1 Department of Pharmacology, University of South Alabama College of Medicine, Mobile, Alabama 36688; 2 MediQuest Therapeutics, Seattle, Washington 98103; and 3 Department of Pathology, Comenius University, 81372 Bratislava, Slovakia
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ABSTRACT |
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Hypoxic pulmonary vascular remodeling in rats is associated with increased polyamine transport in pulmonary artery smooth muscle cells (PASMCs). We therefore defined constitutive and hypoxia-induced polyamine transport properties of rat cultured PASMCs and determined the impact of polyamine transport blockade on hypoxia-induced accumulation of p38 MAP kinase. PASMCs exhibited polyamine transport pathways that were characterized by Michaelis-Menten kinetics. RNA synthesis inhibition attenuated while inhibition of protein synthesis increased polyamine uptake, thus suggesting regulation by ornithine decarboxylase-antizyme. The presence of two transporters with overlapping selectivities, one for putrescine and another for all three polyamines, was inferred by cross-competition studies and by findings that only putrescine uptake was sodium dependent and that hypoxia caused a selective, time-dependent induction of putrescine transport. The pathophysiological significance of augmented putrescine import was suggested by the observation that polyamine transport inhibition suppressed hypoxia-induced p38 MAP kinase phosphorylation. These results indicate that rat PASMCs express two polyamine transporters and that a specific increase in the putrescine uptake pathway is necessary for hypoxia-induced activation of p38 MAP kinase.
pulmonary hypertension; vascular smooth muscle; hypoxia; pulmonary artery smooth muscle cells; mitogen-activated protein
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INTRODUCTION |
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FUNCTIONAL PROPERTIES of the pulmonary arterial wall are controlled by dynamic interactions between resident and itinerant cells, the extracellular matrix, and the chemical signaling environment in which the cells reside. In hypertensive pulmonary vascular remodeling, all of these regulatory components are altered (33). Because so many signals impact on lung cells in pulmonary hypertension, it is unlikely that a single key ligand or receptor could serve as an isolated pharmacological target. Alternatively, intracellular signal transduction processes used to integrate stimuli originating from the local environment seem especially appealing. In this context, our research has focused on the polyamines, putrescine, spermidine, and spermine, a family of low molecular weight organic cations that are essential for DNA, RNA, and protein synthesis (26) and that may regulate other aspects of cell function and signaling (12, 13, 16, 27).
De novo polyamine synthesis, driven by the initial and generally
rate-limiting enzyme ornithine decarboxylase (ODC) (26), appears to be a dominant mechanism underlying most types of lung structural remodeling. Increased lung ODC activity is temporally related to elevations in polyamine content that accompany postnatal lung growth (35), hyperoxic lung injury (7,
36), compensatory lung growth after pneumonectomy
(10), and monocrotaline-induced pulmonary hypertension
(6, 20, 21). Blockade of ODC with the site-specific
suicide inhibitor -difluoromethylornithine forestalls a spectrum of
adaptive structural changes in these rat models of lung development and disease.
Unlike the central role of increased de novo polyamine synthesis in other pulmonary diseases, hypoxic pulmonary vascular remodeling seems to have a unique dependence on augmented polyamine transport. For example, although intact rat lung polyamine contents are elevated by in vivo hypoxia (22), ODC activity under this condition is profoundly decreased (32). ODC activity and mRNA content are also decreased by hypoxia in cultured pulmonary artery smooth muscle (PASMCs) (8, 9) and endothelial cells (5). In contrast, putrescine uptake in intact lungs from chronically hypoxic rats is augmented (32).
We recently began to explore the cellular basis of increased polyamine uptake in chronically hypoxic rat lungs (4). With the use of rat lung and pulmonary arterial explant preparations, we found that both intimal and medial pulmonary arterial cells displayed augmented spermidine transport. Interestingly, the ability of hypoxia to increase medial cell spermidine uptake required the presence of an intact, hypoxic endothelium. Similarly, although cultured main pulmonary artery endothelial cells responded to hypoxia with increased spermidine transport (5), hypoxia failed to increase spermidine uptake by rat PASMCs unless they were cultured in media conditioned by hypoxic endothelial cells (4). These findings suggested that an endothelial-derived regulatory molecule(s) might be required for the augmented smooth muscle transport of spermidine in hypoxia. On the other hand, there is substantial cell type-dependent heterogeneity in terms of polyamine uptake pathways, with many cells, including PASMCs from species other than the rat, exhibiting multiple polyamine transporters (3, 29). Hypoxia could thus exert direct effects on a discrete polyamine transporter in rat PASMCs. Against this background, the current studies determined if there are multiple polyamine transporter activities expressed in rat PASMCs and whether hypoxia discretely regulates one or more pathways. In addition, we also took advantage of the recent availability of a polyamine transport inhibitor (38), ORI1202, to test the hypothesis that a polyamine import pathway is required for the PASMC adaptive response to hypoxia. As a molecular marker of the PASMC response, we evaluated relative abundances of total and phosphorylated p38 MAP kinase, a growth regulatory protein shown in multiple cell types to be involved in adaptation to hypoxia (11, 31, 39, 41).
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MATERIALS AND METHODS |
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PASMC culture.
Main pulmonary arteries were isolated from 300- to 400-g male
Sprague-Dawley rats anesthetized with pentobarbital sodium and placed
in F12 Nutrient Mixture and DMEM mixture 1:1 supplemented with 10%
fetal bovine serum, 100 U/ml penicillin, and 0.1 mg/ml streptomycin
(GIBCO BRL, Grand Island, NY). After 3 days under standard incubator
conditions, the rings were removed and cells that had migrated from the
explants were grown to confluence. Culture medium was changed every 3 days. Cells were harvested using a 0.05% solution of trypsin (GIBCO
BRL). Cells from passages 3-12 were seeded in
12-well plates (Costar, Cambridge, MA), 1 × 104 cells
in 2 ml of media per well. Polyamine transport experiments were
performed after 2-3 days when the cells reached 95-100%
confluence. Smooth muscle phenotype was confirmed by the presence of
smooth muscle-specific -actin (Sigma, St. Louis, MO) as detected by immunofluorescence microscopy. Cell counts were determined by hemocytometry. Cell viability was assessed using trypan blue exclusion using standard techniques.
Hypoxic exposure. When cells reached at least 95% confluency, the plates were placed in incubators (Forma Scientific, Marietta, OH) that were purged with gas mixtures containing either 21% (designated as "control") or 2% oxygen (termed "hypoxic")-5% CO2 and the balance in nitrogen. Periods of exposure to these environments ranged from 6 to 48 h. Media PO2 values, determined using a Radiometer model ABL30 blood-gas analyzer (Copenhagen, Denmark), were >120 Torr for the control cultures and 25-35 Torr for cultures designated as "hypoxic."
Polyamine transport. After incubation under the indicated conditions, cells were rinsed with serum-free DMEM after which 1 ml of DMEM was added to each well and the cells were allowed to acclimate for 30 min. Subsequently, one of the three [14C]polyamines was added to each well in concentrations from 0.1 to 10 µM. In most studies, the cells were then incubated for 30 min, a duration that preliminary experiments indicated was in the linear range of uptake of all polyamines in the concentrations tested. Nonspecific polyamine transport was determined as the uptake occurring in cells incubated at 4°C, as we and others reported previously (1-3). At the appropriate time, media containing residual [14C]polyamines were aspirated, cells were rinsed twice with cold PBS, and overlaid with 1.5 ml of PBS containing 0.5% SDS for 30 min. The cell lysates were then transferred to scintillation vials and mixed with 4 ml of scintillation cocktail (Beckman Instruments, Fullerton, CA), and radioactivity was determined using a Beckman LS 6500 liquid scintillation counter. The [14C]polyamine uptake rates were expressed in terms of uptake per 1,000,000 cells.
Western analysis of phospho- and total p38. Control cells and cells cultured in hypoxia were washed twice with PBS and lysed in 2% SDS electrophoresis buffer after which 25 µg protein were applied to an SDS/12% polyacrylamide gel. After separation, samples were transferred to nitrocellulose filters (BioRad Laboratories, Hercules, CA). Membranes blocked in 5% nonfat dried milk in PBS with 0.1% Tween 20 were incubated overnight at 4°C with primary rabbit polyclonal antibody recognizing phosphorylated p38 MAP kinase or total p38 MAP kinase (Cell Signaling Technology, Beverly, MA), diluted 1:500 in blocking solution. After being washed, the membranes were incubated with 1:10,000 diluted horseradish peroxidase-conjugated goat anti-rabbit IgG (Calbiochem-Novabiochem, San Diego, CA) for 1 h at room temperature and then revealed by chemiluminescence with the ECL detection kit (Amersham).
Experimental protocols. Uptake of each polyamine in concentrations of 0.1-10 µM was determined at temperatures of 4 and 37°C in rat PASMCs incubated under normoxic and hypoxic conditions. Specific uptake was determined as described above and normalized to 1,000,000 cells. GraphPad Prism software was used to fit data to the Michaelis-Menten equation and to derive values of Km and Vmax for each polyamine.
Competition between nonradioactive polyamines, in concentrations of 0.1, 1, and 5 µM, and 0.1 µM [14C]polyamines, for uptake into PASMCs was used to explore the possibility that there were multiple polyamine import pathways. Effects of RNA and protein synthesis inhibition on polyamine transport in normoxia and hypoxia were studied by incubation of the cells with 50 nM actinomycin D and 1 µM cycloheximide, respectively. To examine the sodium dependence of polyamine transport, PASMCs seeded as described above were washed twice and incubated in 1 ml of the following solutions: a sodium-containing buffer composed of 140 mM NaCl, 5.9 mM KCl, 1.2 mM MgSO4, 1.0 mM CaCl2, 1.0 mM Tris adjusted to pH 7.3 with 1 mM HEPES, and 5.6 mM glucose; a sodium-free buffer had an identical composition except NaCl was replaced with 140 mM choline chloride or buffers with intermediate sodium contents composed of NaCl and choline chloride. Cells were incubated in the indicated media for 24 h after which transport rates were determined at a polyamine concentration of 0.3 µM. Finally, to determine if polyamine import was required for hypoxia-induced increases in phospho-p38, cells were pretreated for 30 min with 30 µM ORI1202 or its vehicle (saline) after which they were cultured in the presence of the drug for an additional 3 or 24 h in either normoxia or hypoxia. Cells were harvested at the indicated time, and Western analyses were used to determine total and phosphorylated p38 MAP kinase.Statistics. Data are expressed as means ± SE. Differences in Km and Vmax values between control and hypoxic cells were determined according to the approach described by Motulsky and Ransnas (19). In other experiments, one-way analyses of variance combined with Newman-Keuls tests were used to detect time- or concentration-related differences. P values < 0.05 were taken as evidence of statistical significance.
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RESULTS |
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Time course of polyamine uptake. Uptake of all three polyamines by rat PASMCs in concentrations of 0.1, 3, and 10 µM increased as a function of the duration of incubation and was linear for up to 1 h (data not shown). In subsequent transport experiments, uptake rates were determined after a 30-min incubation period, which was in the linear portion of all of the curves.
Competition studies.
Figure 1 displays competition between 0.1 µM 14C-labeled polyamines and unlabeled polyamines in
concentration ratios 1:1, 1:10, and 1:50 for uptake into PASMCs. Uptake
of [14C]putrescine was suppressed in a
concentration-related manner by both spermidine and spermine. Uptake of
spermidine and spermine exhibited cross-competition, while putrescine
failed to suppress uptake of either polyamine. The extent of inhibition
of [14C]polyamine uptake by the indicated competitors did
not differ significantly between cells cultured under normoxic and
hypoxic conditions (data not shown).
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Effect of cycloheximide and actinomycin D on polyamine transport.
As shown in Fig. 2A, protein
synthesis inhibition with 1 µM cycloheximide increased uptake of 0.1 µM [14C]polyamines more than twofold after 3-h
incubation under standard culture conditions. The effects of the RNA
synthesis inhibitor actinomycin D in a concentration of 50 nM on
polyamine uptake in rat PASMCs are shown in Fig. 2B.
Actinomycin D caused a time-dependent attenuation of the uptake of all
three polyamines.
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Sodium dependence of polyamine transport.
Cultured rat PASMCs exhibited different sodium requirements for
transport of the three polyamines. As shown in Fig.
3, putrescine uptake was nearly abolished
after cells were incubated for 24 h in media containing
Na+ in concentrations less than 70 mM. Uptakes of
spermidine and spermine in media containing 70 mM Na+ did
not differ from control but were reduced by ~50% in sodium-free media.
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Impact of hypoxia on polyamine transport kinetics.
Uptake rate-concentration curves for transport of the three polyamines
in rat PASMCs cultured in either normoxia or hypoxia for 24 h are
shown in Fig. 4. Uptake
rate-concentration relationships for all three polyamines in both
normoxic and hypoxic culture conditions obeyed Michaelis-Menten
kinetics. Values for Km and Vmax for import of the three polyamines in
normoxic and hypoxic culture conditions are shown in Table
1. Hypoxia failed to influence either Km or Vmax for
either spermidine or spermine uptake. In contrast, the
Km for putrescine import was approximately
doubled and the Vmax was increased nearly
threefold in hypoxic conditions.
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Effect of polyamine transport blockade on hypoxia-induced
phosphorylation of p38 MAP kinase.
Initial studies verified that the polyamine transport inhibitor ORI1202
(30 µM) suppressed the hypoxia-induced increase in putrescine import.
As shown in Fig. 6A, 6-h
culture in hypoxia, as expected, increased putrescine import ~30%
above the control level. The polyamine transport inhibitor profoundly
suppressed putrescine uptake assessed in normoxic cells and in cells
cultured in hypoxia for 6 h. Companion studies explored the effect
of hypoxia, alone and in the presence of 30 µM ORI1202, on p38 MAP
kinase. Figure 6B demonstrates that while hypoxia failed to
change the abundance of total p38 MAP kinase, there was an appreciable
increase in the abundance of phospho-p38. Treatment with ORI1201 failed to alter the normoxic level of either total or phospho-p38 but abolished the hypoxia-induced increase in the latter. Densitometry was
used to semiquantify the effect of hypoxia on phospho-p38, normalizing
the indicated treatments to the intensity determined in control PASMCs.
As shown in Fig. 6C, hypoxia increased phospho-p38 by
~50%, and the increase was prevented by the polyamine transport inhibitor. Data presented in Fig. 6 were obtained after 3-h exposure to
hypoxia, and identical results were obtained for a 24-h hypoxic exposure regimen (data not shown).
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DISCUSSION |
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In general terms, regulation of cell polyamine contents involves
four interactive pathways. The first, de novo synthesis, is well
studied and can be manipulated pharmacologically through inhibition of
ODC with the site-specific, enzyme-activated inhibitor -difluoromethylornithine (15, 24). A second regulatory
pathway is transmembrane transport (29). In some cells,
polyamine uptake accompanies proliferation while it is downregulated
during quiescence. In polyamine-depleted cells, transport processes may
restore polyamine levels and revitalize cell functions. A third
regulatory pathway involves polyamine interconversion, catalyzed by
spermidine- and spermine-aminopropyltransferases, another rate-limiting
enzyme spermidine/spermine N1-acetyltransferase, and
polyamine oxidase (25). These latter two enzymes are
responsible for polyamine catabolism by converting spermine back to
spermidine and spermidine to putrescine. Finally, it has been
speculated for many years that polyamine compartmentation is an
important regulatory pathway. Early reports showed high concentrations
of spermine in mitochondria and nuclei that changed during the cell
cycle (14), but the exact role that polyamine
compartmentation plays in the context of regulation of physiological
function has yet to be explored.
Studies in rat models suggest that elevated de novo synthesis is responsible for increases in lung polyamine content required for postnatal lung growth (35), repair of hyperoxic lung injury (7, 36), compensatory lung growth after pneumonectomy (34), and monocrotaline-induced hypertensive pulmonary vascular remodeling (6, 23). Hypoxic pulmonary vascular remodeling in the rat seems to use a different polyamine regulatory paradigm. Rat lung polyamine contents are elevated by hypoxia in a manner that is temporally related to development of sustained pulmonary hypertension (22), but ODC activity is profoundly decreased within 24 h of hypoxic exposure (32). In contrast, putrescine uptake is elevated (32). Activities of S-adenosylmethionine decarboxylase, spermidine/spermine acetyltransferase, and polyamine oxidase are also elevated in intact rat lung (32), suggesting that transported polyamines may be interconverted between pools. A more detailed understanding of this unusual mode of polyamine regulation may reveal opportunities to use agents disrupting polyamine import or interconversion as means to suppress pulmonary vascular remodeling.
Polyamine regulatory pathways operative in hypoxia have been explored in some detail using bovine PASMCs as a model system. Polyamine regulation in this cultured cell type mimics the response of intact rat lung tissue inasmuch as hypoxia inhibits ODC activity, possibly by suppressing ODC mRNA transcription (8). However, unlike rat lung tissue wherein the induction of spermidine import in medial PASMCs by hypoxia requires a soluble factor(s) elaborated from hypoxic endothelial cells (4), hypoxia directly increases the uptake of all three polyamines in cells from the bovine species (3). One explanation for this apparent disparity could relate to the well-recognized cell type- and species-dependent differences in polyamine transport pathways (29, 30). Accordingly, two objectives of the present study were to provide a more complete characterization of polyamine import pathways operative in rat PASMCs and to test the hypothesis that hypoxia could directly regulate discrete uptake pathways without involvement of an endothelial cell-derived permissive factor(s).
In the present study, we found that rat PASMCs were similar in several key respects to bovine PASMCs as well as other cell types so far studied. For example, rat PASMCs exhibit polyamine transport pathways that are time, temperature, and concentration dependent. Polyamine transport in rat and bovine PASMCs requires ongoing RNA synthesis. Protein synthesis inhibition caused a transient increase in polyamine import as it does in other cells (5), presumably as a result of relief from antizyme-mediated inhibition (17, 18). Uptake of putrescine, spermidine, and spermine could be modeled according to Michaelis-Menten kinetics. Values for Km in rat PASMCs were in the low micromolar range and ~10-fold higher than in bovine cells. Values for Vmax were of the same order of magnitude in the two cell types. Finally, competition experiments suggested that in rat PASMCs, like bovine cells, polyamine uptake could occur via a single transport pathway with overlapping specificities for the three polyamines. This suggestion derives from the observation in both cell types that spermidine and spermine effectively compete with each other and with putrescine for uptake while putrescine fails to significantly depress uptake of either spermidine or spermine. However, as discussed below, there are compelling reasons to suspect that there could be two discrete transport pathways operative in rat PASMCs.
We found that hypoxia increased the Km and Vmax only for putrescine transport; Km and Vmax values for spermidine and spermine were unaffected by hypoxia. If a single transporter were involved, then it would be more reasonable to expect that hypoxia would have a similar effect on kinetic parameters for all three polyamines, as is the case in bovine PASMCs (3). Additionally, and as shown in Fig. 3, putrescine uptake, but not that for spermidine and spermine, was markedly impaired in the absence of Na+. Collectively, these observations suggest that in rat PASMCs, there are two polyamine transporters, one for putrescine and another for all three polyamines. Hypoxia evokes a selective increase in putrescine uptake.
As noted above, we found that cycloheximide caused a transient increase in polyamine uptake, most likely reflecting loss of antizyme-mediated repression of the transporter(s). We therefore considered the possibility that the specific increase in putrescine uptake caused by hypoxia could be modulated by antizyme. PASMCs were incubated with the protein synthesis inhibitor for 12 h, a duration sufficient to permit the uptake rate to return to baseline levels after a transient increase. Exposure of the cells to hypoxia after this treatment increased putrescine uptake to an extent greater than in the absence of the protein synthesis inhibitor. This observation suggests that antizyme may suppress the hypoxia-induced increase in putrescine uptake, but further studies will be required to verify this idea.
The reason why hypoxia selectively augments uptake of putrescine is unknown. One explanation is that hypoxia, in intact rat lung PASMCs, causes a profound decrease in ODC activity that would be expected to diminish the putrescine pool (32). Upregulation of putrescine transport may thus serve to offset the effects of ODC depression in depleting putrescine. The transported putrescine may be converted to higher order polyamines and/or shuttled into discrete cellular compartments (28). As shown by our previous study, PASMC uptake of the triamine spermidine also seems to be induced by hypoxia, but in this case, a soluble factor(s) released by hypoxic endothelial cells is required, unlike the direct action of hypoxia on putrescine transport (4).
Despite the well-established facts that polyamines are present in all mammalian cells and that adjustments in their levels are required for cell growth, differentiation, and survival, the precise role(s) that polyamine transport, per se, plays in governing polyamine-dependent events has not been established. This deficiency can be ascribed, in part, to the lack of specific polyamine transport inhibitors. Recently, however, Weeks et al. (38) reported the development of a lysine-polyamine conjugate ORI1202 that specifically inhibited polyamine import in a variety of transformed cells. We used this agent to explore the importance of augmented putrescine transport in the adaptive response to hypoxia. As a molecular marker of the hypoxic response, we assessed the amount of total and phosphorylated p38 MAP kinase, which is centrally involved in the response to hypoxia in a variety of cells (11, 31, 39, 41). The transport inhibitor profoundly reduced both the baseline import and the hypoxia-induced increase in putrescine import. Although neither hypoxia nor ORI1202 altered the abundance of total p38, we found that hypoxia elevated the amount of phospho-p38 after both 3 and 24 h of exposure and, importantly, that these increases were suppressed by ORI1202. Because the transport inhibitor failed to decrease the basal levels of total or phosphorylated p38, these observations suggest that induction of putrescine transport by hypoxia is necessary for activation of p38 in PASMCs.
It seems likely that the increased putrescine transport may be required for other aspects of the adaptive response to hypoxia as well. In this regard, mitochondria express an inner-membrane polyamine "uniporter" that transports all three polyamines into the mitochondrial matrix (37) where they may be linked to maintenance of mitochondrial inner-membrane integrity and antioxidant defense (27), regulation of enzymes involved with oxidative phosphorylation (40), and perhaps for other mitochondrial functions. Transmembrane transport could be the first step in providing polyamines for subsequent transport into the mitochondrial compartment. Additional studies will be required to fully appreciate the specific events in the hypoxic response of PASMCs in which augmented putrescine transport plays a role.
In summary, the present study shows that rat PASMCs are similar to the corresponding bovine cell population in a number of key respects, including the time, temperature, and concentration dependence of polyamine import. Polyamine transport also requires ongoing RNA synthesis and seems to be negatively regulated by a short-lived protein, probably antizyme. Although competition experiments suggest that there may be one transporter with overlapping selectivities for the three polyamines, findings that hypoxia causes a selective upregulation of putrescine transport and that only putrescine transport is markedly affected by Na+ depletion suggest that two discrete transporter systems are operative in rat PASMCs, one for putrescine and another for the three polyamines. The hypoxia-induced increase in putrescine transport is required for phosphorylation p38 MAP kinase, thus implying that transmembrane putrescine transport could govern a range of responses involved in hypoxic adaptation of PASMCs.
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. N. Gillespie, Dept. of Pharmacology, College of Medicine, Univ. of South Alabama, Mobile, AL 36688 (E-mail: mgillesp{at}jaguar1.usouthal.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.
September 27, 2002;10.1152/ajplung.00234.2002
Received 18 July 2002; accepted in final form 23 September 2002.
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