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VASCULAR BIOLOGY
1Department of Biomedical and Pharmaceutical Sciences and the 2International Heart Institute, The University of Montana, Missoula, Montana
Submitted 11 October 2005 ; accepted in final form 28 February 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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reactive nitrogen species; apoptosis mitochondrial dysfunction
Intense study of NO-mediated effects has revealed dual role for NO, functioning both as an anti- and pro-apoptotic agent. At low concentrations, NO can protect cells from the effects of pro-apoptotic stimuli. Conversely, exposure to elevated levels of NO has been shown to induce apoptosis (6). Furthermore, in vitro evidence indicates that acute nitrosative stress causes an increase in intracellular free zinc (Zn2+). This has been shown either by exposing cells to exogenous NO (42, 51) or through increasing endogenous NO synthesis mediated by the upregulation of inducible NO synthase (NOS) activity (49). One proposed mechanism for NO-mediated Zn2+ upregulation involves the interaction of NO-derived RNS with cysteine protein residues in Zn2+ metal-binding domains. As the second-most abundant intracellular metal, zinc-associated proteins are virtually ubiquitous in biological systems. Indeed, we have previously shown that the Zn2+-associated protein, endothelial NOS (eNOS), is directly sensitive to nitrosative stress (44). During normal physiological circumstances, eNOS is thought to be coupled to a regulatory co-factor, tetrahydrobiopterin, and this coupling is Zn2+ dependent. However, exposure to NO has been shown to cause both uncoupling of eNOS from tetrahydrobiopterin (61) and release of Zn2+ through an NO-mediated disruption of Zn2+-coordianated thiolate clusters (44). Given that zinc-binding proteins are the most abundant class of protein of the human cellular proteome (40), a general disruption of zinc-protein interaction could potentially liberate massive amounts of intracellular zinc. Given this potential, we were interested in further examining the effect of modulation of Zn2+ homeostasis on endothelial cell survival.
In this study, we report that loss of zinc homeostasis is a key factor in nitrosative stress in pulmonary artery endothelial cells, and that disrupting normal zinc homeostasis induces endothelial cell death via an apoptotic pathway. Furthermore, adenoviral-mediated overexpression of metallothionein (MT-1) or co-incubation with N,N,N',N'-tetrakis(2-pyridylmethyl)ethylene-diamine (TPEN), a Zn2+ chemical chelator, in endothelial cells (42, 51, 52) confers significantly improved resistance to Zn2+-mediated toxicity. Third, we demonstrate that increased Zn2+ can induce additional oxidative stress, demonstrating a biochemical link that could potentially bridge nitrosative and oxidative stress in endothelial cells.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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50% confluence, and utilized when fully confluent. Before experimental treatment, except as noted, cells were trypsinized, counted with a hemacytometer, replated in 6-, 24-, or 96-well plates (Costar, Corning, NY) at a density of 5 x 105 cells/cm2, and allowed to adhere for at least 18 h. Cells were then serum starved by replacing normal DMEM with serum-free, phenol red-free DMEM (GIBCO-BRL, Gaithersburg, MD) and allowed to incubate overnight. Upon serum deprivation, no exogenous sources of Zn2+ are available to the cells. On the day of experiment, cells were treated up to 4 h with 0–1 mM ZnCl2 (200 mM in PBS, diluted in serum-free, phenol red-free DMEM) or 0–1 mM N-[2-aminoethyl]-N-[2-hydroxy-2-nitrosohydrazino]-1,2-ethylenediamine (spermine NONOate; Calbiochem, San Diego, CA) or a positive control for mitochondrial disruption 100 µM potassium cyanide (KCN) (31) in serum-free, phenol red-free DMEM, and media pH was checked before cell exposure to avoid any spurious pH-related consequences of these compounds. These doses were selected to allow examination of the entire range of potential response in PAECs. After treatment, cells were removed from experimental media and subjected to immediate analysis unless otherwise noted. Statistical comparisons between treatments were carried out as detailed in Statistical analysis. Chemical Zn2+ chelation. To better define the role of Zn2+, immediately following the addition of either spermine NONOate or ZnCl2, 6.25 µM TPEN was added as chemical Zn2+ chelating agent. TPEN has been demonstrated to decrease Zn2+ levels in previous studies using endothelial cells (42, 51, 52). Cells were incubated with TPEN in addition to either spermine NONOate or ZnCl2 for the remainder of the experimental time course. This particular dose was utilized based on initial dose-toxicity experiments utilizing 0–25 µM TPEN for 4 h. This dose of TPEN was the highest tested that did not result in significant cell toxicity (data not shown).
NO exposure levels.
The amiNO-2000 sensor (Innovative Instruments, Tampa, FL) was calibrated using sodium nitrite standard solutions in an acidified solution in the presence of potassium iodide reducing agent. As a result, a stoichiometric conversion of nitrite to NO occurs. Briefly, nitrate standards in the range of 250 nM to 100 µM were prepared in 0.1 M sulfuric acid plus 6 mM KI. NO release was monitored in real time with the use of a NO measuring system (model inNO II; Innovative Instruments) and quantified using inNO II software (version 2.0). Readings were taken in triplicate for each standard, and the average sensitivity of the sensor was calculated. Solutions of 500 µM and 1 mM spermine NONOate (Calbiochem) were prepared in serum-free, phenol red-free DMEM media (Invitrogen), and the NO release was monitored over a 4-h period. To determine NO release by PAECs in culture, cells were washed in DPBS and the same media used to make the spermine NONOate solution was added to minimize variation in pH and temperature. The average sensitivity of the electrode, as determined from the standards, was 0.127 nA/nM. Aliquots of media (200 µl) were removed from cell wells every 15 min and measured immediately with the probe, and measurements were taken up to 4 h. Resting PAECs produced a constant 240 nM NO/min, and the average NO released from a DMEM solution containing 500 µM and 1 mM spermine NONOate was found to be 1.2 ± 0.2 and 3.1 ± 0.5 µM NO/min, respectively. Therefore, the addition of 500 µM and 1 mM spermine NONOate results in PAECs being exposed to a 5-fold and 13-fold increase in NO over endogenous production.
Fluorescence microscopy. A PC-based imaging system consisting of the following components: an Olympus IX51 microscope equipped with a charge-coupled device camera (Hamamatsu Photonics, Hamamatsu City, Japan) was used for acquisition of fluorescent images. Fluorescent-stained cells were observed with the appropriate excitation and emission, measuring at least 300 cells per sample, and the average fluorescent intensities (to correct for differences in cell number) were quantified using ImagePro Plus version 5.0 imaging software (Media Cybernetics, Silver Spring, MD).
Quantification of intracellular Zn2+ levels. Cells were plated in 24-well plates, treated as described above. Following exposure to ZnCl2, cells were washed twice in Zn2+-free DMEM and incubated with 20 µM zinquin ethyl ester; ethyl {[2-methyl-8-[[(4-methylphenyl)sulfonyl]amino]-6-quinolinyl]oxy} acetate (Biotium, Hayward, CA), a live-cell permeant Zn2+-specific fluorophore (14, 58) [20 mM in DMSO, diluted 1:1,000 in Dulbecco’s PBS (DPBS)] for 30 min at room temperature, and washed with fresh DPBS. Zinquin-stained cells were observed at 368 nm excitation and 490 nm emission, and the average fluorescent intensities were quantified as described above.
Analysis of cytotoxicity. Serum-starved PAECs were cultured in 24-well plates and treated with ZnCl2 as described above. After the incubation period, the medium was collected, centrifuged for 5 min at 500 g, and the supernatant was stored at 4°C until assay. Relative cytotoxicity was quantified by measurement of release of the soluble cytoplasmic enzyme lactate dehydrogenase (LDH). LDH activity in cell-free supernatant was measured using a commercial kit and following the manufacturer’s published protocol (Roche Applied Science, Indianapolis, IN).
Detection of apoptotic events. PAECs were seeded onto 96-well plates and treated as described above. Caspase activation was visualized by co-treating cells with 1 µM CaspACE FITC-VAD-FMK In Situ Marker (Promega, Madison, WI). This is a fluorescent analog of the pancaspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK), which readily enters cells and binds irreversibly to activated caspases (5). Fluorescent cells were observed using excitation at 485 nm and emission at 530 nm. After treatment, cells were washed with ZnCl2-free DMEM and then incubated overnight in phenol red-free media with 10% FCS. At 18 h after onset of exposure, cells were incubated in media with 10 µM FITC-conjugated caspase inhibitor (FITC-vad-FMK). After 20 min of incubation at 37°C in dark conditions, cells were washed with fresh media and visualized using fluorescence microscopy.
A second method of determining apoptotic induction involved TdT-mediated dUTP nick end labeling (TUNEL) analysis. Following previously described treatment, PAECs were incubated for 18 h in phenol red-free DMEM supplemented with 10% FBS. Following incubation, analysis was performed as we have previously described (54). TUNEL-positive cells were visualized by indirect immunofluorescence with excitation at 485 nm and emission at 530 nm.
Determination of mitochondrial function. Serum-starved PAECs were cultured in 24-well plates and treated with ZnCl2 as previously described. Mitochondrial function of cells following treatment was quantified using an MTS tetrazolium assay (CellTiter 96, AQueous MTS Reagent, Promega) according to the manufacturer’s instructions. Briefly, functional mitochondria are able to convert the MTS reagent into a readily detectable colored product (12). MTS reagent (20 µl) was added directly to cells in 100 µl of medium, and after a 4-h incubation period at 37°C, the absorbance at 492 nm was read using a Labsystems Multiskan EX plate reader (Fisher, Hampton, NH).
Second, mitochondrial membrane potential was analyzed using the lipophilic cation 5,5'6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (13). This dye fluoresces red in its multimeric form in healthy mitochondria and is the active reagent in the DePsipher Mitochondrial Potential Assay kit (Trevigen, Gaithersburg, MD). PAECs were seeded onto 96-well plates and incubated with 0–1 mM ZnCl2 as described above. DePsipher reagent (25 µg/ml) was added after treatment with ZnCl2 and incubated for a further 20 min. Following an additional wash with DPBS, the cytosolic monomer (green) form was observed and quantified by fluorescence microscopy at 530 nm as described above.
A third method of evaluating mitochondrial function involved the examination of cardiolipin, using changes in 10-N-nonyl acridine orange (NAO; Sigma-Aldrich, St. Louis, MO) fluorescence, which will differentially fluoresce, either 640 and 525 nm, depending on the state of cardiolipin within the cell (22). Serum-starved PAECs were plated into 24-well plates and treated as previously described. NAO (10 mM in ZnCl2, diluted 1:1,000) was added to the media 30 min before the end of the experiment. Cells were washed thoroughly with PBS and imaged using fluorescence microscopy.
Measurement of cellular O2–· levels. O2–· production was measured using electron paramagnetic resonance (EPR) spectroscopy with the spin probe, 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine·HCl (CMH, Alexis Biochemicals, San Diego, CA), as previously demonstrated (26). Each EPR assay was carried out using cells treated with or without polyethylene glycol-conjugated superoxide dismutase (PEG-SOD; 100 U/ml, Sigma-Aldrich) to quantify the SOD-inhibitable formation of CMH. Cells were plated in 6-well plates and treated as described above. In the final hour of incubation with ZnCl2, 20 µl of spin-trap stock solution consisting of CMH (20 µM in DPBS +25 µM desferrioxamine; Calbiochem, La Jolla, CA) and 5 µM diethyldithiocarbamate (Alexis Biochemicals, Lausen, Switzerland) + 2 µl DMSO were added to each well. Upon completion of incubation, adherent cells were trypsinized and pelleted at 500 g. Cell pellet was washed and suspended in a final volume of 35 µl DPBS (+desferrioxamine, diethyldithiocarbamate), loaded into a 50-µl capillary tube and analyzed with a MiniScope MS200 EPR (Magnettech, Berlin, Germany) at a microwave power of 40 mW, modulation amplitude of 3,000 mG, and modulation frequency of 100 kHz. EPR spectra were analyzed measured for amplitude using ANALYSIS software (version 2.02; Magnettech) and experimental groups were compared using statistical analysis described below.
Detection of mitochondrial O2–· levels.
Serum-starved PAECs were plated into 24-well plates and treated as previously described. Mitochondrial superoxide production was measured using MitoSOX Red mitochondrial superoxide indicator (Molecular Probes), a fluorogenic dye for selective detection of superoxide in the mitochondria of live cells (45). Briefly, after treatment with ZnCl2, cells were washed with fresh media, and then incubated in media containing MitoSOX Red (2 µM), for 10 min at 37°C in dark conditions. Cells were washed with fresh serum-free media and imaged using fluorescence microscopy as previously described at an excitation of 510 nm and an emission at 580 nm.
Generation of MT-1 adenoviral expression construct.
An adenoviral vector expressing MT-1 was constructed with the pAd/pENTR System (Invitrogen). Briefly, a pAd/cytomegalovirus plasmid containing an MT-1 cDNA (cDNA purchased from ATCC, Manassas, VA) was created and transfected into 293A cells. A selected clone was propagated and viral lysate was collected and titered per the manufacturer’s protocol. The titer for the AdV.MT-1 preparation was 3.4 x 1010 plaque-forming units/ml. Infections on PAECs were performed for 120 min with virus diluted in normal DMEM to the desired multiplicity of infection. Verification of expression was performed using Western blot analysis as previously described (7) using 20 µg of protein extracts probed with anti-MT-I antibody (1:1,000, QED, Malden, MA) and normalized using 
-actin (1:1,000, Sigma).
Statistical analyses. The results are presented as means ± SE from at least three experiments and statistical analysis between experimental groups was performed using one-way ANOVA. The statistical significance of differences was set at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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5-fold and 13-fold increase in NO over basal levels. Using the Zn2+-specific fluorescent probe zinquin ethyl ester, we observed a significant increase in Zn2+-mediated zinquin fluorescence arising specifically from endogenous sources when we exposed cells to spermine NONOate (Fig. 1A). Quantification of the fluorescence signal indicated that exposure of PAEC to 500 µM or 1 mM of spermine NONOate resulted in an increase in zinquin fluorescence of 30- and 60-fold, respectively (Fig. 1A). We next determined the amount of exogenous ZnCl2 required to stimulate a similar increase in intracellular zinc. Our data indicated that the addition of 250 µM-1 mM ZnCl2 produced similar 30- to 60-fold increases in intracellular Zn2+ (Fig. 1B).
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) by Zn2+ or 100 µM KCN as a positive control (Fig. 3A) using the DePsipher assay. Furthermore, using NAO, we found a significant decrease in the ratio of red multimeric mitochondrial cardiolipin to green monomeric cardiolipin (Fig. 3B). Together, these data indicate that a loss of cardiolipin from the inner leaflet of the mitochondrion is occurring.
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(Fig. 6, C and D). In addition, the overexpression of MT-1 significantly improved cell survival to high-dose ZnCl2 or spermine NONOate as measured by a reduction in LDH release assay (Fig. 7, A and B). Interestingly, the reduction in LDH release was significantly lower for the ZnCl2 treated cells (Fig. 7A) than spermine NONOate-treated cells (Fig. 7B). Finally, we found that MT-1 overexpression significantly reduced both the Zn2+-mediated increase in caspase activation (Fig. 7, C and D) and the accumulation of TUNEL-positive cells, either by the addition of Zn2+ (Fig. 7E) or NO (Fig. 7F). Overall these data suggest that only a portion of the cytotoxic effects of NO are mediated via alterations in Zn2+ homeostasis.
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(Figs. 8C and 9C, for ZnCl2 and NO, respectively), caspase activation (Figs. 8D and 9D, for ZnCl2 and NO, respectively) and increase in percentage of TUNEL-positive cells (Figs. 8E and 9E, for ZnCl2 and NO, respectively) through the addition of TPEN.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; 2) increases in intracellular O2–·, which is generated, at least in part, in the mitochondria; 3) loss of cardiolipin from the inner mitochondrial leaflet; and 4) induction of events associated with the apoptotic pathway. Significantly, the observed effects of increased apoptotic induction as shown here (Fig. 2F) and in previous studies (18), as well as the observed decrease in mitochondrial function we report here could be duplicated through exposure of the cells directly to exogenous Zn2+, and which could be mitigated through overexpression of the Zn2+-sequestering protein, MT-1, or via chemical chelation with TPEN. As a result, we conclude that our data suggest a role for Zn2+ in mitochondrial disruption following acute nitrosative stress, and that as this results in an increase in oxidative stress through increased O2–· production, that this mechanistically connects two seemingly distinct processes. In this study, we selected doses of both spermine NONOate and ZnCl2 to span the entire cellular response range in this particular cell line. While it has been shown that with use of compounds to enhance Zn2+ uptake into cells, notably zinc ionophore pyrithione (52), adverse toxicity by the compound precluded its use in this model system. As such, to achieve a response, we elected to use elevated Zn2+ concentrations while monitoring for potential pH-mediated effects. While the concentrations utilized in this report are somewhat high, similar levels of exogenous Zn2+ have been utilized in previous studies in neuronal cell culture models (29). Interestingly, Zn2+ has been previously shown to be an anti-apoptotic factor in PAECs, where chelation of Zn2+ with TPEN resulted in induction of apoptosis. In our studies with TPEN (Figs. 8 and 9), in similar fashion to MT-1 protein over-expression, we observed statistically significant decreases in both caspase activation and apoptotic cells when added in conjunction with Zn2+ or NO, seemingly in contrast with these previous studies. This suggests that Zn2+ levels may need to be tightly regulated to remain in a relatively narrow physiological window for a given cell type, and that both sub- and super-optimal intracellular Zn2+ can result in similar cellular consequences. With the TPEN studies, however, it is important to note that TPEN is not a Zn2+-specific chelation agent, and as such it is difficult to ascribe the observed effects solely to Zn2+ (3), and we speculate that the seemingly contradictory findings between our findings with TPEN and previously published studies could be the result of chelation and homeostatic disruption of multiple divalent metals, including Fe2+, Cu2+, and possibly Ca2+.
To date, a diverse set of physiological, nutritional, and biochemical functions have been attributed to zinc (43). Intracellular Zn2+ has both catalytic roles and structural roles in proteins, and although Zn2+ is redox inert it can bind to cysteine residues to create protein folds important in protecting the target molecule from oxidation. Numerous reports have concluded that zinc functions as a cytoprotective agent, defending cells against both oxidative insult and against stimuli that induce apoptosis. Proposed explanations for this protective effect include serving as a factor in maintenance of cellular membranes (41), activation of anti-apoptotic signal transduction (2), inhibition proapoptotic NF-
B and AP-1 signaling (38), antagonism of lipid peroxidation (10), as well as inhibition of the proapoptotic enzymatic activities of caspases and endonucleases (11, 36). Pathologies associated with Zn2+ deficiency are well documented (20), and inadequate dietary uptake of zinc remains a nutritional issue of global dimensions (47). However, the dynamics of how and when Zn2+ binds to and is released from proteins, the cellular distribution of Zn2+, the hierarchy of distribution, and the homeostatic control of Zn2+ remain largely unresolved. Cells have a very tight regulatory apparatus in place for zinc, as intracellular concentrations of Zn2+ ions have been measured in pico- or nanomolar concentrations (32), indicative of the requirement for strict sequestration. One major component of the Zn2+ homeostatic mechanism within mammalian cells is the MT family of proteins. Studies over the past two decades have demonstrated that the MT/thionein (T) ratio plays an important role in Zn2+ homeostasis. MT is an intracellular cysteine-rich metal-binding protein that acts to control the concentration of labile Zn2+. On the basis of the Zn2+ binding constant of mammalian MT, 2 x 1012 M–1 (33), MT binds Zn2+ more tightly than most other Zn2+-binding proteins and constitutes a thermodynamic "sink" for Zn2+. In fact, Zn2+ bound to MT is believed to represent at least 5–10% of the total Zn2+ within mammalian cells (9). The MT/T system is believed to function in sequestering and releasing Zn2+ when required, utilizing thiolate clusters within the MT protein (33). There is evidence to indicate that MT/T ratio is important in the ability of cells to tolerate oxidative insult, and fully Zn2+-bound MT may actually contribute to oxidative stress through release of Zn2+ (35). We speculate in these experiments that a significant fraction of the overexpressed protein is relatively zinc depleted and retains the ability to bind Zn2+ despite the nitrosative stress induced by NO-donor exposure. Furthermore, nitrosative stress is a dynamic process. We have previously shown that S-nitrosylation of cysteine residues in eNOS can be prevented by intracellular reducing agents (43). As a consequence, it is likely that a certain fraction of reduced cysteine residues are in a state capable of binding and sequestering Zn2+ at any given time.
Although the protective and beneficial effects of zinc are well documented, evidence also shows that elevated Zn2+ concentrations can be adverse to cells. Excessive levels of Zn2+ have been shown to interfere with metal-dependent processes normally not associated with Zn2+ (15, 16). Elevated Zn2+ can inhibit protein catalytic function, including those in the mitochondria (17, 46). There is also evidence of an increased requirement for zinc in the vascular endothelium during inflammatory situations such as during the progression of atherosclerosis (25). Numerous reports demonstrate a key role for Zn2+ as a damaging agent in neurons (reviewed in Ref. 21). Furthermore, there is in vivo evidence that excessive dietary zinc intake can induce pathological conditions which have been associated with oxidative stress (57). Thus, in the same manner as Ca2+, a loss of intracellular Zn2+ regulation may be equally damaging as situations of zinc deficiency (50). This homeostatic balancing act, where physiological levels of Zn2+ levels are required for normal cell function but excess Zn2+ levels are cytotoxic could provide a potential explanation for the apparent discrepancy between our findings and previous findings in PAECs (42).
Relating our observed Zn2+-mediated effects to nitrosative stress, it is known that RNS react directly with Zn2+-bound proteins, including the tetrathiolate cluster in eNOS, causing Zn2+ to release from the metal-binding domain (44). Given the plethora of Zn2+-binding proteins within the cell, each with a relatively similar mechanism of binding Zn2+, there exists the potential for significant intracellular Zn2+ release, as indicated by our observations with zinquin ethyl ester (Fig. 1A), where NO induced Zn2+-dependent fluorescence increased 60-fold. One proposed mechanism is that Zn2+, in high enough concentrations, has the ability to competitively displace copper and iron from heme domains and other protein metal-binding domains (24, 43). Subsequently, copper and iron ions are highly redox-reactive, and their release into the intracellular space and in the presence of reducing agents allows rapid catalytic conversion of O2, to potentially damaging ROS. These include O2–· and ·OH, and would potentially exacerbate the nitrosative stress condition initially caused by increased levels of NO and RNS.
More evidence to suggest a Zn2+-mediated link between nitrosative and oxidative stress comes from our data demonstrating that O2–· levels increase in the mitochondria in response to Zn2+ exposure (Fig. 4C). During normal cellular respiration, mitochondrial-derived O2–· is spontaneously dismutated to H2O2 or is catalyzed by mitochondrial SOD. We observed a significant increase in mitochondrial O2–· (Fig. 4C) as well as loss of m (Fig. 3A) and cardiolipin (Fig. 3B) from the inner mitochondrial leaflet. This increase in markers for apoptosis suggests that Zn2+ causes a loss of mitochondrial integrity, a well-defined early event in apoptosis (60). However, these data cannot rule out additional sources of the observed increase in O2–· arising from cytosolic or plasma membrane-bound producers of O2–·, including NADPH oxidase and/or ·OH, resulting from lipid peroxidation. Indeed, the EPR signal from CMH oxidation was not completely quenched by the addition of PEG-SOD (Fig. 4A), and we speculate that this may be due to direct stress-related effects of Zn2+ causing production of ·OH not associated with mitochondrial dysfunction. On the other hand, the fact that we were able to significantly protect cells with MT-1 protein overexpression argues for a specific role for Zn2+ in mitochondrial dysfunction and subsequent induction of apoptotic cell death. In addition, it has been proposed that MT-1 and MT proteins can function directly as antioxidant factors within cells (39). However, given that we could not detect a statistically significant reduction in detectable ROS in unchallenged MT-1 overexpressing cells (Fig. 5D) we conclude that MT-1 is not functioning as a direct antioxidant in this particular model system.
On the basis of our data, we hypothesize that if the cellular ability to sequester or otherwise remove free Zn2+ from the cytosol is overwhelmed and/or the ability to detoxify ROS is impaired, the result is a positively reinforced cycle of ROS and RNS creation and intracellular redox disruption. If unchecked, this cycle will ultimately drive the cell into an apoptotic fate, or in severe toxicity, outright necrosis. Adenoviral-mediated over-expression of MT-1 protein in PAECs completely abolished Zn2+-mediated cytotoxicity through induction of apoptosis (Fig. 7, C and E), as well as general cell death as measured by LDH release (Fig. 7A). However, although MT-1 overexpression significantly reduced both LDH release (Fig. 7B) and markers of apoptosis (Fig. 7, D and F) in NO challenged cells there was still significant cell death. This suggests that only a portion of the cytotoxic effects of NO are mediated via alterations in Zn2+-homeostasis.
One primary function of pulmonary circulation is the acquisition of O2 for systemic needs. Thus it is likely that there exists an exquisite sensitivity of pulmonary endothelial cells to O2, NO, and associated ROS and RNS species. It has been proposed that ROS can act as signaling mediators in mechanisms for maintaining adequate gas exchange in the lung by diverting blood flow from areas with low O2 tension. Furthermore, production of ROS in pulmonary systems serves as a functional signal for leukocyte infiltration and induction of tissue defense and wound healing processes (28). As such, a severe acute upregulation of ROS and other signals of oxidative stress in the pulmonary system, including Zn2+ release, could result in inappropriate physiological responses, additional oxidative stress and tissue damage on a systemic level. Thus, in clinical situations where acute oxidative stress occurs, we hypothesize that proper management of intracellular Zn2+ balance must be considered to maximize the potential clinical benefits of zinc and minimize the possibility of zinc-mediated adverse consequences. Although true clinical improvements from antioxidant therapy remain elusive (27), our data suggest the necessity of additional investigation into the timely and appropriate management of factors contributing to oxidative and nitrosative stress, including Zn2+ homeostasis. However, this recommendation should be approached with caution, as creation of high concentrations of MT or other Zn2+ reduction factors could potentially result in a deficiency of Zn2+ (34). Therefore, we propose that a critical balance between liberation and sequestration of Zn2+ must be achieved to avoid adverse physiological consequences. Recent in vivo data are beginning to validate this postulation (30).
In conclusion, our data further establishes a link between increase NO generation and alterations in cellular Zn2+ homeostasis that underlie cell toxicity and induction of apoptosis in ovine PAECs. Our data suggest that Zn2+ scavenging could be a potential therapy to reduce or prevent cardiovascular or other diseases associated with oxidative and nitrosative stress. However, the effect of this type of treatment in vivo is currently unknown and will require additional animal studies to directly assess this issue.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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P < 0.05 vs. previous dose.
P < 0.05 PEG-SOD (100 U/ml, 1 h) pretreatment vs. control cells at identical dose.
400 (A). Ovine PAECs (exposed to adenoviruses for MT-1 or LacZ at an MOI of 1,000) were exposed for 4 h to ZnCl2 (0–1 mM; B) or spermine NONOate (0–3.1 µM/min; C) and the effect on free zinc levels estimated by zinquin ethyl ester fluorescence. The overexpression of MT-1 significantly reduced the level of free zinc in ZnCl2 or NO challenged cells. In addition, the effect on basal oxidative stress was estimated by measuring cellular superoxide levels by electron paramagnetic resonance (EPR) assay using the spin-trap compound CMH (D). There was no significant difference in the level of superoxide in cells transduced with either LacZ or MT-1. All values are mean ± SE (n = 6). *P < 0.05 vs. untreated; 
