Departments of Medical and Surgical Sciences, Pediatrics, and Clinical Medicine and Centro per lo Studio dell' Invecchiamento, University of Padua, 35128 Padua; and Department of Biomedical Sciences, University of Modena and Reggio Emilia, 41100 Modena, Italy
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ABSTRACT |
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Amiodarone may induce lung damage by direct toxicity or indirectly through inflammation. To clarify the mechanism of direct toxicity, we briefly exposed rabbit alveolar macrophages to amiodarone and analyzed their morphology, synthesis, and degradation of dipalmitoylphosphatidylcholine (DPPC); distribution of lysosomal enzymes; and uptake of diphtheria toxin and surfactant protein (SP) A used as tracers of the endocytic pathway. Furthermore, in newborn rabbits, we studied the clearance of DPPC and SP-A instilled into the trachea together with increasing amounts of amiodarone. We found that in vitro amiodarone decreases the surface density of mitochondria and lysosomes while increasing the surface density of inclusion bodies, increases the incorporation of choline into DPPC, modifies the distribution of lysosomal enzymes, and does not affect the uptake and processing of diphtheria toxin but inhibits the degradation of SP-A. In vivo amiodarone inhibits the degradation of SP-A but not of DPPC. We conclude that the acute exposure to amiodarone perturbs the endocytic pathway acting after the early endosomes, alters the traffic of lysosomal enzymes, and interferes with the turnover of SP-A.
endocytic pathway; surfactant protein A
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INTRODUCTION |
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AMIODARONE, A BENZOFURAN DERIVATIVE with class III antiarrhythmic activity, is widely used to control supraventricular and ventricular arrhythmias. Although the therapeutic efficacy of amiodarone is well established, its use is limited by adverse effects concerning the lungs, thyroid gland, liver, gastrointestinal tract, eyes, skin, and nervous system (18).
Amiodarone lung toxicity usually presents as an indolent illness characterized by dyspnea, cough, low fever, malaise, and diffuse parenchymal infiltrates. In a few patients, toxicity develops abruptly, mimicking pneumonia or the acute respiratory distress syndrome. Morphologically, the damage to the lungs is characterized by the accumulation of multilamellar bodies in the cytoplasm of various cell types and by inflammation. In some patients, the inflammatory changes lead to fibrosis (18, 30).
The accumulation of multilamellar bodies in the cytoplasm has been extensively studied and is thought to be due to a decreased degradation of phospholipids because amiodarone is a powerful inhibitor of lysosomal phospholipases A1 and A2 (14, 16, 19, 24). The origin of the inflammation, on the other hand, is less clear, and the literature presents both studies supporting and studies refuting a role for the immune system in the genesis of lung damage (reviewed in Ref. 31). Studies supporting a role for the immune system (17, 32, 33, 37) have shown that amiodarone can activate lung natural killer cells and change the pattern of secretion of cytokines by alveolar macrophages, but it is unclear if the observed changes represent the starting lesion or are the consequence of direct lung damage. In fact, amiodarone could induce direct damage by accumulating in lysosomes and disturbing crucial catabolic processes, changing the physical properties of cell membranes, inhibiting mitochondrial functions like beta-oxidation, disturbing calcium homeostasis, producing reactive oxidant species, inhibiting ionic pumps, influencing the activity of G proteins or phospholipase C, and inducing apoptosis (1, 5, 16, 23, 31, 36). Some of the changes induced by amiodarone could also be due to interference with the turnover of surfactant because it has been shown that amiodarone causes type II cell hyperplasia and deposition of surfactant protein (SP) A in the alveoli (27).
The aim of this study was to clarify the mechanism of direct lung damage induced by an acute exposure to amiodarone. Thus, using rabbit alveolar macrophages, we examined the effect of amiodarone on the ultrastructure, the incorporation of choline into dipalmitoylphosphatidylcholine (DPPC), the degradation of exogenous DPPC, the distribution of lysosomal hydrolases among organelles isolated from the postnuclear supernatant, the release of lysosomal hydrolases into the extracellular milieu, and the targeting of extracellular substrates to the appropriate cellular compartments. The substrates tested were diphtheria toxin and SP-A, two proteins that follow different routes after uptake. In fact, diphtheria toxin binds to a specific receptor and is delivered to the early endosomes where it is hydrolyzed. From the early endosomes, the active fragment is then translocated to the cytosol where it inhibits protein synthesis through ADP-ribosylation of elongation factor 2 (34). SP-A is taken up by alveolar macrophages and moved to the lysosomes where it is degraded (2). Besides these in vitro studies, we analyzed the clearance of surfactant DPPC and SP-A administered into trachea of 3-day-old rabbits together with increasing amounts of amiodarone.
We found that amiodarone at concentrations that do not affect macrophage viability 1) induces the formation of cytoplasmic vacuoles, increases the surface density of inclusion bodies containing electron-dense material and/or multilamellar membranes, and decreases the surface density of mitochondria and lysosomes; 2) increases the incorporation of choline into DPPC while not affecting the ability to degrade exogenous DPPC; 3) changes the distribution of lysosomal enzymes among cytoplasmic organelles; 4) induces the release into the medium of lysosomal enzymes; and 5) does not affect the uptake, processing, and routing of diphtheria toxin but inhibits the degradation of SP-A after uptake. In vivo amiodarone inhibits the degradation of SP-A taken up into tissue compartments but not that of DPPC.
These results indicate that amiodarone perturbs the endocytic pathway acting after the early endosomes and suggest that released lysosomal enzymes may contribute to the genesis of lung damage. They also indicate that the alveolar accumulation of SP-A observed in animals with amiodarone-induced lung toxicity may be due to decreased degradation.
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METHODS |
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Materials.
All reagents were of analytic grade. Na125I,
L--dipalmitoyl[2-palmitoyl-9,10-3H(N)]phosphatidylcholine
([3H]DPPC; specific activity 40.0 Ci/mmol),
[methyl-3H]choline chloride (specific activity
80 µCi/mmol), and L-[4,5-3H]leucine
(specific activity 151.0 Ci/mmol) were from Amersham Pharmacia Biotech
(Little Chalfont, UK). Before use, [3H]DPPC was purified
by preparative thin-layer chromatography. Percoll was from Pharmacia
(Uppsala, Sweden). DPPC, egg phosphatidylcholine (PC), egg
phosphatidylglycerol, and cholesterol were obtained from Sigma (St.
Louis, MO), and their purity was checked by thin-layer chromatography.
Amiodarone and desethylamiodarone, stored at
26°C as 100 mM
solutions in DMSO, were from Sigma and Sanofi, respectively. Diphtheria
toxin, kept frozen in liquid nitrogen in 10 mM potassium HEPES-0.1 mM
EDTA, pH 7.0, at a concentration of 2 mg/ml, was a kind gift from Prof.
Emanuele Papini (University of Padua, Padua, Italy).
Cells. Alveolar macrophages were isolated from bronchoalveolar lavage fluid obtained from adult rabbits. The cells were washed with Ringer buffer (145 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM Na2HPO4, 10 mM glucose, and 10 mM HEPES, pH 7.4), suspended in Ringer buffer plus 1 mg/ml of bovine serum albumin, 50 IU/ml of penicillin, and 50 µg/ml of streptomycin (RBA), and used either as a suspension or after adhesion to Falcon plates or flasks (Becton Dickinson Labware Europe, Meylan, France). We recovered 62.0 × 106 ± 3.7 × 106 (SE) cells/kg body wt (n = 99 rabbits). The cells were over 90% macrophages (May-Grunwald-Giemsa staining) and 94 ± 1% viable as determined by trypan blue exclusion.
Morphology. Adhering macrophages were fixed overnight in 2.5% glutaraldehyde in PBS, scraped, and centrifuged at 10,000 g for 5 min. The pellets were postfixed for 2 h in 1% osmium tetroxide in PBS, dehydrated, and embedded in Spurr resin. Semithin sections obtained through the whole thickness of the pellets were stained with toluidine blue and observed with a Zeiss Axiophot light microscope. Ultrathin sections were stained with uranyl acetate and lead citrate and observed with a Jeol 1200 EXCII electron microscope.
Morphometry was performed on 10 electron micrographs for each experimental condition (i.e., control and amiodarone-treated cells) randomly taken at ×4,000 magnification and then photographically enlarged to ×10,000 magnification to appreciate the details of at least 2-3 cells/micrograph. A total number of 25 cells in each experimental condition were counted. By means of a ruler inserted within an optical magnifier, we measured the surface area covered by the whole cell and by the mitochondria, lysosomes (i.e., electron-dense, membrane-bound organelles), cytoplasmic inclusions (i.e., electron-dense structures containing amorphous and/or multilamellar membranes, not surrounded by any type of membrane), and endoplasmic reticulum. All organelles that were detectable in the section of each of the randomly selected cells were analyzed. Not less than 100 organelles were analyzed for each experimental condition. Results are expressed as percent of the surface area covered by different organelles in relation to the surface area covered by the whole cytoplasm.SP-A labeling.
SP-A was isolated from rabbit surfactant (3) with the
method of Hawgood et al. (13). All preparations of SP-A
were tested for lipopolysaccharide contamination with the
Limulus amebocyte lysate assay (Limusate, Haemachem, St.
Louis, MO), and lipopolysaccharide concentration results were always
<0.125 endotoxin unit/ml (<0.2 pg/µg protein). SP-A labeling was
done according to Goldstein et al. (12). Briefly,
0.3-0.5 mg of SP-A in 0.6 ml of 5 mM Tris · HCl, pH 7.4, was mixed with 0.2 ml of 1 M glycine, pH 10.0, and then with 500 µCi
of 125I. Within 1 min, 0.2 ml of freshly prepared 20 mM ICl
was added. After 5 min on ice, the mixture was applied to a PG10 column
(Bio-Rad) to separate 125I-SP-A from free iodine.
125I-SP-A was then dialyzed against 5 mM
Tris · HCl, pH 7.4. The whole procedure was done at
4°C. 125I- SP-A (specific activity 2-6 × 105
counts · min1 · µg
1) was
stored at 4°C and used within 1 mo.
Viability and phospholipid content of alveolar macrophages. To study the effect of amiodarone on viability, alveolar macrophages (106 cells/ml) were cultured for 1 or 24 h as suspensions in the presence of 0-100 µM amiodarone in DMSO (final DMSO concentration 1 µl/ml), and viability was estimated by trypan blue exclusion. In some experiments, after incubation, we pelleted the macrophages, washed them two times with saline, and measured the phospholipids (4) after extraction according to Bligh and Dyer (7). To further characterize the effect on viability, macrophages seeded on six-well plates were incubated for 1 h with 0-100 µM amiodarone. The cells were then either collected and homogenized immediately to measure cytochrome-c oxidase (35) or were switched to plain RBA to measure the release of lactate dehydrogenase (LDH) during the next hour (2).
Choline incorporation by alveolar macrophages. Macrophages seeded onto six-well plates (2 × 106 cells/well) were incubated for 1 h with 0-50 µM amiodarone in RBA. Without changing the medium, [3H]choline chloride was then added (2 µCi in 100 µl RBA/well), and the incubation was continued for 16 h. At the end, saturated PC associated with the cells was isolated and counted (25). Choline incorporation tended to deviate from linearity during the last 8 h of incubation (data not shown). The cause of the deviation from linearity remains unclear. Less than 5% of added choline was utilized during incubation. Results are presented as femtomoles of choline incorporated into DPPC by 106 cells in 16 h (n = 4 experiments).
Degradation of DPPC by alveolar macrophages.
Suspensions of alveolar macrophages (2 × 106/ml) were
incubated for 1 h with 0-100 µM amiodarone and then for 1 more hour with [3H]DPPC (0.05 µCi/ml) presented as
liposomes prepared by sonication (2) and with the
following composition (in µg/ml): 25 DPPC, 17.5 egg PC, 5 egg
phosphatidylglycerol, and 2.5 cholesterol (2). The
degradation of DPPC was measured by extracting the incubation mixture
(7) and counting the washed water-methanol phase in the
presence of Hyonic Fluor (Packard). Control cells degraded 168 ± 24 (SE) ng DPPC · 106
cells1 · h
1 (n = 17 experiments). Under the present conditions, the generation of
water-soluble by-products was linear up to 100 min (data not shown).
The results are presented as percent of the degradation measured with
control cells (n = 4 experiments).
Distribution of lysosomal enzymes in the postnuclear supernatant.
Macrophages seeded onto six-well plates (2 × 106
cells/well) were incubated with 0 or 10 µM amiodarone for 60 min at
37°C, rinsed in 0.25 M sucrose-1 mM EDTA, pH 7.4 (2),
scraped, and homogenized in the same medium. The homogenate was
centrifuged at 750 g for 7 min to obtain the postnuclear
supernatant. Two milliliters of the postnuclear supernatant were
deposited over a discontinuous gradient made by a cushion of 2.5 M
sucrose in 1 mM EDTA (1.2 ml) and an intermediate layer of 18% Percoll
in homogenization medium (8.5 ml) and were centrifuged for 30 min at
20,000 rpm in a Ti 50 rotor (Beckman, Palo Alto, CA) (2). Fractions obtained from the gradient were analyzed for
-hexosaminidase (21) and arylsulfatase
(11) activities. The content of each fraction is presented
as a percent of the total (n = 4 experiments for
-hexosaminidase and 2 experiments for arylsulfatase).
Release of lysosomal enzymes by alveolar macrophages.
Alveolar macrophages seeded onto six-well plates (2 × 106 cells/well) were incubated with 0-50 µM
amiodarone for 60 min. The medium was then changed with plain RBA, and
the incubation was continued for 1 more hour. At the end, the medium
was collected and centrifuged at 500 g for 10 min.
-Hexosaminidase activity was then measured in the resulting
supernatant and in the cell homogenate (pellet from the
500-g centrifugation plus cells scraped from the plate;
n = 11 experiments).
Effect of amiodarone on the diphtheria toxin-mediated inhibition of protein synthesis. Alveolar macrophages in RBA were seeded onto 24-well plates (5 × 105 cells/well) and allowed to adhere for 2 h at 37°C. Amiodarone was then added (final concentration 0-50 µM), and the incubation was continued for 1 more hour. Afterward, the medium was substituted with RBA containing 0 or 10 nM diphtheria toxin, and the incubation was continued for 2 h. [3H]leucine (200 nCi/well) was then added for 20 min. At the end, the cells were washed one time with 50% methanol, fixed for 30 min with cold methanol, and then washed three times with cold 10% trichloroacetic acid. The final precipitate was dissolved overnight with 1 N NaOH, and aliquots of the lysate were used for protein assay (29) and to count the radioactivity in the presence of Hyonic Fluor (Packard). Protein synthesis is presented as picomoles of leucine incorporated per milligram of protein in 20 min. Data are from three experiments (29). Control macrophages incorporated 0.82 ± 0.09 (SE) pmol leucine/mg protein in 20 min (n = 7 experiments).
Association of SP-A with alveolar macrophages. Suspensions of alveolar macrophages (5 × 106/ml) were incubated for 1 h at 37°C with 0-50 µM amiodarone and then for 0-100 min with 125I-SP-A (1 µg/ml). At the end, the cells were pelleted, transferred to new tubes, and washed two more times with cold RBA before being counted (n = 5 experiments).
Degradation of SP-A by alveolar macrophages.
Suspensions of macrophages (2 × 106/ml) were
incubated for 1 h with 0-100 µM amiodarone, centrifuged,
and resuspended in RBA containing 1 µg/ml of 125I-SP-A.
After 1 h of incubation, the radioactivity soluble in 20% cold
TCA was measured in medium plus cells. Control macrophages degraded
13.6 ± 2.4 (SE) ng SP-A · 106
cells1 · h
1 (n = 11 experiments), and the degradation was linearly related with time.
Results are presented as percent of degradation measured in the control
cells (n = 7 experiments).
Distribution of SP-A among components of the postnuclear supernatant. Alveolar macrophages seeded onto six-well plates (2 × 106/well) were incubated for 1 h with 0 or 50 µM amiodarone in RBA and then exposed for 15 min to 1 µg/ml of 125I-SP-A. Afterward, 125I-SP-A was removed, and adhering cells were rinsed three times with RBA. Then the cells were homogenized immediately or were incubated for 30 more minutes in the presence of 1 µg/ml of unlabeled SP-A (chase) before being homogenized. Each homogenate was centrifuged to obtain a postnuclear supernatant that was then centrifuged as described in Distribution of lysosomal enzymes in the postnuclear supernatant over a Percoll-sucrose gradient (2). Fractions collected from the gradient were counted, and the results are expressed as counts per minute per microgram of homogenate DNA (22).
Clearance of surfactant DPPC and SP-A administered into the
trachea.
Labeled surfactant was obtained by administration of 50 µCi of
[3H]choline into the tracheae of 3-day-old rabbits and
then isolating the surfactant 15 h later as previously reported
(3). The surfactant thus obtained was suspended in saline,
mixed with 125I-SP-A, and then combined with different
amounts of amiodarone dissolved in DMSO. The final mixtures
administered to recipient animals contained 5% of the alveolar pool of
phospholipids normally found in 3-day-old rabbits, 5 × 104 to 105 dpm as DPPC, 1 µg of
125I-SP-A, and different amounts of amiodarone (0, 2, 10, 50, 250, and 1,250 nmol, corresponding to 0, 1.3, 6.5, 32.3, 161.3, and 806.3 µg and concentrations of 0, 10, 50, 250, 1,250, and 6,250 µM,
respectively) suspended in 12.5 µl of DMSO in 200 µl (the volume
instilled). The dose was administered to 3-day-old rabbits (5 rabbits
for each concentration of amiodarone) by puncturing the trachea that
was exposed surgically under local anesthesia (3). After
instillation, the animals were returned to a warm environment and
killed after 3 h with an excess of pentobarbital sodium injected
into the peritoneum. We chose a 3-h interval on the basis of
preliminary experience with control rabbits killed 1 min, 3 h, and
24 h after the tracheal administration of a mixture made by
125I-SP-A and surfactant labeled in vivo with
[3H]choline (Table 1). In
those experiments, we found that 3 h after instillation
1) the administered materials were evenly distributed between alveoli and parenchyma, 2) the lung retains enough
125I-SP-A for counting, and 3)
125I-SP-A leaves the airways with kinetics similar to those
of SP-A labeled in vivo (Table 1) (2). Finally, we
chose a 3-h interval to reproduce in vivo the acute setting of exposure
to amiodarone studied in vitro.
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Statistical analysis. Data are means ± SE. Differences between two groups were analyzed by unpaired t-test (two tailed). Differences between more than two groups were analyzed by ANOVA and with Fisher's protected least significance difference as the post hoc test. The level of significance accepted was 5%.
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RESULTS |
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Morphology.
After 24 h of culture, control macrophages had a normal appearance
(Figs. 1 and
2). On the other hand, macrophages
cultured with 10 µM amiodarone presented an increased number of
vesicles, some occupying most of the cytoplasm, and an increased
surface density of inclusion bodies containing amorphous and/or
multilamellar electron-dense structures. The surface density of
mitochondria and lysosomes had decreased (Figs.
1-3).The endoplasmic reticulum was
not significantly influenced by 24 h of incubation with 10 µM
amiodarone (Fig. 3).
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Viability.
Incubation of alveolar macrophages with 1-20 µM amiodarone for
1 h or with 1-10 µM amiodarone for 24 h had no effect
on viability (Fig. 4). Incubation with 50 and 100 µM amiodarone decreased the viability so that after 24 h, only 31 and 2%, respectively, of the macrophages remained viable
(Fig. 4).
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Macrophage phospholipids. The phospholipid content of alveolar macrophages, expressed as micrograms of phospholipids per microgram of DNA, did not change during the 24-h incubation with 1-10 µM amiodarone (data not shown), in agreement with data from Martin et al. (24). On the other hand, incubation with 50 µM amiodarone for 24 h decreased by 39 ± 9% the cell pool of phospholipids (P < 0.05 by ANOVA).
Even if the total content of phospholipids per cell did not change during the 24-h incubation with 10 µM amiodarone, phospholipid metabolism was deeply affected. In fact, the incorporation of choline into saturated PC increased significantly in the presence of 10 µM amiodarone, whereas at higher concentrations of amiodarone, choline incorporation decreased, likely reflecting unrecoverable injury (Fig. 5).
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Degradation of exogenous DPPC by alveolar macrophages.
One hour of incubation with 0-100 µM amiodarone did not change
the rate of degradation of exogenous DPPC by alveolar macrophages (Fig.
6).
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Distribution of lysosomal enzymes in the postnuclear supernatant.
In this experiment, we centrifuged the postnuclear supernatant over a
gradient of Percoll-sucrose and analyzed the distribution of
-hexosaminidase and arylsulfatase. These enzymes associate with
mature lysosomes, which migrate toward the bottom of the gradient, and
with early and late endosomes, which stay at the top (2,
26).
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Release of -hexosaminidase by alveolar macrophages.
During the 1-h incubation, control macrophages released 0.6 ± 0.1% of the cell pool of
-hexosaminidase. The release increased three times in the presence of 10 µM amiodarone and nine times in the
presence of 50 µM amiodarone (both changes significant; Fig.
8). Because, with 10 µM amiodarone, the
release of LDH was not different from the control value, the observed
effect indicates a specific injury response. On the other hand, the
release of
-hexosaminidase observed with 50 µM amiodarone could be
a nonspecific event of cell death.
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Effects of amiodarone on diphtheria toxin uptake and processing.
Ten micromolar amiodarone did not interfere with the ability of
diphtheria toxin to block protein synthesis (Fig.
9). Results similar to those reported in
Fig. 9 were obtained when macrophages were incubated with 20 and 50 µM amiodarone before being exposed to the diphtheria toxin (data not
shown). This result indicates that in the presence of amiodarone,
diphtheria toxin was correctly internalized and then transferred from
the early endosomes to the cytosol.
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Association of SP-A with alveolar macrophages. Incubation with 1-50 µM amiodarone did not change the association of 125I-SP-A with alveolar macrophages (n = 5 experiments; data not shown).
Degradation of SP-A by alveolar macrophages. Amiodarone inhibited the degradation of 125I-SP-A by alveolar macrophages (Fig. 6). The effect was significant at a concentration of 10 µM, and 50% inhibition was reached between 10 and 20 µM. Interestingly, 50 µM amiodarone decreased SP-A degradation to 15 ± 4% of the control value while decreasing viability to 69 ± 5% of the control value.
The effect of amiodarone on the degradation of SP-A by alveolar macrophages was also analyzed in an experiment in which cells pretreated with 0 or 50 µM amiodarone were exposed to 125I-SP-A for 15 min and then the distribution of radioactivity in the postnuclear supernatant was analyzed immediately or after 30 min. As shown in Fig. 10, after a 15-min incubation with 125I-SP-A, there was no difference between control and treated cells in the recovery and distribution of label in the postnuclear supernatant, indicating that amiodarone did not affect the uptake of SP-A. During the chase, however, the cells pretreated with amiodarone retained a greater fraction of incorporated radioactivity, indicating that amiodarone impairs the degradation of SP-A.
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Effect of amiodarone on the clearance of SP-A and DPPC from the
airways.
The administration of amiodarone into the trachea at the concentrations
used in this experiment did not change the concentration of
phospholipids, the activity of LDH or -hexosaminidase, or the number
of cells present in lung lavage fluid over 3 h (data not shown).
Protein concentration and the viability of the cells from lavage also
remained unchanged except for the highest concentration of amiodarone
(Fig. 11).
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DISCUSSION |
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In alveolar macrophages, amiodarone alters the endocytic pathway. This pathway has been operationally delineated by analyzing the distribution of signal proteins, receptors, or enzymatic activities and by following the movement of tracers that follow specific tracks along it (reviewed in Ref. 26). According to the accepted view, endocytosis results in the delivery of internalized molecules to the early endosomes, which are the site where receptor-ligand complexes dissociate and where unoccupied receptors, free ligands, and other molecules internalized with the fluid phase are sorted. Unoccupied receptors and other components to be returned to the plasma membrane collect into recycling vesicles that shuttle toward the plasma membrane either directly or after migrating to the perinuclear cytoplasm. Materials to be degraded are transferred to the late endosomes and then to the lysosomes. Late endosomes present internal membranes organized as multilayers or as vesicles in a vesicle; have a low density on Percoll gradient centrifugation; are enriched with specific markers like lgp/LAMPs, Rab7, Rab9, syntaxin 7, and the cation-independent mannose 6-phosphate receptor (MPR); and contain active lysosomal hydrolases (26, 28). Lysosomes are distinguished from late endosomes on the basis of their greater density, the lack of MPR, and the property of being the final site of accumulation of internalized macromolecules. Most of newly synthesized lysosomal hydrolases emerge from the trans-Golgi bound to the MPR and in this bound form are transferred to the late endosomes and then, after dissociation from the MPR, to the lysosomes. The late endosomes thus represent a crucial crossroad involved both in the traffic of endocytosed materials and in the biogenesis of the lysosomes.
Amiodarone does not interfere with the ability of macrophages to move molecules from the exterior of the cell to the early endosomes. In fact, in the presence of amiodarone, diphtheria toxin is taken up, processed, and translocated to the cytosol. Because transfer of the toxin to the early endosomes involves clathrin-coated vesicles and because hydrolysis of the toxin in the early endosomes requires a slightly acidic pH (34), it appears that amiodarone does not interfere with formation of clathrin-coated vesicles or with acidification of the early endosomes. Amiodarone at a concentration that does not affect cell viability inhibits the degradation of SP-A by alveolar macrophages. Because amiodarone does not retard the appearance of SP-A in the postnuclear supernatant and does not interfere with the movement of diphtheria toxin from the exterior of the cell to the early endosomes, we conclude that the defect in the degradation of SP-A is likely to be located at the level of the late endosomes and/or the lysosomes. This interpretation is supported by the fact that amiodarone-treated cells present an increased surface density of vesicles containing multilamellar membranes (the hallmark of the late endosomes), have a decreased surface density of normally sedimenting lysosomes, and display a disturbed traffic of lysosomal enzymes. In the present experiments, the inhibition of SP-A degradation and the increase in the release ofAmiodarone inhibits in vivo the degradation of SP-A. To study the effect of amiodarone in vivo, we coadministered labeled surfactant together with increasing amounts of amiodarone into the trachea of 3-day-old rabbits and analyzed the distribution of DPPC and SP-A in alveolar and tissue compartments after 3 h. We used this approach because the tracheal administration of amiodarone (1.25 mg administered two times) is a well-established way to induce pulmonary fibrosis in hamsters (6, 8).
We found that amiodarone impairs the disposal of SP-A by inhibiting the degradation of SP-A in the tissue compartment. The decreased degradation of SP-A could explain the recent observation (27), based on histochemistry, that rats chronically receiving amiodarone accumulate SP-A in the alveoli. As shown in Fig. 12, the minimum amount of amiodarone needed to obtain a significant accumulation of SP-A in the lung was 50 nmol (32.3 µg), a value smaller than that needed to induce fibrosis. This fact and the finding that amiodarone has a direct effect on isolated macrophages suggest that the changes in the turnover of SP-A induced by amiodarone in our animal model represent a direct effect of the drug rather than an effect mediated by inflammation.A mechanism of lung damage by amiodarone. The cationic amphiphilic nature of amiodarone might explain why the terminal organelles of the endocytic pathway are preferentially damaged. Indeed, cationic amphiphilic drugs easily enter the cells and their organelles but, on exposure to an acidic milieu, become insoluble and accumulate to levels that may exceed the extracellular concentration by orders of magnitude (10). Because late endosomes and lysosomes have a strongly acidic interior (26), they are especially prone to accumulate amiodarone. Thus damage to these organelles might impair the ability to degrade exogenous substances like SP-A and direct enzymes normally transported to the lysosomes toward the exterior of the cell. The increased extracellular delivery of lysosomal enzymes might then cause damage and start a cascade of proinflammatory events, but immune or inflammatory mechanisms of lung injury were not explored in this study.
When amiodarone was administered into the trachea of 3-day-old rabbits, we did not find increased levels ofEffect of amiodarone on lung phospholipids. In vitro 10 µM amiodarone increased the surface density of inclusion bodies containing multilamellar membranes and increased the incorporation of choline into DPPC, whereas the ability to degrade exogenous DPPC did not change. Because in vitro amiodarone is a strong inhibitor of lysosomal phospholipase A2 (24), these results suggest that the degradation of exogenous phospholipids by alveolar macrophages may not involve the lysosomal compartment, in agreement with recent findings from our laboratory (2). In vivo amiodarone administered into the trachea over a wide range of concentrations had no effect on the ability to take up and degrade surfactant DPPC. It is therefore tempting to conclude that the intracellular accumulation of membranes induced in macrophages by amiodarone is more likely the result of a derangement in the turnover of cell phospholipids rather than the result of a decreased degradation of surfactant phospholipids. It is worth noting, however, that the tracheal administration may not represent a good model in which to study the effects of amiodarone on the turnover of lung lipids because in some animal models, the oral administration of amiodarone causes accumulation of lung phospholipids, whereas the tracheal administration causes fibrosis (6, 8). Furthermore, the acute effects of amiodarone observed in the present experiment may not reproduce the effects induced by the chronic exposure to the drug.
To summarize, the acute administration of amiodarone inhibits the degradation of SP-A and causes the release of lysosomal enzymes. The alveolar accumulation of SP-A observed in animals with amiodarone-induced lung toxicity could be due to decreased degradation. The release of lysosomal enzymes could contribute to the genesis of lung damage induced by amiodarone. ![]() |
ACKNOWLEDGEMENTS |
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We thank Prof. Gaetano Crepaldi for continuous support and encouragement; Prof. Emanuele Papini for very helpful suggestions; Dr. Mario Plebani for allowing Dr. Martina Zaninotto to do the lactate dehydrogenase assays; Prof. Mariano Ferrari, Prof. Roberto Padrini, and Dr. Donatella Pavan for useful advice; Raffaella Marin for help with subcellular fractionation; and Gervasio and Mariano Bruttomesso for technical help.
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FOOTNOTES |
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Address for reprint requests and other correspondence: A. Baritussio, Clinica Medica I, Policlinico, Via Giustiniani 2, 35128 Padova, Italy (E-mail: aldo.baritussio{at}unipd.it).
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 3 January 2001; accepted in final form 20 July 2001.
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