Departments of Pharmacology, Cell Biology and Physiology, and Environmental Health and Occupational Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
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ABSTRACT |
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Metallothionein (MT) is a low-molecular-weight cysteine-rich protein with extensive metal binding capacity and potential nonenzymatic antioxidant activity. Despite the sensitivity of vascular endothelium to either heavy metal toxicity or oxidative stress, little is known regarding the role of MT in endothelial cells. Accordingly, we determined the sensitivity of cultured sheep pulmonary artery endothelial cells (SPAEC) that overexpressed MT to tert-butyl hydroperoxide (t-BOOH), hyperoxia, or 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN; peroxyl radical generator). Nontoxic doses of 10 µM Cd increased MT levels from 0.21 ± 0.03 to 2.07 ± 0.24 µg/mg and resulted in resistance to t-BOOH and hyperoxia as determined by reduction of Alamar blue or [3H]serotonin transport, respectively. SPAEC stably transfected with plasmids containing either mouse or human cDNA for MT were resistant to both t-BOOH and hyperoxia. In addition, we examined transition metal-independent, noncytotoxic AMVN-induced lipid peroxidation after metabolic incorporation of the oxidant-sensitive fluorescent fatty acid cis-parinaric acid into phospholipids and high-performance liquid chromatography separation. SPAEC that overexpressed MT after gene transfer completely inhibited peroxyl oxidation of phosphatidylserine, phosphatidylcholine, and sphingomyelin (but not phosphatidylethanolamine) noted in wild-type SPAEC. These data show for the first time that MT can 1) protect pulmonary artery endothelium against a diverse array of prooxidant stimuli and 2) directly intercept peroxyl radicals in a metal-independent fashion, thereby preventing lipid peroxidation in intact cells.
cadmium; pulmonary artery endothelial cells; organic hydroperoxides; hyperoxia; peroxyl radicals; lipid peroxidation
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INTRODUCTION |
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METALLOTHIONEINS (MT) are ubiquitous, low-molecular-weight, thiol-containing proteins with unclear functions (21). Because MT not only binds many heavy metals but also is readily induced by these molecules (e.g., Cd, Zn, and Cu), a critical role is apparent in intracellular metal ion homeostasis. An antioxidant role for MT has also been proposed (34) because MT is a redox-sensitive gene (9) that is elevated during various forms of oxidative stress (3), and cells that overexpress MT after chemical induction or after direct gene transfer (25, 35-37) are resistant to several forms of oxidant injury. Furthermore a decrease in MT by gene deletion enhances sensitivity to oxidative injury (20).
The lung is an important target for both heavy metal and oxidant-induced injury, and its vascular endothelium is a critical locus of structural and functional changes in such toxicities (8, 28). Nonetheless, relatively little is known regarding MT and the lung, and there is no information available with respect to pulmonary endothelium. Although intrapulmonary MT levels are low, they are readily induced by exposure of intact animals to Cd (12), high O2 (15, 30, 38), paraquat (3), and tert-butyl hydroperoxide (t-BOOH; see Ref. 3). Surprisingly, pulmonary vascular endothelial MT expression is persistently below the level of detection in situ. For instance, MT protein (immunolocalization) or mRNA (in situ hybridization) is localized to either alveolar type II cells, fibroblasts, chrondrocytes, or alveolar macrophages after exposure to aerosolized Cd (12) or hyperoxia (15, 38). Constitutive intrapulmonary MT expression has been apparent only in saccular fetal lamb lung and is restricted to bronchial epithelium and vascular smooth muscle (31). Exposure of intact animals to Cd produces tolerance to subsequent Cd exposure (16) as well as cross-resistance to ozone (2) and hyperoxia (16). Alveolar macrophages (13) isolated from rats after repeated exposure to Cd or lung fibroblasts adapted to Cd in vitro (14) are each resistant to H2O2. Although such observations are useful first steps in identifying a role for MT, ambiguities of relying solely on chemical generation of MT to deduce its function are apparent (18). Accordingly, additional molecular approaches are often necessary, including those outlined in the current study.
As a first attempt in understanding MT expression in pulmonary vascular endothelium, we isolated and cultured endothelial cells from the proximal pulmonary artery of sheep. We exposed these cells to nontoxic concentrations of Cd and noted concentration- and time-dependent increases in sheep MT mRNA and protein levels. We then demonstrated that preexposure of sheep pulmonary artery endothelial cells (SPAEC) to Cd reduces their sensitivity to oxidative stress, and we provided evidence via direct gene transfer that MT was indeed participating in the antioxidant defenses of pulmonary endothelium to t-BOOH, hyperoxia, and peroxyl oxidation of membrane phospholipids.
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MATERIALS AND METHODS |
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Isolation and Culture of SPAEC
Sheep pulmonary arteries were obtained from a local slaughterhouse. Cells were harvested by collagenase digestion. Early passage primary cultures were incubated with 1,1'-dioactadecyl-1,3,3,3',3'-tetramethyl-indocarbocyanine perchlorate-labeled acetylated low-density lipoprotein (diI-Ac-LDL; Biomedical Technologies, Staughton, MA), and cells preferentially incorporating this substrate were obtained from fluorescent-activated cell sorting (17). SPAEC were grown in Optimem (GIBCO, Grand Island, NY) with 15 µg/ml endothelial cell growth supplement (Collaborative Biomedical Products, Bedford, MA), 10 U/ml heparin sulfate, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% sheep serum (Sigma Chemical, St. Louis, MO) at 37°C in 95% air-5% CO2. Subpassages were routinely homogeneously positive for uptake of diI-Ac-LDL and factor VIII-related antigen, and SPAEC were used between passages 6 and 12.RNA Isolation and Northern Blot Analysis
Total RNA was isolated by the single-step method of Chomczynski and Sacchi (6). Cells were homogenized in a denaturing solution containing 4 M guanidinium isothiocyanate, and RNA was extracted in a phenol-chloroform mixture. After precipitation, RNA was processed and quantified, and 20-µg samples were subjected to 1.0% agarose gel electrophoresis and were transferred by capillary action to Genescreen. Filters were hybridized overnight in 50% formamide, 0.25 M NaHPO4, 0.25 mM NaCl, 1 mM EDTA, 100 µg/ml denatured salmon sperm DNA, and 7% sodium dodecyl sulfate (SDS) to [32P]cDNA probes to either sheep MT-Ib orDetermination of MT Protein Level
Cells were removed from culture dishes with 0.05% trypsin and 0.02% EDTA and were resuspended in 20 mM tris(hydroxymethyl)aminomethane-buffered saline (pH 7.4). The MT protein level of lysate prepared from these cells was determined by a previously described 109Cd binding assay (35). MT was saturated using a solution containing 0.1 µCi 109Cd (1-5 Ci/mg) and 200 ng of CdCl2. Cellular MT content was calculated based on the assumption that 7 mol of Cd bound to 1 mol of MT and was normalized to cellular protein.Transfection
Plasmids pBPVGRPMT and pBPVGRPTM were generous gifts from Dr. Kathyrn Morton (Oregon Health Sciences Center, Portland, OR). Both plasmids contain the genome of the bovine papilloma virus. In pBPVGRPMT, mouse MT-I (mMT-I) is driven by rat glucose-regulated promoter of 78 kDa, and transfected cells are referred to as SPAEC/mMT. In the control plasmid pBPVGRPTM, mMT-I gene is inverted and is removed from its promoter, and cells transfected with this promoter are SPAEC/mTM. Both cell types were cotransfected with pSV2Neo, which contains the aminoglycoside phosphotransferase gene driven by the early SV40 promoter (American Type Culture Collection, Rockville, MD). Cotransfection was performed via lipofectamine, and cells were selected in 250 µg/ml G418, after which they were routinely grown in 150 µg/ml G418 in complete medium. Some cells were transfected with pSV2Neo only, and these stable transfectants are referred to as SPAEC/NEO (35). In some instances, SPAEC were cotransfected with pSV2Neo and a construct (pCMVhMTIIA) containing the human MT II-A (hMT-IIA) cDNA driven by a cytomegalovirus promoter. These cells are referred to as SPAEC/hMT.Cytotoxicity
Cell viability was routinely determined by a quantifying reduction of the fluorogenic indicator Alamar blue (Alamar Biosciences, Sacramento, CA). SPAEC (5 × 104) were allowed to attach to 24-well tissue culture dishes (3-4 wells/condition) and then were exposed to t-BOOH or AMVN. At various times, Alamar blue was added to the medium, and 3-4 h later fluorescence was determined in a cytofluorometer (Cytofluor II; PerSeptive Biosystems, Framingham, MA). It has previously been shown that oxidized Alamar blue is taken up by cells and is reduced by intracellular dehydrogenases, and the water-soluble product is excreted into the medium from which changes in fluorescence emission (590 nm) are utilized as an index of cytotoxicity (29).Cell viability was determined on occasion by a colorimetric assay using diphenyltetrazolium bromide (MTT; Sigma Chemical). Each assay was performed in three to six replicates on confluent cells grown in 96-well plates. After exposure to t-BOOH, SPAEC were incubated in Optimem with MTT (26) for 3 h at 37°C. After removal of MTT, cells were exposed to dimethyl sulfoxide (DMSO) for 5 min, and then absorbance was read at 540 nm on a multiplate reader (Titertek Multiscan Plus, MKII). In separate experiments, for determination of radioactive Cr release (36), cells were incubated with 2 µCi Na251CrO4 (565 mCi/mg; NEN, Boston, MA) for 24 h. Cells were washed three times with Hanks' balanced salt solution to remove unincorporated radioactivity. Cells were then exposed to t-BOOH for 24 h, and endothelial cell 51Cr release was quantified by measuring 51Cr obtained from medium in a gamma counter (LKB 1272, Piscataway, NJ). Cells were lysed with 0.1% Triton X-100, and total releasable 51Cr was quantified in cell lysate. Radioactive Cr release was calculated as the ratio of counts per minute in medium to counts per minute in medium and lysate.
Serotonin (5-HT) uptake was determined by adding
5-hydroxy[G-3H]tryptamine
creatine sulfate (in 2% ethanol and 30 µM EDTA plus ascorbate) to
confluent monolayers of SPAEC in six-well dishes for 60 min at
37°C, as previously described (27). Uptake was terminated by
washing the cells in ice-cold phosphate-buffered normal saline (PBS),
cells were solubilized with 0.1 N NaOH-0.1% Triton X-100, and
cell-associated radioactivity was quantified in a liquid scintillation
counter. Analyses were performed in two to five replicates, an
equivalent additional group of cells was pretreated with
104 M imipramine, and 5-HT
uptake was repeated. Cell number was assessed by a hemacytometer. 5-HT
uptake was calculated by subtraction of the imipramine-insensitive
component and normalization to cell number. Occasional (5 of 20)
subcultures that did not possess detectable imipramine-sensitive 5-HT
transport in room air were not included in the analysis.
Phospholipid Peroxidation
Incorporation of cis-parinaric acid into SPAEC. cis-Parinaric acid (PnA) was incorporated into logarithmically growing SPAEC and SPAEC/hMT (2.5 × 105 cells/ml) by addition of its human serum albumin (hSA) complex(PnA-hSA) to cells (10). The complex was prepared by adding 500 µg PnA (1.8 µmol) in 25 µl of DMSO to 50 mg hSA (760 nmol) in 1 ml of PBS containing (in mM) 137 NaCl, 2.7 KCl, 1.5 KH2PO4, and 8 Na2HPO4 (pH 7.4). PnA-hSA complex was added to the cell suspension to give a final concentration of 5 µg/1 × 106 cells PnA, and the cells were incubated in Dulbecco's modified Eagle's medium (DMEM) with 20% fetal bovine serum (FBS) at 37°C in the dark under aerobic conditions to allow incorporation of PnA into phospholipids. At the end of a specified incubation period, the cells were washed twice with medium with or without 0.5 mg/ml hSA.
2,2'-Azobis(2,4-dimethylvaleronitrile)-induced lipid peroxidation. SPAEC and SPAEC/hMT preloaded with PnA were incubated in DMEM with 20% FBS for 2 h in the presence of 0.5 mM 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN) at 37°C under aerobic conditions in the dark. Cells were then collected with trypsin and were resuspended in PBS.
Extraction of cell lipids. Total lipids were extracted from cells (2.5 × 106 cells in 1 ml) using a slightly modified Folch procedure (10). Methanol (2 ml) containing butylated hydroxytoluene (0.1 mg) was added to the cell suspension and mixed. Chloroform (4 ml) was then added, and the mixture was stored for 1 h at 0°C under N2. One milliliter of 0.1 M NaCl was added to the extract that was vortex mixed under N2. After centrifugation for 5 min at 1,500 g, the upper aqueous methanol layer was aspirated, and the lower chloroform layer was evaporated to dryness under a stream of O2-free dry N2. The lipid extract was dissolved in 0.2 ml of 2-propanol-hexane-H2O (4:3:0.16). Control experiments confirmed that >95% of cell phospholipids were extracted by this procedure.
High-performance thin-layer chromatography analysis of cell lipids. The phospholipid classes in the extracts were separated by two-dimensional high-performance thin-layer chromatography (HPTLC) on silica G plates (5 × 5 cm; Whatman). The plates were first developed with a solvent system consisting of chloroform-methanol-28% ammonium hydroxide (65:25:5). After the plates were dried with a forced air blower to remove the solvent, the plates were developed in the second dimension with a solvent system consisting of chloroform-acetone-methanol-glacial acetic acid-H2O (50:20:10:10:5). The phospholipids were visualized by exposure to iodine vapor and were identified by comparison with migration of authentic phospholipid standards. The spots identified by iodine staining were scraped and were transferred to tubes. Lipid phosphorus was determined as previously described (1).
High-performance liquid chromatography analysis of cell lipids. Lipid extracts were separated by high-performance liquid chromatography (HPLC) using an ammonium acetate gradient (11). The lipid extracts were applied to a 5-µm Supelcosil LC-Si column (4.6 × 250 mm) equilibrated with a mixture of one part solvent A [2-propanol-hexane-H2O (57:43:1)] and nine parts solvent B [2-propanol-hexane-40 mM aqueous ammonium acetate (57:43:10; pH 6.7)]. The column was eluted during the first 3 min with a linear gradient from 10% solvent B to 37% solvent B, then 3-15 min isocratic at 37% solvent B, 15-23 min linear gradient to 100% solvent B, and 23-45 min isocratic at 100% solvent B; the solvent flow rate was maintained at 1 ml/min. The separations were performed using a Shimadzu HPLC system (model LC-600) equipped with an in-line configuration of fluorescence (model RF-551) and ultraviolet (UV)-VIS (model SPD-10A V) detectors. The effluent was monitored by absorbance at 205 nm to detect lipids and fluorescence of PnA by emission at 420 nm after excitation at 324 nm. UV and fluorescence data were processed and stored in digital form with Shimadzu EZChom software. The identity of phospholipids in the chromatogram was established by collecting each of the peak fractions and by subjecting them to HPTLC analysis as described above. Free PnA was used to establish a calibration curve. Integration of the area of each peak from the HPLC chromatogram was used to calculate nanograms of PnA per microgram of total lipid phosphorous.
Determination of lipid phosphorus in lipid extracts. Lipid phosphorus was determined using a modification of the method described by Chalvardjian and Rubnicki (5). Aliquots of lipid extract were pipetted into test tubes, and the solvent was evaporated to dryness under a stream of O2-free dry N2. Fifty microliters of 70% perchloric acid were added to the dry lipid. The samples were incubated for 20 min at 170-180°C. After cooling, 0.4 ml of distilled water was added to each tube followed in succession by 2 ml of sodium molybdate malachite green reagent [4.2% sodium molybdate in 5.0 N HCl-0.2% malachite green (1:3)] and 80 µl of 1.5% Tween 20. The tubes were shaken immediately to stabilize the developed color that was measured at 660 nm in a Shimadzu UV 160U spectrophotometer. Duplicate or triplicate determinations were made on each spot.
Experimental Protocols
Effect of Cd on SPAEC MT expression.
SPAEC (n = 10) were exposed to
CdCl2 (Sigma) in concentrations up
to 1.0 mM for 24 h. Viability was determined by MTT assay. SPAEC
(n = 2) were then exposed to
CdCl2 (0-10 µM; 24 h), and mRNA levels of sheep MT Ib (sMT-Ib) (and -actin) were
determined. In the next series of experiments, SPAEC
(n = 4) were exposed to 10 µM
CdCl2 for 6-48 h. At various
times, protein was isolated from cell cultures, and levels of MT were
determined.
Cd pretreatment and sensitivity to t-BOOH or hyperoxia. SPAEC (n = 11) were exposed to 0.0-0.5 mM t-BOOH for 24 h, and cell viability was determined by Alamar blue assay. An additional six subcultures of SPAEC were pretreated with 10 µM CdCl2 for 24 h, and cytotoxicity to t-BOOH was assessed. Similar experiments were performed in which either MTT (n = 8) or 51Cr (n = 4) release was used as end points of cytotoxicity.
SPAEC (n = 10) were exposed to 95% O2-5% CO2 for 48 h, and then 5-HT transport was determined. A comparable number of replicates from the same cell culture were pretreated with 10 µM CdCl2, and the effects of hyperoxia on 5-HT transport were quantified.Effect of t-BOOH or hyperoxia on stable transfectants of SPAEC that overexpress MT. The effect of t-BOOH or hyperoxia on SPAEC that overexpress MT was contrasted to the effects of t-BOOH or hyperoxia on wild-type SPAEC or SPAEC that expressed either a neomycin resistance gene (SPAEC/NEO) or a promoterless inverted vector (SPAEC/mTM). Stable MT transfectants were obtained by using either pBPVGRPMT or pCMVhMT-IIA and are referred to as SPAEC/mMT and SPAEC/hMT, respectively. Cytotoxicity to t-BOOH was determined using Alamar blue in SPAEC/mMT (n = 6), SPAEC/hMT (n = 8), SPAEC/NEO (n = 3), and SPAEC/mTM (n = 3). The effects of hyperoxia on 5-HT transport were determined in SPAEC/hMT (n = 2), SPAEC/mMT (n = 2), SPAEC/mTM (n = 3), and an additional three wild-type SPAEC.
Effect of MT overexpression on AMVN-induced lipid peroxidation in SPAEC. AMVN-induced lipid peroxidation was analyzed in wild-type SPAEC and SPAEC/hMT. After incubation of SPAEC with PnA-hSA complex for 2 h, 500 µM AMVN was added, and, 2 h later, total lipids were extracted for HPLC analysis of oxidation of phospholipids. We initially determined that there was no cytotoxicity to either wild-type SPAEC or SPAEC/hMT to 500 µM AMVN or 4 µg/ml PnA-hSA complex for 2 h.
Statistical Analyses
All values are means ± SE unless otherwise indicated. The effect of Cd or t-BOOH on cell viability was determined by two-way analysis of variance. The effect of hyperoxia was determined by Student's t-test. The effect of pretreatment with Cd or overexpression of MT on sensitivity to either t-BOOH or hyperoxia was determined by one-way analysis of variance. Multiple means were compared by Neuwman-Keul's test, and significance was established at P < 0.05 (39). ![]() |
RESULTS |
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Effect of Cd on SPAEC MT Expression
Cd caused a concentration-dependent cytotoxicity in SPAEC as shown in Fig. 1. A threshold was noted such that there was no significant change in the capacity of SPAEC to reduce MTT after 24 h of exposure to 1-10 µM CdCl2. This lower range of Cd resulted in a concentration-dependent increase in steady-state mRNA of sMT-Ib (Fig. 2, A and B). CdCl2 (10 µM) caused a time-dependent increase in MT such that MT increased, on average, between 1.6 and 2.1 µg/mg from 24 to 48 h after Cd exposure (Fig. 3).
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Cd Pretreatment and Sensitivity of SPAEC to t-BOOH or Hyperoxia
Pretreatment with 10 µM CdCl2 decreased the sensitivity of SPAEC to either t-BOOH or hyperoxia in the ensuing 24-48 h. In Fig. 4, we show that t-BOOH caused a concentration-dependent cytotoxicity (as determined by Alamar blue assay) in control SPAEC; there was a significant (P < 0.001) decrease in cell survival at t-BOOH concentrations ([t-BOOH]) >10 µM. After 24 h of Cd pretreatment, there was no significant effect on cell survivability at [t-BOOH] <300 µM, and Cd-pretreated SPAEC were >20-fold more resistant than untreated SPAEC, based on the [t-BOOH] required to kill 50% of the cell population. Survivability in Cd-pretreated SPAEC was significantly greater than control SPAEC at [t-BOOH] from 10 to 150 µM. Similar results were noted in separate subcultures of Cd- pretreated SPAEC exposed to t-BOOH and in which either MTT or 51Cr release was used to quantify cytotoxicity (data not shown). In Fig. 5, we show that 48 h of hyperoxia significantly decreased (P < 0.05) 5-HT transport in SPAEC. Pretreatment with 10 µM CdCl2 by itself did not significantly affect 5-HT transport, but it completely inhibited subsequent hyperoxic-induced changes in this function (Fig. 5). Data in Fig. 5 are normalized to control values in room air (without Cd pretreatment) and are expressed as a percentage of this control.
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Reduced Sensitivity of Stable MT Transfectants of SPAEC to t-BOOH or Hyperoxia
Stable transfectants of SPAEC (SPAEC/mMT) after gene transfer with pBPVGRPMT had MT levels of 0.54 µg/mg and were resistant to the cytotoxic effects of t-BOOH (Fig. 6). Similar results were obtained with transfectants (SPAEC/hMT) overexpressing hMT-IIA after gene transfer with pCMVhMTIIA (Fig. 6). At respective [t-BOOH] from 30 to 80 µM, SPAEC/hMT or SPAEC/mMT were each significantly (P < 0.01) different from SPAEC/NEO (or wild-type control). At 100 µM t-BOOH, SPAEC/hMT (but not SPAEC/mMT) were resistant to t-BOOH compared with SPAEC/NEO, SPAEC/mTM, or wild-type cells (P < 0.01). The sensitivities of SPAEC that were transfected with neomycin resistance gene (SPAEC/NEO) only or with a promoterless expression vector (SPAEC/mTM) to t-BOOH were not different from each other (Fig. 6) and were similar to wild-type SPAEC (Fig. 4). In Fig. 7, we note that 48 h of hyperoxia decreased 5-HT transport in either SPAEC/NEO or SPAEC/mTM to a similar extent (~50%) to that noted in Fig. 5. In contrast, after 48 h of hyperoxia, there was a significant (P < 0.01) difference between wild-type (or SPAEC/NEO) SPAEC and either SPAEC/mMT or SPAEC/hMT. Hyperoxia decreased cell growth (as determined by hemacytometer) in SPAEC/hMT, SPAEC/mMT, or control cells (data not shown).
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Overexpression of MT Inhibits Peroxyl Oxidation of Selective Phospholipids in SPAEC
Effect of AMVN on the phospholipid composition of SPAEC and
SPAEC/hMT.
To compare the pattern of susceptibility of individual phospholipid
classes to oxidation in SPAEC and SPAEC/hMT, samples of each cell type
were incubated for 2 h at 37°C in the presence of 0.5 mM AMVN.
After incubation, total lipid extracts were prepared, and lipids were
separated by HPTLC as described in MATERIALS AND METHODS. A typical chromatogram is illustrated in Fig.
8. In both cell lines, phosphatidylcholine
(PC) represents about one-half of the total phospholipid with
phosphatidylethanolamine (PEA), the next most prominent phospholipid
(~25%). Additionally, the phospholipids in the order of their
abundance, phosphatidylserine (PS) > sphingomyelin (SPH) > phosphatidylinositol (PI) > diphosphatidylglycerol (DPG) lysophosphatidylcholine (LPC), were detectable on the HPTLC plates. The
results of three independent experiments are summarized in Table
1. It is clear that there were no
significant differences in the pattern of distribution of phospholipid
classes between SPAEC and SPAEC/hMT. Furthermore, no significant
differences in phospholipid distribution were detected in either cell
line after oxidative stress was imposed by incubation of the cells in
the presence of AMVN. A slight increase the in content of LPC, a
relatively minor phospholipid, in AMVN-treated cells may be due to
hydrolysis exacerbated by oxidative stress.
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Incorporation of PnA into SPAEC lipids. A more sensitive method to detect oxidative stress in SPAEC was developed that consisted of metabolically incorporating PnA into the constituent phospholipids and monitoring oxidative processes by HPLC with fluorescence detection (10, 33). SPAEC and SPAEC/hMT were incubated in the presence of PnA-hSA for 2 h to incorporate PnA into cellular phospholipids. HPLC analysis detected fluorescence peaks that included DPG, PI, PEA, PS, PC, and SPH (Fig. 9). The identity of the fluorescence peaks was also confirmed by HPTLC of the collected HPLC fractions. Control incubations of cells with hSA alone showed no fluorescent HPLC components under the excitation and emission conditions used, indicating that PnA incorporated into the phospholipids was responsible for the fluorescence.
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DISCUSSION |
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This is the first study to report induction of MT gene expression in pulmonary endothelial cells (Figs. 2 and 3) and to identify a potential physiological role for MT as an antioxidant in these cells. An association between Cd-induced MT expression in SPAEC and resistance to oxidant stress is apparent (Figs. 4 and 5). A role for MT as a component of pulmonary endothelial antioxidant defense is more directly shown in resistance of stable transfectants of SPAEC that overexpress MT to t-BOOH (Fig. 6) and hyperoxia (Fig. 7). In addition, these genetically modified cells were capable of inhibiting peroxyl oxidation of selective phospholipids (Fig. 10). This latter observation made use of the oxidant-sensitive fatty acid analog PnA to metabolically label phospholipids of intact cells and allowed us to assess lipid peroxidation from grossly uninjured endothelial cells for the first time. Basic information regarding pulmonary endothelial cell MT expression is particularly useful in light of the sensitivity of these cells to both heavy metals (28) and O2 free radicals (8).
Pulmonary Endothelial Cell MT Expression
Intrapulmonary levels of MT are usually low, and constitutive expression of immunoreactive MT has only been noted in the saccular fetal lamb lung (31). When levels are elevated, either during fetal development (31) or after Cd (12) or hyperoxic (15, 38) exposure, pulmonary MT protein or mRNA has been primarily localized to airway epithelium, fibroblasts, macrophages, or chondrocytes; there are no reports of induction of pulmonary endothelial cell MT in situ, even when bulk lung mRNA levels increase >100-fold (38). We may assume that the stimulus used in the current study (10 µM CdCl2) was either greater or more effective for pulmonary endothelial cell MT induction (Figs. 2 and 3) than that produced by inhalation of Cd in intact rats (12) or during hyperoxic exposure in rats or rabbits (15, 38). It is possible that lack of induction of MT in pulmonary endothelial cells in situ contributes to the sensitivity of this segment of lung to metal and oxidant-mediated toxicity.Cd Pretreatment Reduces Sensitivity of Pulmonary Endothelial Cells to Oxidant Injury
A large number of studies have shown that various stimuli that induce MT result in cells or tissues that are subsequently cross-resistant to a second heavy metal exposure or partially reduced oxidant species (21, 34). In lung, exposure of rats to aerosolized Cd produced cross-resistance to ozone (2) and hyperoxia (16). Alveolar macrophages (13) isolated from these animals were resistant to H2O2. In the present study, we show that pretreatment of SPAEC with nontoxic (Fig. 1) doses of Cd greatly reduces the sensitivity of these cells to two forms of oxidant injury, t-BOOH (Fig. 4) and hyperoxia (Fig. 5). These results support observations noted above but were performed in a simpler model system, obviating complexities secondary to attendant inflammation. Nonetheless, others have noted the difficulties in defining a functional role for MT after its chemical induction (18). For example, the original observation that MT was likely responsible for resistance to oxidative stress in V79 Chinese hamster cells previously rendered resistant to high concentrations of Cd (25) subsequently was reassessed to include a role for glutathione (7). Thus we pursued an alternative approach with the use of direct gene transfer protocols to provide less ambiguous information about the functional role of MT in SPAEC. Nonetheless, it is possible that overexpression of MT after either Cd exposure or direct gene transfer affected intracellular metal ion homeostasis and pools of cysteine in such a fashion that secondary changes in other antioxidant activities may have occurred.Direct Gene Transfer of MT Reduces the Sensitivity of SPAEC to Oxidant Stress
In the current study, we show that overexpression of MT in SPAEC reduces the sensitivity of these cells to t-BOOH (Fig. 6), hyperoxia (Fig. 7), and AMVN-induced peroxyl generation (Figs. 9 and 10). The apparent greater protection to t-BOOH after Cd induction vs. direct gene transfer (Fig. 4 vs. Fig. 6, respectively) may be due to higher levels of MT or induction of other protective gene products after Cd exposure. These data extend original observations about the role of MT in lower eukaryotes (37) as well as our own work in cultured murine NIH/3T3 (35, 36) and embryonic cells (20).Tamai et al. (37) reported that yeast and simian MT could functionally substitute for Cu,Zn-containing superoxide dismutase in superoxide dismutase-1-deleted strains of Saccharomyes cerevisiae when these mutants were forced to respire when grown on agar containing lactate. The protection was limited to this form of oxidant stress because expression of MT in these mutants did not restore wild-type resistance to either paraquat or hyperoxia. Conversely, yeast lacking MT were no more sensitive than wild-type yeast to oxidative stress encumbered by long-term stationary phase growth (23). Differences between yeast and mammalian MT are profound and may contribute to these discrepancies. For instance, S. cerevisiae MT has only one metal-binding domain in contrast to two such domains in mammalian MT (37). These data reinforce the importance of examining multiple forms of oxidative stress before generalizing about the role of one particular antioxidant in a more differentiated higher eukaryotic cell such as SPAEC. In this regard, it is noteworthy that we utilized three distinctly different forms of oxidative stress to identify such a functional role for MT in SPAEC.
Not all cells that overexpress MT are resistant to all forms of oxidative stress. In addition to such conclusions in yeast (see above), Chinese hamster ovary (CHO) cells that overexpress hMT-IIA were resistant to alkylating agents (19) but not to ionizing irradiation, bleomycin, paraquat, or H2O2 (22). It appears that the above-noted MT-transfected CHO cells may have fundamental differences in DNA repair mechanisms (19), obfuscating the role of MT in these cells. Thus, in some instances, it may be advantageous to initiate experiments in primary cell culture.
We utilized several techniques to assess endothelial cell membrane function. The initial effects of hyperoxia on cultured endothelial cells are subtle, and thus we used 5-HT transport as an indicator. We confirmed original observations by Block et al. (4) that 95% O2 reduces 5-HT transport by 50% in cultured endothelial cells. At 48 h, any small changes in either 51Cr release or reduction of MTT could be explained by lack of cell growth in hyperoxia. Thus 5-HT transport appears to be a useful indicator of oxidant-mediated changes in endothelial cell membrane function. The use of the oxidant-sensitive fluorescent probe PnA in the current study is also of interest. This reporter molecule metabolically labels phospholipids of intact cells and then is sensitive enough to detect oxidation of phospholipids in otherwise uninjured cells. In our study, we used a nontoxic dose of AMVN to generate peroxyl radicals that are well known to target phospholipids without the requirement for transition metals. Thus we could exclude the confounding role of potential Fe-MT, Cu-MT, or Zn-MT interactions contributing to the antioxidant activity of MT. Detection of oxidized phospholipids with PnA and HPLC technology is considerably different from information obtained with other methods such as thiobarbituric acid reactive substances (TBARS). In this latter example, the product is neither sensitive nor specific and usually reaches detectable levels only during extensive cell damage. Thus it becomes ambiguous whether lipid peroxidation as assessed by TBARS assay is a component of the cellular response to injury or whether it occurs artifactually due to DNA damage or lipid peroxidation after cell death.
We demonstrated that overexpression of MT in SPAEC provided almost complete protection of all the phospholipid classes except PEA against peroxidation induced by a lipid-soluble azo-initiator of peroxyl radical AMVN. Because 2 h of incubation with AMVN did not result in any significant cell kill, we conclude that both AMVN-induced peroxidation and protection afforded by AMVN occurred in live cells. One has to keep in mind that a significant depletion of PnA fluorescence corresponded to a relatively small level of intracellular peroxidation. This is because PnA-labeled phospholipids represent <1% of the total pool of membrane phospholipids. This may explain, at least to some extent, why compartments containing PnA-labeled PEA (which represented only 1.6% of the total pool of PEA in SPAEC; see Table 2) were not effectively protected by MT overexpression. One may assume that expression of MT in these particular compartments was relatively low. In line with this, our studies and those from other laboratories demonstrated a pronounced compartmentalization of MT expression in cells.
Generation of carbon-centered radicals and subsequently formed peroxyl and alkoxyl radicals from AMVN is not dependent on the presence of transition metals, but it is strictly dependent on the temperature. This means that the protective effect due to MT overexpression was not associated with their ability to chelate transition metals and to form redox-inactive complexes. It is, rather, caused by direct scavenging of alkoxyl and peroxyl radicals by MT and/or other scavengers associated with MT overexpression. Certainly, a sulfhydryl group of one or more MT cysteines might be involved in the scavenging. Further studies may reveal whether only free or also metal-coordinated cysteinyl residues may be effective in radical scavenging.
The mechanism by which MT acts as an antioxidant remains unclear. In vitro experiments clearly show that MT can scavenge superoxide anions or hydroxyl radicals (34), phenoxyl radicals (35), and nitric oxide radicals (36), and thus MT can act as an expendable target for oxidants due to its highly enriched cysteine residue structure. Because these cysteines are normally involved in thiolate clusters with various essential metals, one of the more remarkable features of MT is the facility by which these occupied groups still can interact with electrophiles (24). This was recently confirmed in intact cells by demonstrating that H2O2 could interact with Zn-MT in HL-60 cells oxidizing the MT sulfhydryl groups and releasing Zn (32). Although we are unaware of the presence of MT reductases, it is nonetheless quite remarkable that modest increases in intracellular MT levels (i.e., after direct gene transfer) provide protection against rather large concentrations of t-BOOH (100-300 µM). Thus, if MT is indeed an expendable target for various free radicals, it is possible that an unknown MT-regenerating system exists in cells. Alternatively, MT may function as an antioxidant indirectly by affecting two important metals, Zn and Cu. Zn in itself may be an antioxidant, whereas Cu is an important transition metal for Fenton reactions. The ability of MT to inhibit AMVN metal-independent lipid peroxidation, however, suggests that MT-metal interactions do not exclusively underlie the antioxidant role of MT.
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ACKNOWLEDGEMENTS |
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We acknowledge the technical assistance of Gina Steve, Laura Chesny, and Joseph DeFillippo. In addition, Dr. Arthur Atlas was helpful in the initial design of these experiments. We are grateful to Dr. Julian F. B. Mercer (Victoria, Australia) for the gift of the sheep MT-Ib cDNA probe and to Dr. Kathyrn Morton (Oregon Health Sciences Center, Portland, OR) for the gift of the plasmid expression vectors pBPVGRPMT and pBPVGRPTM.
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FOOTNOTES |
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This work was supported in parts by National Institutes of Health Grants HL-32154 (to B. R. Pitt), P50-GM-53789 (to B. R. Pitt), HL-08614 (to R. J. Mannix), HL-09368 (to E. Yee), and CA-61299 (to J. S. Lazo) and by Grant 96008 from The Johns Hopkins Center for Alternatives to Animal Testing (to V. E. Kagen).
Address for reprint requests: B. R. Pitt, Dept. of Pharmacology, Univ. of Pittsburgh School of Medicine, Pittsburgh, PA 15261.
Received 17 January 1997; accepted in final form 2 July 1997.
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