-exposed roots phospholipid and glycolipid contents decreased about 50 %, while sterols content was reduced about 22 %. Proton extrusion was inhibited by Cd. Lipid peroxidation and proton extrusion inhibition were also detected by histochemical analysis. This work's findings demonstrate that Cd affects sucrose partitioning and invertase activities in apoplastic and symplastic regions in Rangpur lime roots as well as the plasma membrane functionality and H (+)-ATPase activity.     

The properties of the Mn, Ni and Pb transport operating at plasma membranes of cucumber roots

To avoid metal toxicity, plants have developed mechanisms including efflux of metal ions from cells and their sequestration into cellular compartments. In this report, we present evidence for the role of plasma membrane efflux systems in metal tolerance of cucumber roots. We have identified the plasma membrane-transport system participating in Cd, Pb, Mn and Ni efflux from the cytosol. Kinetic characterization of this proton-coupled transport system revealed that it is saturable and has a different affinity for each of the metal ions used (with Km 5, 7.5 and 0.1 m M for Mn, Ni and Pb, respectively). Treatment of cucumber roots with 100 μ M Cd prior to the transport measurements caused a great increase (over 250%) in Cd antiport activity in plasmalemma vesicles. After decreasing the metal concentration to 50 μ M we still observed a large increase (over 150%) of this activity in comparison with the control. Moreover, the addition of 50 μ M Cd to the external solution stimulated not only Cd antiport in the plasmalemma vesicles but also the antiport of other metals used in the experiments. Treatment of cucumber roots with 50 μ M Ni revealed a similar effect: the antiport activity of Cd, Mn, Ni and Pb was stimulated, although to a lesser extent in comparison with stimulation by Cd. The data indicate that the root plasma membrane antiporter system is stimulated by the exogenous presence of heavy metals.     

A Ca2(+)-activated, Mg2(+)-dependent ATPase with high affinities for both Ca2+ and Mg2+ in vascular smooth muscle microsomes: comparison with plasma membrane Ca2(+)-pump ATPase

A Ca2(+)-ATPase with a high affinity for free Ca2+ (apparent Km of 0.13 microM) was found and characterized in membrane fractions from porcine aortic and coronary artery smooth muscles in comparison with the plasma membrane Ca2(+)-pump ATPase purified from porcine aorta by calmodulin affinity chromatography. The activity of the high-affinity Ca2(+)-ATPase became enriched in a plasma membrane-enriched fraction, suggesting its localization in the plasma membrane. The enzyme was fully active in the absence of exogenously added Mg2+, but required a minute amount of Mg2+ for its activity as evidenced by the findings that it was fully active in the presence of 0.1 microM free Mg2+ but lost the activity in a reaction mixture containing trans-cyclohexane-1,2-diamine-N,N,N',N'-tetraacetic acid as a divalent cation chelator which has, unlike EGTA, high affinities for both Ca2+ and Mg2+. It was able to utilize a variety of nucleoside di- and triphosphates as substrates, such as ADP, GDP, ATP, GTP, CTP, and UTP, showing a broad substrate specificity. The activity of the enzyme was not modified by calmodulin (5, 10 micrograms/ml). Trifluoperazine, a calmodulin antagonist, had a partial inhibitory effect on the activity at 30 to 240 microM, but this inhibition could not be reproduced by a more specific calmodulin antagonist, W-7, indicating that this inhibition by trifluoperazine was not specific. Furthermore, the high-affinity Ca2(+)-ATPase activity was not modified either by low concentrations (0.5-9 microM) of vanadate or by 1-100 microM p-chloromercuribenzoic acid. Cyclic GMP, nitroglycerin, and nicorandil did not have any effect on the enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in the level and activation state of the plasma membrane H(+)-ATPase during aging of red beet slices.

The effect of aging on the plasma membrane (PM) H(+)-ATPase of red beet (Beta vulgaris L.) parenchyma discs was analyzed in PM purified by aqueous two-phase partitioning. Aging increased both the activity in the amount of immunodetectable H(+)-ATPase in the PM. The activity assayed at slightly alkaline pH values increased earlier and more strongly than that assayed at acidic pH values, so that the pH curve of the enzyme from aged beet discs was shifted toward more alkaline values. Aging decreased the stimulation of the PM H(+)-ATPase activity by controlled trypsin treatments or by lysophosphatidylcholine. After trypsin treatment the pH dependence of H(+)-ATPase from dormant or aged beet discs became equal. These results indicate that aging not only increases the level of H(+)-ATPase in the PM, but also determines its activation, most likely by modifying the interaction between the autoinhibitory carboxyl-terminal domain and the catalytic site. When the PM H(+)-ATPase activity was assayed at a slightly alkaline pH, the tyrosine modifier N-acetylimidazole inhibited the H(+)-ATPase in the PM from dormant beet discs much less than in the PM from aged discs, suggesting that modification of a tyrosine residue may be involved in the activation of the PM H(+)-ATPase induced by aging. The results are discussed with regard to aging-induced development of transmembrane transport activities.     

Radical-induced inactivation of kidney Na+,K(+)-ATPase: sensitivity to membrane lipid peroxidation and the protective effect of vitamin E

The Na+,K(+)-ATPase is a membrane-bound, sulfhydryl-containing protein whose activity is critical to maintenance of cell viability. The susceptibility of the enzyme to radical-induced membrane lipid peroxidation was determined following incorporation of a purified Na+,K(+)-ATPase into soybean phosphatidylcholine liposomes. Treatment of liposomes with Fenton's reagent (Fe2+/H2O2) resulted in malondialdehyde formation and total loss of Na+,K(+)-ATPase activity. At 150 microM Fe2+/75 microM H2O2, vitamin E (5 mol%) totally prevented lipid peroxidation but not the loss of enzyme activity. Lipid peroxidation initiated by 25 microM Fe2+/12.5 microM H2O2 led to a loss of Na+,K(+)-ATPase activity, however, vitamin E (1.2 mol%) prevented both malondialdehyde formation and loss of enzyme activity. In the absence of liposomes, there was complete loss of Na+,K(+)-ATPase activity in the presence of 150 microM Fe2+/75 microM H2O2, but little effect by 25 microM Fe2+/12.5 microM H2O2. The activity of the enzyme was also highly sensitive to radicals generated by the reaction of Fe2+ with cumene hydroperoxide, t-butylhydroperoxide, and linoleic acid hydroperoxide. Lipid peroxidation initiated by 150 microM Fe2+/150 microM Fe3+, an oxidant which may be generated by the Fenton's reaction, inactivated the enzyme. In this system, inhibition of malondialdehyde formation by vitamin E prevented loss of Na+,K(+)-ATPase activity. These data demonstrate the susceptibility of the Na+,K(+)-ATPase to radicals produced during lipid peroxidation and indicate that the ability of vitamin E to prevent loss of enzyme activity is highly dependent upon both the nature and the concentration of the initiating and propagating radical species.     

Cadmium-mediated oxidative stress in kidney proximal tubule cells induces degradation of Na+/K(+)-ATPase through proteasomal and endo-/lysosomal proteolytic pathways.

The mechanisms of cadmium (Cd)-dependent nephrotoxicity were studied in a rat proximal tubule (PT) cell line. CdCl(2) (5 microM) increased the production of reactive oxygen species (ROS), as determined by oxidation of dihydrorhodamine 123 to fluorescent rhodamine 123. The levels of ubiquitin-conjugated cellular proteins were increased by Cd in a time-dependent fashion (maximum at 24-48 h). This was prevented by coincubation with the thiol antioxidant N-acetylcysteine (NAC, 15 mM). Cd also increased apoptosis (controls: 2.4+/-1.6%; Cd: 8.1+/-1.9%), but not necrosis (controls: 0.5 +/- 0.3%; Cd: 1.4+/- 2.5%). Exposure of PT cells with Cd decreased protein levels of the catalytic subunit (alpha1) of Na+/K(+)-ATPase, a long-lived membrane protein (t(1/2)>48 h) that drives reabsorption of ions and nutrients through Na(+)-dependent transporters in PT. Incubation of PT cells for 48 h with Cd decreased Na+/K(+)-ATPase alpha1-subunit, as determined by immunoblotting, by approximately 50%, and NAC largely prevented this effect. Inhibitors of the proteasome such as MG-132 (20 microM) or lactacystin (10 microM), as well as lysosomotropic weak bases such as chloroquine (0.2 mM) or NH(4)Cl (30 mM), significantly reduced the decrease of Na(+)/K(+)-ATPase alpha1-subunit induced by Cd, and in combination abolished the effect of Cd on Na+/K(+)-ATPase. Immunofluorescence labeling of Na+/K(+)-ATPase showed a reduced expression of the protein in the plasma membrane of Cd-exposed cells. After addition of lactacystin and chloroquine to Cd-exposed PT cells, immunoreactive material accumulated into intracellular vesicles. The data indicate that micromolar concentrations of Cd can increase ROS production and exert a toxic effect on PT cells. Oxidative damage increases the degradation of Na+/K(+)-ATPase through both the proteasomal and endo-/lysosomal proteolytic pathways. Degradation of oxidatively damaged Na+/K(+)-ATPase may contribute to the 'Fanconi syndrome'-like Na(+)-dependent transport defects associated with Cd-nephrotoxicity.