Vascular release of nonheme iron in perfused rabbit lungs

Yuh-Chin T. Huang1, Andrew J. Ghio2, Eva Nozik-Grayck1, and Claude A. Piantadosi1

1 Departments of Medicine and Pediatrics, Duke University Medical Center, Durham 27710; and 2 United States Environmental Protection Agency, Research Triangle Park, North Carolina 27211


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we hypothesized that the lung actively releases excess iron into the circulation to regulate iron homeostasis. We measured nonheme iron (NHFe) in the perfusate of control isolated perfused rabbit lungs and lungs with ischemia-reperfusion (I/R) ventilated with normoxic (21% O2) or hypoxic (95% N2) gas mixtures. Some were perfused with bicarbonate-free (HEPES) buffer or treated with the anion exchange inhibitor DIDS. The control lungs released ~0.25 µg/ml of NHFe or 20% of the total lung NHFe into the vascular space that was not complexed with ferritin, transferrin, or lactoferrin or bleomycin reactive. The I/R lungs released a similar amount of NHFe during ischemia and some bleomycin-detectable iron during reperfusion. NHFe release was attenuated by ~50% in both control and ischemic lungs by hypoxia and by >90% in control lungs and ~60% in ischemic lungs by DIDS and HEPES. Reperfusion injury was not affected by DIDS or HEPES but was attenuated by hypoxia. These results indicate that biologically nonreactive nonheme iron is released rapidly by the lung into the vascular space via mechanisms that are linked to bicarbonate exchange. During prolonged ischemia, redox-active iron is also released into the vascular compartment by other mechanisms and may contribute to lung injury.

anion exchange protein; 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; ferritin; bleomycin


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IRON IS AN ESSENTIAL transition metal integrally involved in the active centers of many metalloproteins and enzymes that serve as electron carriers. During catalytic reactions, iron is cycled between oxidized [Fe(III)] and reduced [Fe(II)] states. The oxidation-reduction properties of iron, however, also make it a powerful potential prooxidant. For example, when iron is mobilized from intracellular iron stores during ischemia-reperfusion (I/R), it may participate in the generation of highly reactive molecules, such as hydroxyl radical, via the metal-catalyzed Haber-Weiss reaction and/or ferryl or perferryl ions via reactions with oxygen (9, 14). Therefore, the cell benefits from tightly regulating cellular iron homeostasis.

In most cells, defenses against excessive intracellular iron occur at multiple levels: decreased uptake of extracellular iron, increased release of intracellular iron into the vascular compartment, and altered expression of ferritin and heme proteins. Among these mechanisms, iron release into the vascular space is most rapid (within seconds), and intravascular iron is chelated by circulating iron-binding proteins, e.g., transferrin and lactoferrin. Such efflux of nontransferrin-bound iron from the cells has been observed in perfused liver (18), isolated hepatocytes (2, 21, 22), and erythroid cells (19). The exact release pathways for non-transferrin-bound iron, however, are not entirely clear. Iron-releasing pathways are saturable, inhibited by other transition metal ions, and show calcium and temperature dependency (2, 3, 5, 15, 19, 20). These characteristics indicate the presence of membrane transporters for the metal. In addition, because of the positive charge of iron, efflux of intracellular non-protein-bound iron must be linked to influx of extracellular cations and/or concomitant efflux of intracellular anions to maintain electroneutrality across the plasma membrane.

Because mammalian lungs are exposed continuously to oxidant stimuli, it is logical that lung cells are able to decrease intracellular iron loads by releasing it into the vascular space. Specific biochemical information related to iron release pathways in the lung, however, is lacking. Therefore, we designed the present study to measure release of nonheme iron from the intact rabbit lung and to determine whether anion exchange function was important in the regulation of the release process. We hypothesized that bicarbonate-dependent anion exchange function was required for intravascular release of nonheme iron because this process generates superoxide anion involved in iron reduction, which is required to transport the metal out of the cell. The study was performed in perfused lungs and lungs subjected to I/R, representing low- and high-intracellular iron stress, respectively. Total nonheme iron in samples of perfusate was measured by inductively coupled plasma emission spectroscopy (ICPES). Oxygen-dependent mechanisms were investigated by ventilating the lungs with air or nitrogen gas. Anion exchange function was inhibited using two mechanistically different approaches: instilling 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) intratracheally and perfusing the lung with bicarbonate-free buffer (16). We found that the uninjured rabbit lung releases nonheme iron in a bicarbonate-dependent manner. This iron release was attenuated by hypoxia and augmented by ischemia; the latter was associated with both injury and release of redox-active iron from the lungs.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents and Pharmaceuticals

All chemicals including DIDS, bleomycin, sodium, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and human ferritin were purchased from Sigma (St. Louis, MO) unless otherwise indicated.

Isolated Perfused Lung Preparation

The model has been described previously (8, 13). Male New Zealand White rabbits (May's Farm) weighing 2.5-3.0 kg were heparinized and anesthetized with pentobarbital sodium. After the chest wall was opened, the animal was killed by rapid exsanguination from the left ventricle. Stainless steel cannulas were tied into the left atrium and the main pulmonary artery. The ligature at the pulmonary artery also passed around the aorta to prevent loss of perfusate into the systemic circulation. The pulmonary circulation was washed free of blood before a recirculating flow at 100 ml/min was established using Krebs-Henseleit buffer containing 3% bovine serum albumin at a temperature of 37-38°C and pH 7.3-7.4. The perfusion circuit consisted of a reservoir, a roller pump (Sarns, Ann Arbor, MI), a bubble trap, and a heat exchanger connected with Tygon tubing. The reservoir for collecting perfusate from the left atrium was suspended freely from a force displacement transducer (model FT100, Grass Instrument, Quincy, MA). Weight gain of the lung was measured by recording the loss of fluid from the reservoir. The reservoir was placed at the lowest portion of the lung to maintain a left atrial pressure of zero. The volume of the system was ~250 ml. The lung was ventilated with 21% O2-5% CO2 through a tracheotomy tube with an animal respirator (Harvard Apparatus, S. Natick, MA) delivering 30 breaths/min at 2 cmH2O positive end-expiratory pressure. The tidal volume was adjusted to achieve a peak tracheal pressure of 7-10 mmHg (~20 ml). The mean pulmonary artery pressure (Ppa) and peak airway pressure were recorded on a four-channel recorder (model 2450S, Gould, Cleveland, OH) continuously with pressure transducers (P231 D, Gould Statham Instruments, Hato Rey, PR). Only isolated lungs with stable Ppa (<20 mmHg) and no loss of perfusate from the reservoir during the 10-min stabilization period were used in the study.

The Krebs-Henseleit-3% albumin buffer contains (in mM) 82.8 sodium chloride, 4.7 potassium chloride, 2.4 monobasic potassium phosphate, 25 sodium bicarbonate, 1.2 magnesium sulfate, 2.7 calcium chloride, and 11.1 dextrose and 3% (wt/vol) bovine serum albumin, fraction V. For experiments with HEPES buffer, bicarbonate in the perfusion buffer was substituted with 6 mM HEPES, and the concentration of sodium chloride was increased to 107 mM to maintain the osmolarity.

Measurements of Nonheme Iron in Perfusate and Lung Tissue

Nonheme iron concentrations in the perfusate and lung tissue were measured using ICPES (4). The lung was homogenized in NaCl solution (100 mg/ml) and centrifuged. Supernatant or perfusate (1 ml) was hydrolyzed in 1.0 ml of 3 N HCl-10.0% trichloroacetic acid at 70°C for 16 h. After centrifugation at 1,200 g for 10 min, iron in the supernatant was quantified using ICPES (model P40; Perkin Elmer, Norwalk, CT) at a wavelength of 238.2 nm. To minimize iron contamination, plastic containers rather than glassware were used in these studies. Double-distilled, deionized water also was used for all aqueous medium. Standards of commercially available iron in 3 N HCl and 10% trichloroacetic acid were used. The detection limit for iron by ICPES was ~5 ng/ml.

Measurements of Bleomycin-Detectable Iron

Bleomycin-detectable iron in the perfusate was measured using the methods of Halliwell and Gutteridge (11). All reagents except bleomycin were pretreated with chelex-100 to remove as much contaminant iron as possible. The reaction mixture contained 0.5 ml of calf thymus DNA (1 mg/ml), 0.05 ml of bleomycin sulfate (1 U/ml), 0.1 ml of MgCl2 (50 mM), 0.1 ml of sample or iron standard, 0.1 ml of chelex-treated pyrogen-free water, and 0.1 ml of ascorbic acid (0.75 mM). Sample blanks were identical except that water was used instead of samples. Additional negative controls were also performed without adding bleomycin. The mixtures were incubated at 37°C for 1 h with shaking. The reaction was stopped with 0.1 M EDTA, and the sample was mixed with 1 ml of thiobarbituric acid [1% (wt/vol) in 50 mM NaOH] and 1 ml of HCl (25% vol/vol). This reaction mixture was heated to 80°C for 20 min and cooled, and the resulting chromogen was extracted in 3 ml of butanol. After the sample was centrifuged at 2,500 g for 20 min, the supernatant was removed and its absorbance was measured at 532 nm with a spectrophotometer (Shimadzu, UV160U, Kyoto, Japan). A standard curve was prepared from dilutions of molecular iron standard (Sigma). The assay was linear for iron concentrations between 0.1 and 5 µM. The amount of bleomycin-detectable iron in unknown samples was calculated from the standard curve.

Measurements of Lactoferrin

Perfusate was vacuum slot blotted onto 0.45-mm nitrocellulose (Schleicher and Schuell, Keene, NH) in a saline buffer containing 100 mM Tris, pH 8.0. The blots were allowed to air-dry, blocked with bovine serum albumin for 30 min, and then incubated with a 1:1,000 dilution of rabbit anti-human lactoferrin antibody (Sigma) in 5% bovine serum albumin for 2 h. The blots were washed in phosphate-buffered saline-Tween 0.05% and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG in 5% bovine serum albumin for 1 h. Detection was accomplished on film using enhanced chemiluminescence (ECL) reagents as per the manufacturer's instructions (Amersham, Arlington Heights, IL). Band optical densities were quantified using a Millipore Digital Bioimaging System (Bedford, MA).

Measurements of Transferrin

Transferrin protein concentrations were measured using commercially available kits (an immunoprecipitin analysis), controls, and standards from INCSTAR (Stillwater, MN). This assay was modified for use in the Cobas Fara II centrifugal spectrophotometer (Hoffman-LaRoche, Branchburg, NJ).

Measurements of Ferritin

Perfusate samples were vacuum slot blotted onto a 0.45-mm nitrocellulose membrane in 100 mM Tris-buffered saline (pH 8.0). The blots were blocked for 30 min with Tris-buffered saline with 0.05% Tween 20 and 5% milk and then incubated with goat anti-rabbit liver ferritin antibody (Kent Laboratories, Redmond, WA) at 4°C overnight. This was followed by incubation with horseradish peroxidase-labeled secondary antibody. The signals in each slot were detected by an ECL kit and quantified by densitometry. The concentrations of ferritin in perfusate samples were determined by comparison to human ferritin standards.

Experimental Protocols

Control lungs. Control lungs were perfused for 40 min and ventilated with either 21% O2-5% CO2 (normoxia) or 95% N2-5% CO2 (hypoxia). In hypoxic lungs, nitrogen ventilation was begun after the time 0 sample was taken.

Ischemia-reperfusion. Ischemia was initiated by stopping the perfusion pump. The ischemia time was 90 min. At the end of ischemia, the lung was reperfused. The reperfusion time was 40 min. For normoxic I/R, the lung was ventilated with 21% O2-5% CO2 throughout ischemia and reperfusion. Adequate lung oxygenation could be maintained during normoxic ischemia because oxygen was delivered to the lung by continuous ventilation, and the isolated lungs showed no evidence of atelectasis by inspection. Adequate oxygenation of the lung was also reflected by measurement of PO2 of 120-130 mmHg in perfusate obtained immediately after reperfusion (8), and oxygen consumption of the lungs was minimal compared with the oxygen supply in the alveolar space. For hypoxic I/R, the ventilation gas mixture was 95% N2-5% CO2 during ischemia and 21% O2-5% CO2 during reperfusion. Hypoxic ventilation was begun after the baseline (preischemia) perfusate sample was taken.

Anion exchange function and intravascular iron release. The effects of bicarbonate exchange on intravascular iron release by the control and the I/R lungs were investigated by inhibiting anion exchange function using two different mechanisms.

DIDS. DIDS is a water-soluble stilbene inhibitor that was administered intratracheally after the baseline (preischemia) sample was taken. Immediately before intratracheal instillation, the tracheostomy tube was disconnected briefly from the ventilator circuit to allow the lung to deflate completely. DIDS, dissolved in 2 ml of 0.9% NaCl at a final concentration of 25 µM, was then instilled into the distal trachea, the lung was reinflated, and ventilation reestablished to allow DIDS to be distributed to the distal lung regions. Control lungs received 2 ml of saline intratracheally.

PERFUSION WITH BICARBONATE-FREE BUFFER. HEPES was used to determine whether extracellular bicarbonate was required for iron release from the lung. Bicarbonate-free buffers inhibit the exchange function of the major anion exchange protein (AE2) in the lung (1, 16). Lungs were perfused with HEPES-containing buffer at the start of lung preparation so that all perfusate samples were made in HEPES buffer. Additional experiments were also conducted to examine the effects of DIDS on iron release from HEPES-perfused lungs.

Sample collection. For nonheme iron in the circulation in control lungs, perfusate samples (2 ml) were obtained at 0, 10, 20, 30, and 40 min. To measure nonheme iron in lung tissue, freeze-clamped lung samples were obtained at the end of the experiments and kept in -70°C freezer. For nonheme iron in the circulation in I/R, perfusate samples (2 ml) were obtained at baseline (preischemia), and 1, 20, and 40 min after reperfusion was begun.

Statistical Analysis

Data in the text and figures are shown as means ± SE. Time-series data were analyzed using repeated-measures analysis of variance (ANOVA). Differences among groups at a specific time point were evaluated using one-way ANOVA followed by Tukey's test. The statistical analysis was performed using commercially available software (Statview 4.01, Brainpower, Calabasas, CA). P <=  0.05 was considered statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Control Lungs

Physiological parameters of perfused lungs. The average weight gain and Ppa of control lungs with different interventions are shown in Table 1. Neither DIDS nor HEPES affected weight gain of the lung. DIDS treatment increased Ppa at 40 min of perfusion. Hypoxia produced an increase in Ppa and doubled the rate of weight gain.

                              
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Table 1.   Weight gain and mean pulmonary artery pressure for control lungs ventilated with normoxia or hypoxia

Intravascular release of nonheme iron. Basal nonheme iron concentration in the perfusate 10 min after preparation of the lung was 0.30 ± 0.01 µg/ml. The perfusate nonheme iron increased nonlinearly, with ~80% of the total increase occurring during the first 20 min. By 40 min, the total increase in perfusate nonheme iron was 0.23 ± 0.01 µg/ml (Fig. 1). The average nonheme iron content of whole rabbit lung after 40 min perfusion was 25.9 ± 0.9 µg/g wet tissue.


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Fig. 1.   Changes in nonheme iron concentration (Delta [Fe]) in the perfusate in normoxic lungs without ischemia-reperfusion (I/R). All lungs were ventilated with 21% O2-5% CO2 for 40 min. Control lungs were treated with 2 ml of intratracheal normal saline (, n = 3). DIDS (in normal saline at a final concentration of 25 µM) was given by intratracheal instillation after the time 0 perfusate sample was taken (, n = 3). In the HEPES group, the lungs were perfused with bicarbonate-free (HEPES-containing) Krebs-Henseleit-3% albumin buffer (black-triangle, n = 3). The SEs for the HEPES group were very small and contained within the symbols. *P < 0.05 compared with control lungs.

Effects of anion exchange inhibition on intravascular release of nonheme iron. DIDS. Intratracheal administration of DIDS (a final concentration of 25 µM) inhibited iron efflux in perfused lungs by >90% under control conditions. Basal nonheme iron concentration was 0.28 ± 0.05 µg/ml at baseline and increased to no more than 0.30 µg/ml in the perfusate during the 40-min perfusion period (Fig. 1; P < 0.01 vs. control lungs without DIDS).

PERFUSION WITH HEPES BUFFER. Basal nonheme iron in control lungs perfused with HEPES buffer was <0.1 µg/ml at time 0 and remained essentially unchanged during the 40-min perfusion (Fig. 1; P < 0.001 vs. control lungs perfused with bicarbonate buffer).

Effects of hypoxia on intravascular release of nonheme iron. Hypoxia inhibited intravascular release of nonheme iron by ~50%. Mean increase in perfusate nonheme iron concentration was 0.11 ± 0.02 µg/ml at the end of 40-min perfusion period (P < 0.01 compared with 0.23 ± 0.01 µg/ml in normoxia; Fig. 2).


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Fig. 2.   Changes in nonheme iron concentration (Delta [Fe]) in the perfusate in lungs without I/R ventilated with normoxic or hypoxic gas mixture. In normoxic lungs (), lungs were ventilated with 21% O2-5% CO2 for 40 min (n = 3). In hypoxic lungs (), lungs were ventilated with 95% N2-5% CO2 for 40 min (n = 3). *P < 0.05 compared with normoxic lungs.

Perfusate lactoferrin and transferrin. Because most nonheme iron in the circulation is protein bound, we measured the appearance of three major iron-containing nonheme proteins (lactoferrin, transferrin, and ferritin). Lactoferrin concentration in the perfusate was consistently <80 ng/ml, and it did not increase during 40 min of perfusion. The concentration of transferrin in the perfusate was also low, and the increase in transferrin was <50 µg/ml during 40 min of perfusion.

Perfusate ferritin. Ferritin level in the perfusate was 0.44 ± 0.01 µg/ml under basal conditions. Changes in ferritin concentration over time were small and tended to fluctuate around the basal level (Fig. 3). In lungs treated with DIDS or perfused with HEPES buffer, the changes in ferritin followed a pattern similar to that of the control lungs (Fig. 3).


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Fig. 3.   Changes in ferritin levels (Delta ferritin) in the perfusate of control lungs perfused with bicarbonate-containing Krebs-Henseleit buffer and ventilated with 21% O2 (control, n = 5), treated with DIDS given intratracheally (final concentration 25 µM, n = 3) or perfused with HEPES-containing (bicarbonate-free) buffer for 40 min (n = 3).

Bleomycin-detectable iron. To determine whether the rabbit lung released biologically reactive iron, we measured bleomycin-detectable iron in the perfusate. Bleomycin-detectable iron in the perfusate of control lungs averaged 1.29 ± 0.23 nmol/ml at time 0, 1.48 ± 0.11 nmol/ml after 10 min, and 0.92 ± 0.13 nmol/ml after 40 min of perfusion (P = not significant).

Ischemia-Reperfusion

Physiological parameters of I/R lungs. The effects of I/R on average weight gain and Ppa with and without interventions are shown in Table 2. Ischemia for 90 min produced reperfusion pulmonary hypertension and weight gain. Neither DIDS nor HEPES affected these responses. Hypoxia during ischemia decreased weight gain during reperfusion by about 50% without affecting Ppa.

                              
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Table 2.   Weight gain and mean pulmonary artery pressure during normoxic I/R with or without interventions

Release of nonheme iron during I/R. Before ischemia, nonheme iron concentration in the perfusate was 0.39 ± 0.03 µg/ml. After 90 min of ischemia, perfusate nonheme iron concentration increased by 0.27 ± 0.07 µg/ml almost immediately on reperfusion. There were no further increases in perfusate nonheme iron during the subsequent 40 min of reperfusion (Fig. 4). Thus the perfusate nonheme iron in I/R lungs during 90 min of ischemia increased by an amount similar to that in control lungs during 40 min of continuous perfusion (0.23 ± 0.01 µg/ml).


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Fig. 4.   Changes in nonheme iron concentration (Delta [Fe]) in the perfusate during normoxic I/R. All lungs were ventilated with 21% O2-5% CO2 during 90-min ischemia and 40-min reperfusion. In normoxic I/R lungs (, n = 6), intratracheal 0.9% NaCl (2 ml) instillation had no effect on nonheme iron release; therefore, data from normoxic I/R lungs with and without intratracheal saline were pooled to serve as control for DIDS and HEPES. In normoxic I/R with DIDS, DIDS (in normal saline at a final concentration of 25 µM) was given by intratracheal instillation before ischemia after the first perfusate sample was taken (, n = 5). In the HEPES group, the lungs were perfused with bicarbonate-free (HEPES-containing) Krebs-Henseleit-3% albumin buffer (black-triangle, n = 5). *P < 0.01 compared with I/R without interventions.

Bicarbonate dependence of intravascular iron release. DIDS. When DIDS was given before ischemia, nonheme iron release was inhibited on reperfusion. Perfusate nonheme iron before ischemia was 0.30 ± 0.03 µg/ml and increased by only 0.06 ± 0.03 µg/ml on reperfusion; this represented 75% inhibition compared with normoxic I/R without DIDS (Fig. 4). At the end of 40-min reperfusion, the nonheme iron concentration in the perfusate only increased by 0.11 ± 0.01 µg/ml from the preischemic baseline (55% inhibition compared with normoxic I/R lungs without DIDS).

PERFUSION WITH HEPES BUFFER. To determine whether nonheme iron release by the I/R lung also requires extracellular bicarbonate, I/R experiments in lungs perfused with bicarbonate-free buffer were performed in which bicarbonate was replaced with HEPES. The nonheme iron in the perfusate before ischemia was ~50% lower in HEPES-buffer perfused lungs (0.17 ± 0.03 µg/ml) compared with bicarbonate-buffer perfused lungs. The increase in nonheme iron on reperfusion was only about 0.1 µg/ml (Fig. 4). When DIDS was administered to HEPES-perfused lungs, inhibition of nonheme iron release was not greater than with HEPES alone (n = 2). In I/R, nonheme iron release increased during reperfusion by ~0.04 µg/ml compared with 0.05-0.10 µg/ml with either agent alone. These comparable results indicate that iron release is inhibited at the same sites or by the same processes with DIDS and HEPES.

Effects of hypoxia on iron release during ischemia. I/R experiments were performed in which lungs were ventilated with nitrogen during ischemia. This procedure produced a perfusate PO2 of 20-25 mmHg. The hypoxic ischemia was followed by reoxygenation of the lung with 21% O2 during reperfusion. Because the bulk of the nonheme iron was released during ischemia in normoxic I/R experiments, hypoxia was given only during lung ischemia. Perfusate nonheme iron concentration before hypoxic ischemia was 0.34 ± 0.05 µg/ml and increased by 0.12 ± 0.05 µg/ml on reperfusion. This represented 50% inhibition compared with normoxic I/R. With reoxygenation and reperfusion, the perfusate nonheme iron concentration gradually rose toward the level seen in normoxic I/R (Fig. 5).


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Fig. 5.   Changes in nonheme iron concentration (Delta [Fe]) in the perfusate during I/R. All lungs were ventilated with 21% O2-5% CO2 during 90-min ischemia and 40-min reperfusion. As in Fig. 4, data from normoxic I/R lungs with and without intratracheal saline () were pooled to provide the control group for hypoxic I/R. During hypoxic I/R (, n = 5), lungs were ventilated with 95% N2-5% CO2 during ischemia and reoxygenated with 21% O2-5% CO2 during reperfusion. *P < 0.05 compared with normoxic I/R.

Bleomycin-detectable iron. In I/R, the bleomycin-detectable iron in the perfusate increased from a baseline value of 1.48 ± 0.28 to 2.36 ± 0.22 nmol/ml at 5 min of reperfusion (a 60% increase, P < 0.01). It then declined to 1.45 ± 0.02 nmol/ml after 40 min of reperfusion.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study clearly demonstrates that rabbit lungs perfused with physiological buffer release a significant amount of nonheme iron into the vascular space. Basal nonheme iron concentration in the perfusate in our system averaged 0.30 µg/ml immediately after the preparation had been established. Nonheme iron concentration increased rapidly for the next 20 min, reaching a plateau at nearly twice the basal iron concentration after 40 min of perfusion. This amount of nonheme iron averaged ~58 µg from each set of lungs (250 ml of perfusate × 0.23 µg/ml). When this amount of released iron is added to the nonheme iron remaining in the lungs after the studies (58 µg + 259 µg), the total amount of nonheme iron in the pool was about 317 µg. Therefore, the lungs released ~20% of their total available nonheme iron into the vascular space during buffer perfusion.

The method we used to measure perfusate iron (ICPES) will detect both metal bound to nonheme proteins and free (non-protein-bound) iron. To determine the biochemical nature of nonheme iron in the perfusate, we quantified three major iron-binding proteins as well as the amount of bleomycin-reactive iron. Lactoferrin concentration was low at baseline (<80 ng/ml) and did not change over the experimental period. Transferrin was not detectable initially and increased by no more than 50 µg/ml. Assuming each molecule of transferrin contains two molecules of iron, this amount of transferrin would account for no more than 0.02 µg/ml of nonheme iron in the perfusate. Therefore, both lactoferrin and transferrin can be excluded as major sources of nonheme iron leaving the lung and entering the perfusate. Perfusate ferritin concentration was initially 0.44 µg/ml. Assuming an average molecular mass for ferritin of 400 kDa and that each molecule of ferritin binds 4,000 molecules of iron at saturation (12), this amount of ferritin could account for about 0.24 µg/ml of nonheme iron or about 80% of the baseline nonheme iron concentration in perfusate. Because ferritin levels fluctuated very little after perfusion, ferritin release was not an important source of nonheme iron; however, incompletely saturated ferritin in the perfusate may have taken up nonheme iron released from the lungs during the studies. We also excluded contributions to the circulating iron pool from low molecular mass iron compounds (Fe2+) that could react with bleomycin because bleomycin-detectable iron comprised a very small fraction of the initial iron content of perfusate (<0.01 µg/ml). Therefore, most of the nonheme iron measured by ICPES at the start of the experiments could have been ferritin-bound iron but the increase in nonheme iron during 40 min of perfusion could not be attributed to changes in release of any of the three iron-binding proteins or to bleomycin-reactive species. The nonheme iron is most likely bound to ferritin or chelated with low molecular mass compounds such as citrate or glycine. Although the exact biochemical species of nonheme iron in the perfusate remain unidentified, this iron should have relatively low oxidative potential in continuously perfused lungs because it is probably in the ferric [Fe(III)] state. The finding that inhibition of nonheme iron release by DIDS or HEPES did not correlate with physiological lung injury supports this conclusion.

Release of nonheme iron from intracellular to extracellular spaces has been noted previously primarily in hepatocytes and hematopoietic cells. In cultured cells, the characteristics of the release process of iron resemble its uptake in many ways, e.g., saturability, inhibition by other transition metal ions, and calcium and temperature dependency (2, 3, 5, 15, 19, 20). The presence and importance of these release mechanisms, however, has never been demonstrated in the lung. Considering that the lungs are constantly exposed to oxidant stress from the ambient environment, it is not too surprising that the lung readily releases intracellular iron. Our data support this hypothesis and demonstrate a significant capacity of the lungs to rapidly release iron at normal oxygen tensions and presumably at normal levels of oxidative stress. Although it is unknown whether nonheme iron was released via the same or similar transport mechanisms as those demonstrated in hepatocytes and red blood cells, our data clearly show that iron release in lungs is sensitive to hypoxia and requires extracellular bicarbonate. Because both DIDS and depletion of extracellular bicarbonate are known to inhibit anion exchange function of the cells, we speculate that membrane iron transport mechanisms in lung cells are linked functionally to anion exchangers such as AE2 (7, 16). The most likely cellular source of nonheme iron released into the perfusate are endothelial cells that express AE2 adjacent to the vascular space. Some nonheme iron in alveolar epithelial cells that express AE2 may also gain access to the vascular space through relatively permeable capillary endothelial junctions.

In lungs subjected to I/R, the increase in nonheme iron after 90 min of ischemia reached a level (0.27 µg/ml) similar to that in control lungs after 40 min; however, no further increase occurred during 40 min of reperfusion. This indicates that most of the nonheme iron was released into the vascular lumen during ischemia, and the release process was not dependent on perfusion. The intravascular release of nonheme iron during ischemia was inhibited by hypoxia by 50%, an amount comparable to that of control lungs. Hypoxia may inhibit intravascular nonheme iron release by inhibiting transmembrane iron transport mechanisms or decreasing levels of intracellular iron. On the other hand, inhibition of intravascular nonheme iron release by DIDS and HEPES buffer in I/R lungs was significantly less (60%) than that in control lungs (90%), and the combination of DIDS and HEPES did not increase the extent of inhibition. These results indicate that DIDS and HEPES, despite administration by different routes, probably act on the same cellular sites or processes in rabbit lungs. AE-related mechanisms for nonheme iron release were probably partially impaired in I/R lungs, and some nonheme iron may have leaked out of the cells via injured plasma membranes.

In I/R lungs, intravascular nonheme iron was not all biologically inactive as found in the control lungs because bleomycin-detectable iron increased during early reperfusion. The increase in bleomycin-detectable iron would be equivalent to about 0.05 µg/ml or 12.5 µg of nonheme iron. This finding agrees with increases in hydroxyl radical activity detected by salicylate trapping during reperfusion in a previous study in rabbit lungs from our laboratory (8). Bleomycin-reactive iron is not likely to have been bound to proteins (e.g., transferrin, lactoferrin, or ferritin) because this iron needs labile coordination sites (10). Iron bound to low molecular mass chelators could react with bleomycin during I/R because redox cycling between ferrous and ferric iron is significantly enhanced by reductive stress (17).

Previous studies in isolated perfused lung have shown global increases in low molecular mass iron in nonventilated I/R (6) and desferrioxamine-chelatable iron in normoxic I/R lungs (23). Desferrioxamine-chelatable iron, however, was not detected in perfusate in the latter study. The minor discrepancy between our results and those of the latter study (23) for vascular release of reactive iron can be attributed to different methods and sensitivity for detecting reactive iron in situ. Alternatively, the more severe ischemic insult produced by our I/R protocol [90-min ischemia vs. 60 min in the study by Zhao et al. (23)] could produce higher levels of intracellular reactive iron species during ischemia, which was released subsequently into the vascular compartment. Taken together, these results imply that when ischemia is prolonged, excessive redox-active Fe is mobilized reductively from intracellular iron stores and transported to the vascular space. Iron stress in the vascular space could enhance generation of highly damaging reactive oxygen species extracellularly.

In summary, our study shows that perfused rabbit lungs rapidly release ~20% of their total nonheme iron into the circulation as nonreactive forms. Most of the nonheme iron is released via transport mechanisms that are O2 sensitive and require extracellular bicarbonate. These processes may represent physiological mechanisms by which the lung can eliminate excessive intracellular iron by releasing it into the circulation where it can be safely bound to abundant plasma iron-binding proteins. In I/R, however, additional redox-active iron is released from the lung after ischemia and may exacerbate lung injury by enhancing production of highly reactive oxygen species in the vascular compartment.


    ACKNOWLEDGEMENTS

We thank Susan Fields for excellent technical support.


    FOOTNOTES

The study was supported by research grants from the National Heart, Lung, and Blood Institute and the Institute of Medical Research and a Grant-in-Aid from the American Heart Association.

Portions of this work were presented at the meeting of American Thoracic Society in Chicago, IL, in 1998 (Am J Respir Crit Care Med 157: A353, 1998).

Address for reprint requests and other correspondence: Y.-C. T. Huang, Box 3315, Duke Univ. Medical Center, Durham, NC 27710 (E-mail: huang002{at}mc.duke.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.

Received 28 January 2000; accepted in final form 4 October 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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