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Manganese and iron transport across pulmonary epithelium

¹Department of Genetics and Complex Diseases, ²Department of Environmental Health, ³Harvard NIEHS Center for Environmental Health Proteomics Facility, Harvard School of Public Health, Boston, Massachusetts

Submitted 27 October 2005 ; accepted in final form 13 January 2006

	ABSTRACT

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Pathways mediating pulmonary metal uptake remain unknown. Because absorption of iron and manganese could involve similar mechanisms, transferrin (Tf) and transferrin receptor (TfR) expression in rat lungs was examined. Tf mRNA was detected in bronchial epithelium, type II alveolar cells, macrophages, and bronchus-associated lymphoid tissue (BALT). Tf protein levels in lung and bronchoalveolar lavage fluid did not change in iron deficiency despite increased plasma levels, suggesting that lung Tf concentrations are regulated by local synthesis in a manner independent of body iron status. Iron oxide exposure upregulated Tf mRNA in bronchial and alveolar epithelium, macrophages, and BALT, but protein was not significantly increased. In contrast, TfR mRNA and protein were both upregulated by iron deficiency. To examine potential interactions with lung Tf, rats were intratracheally instilled with ⁵⁴Mn or ⁵⁹Fe. Unlike ⁵⁹Fe, interactions between ⁵⁴Mn and Tf in lung fluid were not detected. Absorption of intratracheally instilled ⁵⁴Mn from the lungs to the blood was unimpaired in Belgrade rats homozygous for the functionally defective G185R allele of divalent metal transporter-1, indicating that this transporter is also not involved in pulmonary manganese absorption. Pharmacological studies of ⁵⁴Mn uptake by A549 cells suggest that metal uptake by type II alveolar epithelial cells is associated with activities of both L-type Ca²⁺ channels and TRPM7, a member of the transient receptor potential melastatin subfamily. These results demonstrate that iron and manganese are absorbed by the pulmonary epithelium through different pathways and reveal the potential role for nonselective calcium channels in lung metal clearance.

transferrin; bronchoalveolar lavage fluid; macrophages

The mechanisms involved in metal uptake by pulmonary epithelial cells, and transport of trace metals across the air-blood barrier, are not fully understood. For manganese and iron, understanding the interplay between regulation of transporters by iron status, and competition (or lack thereof) for different transport mechanisms, would be of significant benefit in risk assessment for mixtures of iron and manganese particles, as found in welding fume.

Divalent metal transporter-1 (DMT1/Nramp2/SLC11A2) is expressed by pulmonary epithelial cells and appears to play an important role in regulation of iron homeostasis in the lungs. Belgrade rats, which possess a naturally occurring point mutation in DMT1 (G185R) that results in protein misfolding and degradation, exhibit reduced clearance of iron and vanadium from the lungs after instillation with residual oil fly ash (ROFA) (25). DMT1 expression is upregulated in bronchial epithelium of rats exposed to ferric ammonium citrate, supporting a role in detoxification (56). DMT1 can transport several other divalent metal ions in addition to iron, including manganese (26).

The iron-binding proteins transferrin (Tf), ferritin, and lactoferrin are also synthesized by pulmonary epithelial cells. These soluble iron-binding factors are found in pulmonary fluid and are thought to contribute to regulation of free iron levels in the lungs and the prevention of oxidative damage (24, 39, 51, 60). The ferroxidase ceruloplasmin is also present in pulmonary fluid and could potentially oxidize Fe²⁺ to Fe³⁺, thereby limiting oxidative damage caused by highly reactive ferrous iron and promoting interaction of ferric iron with Tf (39).

Tf not only binds Fe³⁺, but can also bind Mn³⁺, albeit with lower affinity (2). Receptor-mediated endocytosis of Tf by transferrin receptor (TfR) expressed on the apical surface of pulmonary epithelial cells therefore could be an important mechanism for clearance of iron, manganese, and other metals from airway and alveolar fluid. DMT1 also plays a key role in TfR-mediated iron uptake as the transporter of iron released from Tf in the endocytic vesicle into the cytoplasm and would be envisioned to have an analogous role in uptake of Mn-Tf via this pathway. As for iron, the speciation of manganese would be an important determinant for transport mediated by DMT1 across the plasma membrane (Mn²⁺) or for uptake by TfR-mediated endocytosis of Tf-bound metal (Mn³⁺). Whether Mn²⁺ would be converted to Mn³⁺ by lung fluid oxidases (e.g., ceruloplasmin) remains unknown.

In addition to the Tf/TfR/DMT1-mediated pathway for metal uptake, there is mounting evidence that nonselective calcium channels transport divalent iron and manganese. Fe²⁺ and Mn²⁺ uptake by cardiomyocytes in vitro (52) and uptake of Fe²⁺ by in situ-perfused heart (33) are both partially blocked by inhibitors of L-type voltage-gated channels (LVGCs) and increased by LVGC activators. Mn²⁺ uptake by liver was found to be sensitive to the LVGC inhibitor nifedipine (12). Similar analysis of isolated blood-brain-barrier-derived cells, and in situ brain perfusion assays, has demonstrated sensitivity of Mn²⁺ uptake to store-operated calcium channel (SOC) inhibitors and activators (17). Several members of the transient receptor potential (TRP) family of receptor- and store-operated calcium channels have also been implicated in transport of multiple divalent metal ions. For example, use of Mn²⁺ as a proxy for Ca²⁺ in patch-clamp experiments has demonstrated that TRPC1, TRPC3, TRPC4, TRPC5, and TRPC6 are permeable to manganese ions (9, 29).

Previously, we observed that the in vivo pharmacokinetics for intratracheally instilled ⁵⁴Mn and ⁵⁹Fe were remarkably different, suggesting that these metals cross the pulmonary epithelium through different molecular mechanisms (28). Here, we further examine pathways for transport of these metals by pulmonary epithelial cells. Our results demonstrate that after instillation of soluble divalent forms, iron and manganese appear to be cleared from the lungs by distinct mechanisms. Evidence from in vitro studies of ⁵⁴Mn transport by A549 type II alveolar epithelial cells suggests that nonselective calcium channels may play an important role in uptake of manganese ions by pulmonary epithelial cells.

	MATERIALS AND METHODS

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Animal care and diets. Animal protocols for this study were approved by the Harvard Medical Area Animal Care and Use Committee of Harvard University. Male CD/HSD rats (21 days) were purchased from Harlan Sprague Dawley (Indianapolis, IN). Rats were maintained on a 12:12-h light-dark cycle and were given food and water ad libitum. To induce iron deficiency, rats were fed a low-iron diet containing 20–25 ppm Fe for 3 wk (Purina test diet no. 7444; PharmaServ, Framingham, MA). Age-matched control rats were fed a standard diet containing 200 ppm Fe (Purina diet no. 5053). Iron deficiency was confirmed by measurement of hematocrit as described previously (28); hematocrits of Fe-deficient rats were significantly lower than in control animals (27.7 vs. 44.8%, respectively). For iron oxide particle exposure, rats were intratracheally instilled with a suspension of iron oxide particles (mass median aerodynamic diameter of 0.68 µm) generated by the combustion of iron pentacarbonyl vapor (55). Rats were placed on a slanted platform, supported by an elastic band placed under the upper incisors. Iron oxide particle suspension in sterile PBS was delivered to the lungs via a blunt 18-gauge needle inserted between the laryngeal folds and into the trachea. Transillumination of the larynx was provided by a microscope lamp shining on the neck (8). Rats were instilled with 7.5 mg iron oxide·kg^–1·day^–1 every 3 days for a total of five instillations in 2 wk and were humanely killed 3 wk after commencement of instillations. Lung nonheme iron levels were significantly elevated in these animals as previously determined (7).

To study metal binding by lung fluid proteins, ⁵⁴MnCl₂ and ⁵⁹FeCl₃ were purchased from Perkin Elmer/New England Nuclear (Boston, MA). ⁵⁹Fe was diluted with 1:20 molar excess of ascorbic acid immediately before the experiment. Radioisotopes were further diluted to 13.3 µCi ⁵⁹Fe/ml and 14.7 µCi ⁵⁴Mn/ml in sterile PBS immediately before instillation. To deliver radioisotope to the lungs, rats were anesthetized with vaporized halothane (Halocarbons Lab, North Augusta, SC) and intratracheally instilled as described above with a delivery volume of 1.5 ml per kilogram of body weight. Ten minutes or 1 h postinstillation, rats were anesthetized with pentobarbital sodium and killed by exsanguination. The trachea was then cannulated and the lungs were lavaged once with 3 ml of sterile PBS. Bronchoalveolar lavage (BAL) samples were centrifuged at 3,000 g for 10 min at 4°C to remove cells and supernatants were immediately processed for fast protein liquid chromatography (FPLC) studies or stored at –80°C for Western blot analysis.

A Belgrade rat colony was established with animals obtained from Dr. M. Garrick (SUNY-Buffalo). Mating pairs of female +/b and male b/b rats were maintained on an iron-supplemented diet containing 500 ppm ferrous iron (TD02385, Harlan Teklad, Madison, WI). Female +/b rats were fed the iron-supplemented diet throughout pregnancy. At postnatal day 6, litters were cross-fostered onto F344 Fischer dams (Harlan Sprague Dawley) fed a standard diet containing 200 ppm iron (Purina diet no. 5053, PharmaServ). On weaning, b/b and +/b rats were fed the iron-supplemented diet (500 ppm). Belgrade (b/b) pups were identified at birth by pale, anemic features and genotype was verified by PCR as described by Fleming et al. (21).

To determine the pharmacokinetics of manganese absorption after intratracheal instillation, b/b and +/b rats were anesthetized with vaporized halothane. ⁵⁴MnCl₂ diluted to 10 µCi/ml in PBS was administered by intratracheal instillation as described above (final delivery volume of 1.5 ml/kg body wt). Blood samples were collected from the tail vein at 5, 15, and 30 min and at 1, 2, and 4 h postinstillation. Rats were killed 4 h postinstillation and brain, heart, liver, lung, bone marrow, muscle, kidney, stomach, small intestine and large intestine were collected for measurement of associated ⁵⁴Mn. All tissue and blood samples were weighed and radioactivity was measured in a Packard gamma counter (Cobra Quantum, Packard Instrument, IL), and ⁵⁴Mn level was calculated as a percentage of the instilled dose.

In situ hybridization. After death, the tracheas of control and iron-deficient rats were cannulated with a blunt 18-gauge needle and syringe containing optimum cutting temperature (OCT) compound (Sakura Finetek USA, Torrance, CA) prewarmed to 37°C. Lungs were filled with OCT, cut laterally into sections, mounted in OCT, snap-frozen in 2-methylbutane chilled on dry ice, and stored at –80°C. Ten-micrometer-thick sections were cut on a cryotome and stored at –20°C until processed. Digoxigenin-labeled sense and antisense cRNA probes were transcribed from a full-length rat Tf cDNA (generously supplied by Dr. S. Hisayasu, Tokyo, Japan) or a 594-bp rat TfR EST (Resgen/Invitrogen, Carlsbad, CA), flanked by T7 and T3 promoter sites. Transcripts were shortened to an average length of 200–400 bp by alkali hydrolysis. Sections were incubated with sense or antisense probe (200 ng/ml) in hybridization buffer (50% formamide, 5x SSC, 2% blocking reagent, 0.02% SDS, 0.1% N-laurylsarcosine). Hybridized probes were detected using anti-digoxigenin-alkaline phosphatase-coupled Fab fragments and bromo-4-chloro-3-indoxyl-phosphate and nitro blue tetrazolium as a substrate. Sections were incubated in substrate solution for 42 h, rinsed in TE buffer (10 mM Tris and 1 mM EDTA, pH 8.0), and mounted in 50% PBS-glycerol.

Detection of proteins by Western blotting. Snap-frozen lung tissue from control, Fe-deficient, or Fe oxide-exposed rats was homogenized on ice in 20 mM HEPES, pH 7.4, 100 mM KCl, 85 mM sucrose, 20 µM EGTA, 1% Triton X-100, with Complete Mini protease inhibitor cocktail (Roche, Indianapolis, IN) added. Homogenates were incubated at 4°C with rotation for 45 min and then clarified by centrifugation for 10 min at 12,000 g. Protein concentration in supernatants was measured by Bradford assay (6). Samples of 120 µg lung protein or 20 µg BAL protein were electrophoresed on 10 or 7% SDS-polyacrylamide gels, respectively, and transferred to nitrocellulose for immunoblotting. Membranes were blocked with 5% nonfat dry milk in TBST (50 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.4) for 1 h at room temperature, then incubated with mouse anti-human TfR (1:1,000; Zymed, San Francisco, CA), rabbit anti-human Tf (1:2,000, Rockland), sheep anti-human ferritin (1:1,000, The Binding Site, Birmingham, UK) or mouse anti--tubulin (1:10,000, Sigma, St. Louis, MO) overnight at 4°C. Secondary horseradish peroxidase-labeled goat anti-mouse IgG (1:40,000, Pierce, Rockford, IL) or donkey anti-rabbit IgG (1:40,000, Pierce) was used to detect immunoreactivity by enhanced chemiluminescence using SuperSignal West Pico Reagent (Pierce). Intensity of bands was quantified with Bio-Rad QuantityOne software.

FPLC fractionation of BAL fluid. After intratracheal instillation of ⁵⁴Mn or ⁵⁹Fe as described above, BAL supernatant was centrifuged at 12,000 g for 15 min to remove insoluble material and debris. Two milliliters were then dialyzed (Spectra/Por, MWCO 12,000–14,000; Spectrum Laboratories, Rancho Dominguez, CA) against FPLC buffer A (25 mM Tris, pH 8.5) for 4–6 h at 4°C to lower salt concentration. Samples were loaded on a MonoQ anion-exchange column and proteins were eluted with a two-step linear salt gradient (40 ml of 0–0.5 M NaCl, followed by 20 ml 0.5–1.00 M NaCl). Radioactivity in 1-ml fractions from FPLC, along with cpm in lungs, BAL cell pellet, and BAL (pre- and postdialysis) was measured by gamma counting.

For FPLC fractionation of ⁵⁴Mn-Tf, a 100-µl solution containing 12.5 µM human apo-Tf (Sigma), 125 µM MnCl₂, and 125 nM ⁵⁴MnCl₂ (0.3 µCi) in 15 mM NaHCO₃ (pH 8.0) was incubated at 37°C for 5 h. The binding of manganese to Tf was confirmed by measuring the absorbance ratio at 300 nm/280 nm (16), and ⁵⁴Mn-Tf was subsequently dialyzed (molecular weight cutoff 12,000–14,000) against FPLC buffer A for 12–16 h at 4°C. ⁵⁴Mn-Tf was fractionated by FPLC on a MonoQ column exactly as described above.

Protein fractions were precipitated overnight at 4°C (25% trichloroacetic acid, 0.02% casamino acids), washed twice with ice-cold acetic acid, and resuspended by boiling for 10 min in 1x Laemmli buffer containing 1% -mercaptoethanol. Fractions were electrophoresed on 5–20% SDS-polyacrylamide gels and proteins were visualized by either Coomassie blue or silver staining. Bands of interest were excised and digested overnight at 37°C in 50 mM NH₄HCO₃ containing 10–20 µg/ml trypsin. Tryptic peptides were extracted with one change of 20 mM NH₄HCO₃, two changes of 5% formic acid in 50% acetonitrile, and one change of 5% formic acid in 50% isopropanol at room temperature. For identification of proteins by nano-liquid chromatography (LC) electrospray ionization tandem mass spectrometry, digests were injected onto a C18 solid-phase extraction column for desalting, eluted onto and separated on a nano-LC column with a linear gradient of acetonitrile in 0.1% formic acid, and the eluent was introduced into a LCQ Deca XP Plus mass spectrometer by nanoelectrospray. A full MS scan between 400 and 1,800 m/z was performed, followed by five MS/MS scans for the five most intense ions from the MS scan acquired in data-dependent MS/MS scanning experiments. Data files were converted to .dta files using Bioworks 3.2 software (ThermoElectron) and submitted to the database search against a composite [forward and reversed (40)] protein sequence database derived from NCBI rat database on a Sorcerer search station (Sage-N, Thermo Electron, Philadelphia, PA) using SEQUEST algorithm. SEQUEST output (.out files) was analyzed using DTASelect software (provided by Dr. J. Yates, Scripps Research Institute).

⁵⁴Mn and ⁵⁵Fe uptake assays. A549 cells (ATCC no. CCL-185; kindly provided by Dr. D. Tschumperlin, Harvard School of Public Health) were maintained in DMEM/F12 (1:1) media (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum (Gemini Bio Products, Woodland, CA) and seeded at a density of 3.6 x 10⁵ cells per well in 6-well plates, or 1.6 x 10⁵ cells per well in 12-well plates, 24 h before uptake assays. Cells were subconfluent (70–80%) at time of assays. Cells were washed 3x with HeBS (20 mM HEPES, 137 mM NaCl, 0.5 mM Na₂HPO₄, 0.5 mM KCl, pH 7.4) and then incubated with 1–2 µCi ⁵⁴MnCl₂ (Perkin Elmer/New England Nuclear) in HeBS with drugs or vehicle for 4 min at 37°C unless otherwise indicated. After being washed 3x with HeBS, cells were incubated with 100 mM MnCl₂ in HeBS for 1 h at 4°C to eliminate nonspecific binding of ⁵⁴Mn. Cells were then washed 3x in HeBS and lysed in 600 µl of solubilization buffer (0.02 M NaOH, 0.1% Triton X-100). Simultaneous control assays were performed at 4°C for all treatments. For assays with ⁵⁵Fe, a stock solution of ⁵⁵Fe-nitrilotriacetic acid (1:4) was diluted with a 1:50 molar ratio of freshly made ascorbic acid immediately before each assay, then diluted further in HeBS uptake buffer at indicated pH. Radioactivity of lysates was measured in a gamma (⁵⁴Mn) or beta (⁵⁵Fe) counter, and protein concentration was determined by Bradford assay. Specific uptake was taken as the difference between uptake measured at 37 and 4°C and was calculated as picomole Mn per microgram of protein. MgCl₂, NiCl₂, and CaCl₂ stocks were 0.1–1.0 M in H₂O. FeCl₃ was dissolved in water and mixed with a 1:50 molar excess of ascorbic acid immediately before use. Drug stocks were prepared as follows: verapamil, 10 mM in H₂O; amiloride, 10 mM in DMSO; thapsigargin, 3 mM in DMSO; nifedipine, 10 mM in ethanol; BAY K 8644, 0.1 mM in ethanol; 2-aminoethoxydiphenyl borate (2-APB), 10 mM in DMSO; and oleylacetylglycerol (OAG), 20 mM in DMSO.

RT-PCR. RNA was isolated from subconfluent A549 cells with RNA-Bee (Tel-Test, Friendswood, TX) following the manufacturer's instructions. RNA was treated with DNase I (Promega, Madison, WI) to remove genomic DNA, extracted with phenol-chloroform-isoamyl alcohol (25:24:1) mixture, precipitated, resuspended in diethyl pyrocarbonate-treated water, and quantified by UV spectrophotometry. cDNA was synthesized from 2 µg RNA in reactions containing 1x Moloney murine leukemia virus-reverse transcriptase (MMLV-RT) buffer, 0.5 mM dNTP mix, 25 µg/ml oligo(dT)15 primer, 5 mM MgCl₂, 10 mM dithiothreitol, 1 U of RNasin, and 200 U of MMLV-RT enzyme (Promega). MMLV-RT enzyme was ommitted for RT-control reactions. Amplification reactions were carried out with 1 µl of cDNA in 1x PCR buffer (10 mM Tris·HCl, pH 9.0, 50 mM KCl, 2.0 mM MgCl₂; Promega). Sequences of primers for TRPC1–6 were previously published elsewhere (53). Sequences of other primers used are as follows: TRPC7 forward, 5'-CTGCCAGCCAACGAGAGT-3'; TRPC7 reverse, 5'-TCATCAAAGTAAGACAGCCAGAGT-3'; TRPM7 forward, 5'-TCCAGCACAGTTAAAAGGTCAATA-3'; TRPM7 reverse, 5'-GTCTTCAGTTCAGGCTTCTCATAC-3'; LVGC (-subunit) forward, 5'-AGAATCTGGGCAGAGTATGAC-3'; and LVGC reverse, 5'-CAGGAACGTGGCGTAGAACTT-3'.

Fifteen microliters of each reaction were separated on a 1.2% agarose gel containing ethidium bromide, and band intensity was quantified with QuantityOne software (Bio-Rad, Hercules, CA).

Statistical analysis. All values are means ± SE. Comparisons of protein expression measured by Western blotting and A549 cell metal uptake assays were evaluated by unpaired Student's t-test. Characteristics of the pharmacokinetics of manganese transport and tissue distribution of ⁵⁴Mn were evaluated by multivariate ANOVA (MANOVA) using the general linear model procedure (SAS statistical analysis software; SAS Institute, Cary, NC). Statistical significance was based on an level of 0.05.

	RESULTS

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Expression of transferrin in lungs. Expression and iron-responsive regulation of Tf and TfR were examined by in situ hybridization and Western blot analysis of lung tissue from control, iron-deficient, and iron oxide-exposed rats. In control animals, Tf mRNA was detected in type II alveolar epithelial cells, alveolar macrophages, bronchial epithelium, and in localized regions of bronchus-associated lymphoid tissue (BALT; Fig. 1, A and D). Tf mRNA expression in iron-deficient rats exhibited the same localization pattern and staining intensity as in controls (Fig. 1, B and E). In iron oxide-exposed lung, staining for Tf mRNA was greater in BALT and bronchial epithelium than in control lung (Fig. 1F) and appeared more intense in alveolar epithelial cells and macrophages in regions where iron particles were deposited (Fig. 1C).

View larger version (129K): [in this window] [in a new window]   Fig. 1. In situ hybridization detecting lung transferrin (Tf) mRNA expression. Ten-micrometer-thick cryosections of lungs from control (A, D), iron-deficient (B, E), and iron oxide-exposed (C, F) rats embedded in optimum cutting temperature (OCT) were hybridized with digoxigenin-labeled sense and antisense Tf probes as described under MATERIALS AND METHODS. Tf mRNA is detected by an alkaline phosphatase-coupled reaction (bluish-purple staining) and iron oxide particles appear brown (C, F). AE, alveolar epithelial cell; M, macrophage; P, particle-containing macrophage; BALT, bronchus-associated lymphoid tissue; BE, bronchial epithelium. Magnification x10.

View larger version (19K): [in this window] [in a new window]   Fig. 2. Transferrin receptor (TfR), Tf, and ferritin protein levels in control, Fe-deficient, and Fe oxide-exposed rat lung. A: Western blot analysis was performed as described under MATERIALS AND METHODS. Lanes contain samples from individual animals. Separate experiments were conducted for Fe-deficient and Fe oxide-exposed groups, with age-matched controls. Note that ferritin levels were below limit of detection in control and Fe-deficient lung. B: Tf and TfR protein expression levels in A were quantified (QuantityOne imaging software, Bio-Rad). Values are expressed as % control and normalized to -tubulin expression level. *P < 0.0005 (n = 4 to 5; means ± SE).

View larger version (13K): [in this window] [in a new window]   Fig. 3. Tf levels increase in plasma but not bronchoalveolar lavage (BAL) of iron-deficient rats. A: Western blot analysis was performed to determine Tf levels in BAL and plasma of control and Fe-deficient rats. B: band intensity detected by chemiluminescence was quantified using QuantityOne imaging software (Bio-Rad). *P < 0.05 (n = 5 to 6; means ± SE).

View larger version (122K): [in this window] [in a new window]   Fig. 4. In situ hybridization detecting lung TfR mRNA expression. In situ hybridization for TfR transcripts present in control (A, D), iron-deficient (B, E), and iron oxide-exposed (C, F) rat lungs was detected as described for Fig. 1. Magnification x25 (A–C) and x10 (D–F).

Interaction of ⁵⁴Mn and ⁵⁹Fe with proteins in BAL fluid. To determine whether soluble manganese or iron introduced into the lungs interacts with Tf in pulmonary fluid, rats were intratracheally instilled with ⁵⁴Mn or ⁵⁹Fe and lavage fluid was collected 10 min postinstillation. Although the levels associated with lung tissue were comparable for ⁵⁴Mn and ⁵⁹Fe (55% instilled dose), significant differences were observed in the distribution of the radioisotopes in BAL fluid (Table 1). Only 14% of instilled ⁵⁴Mn remained in BAL fluid after 10 min of which 1% was found to be associated with protein (as determined by dialysis). In contrast, 27% of instilled ⁵⁹Fe was found in BAL, of which 22% was bound to protein. To examine the association of the radioisotopes with Tf in lung fluid, in vivo-radiolabeled BAL samples were fractionated by anion-exchange chromatography using fast-performance liquid chromatography. The two major chromatographic peaks were identified as Tf (fraction 17) and albumin (fractions 26-27) by Western blotting (data not shown). After dialysis of BAL fluid, ⁵⁹Fe was found predominantly in fraction 17 containing the peak for Tf (Fig. 5A). In contrast, ⁵⁴Mn was detected only in fraction 15 (Fig. 5B). Fractionation of BAL fluid collected 1 h after instillation of ⁵⁴Mn demonstrated that only trace amounts of ⁵⁴Mn are associated with protein in pulmonary fluid after longer in vivo incubation periods (Fig. 5C). To demonstrate that ⁵⁴Mn bound to Tf would remain associated during fractionation, apoTf was loaded with ⁵⁴Mn in vitro and subjected to dialysis and anion exchange FPLC procedures. In vitro-radiolabeled ⁵⁴Mn-Tf was only detected in fraction 17, confirming that if present in BAL fluid, this species would be detected under our experimental conditions (Fig. 5D).

View larger version (12K): [in this window] [in a new window]   Fig. 5. Fast protein liquid chromatography (FPLC) fractionation of BAL. Lung fluid was collected by lavage 10 min after intratracheal instillation of 59Fe (A) or 54Mn (B), and 1 h after instillation of 54Mn (C). Samples were dialyzed and proteins were fractionated by FPLC over an anion-exchange column as described under MATERIALS AND METHODS. To demonstrate detection of 54Mn-Tf by this method, in vitro-labeled 54Mn-Tf was also subjected to fractionation (D). Radioactivity in fractions was measured by gamma counting. The 2 major protein peaks in BAL were identified as transferrin and albumin (see Fig. 6). Solid line is trace of UV absorbance and dashed line shows cpm with background subtracted. AU, arbitrary units. *Flow-through fraction.

View larger version (32K): [in this window] [in a new window]   Fig. 6. Detection of proteins in BAL fluid. After anion- exchange FPLC chromatography, 1-ml fractions (as shown in Fig. 5) were concentrated. A: after electrophoresis on an 8% SDS-polyacrylamide gel, proteins were stained with Coomassie blue. B: proteins in fraction 15 (F15) were concentrated, electrophoresed on a 5–20% gradient gel, and silver stained. Indicated bands were excised and identity of proteins was determined by mass spectrometry.

View larger version (11K): [in this window] [in a new window]   Fig. 7. Vascular kinetics of intratracheally instilled 54Mn in Belgrade rats and heterozygous littermates. Heterozygous (b/+) rats () and Belgrade homozygous (b/b) rats () were administered 54Mn by intratracheal instillation, and blood samples were drawn at the times indicated postinstillation. Blood 54Mn levels are expressed as % of instilled dose. Each point is mean ± SE of 5 (b/b) or 7 (b/+) rats.

View larger version (11K): [in this window] [in a new window]   Fig. 8. Characteristics of 54Mn uptake by A549 cells. A549 cells were grown to 70–80% confluency, washed with HeBS, incubated with 1–2 µCi 54Mn at 4°C (squares) or 37°C (diamonds), and then incubated on ice for 1 h at 4°C to displace nonspecific binding of radioisotope. After being washed in HeBS, cells were lysed in solubilization buffer and the uptake was determined as pmol Mn/µg cell protein as described under MATERIALS AND METHODS. A: A549 cells were incubated with 0.68 µM 54MnCl2 and the time course of uptake was determined. B: rate of 54Mn2+ uptake was determined as a function of [Mn]. Assays similar to A were performed to measure uptake on incubation with the indicated concentrations of Mn for 4 min. The averages of duplicate determinations are shown.

View larger version (7K): [in this window] [in a new window]   Fig. 9. pH dependence of 54Mn and 55Fe uptake by A549 cells. Cells were incubated with 1 µM 55Fe (A) or 54Mn (B) for 20 min in uptake buffer adjusted to pH 6.75 or 7.4 as described for Fig. 8 and in MATERIALS AND METHODS. *P < 0.05 (n = 4; means ± SE).

View larger version (17K): [in this window] [in a new window]   Fig. 10. Pharmacological profile of 54Mn uptake by A549 cells. A: cells were incubated with 0.68 µM 54Mn2+ in the presence or absence of other divalent cations at the concentrations indicated. B: transport assays with 0.34 µM 54Mn2+ in the presence and absence of inhibitors/activators of L-type calcium channels, TRPC channels, and TRPM7. Cells were incubated with 54Mn ± treatment or vehicle for 4 min. *P < 0.05 (n = 3–4; means ± SE).

Identification of TRP channels expressed by A549 cells. Electrophysiological studies in which Mn²⁺ was substituted for Ca²⁺ have demonstrated that several members of the TRPC (transient receptor potential canonical) subfamily are permeable to manganese ions (9, 29). TRPM7, a member of the TRPM (melastatin) subfamily, is also permeable to several divalent metal ions, including Mn²⁺. To determine which TRP channels are expressed by A549 cells, and to verify expression of LVGCs, RT-PCR was performed (Fig. 11). Expression of TRPC1, 3, 6, and 7, TRPM7, and the ₁-subunit of LVGC was detected. Interestingly, primers for TRPC7 amplified two products, one of the expected size (428 bp), and a smaller amplicon of 200 bp. Because the 5' and 3' primers are located in different exons (exons 1 and 2, respectively), the 200-bp amplicon may be the result of alternative splicing of TRPC7 mRNA in A549 cells.

View larger version (21K): [in this window] [in a new window]   Fig. 11. Expression of transient receptor potential (TRP) channels and L-type voltage-gated calcium channel (LVGC) in A549 cells. To detect expression of indicated channels, reverse transcription-coupled PCR reactions were carried out with primers for TRPC1, 3, 6, and 7, TRPM7, and LVGC (1-subunit) as described under METHODS AND MATERIALS. Top: amplified products from A549 cDNA. Bottom: control reactions for which reverse transcriptase was omitted during cDNA synthesis.

	DISCUSSION

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TfR mRNA was detected primarily in BALT, with weak staining in bronchial epithelium. TfR mRNA expression was enhanced in type II alveolar epithelial cells by iron deficiency, and TfR protein levels in whole lung were significantly increased. Iron oxide exposure also resulted in induction of TfR mRNA in lung; however, TfR protein levels did not change. Because ferritin was upregulated in iron oxide-exposed lung, it is unlikely that IRP activity accounts for posttranscriptional regulation of TfR mRNA. These findings suggest that while TfR gene expression may be induced at the transcriptional level by inflammatory stimuli (54), protein expression of lung TfR does not change when iron concentration is very high as a defense against cytotoxicity.

To test whether the Tf/TfR pathway might be involved in uptake of manganese, ⁵⁴Mn²⁺ was administered to rats by intratracheal instillation. At 10 min postinstillation, 85% of the dose of ⁵⁴Mn had been absorbed from pulmonary fluid and only a small fraction (1%) of the remaining ⁵⁴Mn was associated with BAL fluid protein. In contrast, 27% of the dose of intratracheally instilled ⁵⁹Fe²⁺ remained in BAL fluid, the majority of which was associated with Tf. These data suggest that while soluble ⁵⁹Fe²⁺ is oxidized to Fe³⁺ after instillation into the lungs and rapidly binds to Tf, ⁵⁴Mn²⁺ is directly taken up by pulmonary epithelial cells via Tf-independent pathways. This idea is confirmed by the fact that 1 h postinstillation, very little of the instilled ⁵⁴Mn remains bound to BAL fluid protein. It is likely that association of instilled soluble iron with Tf present in lung fluid is a first step in the process of pulmonary uptake of this metal and that this key difference contributes to the different pharmacokinetic profiles we previously observed for absorption of iron and manganese from the lungs to the blood (28). It should be noted that our experiments are limited to instillation studies of soluble forms of divalent ⁵⁴Mn and ⁵⁹Fe and that metals present in particulate matter inhaled into the lungs may be handled quite differently. For example, it is possible that trivalent manganese can associate with lung Tf after particle dissolution. Further investigation is necessary to test this idea.

Although DMT1 is known to play a role in the endocytic Tf/TfR pathway, the function of this divalent metal transporter at the plasma membrane could also provide a potential pathway for the direct uptake of Mn²⁺. However, two separate lines of evidence argue against this possibility. In vivo experiments with Belgrade rats demonstrate that the impaired DMT1 activity of homozygous animals does not modify the pharmacokinetics of pulmonary ⁵⁴Mn absorption from the lungs to the blood. Moreover, in vitro studies of ⁵⁴Mn²⁺ uptake by A549 cells show that transport is not enhanced by low pH, indicating that DMT1 cannot be the predominant Mn transport activity. These results prompted us to consider alternative pathways for pulmonary manganese uptake. Pulmonary epithelial and endothelial cells express several calcium channels that are essential in maintenance of fluid balance in the lungs (34). Several recent publications have demonstrated that uptake of manganese into heart, liver, and brain is sensitive to modulators of LVGCs (12, 17, 52). Furthermore, patch-clamp studies have shown that several members of the TRPC subfamily of nonselective calcium channels are permeable to Mn²⁺ (9, 29). TRPC proteins are store-operated and/or receptor-operated calcium channels that can be activated by intracellular store depletion and/or by stimulation of G protein- or tyrosine kinase-coupled receptors (36). Formation of heteromultimeric complexes within subfamilies (e.g., TRPC4/5, TRPC3/6, TRPC1/3/7) produces channels with different characteristics and contributes to functional variability in vivo (48). Whether TRPC channels are permeable to Mn²⁺ in vivo is not clear. A widely expressed member of the TRP melastatin-related (TRPM) subfamily, TRPM7, is also implicated in Mn²⁺ transport. TRPM7 is important in cellular magnesium homeostasis and is involved in human disorders of magnesium metabolism (10). TRPM7 transports several divalent metal ions under physiological conditions, including Mn²⁺ (35, 37).

Analysis of gene expression of TRPC family members by RT-PCR and Northern blotting has shown that TRPC1, 3, 4, 5, 6, and 7, and TRPM7 are expressed in lung (43, 45). TRPC4 and 6 have been found in differentiated human bronchial epithelial cells, whereas TRPC3 and TRPM7 are expressed by bronchial epithelium-derived BEAS-2B cells (10, 13). Expression of TRPC1 and TRPC6 in A549 cells was previously reported (59). We have verified by RT-PCR that A549 cells express TRPC1, TRPC6, and LVGC and have additionally determined that TRPC3, TRPC7, and TRPM7 are also expressed in this cell line.

Pharmacological features of manganese transport in A549 cells were defined in our study by the effects of several calcium channel inhibitors and activators on ⁵⁴Mn²⁺ uptake. Treatment with verapamil, a phenylalkylamine antagonist that binds to the ₁-subunit of LVGC, caused a significant dose-responsive reduction in ⁵⁴Mn uptake. Treatment with the DHP antagonist nifedipine or the activator BAY K 8644, both of which bind to a specific site on the ₁-subunit distinct from the binding site of verapamil, did not have an effect. Because the efficacy of DHP drugs appears to be voltage dependent, their lack of effect in A549 cells could be due to the fact that these cells are at resting potential (4, 47). Alternatively, verapamil could potentially inhibit Mn influx through TRPC3 (63) or by another uncharacterized mechanism.

To determine whether manganese influx might also occur through SOCs, including TRPC1 and TRPC7 (31), A549 cells were treated with thapsigargin, an inhibitor of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase that produces a rapid increase in cytosolic [Ca], triggering opening of SOCs on the plasma membrane. Treatment with thapsigargin has previously been shown to increase Ca²⁺ influx and trigger Mn²⁺-mediated quenching of fura-2 fluorescence in A549 cells (59). We did not observe any effect of thapsigargin on ⁵⁴Mn uptake, but we note that the concentration of Mn²⁺ studied by Xue et al. (59) was 1.2 mM, 2,000-fold greater than the concentrations used in our assays.

2-APB is a noncompetitive inhibitor of the IP₃ receptor which blocks activation of ROCs in response to G protein-coupled receptor (GPCR)-mediated phospholipase C (PLC) activation (32). 2-APB can also act directly on TRPC3, TRPC5, TRPC6, and TRPC7, possibly by binding in the pore region of these channels (31, 58). Inhibition of ⁵⁴Mn uptake by 2-APB suggests involvement of one or more of the 2-APB-sensitive TRPC proteins that behave as ROCs. However, we found that OAG, a membrane-permeable diacylglycerol (DAG) analog that activates TRPC3, 6, and 7, had no effect on ⁵⁴Mn uptake. The subunit composition of TRPC heteromeric channels in vivo modulates sensitivity to DAG (50) and could explain this apparently contradictory finding. A second explanation might be that TRPM7 is responsible for manganese uptake. One report has indicated that calcium influx through TRPM7 is also sensitive to 2-APB treatment (27). Unlike the TRPC channels, TRPM7 is inhibited by PLC-mediated depletion of PIP₂ following GPCR stimulation and is not activated by OAG (1, 46). Depletion of intracellular Mg²⁺ and Mg-ATP activates TRPM7, and high concentrations of either form of magnesium inhibit channel activity (38). In our assays, addition of 4 mM MgCl₂ resulted in a 75% inhibition of ⁵⁴Mn²⁺ uptake by A549 cells, supporting a key role for this channel. The preference of TRPM7 for manganese and other divalent metals over calcium make this channel a plausible candidate for manganese transport in vivo (35).

In conclusion, transition metals in ambient particulate matter from diesel exhaust, ROFA, and coal dust are thought to be the component(s) responsible for increased airway hyperresponsiveness, inflammation, and oxidative stress from these exposures (23, 42). We have characterized changes in Tf and TfR expression in the lungs in response to iron status that may reflect how the lungs handle metal burden to minimize oxidative damage. Of equal importance are the mechanisms by which potentially toxic inhaled metals enter the blood stream. While a role for lung fluid Tf in iron absorption should be tested more rigorously, our results suggest that ferrous iron can be readily oxidized to bind Tf and that elements of the Tf/TfR/DMT1 pathway may mediate its absorption from the lungs. In contrast, our instillation studies indicate that soluble Mn²⁺ does not interact with Tf in BAL fluid, suggesting alternative pathways for uptake are responsible for its transport from the lungs. One caveat is that the potential role for the Tf/TfR/DMT1 pathway in the uptake of trivalent Mn cannot be rigorously ruled out as it is possible that on dissolution, metal from particulate matter could be oxidized and cleared by this pathway. Our findings do support a potential role for TRPM7, LVGCs, and possibly other TRP channels in the process of lung Mn²⁺ absorption. It will be of great interest to determine whether such channels function in transport of trace metals in the lungs in vivo and whether exposure to components of particulate matter and/or resulting inflammation might affect channel activity.

	GRANTS

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This work was supported by National Institutes of Health Grant DK-60528 to M. Wessling-Resnick and grants from the American Welding Society to J. D. Brain. Support for proteomic analysis was provided by the Harvard School of Public Health-National Institute of Environmental Health Sciences (NIEHS) Center for Environmental Health (ES-00002). Additional support for the interpretation of results and authorship of this publication was made possible by Grant P01-ES-012874 from NIEHS and from STAR Research Assistance Agreement No. RD-83172501 awarded by the US Environmental Protection Agency (EPA).

	DISCLOSURES

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This work has not been formally reviewed by either the NIEHS or EPA. The views expressed in this document are solely those of the authors and do not necessarily reflect those of either the NIEHS or the EPA. Neither NIEHS nor the EPA endorses any products or commercial services mentioned in this publication.

	ACKNOWLEDGMENTS

 
We thank T. Donaghey and K. Doud for excellent technical help.

	FOOTNOTES

 

Address for reprint requests and other correspondence: M. Wessling-Resnick, Dept. of Genetics and Complex Diseases, Harvard School of Public Health, 665 Huntington Ave., Boston, MA 02115 (e-mail: wessling{at}hsph.harvard.edu)

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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