Sulpiride

We have previously reported that dopamine increased active Na+ transport in rat lungs by upregulating the alveolar epithelial Na,K-ATPase. Here we tested whether alveolar epithelial cells produce dopamine and whether increasing endogenous dopamine production by feeding rats a 4% tyrosine diet (TSD) would increase lung liquid clearance. Alveolar Type II cells express the enzyme aromatic-L-amino acid decarboxylase (AADC) and, when incubated with the dopamine precursor, 3-hydroxy-L-tyrosine (L-dopa), produce dopamine. Rats fed TSD, a precursor of L-dopa and dopamine, had increased urinary dopamine levels, which were inhibited by benserazide, an inhibitor of AADC. Rats fed TSD for 15, 24, and 48 hours had a 26, 46, and 45% increase in lung liquid clearance, respectively, as compared with controls. Also, dopaminergic D^sub 1^ receptor antagonist-but not dopaminergic D^sub 2^ receptor antagonist-inhibited the TSD-mediated increase in lung liquid clearance. Alveolar Type II cells isolated from the lungs of rats after they had been fed TSD for 24 hours demonstrated increased protein abundance of Na,K-ATPase [alpha]^sub 1^ and [beta]^sub 1^ subunits. Basolateral membranes isolated from peripheral lung tissue of tyrosine-fed rats had increased Na,K-ATPase activity and Na,K-ATPase [alpha]^sub 1^ subunit. These data provide the first evidence that alveolar epithelial cells produce dopamine and that increasing endogenous dopamine increases lung liquid clearance.

Keywords: dopamine; lung epithelium; Na,K-ATPase; tyrosine diet

Clearance of pulmonary edema occurs via vectorial Na+ transport in which water follows the Na+ gradients from the alveoli into the interstitium and the pulmonary circulation (1, 2). Na+ enters the alveolar epithelial cells mainly via apical amiloride-sensitive Na+ channels and is extruded by the basolaterally located Na,K-ATPases (1-8). Recent studies have demonstrated that exogenous dopamine (DA) increases lung liquid clearance in rats by about 40-70% above control levels by upregulating alveolar epithelial Na,K-ATPase (9, 10). Dopamine synthesis begins with the amino acid tyrosine derived from the diet or from hydroxylation of phenylalanine in the liver. Tyrosine is converted to 3-hydroxy-L-tyrosine (L-dopa) by tyrosine hydroxylase, which is found in all chromaffin tissue and L-dopa is then decarboxylated by the enzyme aromatic-L-amino acid decarboxylase (AADC) to produce DA (11-14). Previous studies have shown that dietary tyrosine regulates the rate of DA synthesis and that animals fed a tyrosine-supplemented diet (TSD) have increased DA synthesis (15, 16).

We reasoned that alveolar epithelial cells may express AADC and convert the circulating L-dopa to DA. Thus, we studied whether a 4% tyrosine-enriched diet would increase endogenous dopamine production and upregulate alveolar epithelial Na,K-ATPase and thus lung liquid clearance.

METHODS

Pathogen-free male Sprague-Dawley rats weighing 275-300 g were purchased from Harlan (Indianapolis, IN). All animals were, unless otherwise specified, provided food and water ad libitum and were maintained on a 12-hour:12-hour light:dark cycle. Amiloride, ouabain, and benserazide were purchased from Sigma (St. Louis, MO). Animals were treated according to National Institutes of Health guidelines and the protocol was approved by the institutional animal care and use committee.

Feedings

Rats were fed after overnight fast with a normal chow diet, TD 85172, or a 4% tyrosine-supplemented diet (Harlan Teklad, Indianapolis, IN), for 6, 15, 24, and 48 hours. The 4% tyrosine diet is a modification of diet TD 85172, composed of NIH-07 open formula rat/mouse (7022), ground (960 g), and L-tyrosine (40 g).

Isolated Perfused Lung Experiments

Isolated lung was prepared as previously described (2, 17, 18). Briefly, rats were anesthetized with pentobarbital (50 mg/kg body weight). A tracheotomy was performed and the rats were mechanically ventilated with a tidal volume of 2.5 ml, a peak airway pressure of 8 to 10 cm H2O, and 100% oxygen for 5 minutes. The chest was opened via a median sternotomy, after which 400 U of heparin sodium was injected into the right ventricle. After exsanguination, the heart and lungs were removed en bloc. The pulmonary artery and left atrium were catheterized, and the pulmonary circulation was flushed of remaining blood by perfusing with buffered salt albumin (BSA) solution containing 135.5 mM Na+, 119.1 mM Cl-, 25 mM HCO^sup -^^sub 3^, 4.1 mM K+, 2.8 mM Mg^sup 2+^, 2.5 mM Ca^sup 2+^, 0.8 mM SO^sub 4^^sup 2-^, 8.3 mM glucose, and 3% bovine albumin, and an osmolality of 300 mOsm/kg H2O. The solution was maintained at pH 7.40 by bubbling a mixture of 5% CO2 and 95% O2 as needed. The lungs were then instilled with the volume necessary to leave 5 ml of BSA solution containing Evans blue dye (EBD, 0.1 mg/ml; Sigma), ^sup 22^Na+ (0.02 µCi/ml; PerkinElmer Life and Analytical Sciences, Boston, MA), and [^sup 3^H]mannitol (0.12 µCi/ml; PerkinElmer Life and Analytical Sciences) in the airspace. Finally, the lungs were immersed in a "pleural bath" reservoir containing 100 ml of BSA solution maintained at 37°C. This allowed us to monitor markers that had moved across the pleural membrane or were drained by the lung lymphatics.

Perfusion of the lungs was performed with 90 ml of the same BSA solution containing a 0.16-mg/ml fluorescein isothiocyanate-tagged albumin (FITC-albumin; Sigma). The perfusate was pumped from a lower reservoir to an upper reservoir by a peristaltic pump, and from there flowed through the pulmonary artery and exited via the left atrium. Left atrial and pulmonary artery pressures were maintained at 0 and 12 cm H2O, respectively, and continuously recorded via a pressure transducer with a zero reference point at the level of the left atrium. Samples were drawn from the three reservoirs: airspace instillate, "pleural bath," and perfusate at 10 and 70 minutes. To ensure homogeneous sampling from the airspaces, 2 ml of instillate was aspirated and reintroduced into the airspace three times before removing each sample. This has been shown to provide a reproducibly mixed sample in our laboratory (17-20). All samples were centrifuged at 3,000 × g for 15 minutes and the colorimetric analysis of the supernatant for EBD (absorbance at 620 nm) was performed in a Hitachi model U2000 spectrophotometer (Hitachi Research Institute, San Jose, CA). Analysis of FITC-albumin (excitation, 487 nm; emission, 520 nm) was performed in a PerkinElmer fluorescence spectrometer (model LS-3B; Perkin-Elmer Analytical Instruments, Oakbrook, IL). ^sup 22^Na+ and [^sup 3^H]mannitol were measured in a [beta] counter (Tri-Carb; Packard BioScicnce/PerkinElmer, Downers Grove, IL).

Experimental Protocols

Ninety Sprague-Dawley male rats were studied: 66 for physiologic studies and 24 for isolated cell studies.

Group A: Rats were fed normal chow after an overnight fast (n = 6).

Group B: Rats were fed 4% tyrosine-supplemented diet (TSD) for 6, 15, 24, and 48 hours after an overnight fast (n = 6 at each time point, for a total of 24).

Groups C and D: Rats were given benserazide (to inhibit the conversion of L-dopa to dopamine) (30 mg/kg, per os) and then maintained on normal chow or 4% TSD for 24 hours (n = 6, for a total of 12).

Group E: Rats were given benserazide (30 mg/kg, per os) and then maintained on normal chow for 24 hours. The rats were then treated with isoproterenol (10^sup -6^ M) via the perfusate (n = 3).

Groups F-I: Rats were fed a 4% tyrosine diet for 24 hours (after an overnight fast) or normal chow and were treated with amiloride (10^sup -6^ M) via the airway instillate or ouabain (5 × 10^sup -4^ M) via the perfusate (n = 3 in each group, for a total of 12).

Groups J and K: Rats fed a 4% tyrosine diet for 24 hours after an overnight fast were treated with SCH 23390 (10^sup -6^ M) (D^sub 1^ receptor antagonist), S-sulpiride (10^sup -6^ M) (D^sub 2^ receptor antagonist), and propranolol (10^sup -4^ M) ([beta]-adrenergic receptor blocker) via the airway instillate (n = 9).

Calculations

Albumin flux from the pulmonary circulation into the alveolar space was determined from the fraction of FITC-albumin that appeared in the alveolar space during the experimental protocol. These calculations were performed for each sampling period.

Alveolar Type II Cell Isolation

Alveolar Type II (ATII) cells were isolated from male Sprague-Dawley rats as previously described (5, 19). Briefly, the lungs were perfused via the pulmonary artery, lavaged, and digested with elastase (30 U/ml; Worthington Biochemical, Lakewood, NJ) for 20 minutes at 37°C. The tissue was minced and filtered through sterile gauze and 70-µm nylon mesh. The crude cell suspension was purified by differential adherence to immunoglobulin G (IgG)-pretreated dishes. Total cell lysate was then obtained for Western blot analysis.

Western Blot Analysis

Na,K-ATPase [alpha]^sub 1^ and [beta]^sub 1^ subunit abundance was determined by Western blot analysis of ATII cells isolated from the lungs of control rats and rats fed tyrosine for 24 hours. Total cell lysates were resolved in a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred onto nitrocellulose membranes (Optitran; Schleicher & Schnell BioScience, Keene, NH). Incubation with a specific anti-Na,K-ATPase [alpha]^sub 1^ subunit monoclonal antibody (Upstate Cell Signaling Solutions, Lake Placid, NY) and with SpET[beta]1 polyclonal antibody (kind gift from P. M. Vasallo, University of La Laguna, La Laguna, Canary Islands, Spain) at dilutions of 1:5,000 and 1:2,500, respectively, were performed overnight at 4°C. Blots were developed with an enhanced chemiluminescence (ECL+; Amersham Biosciences, Piscataway, NJ) detection kit used as recommended by the manufacturer. The bands obtained were quantified by densitometric scan (Eagle Eye II; Stratagene, La Jolla, CA) and compared with the control group. The presence of AADC in ATII cells was determined by Western blot analysis. A specific monoclonal antibody (Alpha Diagnostic International, San Antonio, TX) at a 1:400 dilution was used.

Na,K-ATPase Activity and Protein Abundance at the Basolateral Membranes

Basolateral membranes were isolated from peripheral lung tissue from lyrosine-fed rats, tyrosine-fed rats pretreated with benserazide (30 mg/kg), and control rats as previously described (20). Triplicate samples of basolateral membrane (BLM) protein (20 µg) were resuspended in a high-[Na+]/low-[K+] reaction buffer (50 mM NaCl, 5 mM KCl) with [[gamma]-^sup 32^P]-ATP as described previously (20). Na,K-ATPase activity was calculated as the difference in liberation of ^sup 32^P from ATP between the test samples (total ATPase activity) and samples assayed in reaction buffer with 2.5 mM ouabain and devoid of Na+ and K+ (ouabain-insensitive ATPase activity). Results were expressed as nanomoles of orthophosphate per milligram of protein per hour. Na,K-ATPase [alpha]^sub 1^ subunit abundance was determined by Western blot analysis of BLMs isolated from peripheral lung tissue of control, tyrosine-fed rats and tyrosine-fed rats pretreated with benserazide (30 mg/kg).

Dopamine Production by ATII Cells

ATII cells were isolated from rats fed with normal chow. On Day 3 after isolation and plating the cells were incubated with the dopamine precursor L-dopa (30 minutes, 37°C) at concentrations of 1.0 and 5 mM. After 30 minutes the ATII cells and the media were collected and homogenized and dopamine levels were measured.

Urine Collection

Urine was collected in control and tyrosine-fed rats for 24 hours, followed by a single time direct aspiration of the urinary bladder in the presence of 1.0 ml of 2 N HCl and 40 mg of sodium metabisulfite. The internal standard 3,4-dihydroxybenzylamine hydrobromide (Sigma) was added to the urine sample before processing, and the pH was adjusted to between pH 2 and 3, before storage at 20°C. Samples were subsequently analyzed.

Determination of Catecholamine Levels in Urine

Analysis of catecholamine was performed according to the method of Smedes and coworkers (21) as modified by Macdonald and Lake (22). The pH of the samples was adjusted to pH 7.5, and this mixture was then added to 30-50 mg of Analytichem Bondesil SCX (Varian, Palo Alto, CA), previously activated by exposure to 1 ml of 0.2 M NaH^sub 2^PO^sub 4^, pH 7.5, and mixed vigorously. After removal of the supernatant, the SCX was washed twice with water. Catecholamines were eluted with 1 ml of 1 M NaH^sub 2^PO^sub 4^, pH 2.9. This supernatant was transferred quantitatively into screw-topped glass tubes. Approximately 1.0 ml of NH^sub 4^Cl-NH^sub 4^OH buffer containing 0.2% (wt/vol) diphenylboric acid ethanolamine complex (Aldrich Chemical, Milwaukee, WI) and 0.5% EDTA, 2.0 ml of 2 M (NH^sub 4^)^sub 2^HPO^sub 4^, pH 9.0, and about 150 µl of 5 N NaOH were added to bring the pH to 8.6. After addition of 2.5 ml of n-heptane containing 1% (vol/vol) octanol and 0.35% (wt/vol) tetraoctylammonium bromide (Fluka, Buchs, Switzerland), tubes were capped and shaken. The heptane supernatant was then transferred to a centrifuge tube, and 1 ml of octanol and 0.2 ml of 0.4 N acetic acid containing 10-15 mg of glutathione were added. After mixing and centrifugation, aliquots of the acetic acid phase were injected onto a Chromatographic system for quantitation of catecholamines. For the urine assay results presented here, intraassay coefficients of variation were 2-3% and interassay coefficients of variation were 2-4% for norepinephrine.

Statistical Analysis

Data are presented as means ± SEM. When comparisons were made between two experimental groups an unpaired Student's t-test was used. When multiple comparisons were made one-way analysis of variance was used, followed by a multiple comparison test (Tukey) when the F statistic indicated significance. Results were considered significant when p

RESULTS

Dopamine Levels

To determined whether ATII cells have the ability to produce dopamine we first examined the presence and catalytic activity of AADC. Western blot analysis demonstrated the presence of AADC in ATII cell lysates (Figure 1A), whereas incubation of ATII cells with L-dopa resulted in increased dopamine production at 30 minutes (Figure 1B).

We studied whether feeding 4% enriched tyrosine diet to rats would increase DA levels in their urine as compared with control rats and rats pretreated with benserazide, which inhibits DA synthesis by blocking the enzyme AADC, which converts L-dopa to dopamine. As shown in Figure 1C, urinary dopamine levels increased in TSD-fed rats as compared with control rats, whereas benserazide prevented the increase in urinary dopamine levels. Urinary dopamine levels were used as a surrogate for TSD-mediated systemic increase in dopamine production, as previously reported (23). No significant increases were found in urinary norepinephrine levels: 435.41 ± 58.33 pmol/ml in control rats (n = 4) and 534.58 ± 74 pmol/ml in tyrosine-fed rats (n = 5).

Lung Liquid Clearance

In control rat lungs, the rate of lung liquid clearance was about 10% of the instillate at 1 hour (0.50 ± 0.02 ml/hour). Rats fed a TSD for 6 hours did not have increased lung liquid clearance (0.53 ± 0.02 ml/hour); however, after 15, 24, and 48 hours of TSD lung liquid clearance increased by about 25, 46, and 45%, respectively (Figure 2). As such, further experiments were conducted after feeding rats TSD for 24 hours. To study whether TSD resulted in increased lung liquid clearance by increasing dopamine production we pretreated rats with benserazide (30 mg/kg via nasogastric tube), which prevented the TSD-mediated stimulation of lung liquid clearance but did not inhibit the stimulatory effect of isoproterenol (Figure 3).

To investigate the role of dopaminergic receptor activation in TSD-fed rats, we performed studies using either specific D^sub 1^ or D^sub 2^ receptor antagonists. The D^sub 1^ receptor antagonist, SCH 23390, inhibited whereas the specific D^sub 2^ receptor antagonist, S-sulpiride had no effect on the TSD-mediated increase in lung liquid clearance. To determine whether the increase in lung liquid clearance was mediated by [beta]-adrenergic stimulation we conducted studies with propranolol a [beta]-adrenergic receptor antagonist which did not inhibit the increase in lung liquid clearance (see Figure 4).

As shown in Figure 5, when lungs were instilled with 10^sup -6^ M amiloride, lung liquid clearance decreased by about 30% and by about 40% in lungs of control rats and tyrosine-fed rats, respectively, and when rat lungs were perfused with ouabain (5 × 10^sup -4^ M) lung liquid clearance decreased by about 50% in control rats and by about 65% in TSD-fed rats.

Epithelial Permeability

The movement of FITC-albumin and small solute (^sup 22^Na and [^sup 3^H]mannitol) flux across the alveolar epithelial barrier were similar and did not change under any of the experimental conditions as compared with controls (data not shown). The perfusion flow rate was about 12 ml/minute and Na+, at about 135 mM, did not change in any of the experimental groups.

Na,K-ATPase Activity and Protein Abundance

Membrane-bound Na,K-ATPase activity was quantified by measurement of ouabain-sensitive ATP hydrolysis (Na,K-ATPase activity) at BLMs isolated from the peripheral lung of rats fed with 4% TSD and rats pretreated with benserazide as compared with control rats. Activity was measured in the presence of low [K+], high [Na+], and [ATP] to measure ATPase function per molecule at Vmax. These experiments revealed that Na,K-ATPase activity increased by about 60% in parallel with increases in Na,K-ATPase [alpha]^sub 1^ protein abundance at the BLMs of tyrosine-fed rats as compared with control rats. Benserazide, which blocks the conversion of L-dopa to DA, inhibited the TSD-mediated increase in Na,K-ATPase activity (Figure 6A) and the increase in Na,K-ATPase [alpha]^sub 1^ protein abundance at the BLMs of tyrosine-fed rats as compared with control rats (Figure 6B). As depicted in Figure 7, the abundance of both Na,K-ATPase [alpha]^sub 1^ subunit protein and [beta]^sub 1^ subunit protein increased in ATII cells isolated from rats fed with tyrosine for 24 hours as compared with controls.

DISCUSSION

Patients with hypoxemic respiratory failure whose lungs do not clear edema effectively have worse outcomes (24, 25). The alveolar epithelium reabsorbs fluid from the alveolar space via active sodium transport by generating a gradient of Na+, which enters the alveolar epithelial cell through apical amiloride-sensitive Na+ channels and is actively extruded out of the cell by the Na,K-ATPases located at the cell basolateral membranes (1-8, 26). Exogenous [beta]-agonists, including terbutaline, isoproterenol, salmeterol, epinephrine, and dobutamine, can also stimulate alveolar fluid clearance (7, 20, 27-31). Endogenous epinephrine has been shown to upregulate lung liquid clearance in an experimental model of septic shock in rats (32) and neurogenic pulmonary edema in dogs (33). However, the plasma levels of catecholamines did not correlate with lung liquid clearance in patients with acute lung injury (24). Exogenous dopamine increases Na,K-ATPase activity and lung liquid clearance (short term) via activation of dopamine D, receptors, which are expressed on the basolateral membranes of alveolar Type II cells (9,10). Furthermore, activation of the D2 receptor increases (long term) Na,K-ATPase abundance and enzymatic activity (34). However, there are no previous studies exploring whether increased endogenous levels of dopamine modulate lung edema clearance.

Epithelial cells that express high levels of AADC activity are able to convert circulating L-dopa into DA (35-37). As depicted schematically in Figure 8, tyrosine is converted to L-dopa by tyrosine hydroxylase, which is decarboxylated by the enzyme AADC to produce dopamine at the peripheral tissue level (15, 16, 38-41). We reasoned that if dopamine is also produced by the alveolar epithelial cells, then it may act as a paracrine or autocrine substance thus contributing to the regulation of lung liquid clearance.

We present data that ATII cells express AADC, which converts L-dopa to dopamine in ATII cells and that ATII cells produce dopamine. The significance of our finding is that by increasing the availability of dopamine precursors we may increase lung dopamine levels and modulate lung liquid clearance. We present evidence that increasing the production of dopamine (by feeding rats a 4% enriched tyrosine diet) increased Na, K-ATPase [alpha]^sub 1^ and [beta]^sub 1^ subunit abundance in alveolar Type II cells and increased Na,K-ATPase activity and protein abundance at the basolateral membranes of peripheral lung tissue. We also found that rats fed a 4% tyrosine-supplemented diet for 24 hours had increased urinary dopamine levels and that benserazide, which inhibits the conversion of L-dopa to DA, prevented the increase in dopamine in tyrosine-fed rats (see Figure 1). There was no increase in other catecholamines in the urine of tyrosine-fed rats (neither epinephrine nor epinephrine), which concords with previous reports (15). No significant levels of dopamine or other catecholamines were found in the peripheral blood of either control or tyrosine-fed rats. These data support the notion that TSD increases L-dopa production in chromaffin tissue and that L-dopa was then released into the circulation and taken up by alveolar epithelial cells to produce dopamine (see Figure 8).

A tyrosine-enriched diet increased lung liquid clearance in rat lungs within 15 hours, reaching a plateau by 24 hours (see Figure 2). The rate of lung liquid clearance was similar to rates in previously reported studies, in which exogenous dopamine was perfused through the pulmonary circulation or instilled into rat lung airspaces (9, 10, 42). Prelreatment with benserazide, which blocks endogenous dopamine production, inhibited the tyrosine-enriched diet effect on lung liquid clearance (see Figure 3). We did not observe increases in epinephrine or norepinephrine levels in TSD-fed rats. Benserazide prevented the increase in lung liquid clearance in tyrosine-fed rats but not the effects of the [beta]-adrenergic agonist, isoproterenol. Propranolol, a [beta]-adrenergic receptor antagonist, did not inhibit the TSD-mediated increase in lung liquid clearance. Taken together, these data suggest that the increase in clearance in TSD-fed rats was due to endogenous production of dopamine and was not due to the action of other catecholamines.

Both amiloride and ouabain inhibited in part the stimulatory effects of a tyrosine-enriched diet on lung liquid clearance, suggesting that the observed increases in active Na+ transport were due to upregulation of apical sodium channels and basolateral Na,K-ATPase function (see Figure 5).

The short-term effects of dopamine on lung edema clearance occur within minutes as a result of the recruitment/translocation of Na,K-ATPase molecules from intracellular compartments into the cell plasma membrane via protein phosphatase 2A and a novel protein kinase C-dependent pathway (43-45). We report here a significant increase in total Na,K-ATPase [alpha]^sub 1^ and [alpha]^sub 1^ subunit abundance in alveolar Type II cells isolated from rat lungs after 24 hours of tyrosine-enriched diet and a significant increase in Na,K-ATPase activity and Na,K-ATPase [alpha]^sub 1^ subunit protein abundance at BLMs isolated from peripheral lungs of tyrosine-fed rats as compared with control rats. SCH 23390, a specific D^sub 1^ receptor antagonist, inhibits whereas S-sulpiride, a specific D^sub 2^ receptor antagonist, does not inhibit the increased lung liquid clearance in tyrosine-fed rats. These data suggest that exposure to increased endogenous dopamine levels increases Na,K-ATPase protein abundance via the D^sub 2^ dopaminergic receptor and that activation of the D^sub 1^ dopaminergic receptor causes the recruitment of Na,K-ATPase from intracellular pools into the basolateral membrane and results in increased Na,K-ATPase activity.

In summary, we provide new evidence that feeding tyrosine-enriched diet to rats increases endogenous dopamine production, which causes increased Na,K-ATPase protein abundance in alveolar epithelial cells, leading to increased Na,K-ATPase activity via the activation of D^sub 1^ dopaminergic receptors (see Figure 8). Conceivably, the ability of alveolar epithelial cells to produce dopamine may constitute a physiologic mechanism favoring alveolar fluid reabsorption. We also reason that, by increasing endogenous dopamine production, lung liquid clearance can be accelerated, which may be of relevance for the treatment of patients with pulmonary edema.

Conflict of Interest Statement: Y.A. has no declared conflict of interest; Z.S.A. has no declared conflict of interest; E.L. has no declared conflict of interest; S.L. has no declared conflict of interest; L.P. has no declared conflict of interest; V.D. has no declared conflict of interest; A.M.B. has no declared conflict of interest; P.F. has no declared conflict of interest; J.B.Y. has no declared conflict of interest; K.M.R. has no declared conflict of interest; J.I.S. has no declared conflict of interest.

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Yochai Adir, Zaher S. Azzam, Emilia Lecuona, Sergio Leal, Liuska Pesce, Vidas Dumasius, Alejandro M. Bertorello, Phillip Factor, James B. Young, Karen M. Ridge, and Jacob Iasha Sznajder

Department of Medicine, Northwestern University, Feinberg School of Medicine, Chicago, Illinois; Technion, Israel Institute of Technology, Haifa, Israel; Department of Medicine, Atherosclerosis Research Unit, Karolinska Institutet, Karolinska Hospital, Stockholm, Sweden; and Pulmonary, Allergy, and Critical Care Medicine, Columbia University College of Physicians and Surgeons, New York, New York

(Received in original form July 24, 2002; accepted in final form December 23, 2003)

Correspondence and requests for reprints should be addressed to Jacob I. Sznajder, M.D., Pulmonary and Critical Care Medicine, Northwestern University, 303 East Chicago, Tarry 14-707, Chicago, IL 60611-3010. E-mail: j-sznajder@northwestern.edu

Am J Respir Crit Care Med Vol 169. pp 757-763, 2004

Originally Published in Press as DOI: 10.1164/rccm.200207-744OC on December 30, 2003

Internet address: www.atsjournals.org

Copyright American Thoracic Society Mar 15, 2004
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