INTRODUCTION

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The concept that clearance of lung edema could be accelerated by enhancing the sodium-transporting ability of the pulmonary alveolar epithelium, formulated only in the last decade (1), has potential clinical significance. Upregulation of sodium transport systems associated with enhancement of lung edema clearance has been demonstrated for pathological conditions in which lung fluid balance promotes edema formation, such as oxygen toxicity, sepsis, or hemorrhagic shock, as well as in normal lungs stimulated by catecholamines and other mechanisms (1, 6).

It has been reported that lung edema clearance can be increased by -adrenergic stimulation (1, 10). We have recently reported that dopamine (DA) also increases lung edema clearance in rat lungs, and that this clearance is dependent upon sodium transport (14). In the present study, we demonstrate that DA mediates an increase in active sodium transport not through the previously described -adrenergic pathway, but via a novel mechanism where activation of dopaminergic D₁ type receptors results in increased lung edema clearance.

	METHODS

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Isolated Perfused Lungs

The isolated perfused lung preparation used in our laboratory has been described in detail (7, 8, 14, 15). Briefly, lungs were isolated from anesthetized rats (65 mg/kg pentobarbital sodium [Nembutal] intraperitoneally) after a 10-min ventilation with 100% O₂. The pulmonary artery and left atrial appendage were cannulated and perfused with a solution of 3% bovine serum albumin (BSA) in buffered physiological salt solution. Fluorescein (FITC)-labeled albumin was added to the perfusate to monitor leakage of protein from the vascular space into the airways. The recirculating volume of the constant pressure perfusion system was 90 ml; arterial and venous pressures were set at 12 and 0 cm H₂O, respectively. The lungs were excised from the thoracic cavity and placed in a "pleural" bath (100 ml) filled with the same BSA solution. The entire system was maintained at 37° C in a water bath. The lungs were then instilled via the tracheal catheter with 5 ml BSA containing Evans blue dye (EBD)-labeled albumin, ²²Na⁺, and ³H-mannitol. Samples were taken from the instillate, perfusate, and bath solutions after a 10-min equilibration period and 60 min later. Absorbance at 620 nm (for EBD-labeled albumin), fluorescence (excitation 487 nm, emission 520 nm; for FITC-labeled albumin), and scintillation counting (for ²²Na⁺ and ³H-mannitol) were measured in centrifuged samples from each compartment.

Calculations

The derivation of all equations involved in the mathematical model of edema clearance has been previously described in detail (14, 15). Concentration of EBD-labeled albumin was used to estimate airspace volume. As virtually all EBD-labeled albumin remains in the airspace, instillate volume (V) at a given time can be calculated from the increase in airspace protein concentration. The total unidirectional outflux of Na⁺ from the alveolar space, a result of active transport and passive movement, was calculated from the rate of loss of ²²Na⁺ from the airspaces. Passive sodium flux was calculated by subtracting the active sodium flux, calculated from the rate of net fluid clearance, from the total. Similarly, the unidirectional volume flux of mannitol was calculated from the rate of loss of ³H-mannitol from the airspaces. Albumin flux from the pulmonary circulation into the alveolar space was determined from the fraction of FITC-labeled albumin that appeared in the alveolar instillate during the experimental protocol. For comparison, fluxes are reported as volume fluxes (volume/time) by using the appropriate solute concentrations.

Alveolar Type II (ATII) Cell Isolation and Culture

Lungs from anesthetized rats were perfused free of blood and excised from the thoracic cavity. ATII cells were isolated as previously described (16). Lungs were filled and incubated with an elastase solution (15 U/ml) for 20 min at 37° C. The tissue was minced and sequentially filtered through sterile 150 µm and 15 µm nylon mesh, then purified by differential adherence to IgG-coated plastic dishes. Viability of ATII cell preparations was > 95% as assessed by exclusion of trypan blue. The yield was approximately 3 × 10⁷ cells/animal with a purity of 85 to 90% as determined by staining for lamellar bodies with Papanicolaou or phosphine 3R and tannic acid (16). Isolated cells were suspended in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum with 2 mM L-glutamine, 40 mg/ml gentamicin, 100 U/ml penicillin, and 100 mg/ml streptomycin and plated at a density of 4 × 10⁵/cm² in plastic tissue-culture wells. Nonadherent cells were removed after 24 h incubation in a humidified atmosphere of 5% CO₂/95% air at 37° C. Adherent cells were used by the third day of culture for all further studies.

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)

The reverse transcriptase (RT) reaction was performed using the Superscript preamplification system by GIBCO-BRL (Gaithersburg, MD) following the manufacturer's instruction. One µg of total RNA is converted into complementary DNA (cDNA), after denaturing at 70° C for 15 min, by incubation with a buffer containing oligo-dT primers, the RT enzyme, and deoxynucleoside triphosphates (dNTPs) mix for 50 min at 42° C. The RT enzyme is then inactivated by incubation at 70° C for 15 min and the RNA removed by incubation with ribonuclease H (RNase H) for 20 min at 37° C. The resultant cDNAs are amplified by polymerase chain reaction (PCR) using D1-specific primers, then analyzed by 2% agarose gel electrophoresis (17).

Western Blot Analysis

Dopaminergic receptor characterization was determined by Western blot analysis. Proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 6 to 10% polyacrylamide gradient gel and transferred to nitrocellulose membranes (Hybond C; Amersham, Arlington Heights, IL). After transfer was completed (3 h at 1 A), the membranes were quenched at room temperature for 1 h with 7.5% casein in phosphate-buffered saline (PBS) containing 0.1% Tween 20. Incubation with specific dopaminergic antibodies was performed overnight at 4° C. The D1 receptor antibodies were purchased from Santa Cruz Biochemicals (Santa Cruz, CA). The membranes were rinsed five times with PBS, 1% Tween 20 followed by incubation with a horseradish peroxide-conjugated goat anti-mouse secondary antibody (Bio-Rad, Richmond, CA) for 1 h. Blots were developed as previously described with an enhanced chemiluminescence (ECL) detection kit (Amersham) used as recommended by the manufacturer.

Binding Assays

To determine saturability of binding, a modified method of Felder and colleagues was used (18). All studies were performed on ATII cells grown in 12-well tissue culture plates for 2 d. Cells were incubated with ¹²⁵I-SCH 23982 (0.1 nM to 100 nM) (NEN Life Science Products, Boston, MA) in the presence and absence of a 1,000-fold excess of unlabeled ligand (SCH 23390, 0.1 to 100 µm) to determine nonspecific binding. Cells were rinsed three times with excess incubation buffer at 4° C, solubilized, and placed in scintillation vials with scintillation cocktail for radioactive counting. Specific binding was defined as the difference between radiolabeled ligand bound in the absence and in the presence of unlabeled ligand. The incubation time of 60 min was chosen for equilibrium based on preliminary binding kinetics studies.

Experimental Protocols

A total of 78 experiments were conducted in the isolated perfused rat lung model. Drugs were added to the alveolar instillate and the compounds were present at the final concentration from the initial instillation.

Miscellaneous

DA (Sigma Chemical Co., St. Louis, MO) was freshly prepared before each experiment as a 1,000× stock in normal saline. SCH 23390 (RBI, Natick, MA) and fenoldopam (a gift from Neurex Pharmaceutical, Menlo Park, CA) were prepared as 10 mM stocks in water, quinpirole (RBI) was prepared as a 10 mM stock in 0.1 N HCl, and S-sulpiride (RBI) was prepared as a 10 mM stock in ethanol.

Statistical Analysis

All data are presented as mean ± SEM. Analysis of variance (ANOVA) was used to test for differences between groups, followed by a multiple comparison test (Tukey) when the F statistic indicated significance.

	RESULTS

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Instilled DA (Figure 1, 10⁴ M; Figure 2, 10⁶ M) increased edema clearance out of the alveoli of isolated perfused lungs. Because DA at high concentrations could potentially activate -adrenergic receptors, we carried out studies using the -adrenergic antagonist propranolol. Instillation of 10⁴ M propranolol alone had no effect on basal lung edema clearance, and coinstillation of propranolol with 10⁴ M DA produced an increase in clearance comparable to the increase seen with DA alone (Figure 1).

View larger version (14K): [in this window] [in a new window]   Figure 1.   The stimulation of lung liquid clearance by DA is not mediated by -adrenergic receptor stimulation. 104 M DA increased clearance from the isolated perfused rat lung (n = 6). Instillation of 104 M propranolol (PRO), a -adrenergic receptor antagonist, did not affect either baseline (n = 4) or DA-stimulated clearance (n = 5). *p < 0.05.

View larger version (15K): [in this window] [in a new window]   Figure 2.   A dopaminergic D1 receptor antagonist (SCH 23390) inhibits DA-stimulated clearance. When instilled intratracheally, 106 M DA increased clearance from the isolated perfused rat lung. Coinstillation with 106 SCH 23390 (n = 6) or 108 M SCH 23390 (n = 5) inhibited this effect. The inhibitory effect was not seen with 1010 M SCH 23390 (n = 5). Lungs instilled with SCH 23390 alone were not different from controls. *p < 0.05 by ANOVA compared with control. +p < 0.05 compared with 106 M DA alone.

Dopamine or propranolol did not affect passive sodium or mannitol flux, or increase epithelial permeability to albumin (data not shown). Perfusate flow did not change in any of the experimental protocols (data not shown).

To investigate the role of dopaminergic receptor activation, we carried out studies using either specific D₁ or D₂ receptor antagonists coinstilled with DA, or specific D₁ or D₂ receptor agonists. Coinstillation of a dopaminergic D₁ receptor antagonist, SCH 23390 (19) (10⁶ or 10⁸ M), with 10⁶ M DA inhibited the increased clearance observed with DA alone, whereas SCH 23390 (10⁶ M) instilled alone had no effect on baseline clearance (Figure 2). Loss of the inhibitory effect of SCH 23390 was observed at 10¹⁰ M. In contrast, coinstillation of a dopaminergic D₂ receptor antagonist, S-sulpiride (10⁶ M), with 10⁶ M DA did not inhibit the increased clearance observed with DA alone, and S-sulpiride instilled alone had no effect on baseline clearance (Figure 3). None of the instilled agonists or antagonists had an effect on passive mannitol or sodium flux, epithelial permeability for albumin, or flows (Table 1).

View larger version (13K): [in this window] [in a new window]   Figure 3.   A dopaminergic D2 receptor antagonist (S-sulpiride) does not inhibit DA-stimulated clearance. When instilled intratracheally, 106 M DA increased clearance from the isolated perfused rat lung. Coinstillation with 106 M S-sulpiride (S-SUL) did not inhibit DA-stimulated clearance (S-sulpiride + DA was not significantly different from DA alone by ANOVA, n = 10). Lungs instilled with 106 M S-sulpiride alone showed a clearance similar to that of controls (n = 4). *p < 0.05.

When the specific dopaminergic D₁ agonist fenoldopam (10⁶ M) was instilled there was a significant increase in lung edema clearance, comparable to that seen with DA. In contrast, and in agreement with the antagonist studies, when the dopaminergic D₂ agonist quinpirole (10⁶ M) (20) was instilled into the lungs, there was no increase in clearance (Figure 4). Again, there was no effect on passive mannitol or sodium flux, epithelial permeability for albumin, or flows (data not shown).

View larger version (12K): [in this window] [in a new window]   Figure 4.   A dopaminergic D1 receptor agonist but not a D2 receptor agonist stimulated lung liquid clearance. The effect of DA (106 M), is shown for comparison. Clearance increased significantly in lungs instilled with 106 M fenoldopam (FEN) (n = 4), a specific dopaminergic D1 agonist, but did not change significantly with 106 M quinpirole (QP) (n = 6), a specific dopaminergic D2 agonist. *p < 0.05.

RT-PCR demonstrated the presence of messenger RNA for the D₁ receptor in ATII cells. An amplification product of the predicted size (247 bp) for the D₁ receptor was detected in the RT-PCR reactions using extracted ATII cell RNA (Figure 5). Expression of the D₁ receptor protein in ATII cell basolateral membranes and whole cell homogenates was analyzed by Western blot. As shown in Figure 6, rat lung ATII cell basolateral membranes reacted with antibodies specific for D₁ DA receptors.

View larger version (81K): [in this window] [in a new window]   Figure 5.   Size analysis of the PCR products in 2% agarose gel stained with ethidium bromide. An amplification product of the predicted 247 bp for the D1 receptor was evident in the RT-PCR reactions using extracted ATII cell RNA. Mardin Darby canine kidney (MDCK) cell RNA was used as a positive control for the D1 receptor.

View larger version (35K): [in this window] [in a new window]   Figure 6.   Representative Western blot of ATII cell basolateral membranes and whole cell homogenates probed for the D1 dopaminergic receptors demonstrating that D1 receptors are expressed. ATII cell homogenates (ATII-H, 70 µg), ATII cell basolateral membranes (ATII BLM, 50 µg), striatum homogenates (Striatum-H, 125 µg).

D₁ receptors were then quantitated by the specific binding of ¹²⁵I-SCH 23982, a D₁ antagonist, in ATII cells. Binding of ¹²⁵I-SCH 23982 was time- and concentration-dependent, saturable and reversible (Figure 7). The nonlinear regression analysis of the data suggested binding to a single site with an apparent dissociation constant (K_d) of 4.4 nM and binding maximum (Bmax) of 9.8 pmol/mg protein. These data are comparable with results reported in the kidney and heart.

View larger version (16K): [in this window] [in a new window]   Figure 7.   Dose-dependent 125I-SCH 23982 binding in cultured ATII cells. ATII cells were incubated with various concentrations of 125I-SCH 23982 in the presence (nonspecific) or absence (total) of a 1,000-fold excess of SCH 23390. Binding of 125I-SCH 23982 was time- and concentration dependent, saturable, and reversible. Using nonlinear regression analysis, dissociation constant (Kd) of 4.4 nM and a binding capacity of 9.8 pmol/mg protein were calculated.

	DISCUSSION

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Decreased lung edema formation and/or increased clearance have been associated with improved outcome in patients with hypoxemic respiratory failure (21). Critically ill patients are often treated with DA to increase natriuresis and diuresis (22). These effects reflect, in part, inhibition of renal tubular Na,K-ATPase that leads to decreased sodium reabsorption (23, 24). We have previously reported that the effect of DA in the alveolar epithelium is opposite to that in renal tubular epithelium, or most other previously studied epithelia, as DA increased active sodium transport and improved the ability of the lung to clear edema (14).

DA can activate both -adrenergic and dopaminergic receptors in a concentration-dependent manner (22). Thus, we carried out three sets of studies in the isolated perfused lung to define the population of receptors involved in DA-stimulated lung edema clearance: the first using DA with propranolol to determine whether -adrenergic receptors had a role in DA-stimulated lung edema clearance (Figure 1), a second using DA and a specific D₁ or D₂ antagonist (SCH 23390 or S-sulpiride, respectively, Figures 2 and 3), and a third using a specific D₁ or D₂ agonist (fenoldopam or quinpirole, respectively, Figure 4). Although previous studies have demonstrated that -adrenergic agonists upregulate Na,K-ATPase and increase lung edema clearance in several models (1, 2, 10- 13, 25), our data indicate that DA is not acting via activation of -adrenergic receptors, suggesting a novel mechanism of sodium transport and edema clearance regulation.

The dopaminergic receptor antagonist and agonist studies strongly suggest that D₁ receptor activation mediates the effects observed with dopaminergic stimulation of lung edema clearance. When coinstilled with DA, SCH 23390 (a D₁ antagonist) inhibited DA-stimulated clearance to levels not different from control rat lungs, and fenoldopam (a D₁ agonist) significantly increased clearance to an extent similar to that seen with DA. The D₁ antagonist, SCH 23390, had no effect on the baseline reabsorption of fluid. In contrast, the results do not support a role for D₂ receptor short-term regulation of sodium transport in the pulmonary alveolar epithelium, as S-sulpiride (a D₂ antagonist) did not inhibit the DA-stimulated clearance, and quinpirole (a D₂ agonist) did not stimulate it.

RT-PCR demonstrated the presence of D₁ receptor messenger RNA (mRNA) in ATII cells (Figure 5). Western blot analysis confirmed that the D₁ receptor protein is present in ATII cell basolateral membranes (Figure 6). D₁ receptors were quantitated by the specific binding of ¹²⁵I-SCH 23982, a D₁ antagonist, in ATII cells (Figure 7). Taken together, these results suggest that in ATII cells DA mediates its short-term effects via the D₁ receptor.

Our observations demonstrate for the first time that the dopaminergic D₁ receptor is mainly expressed in the basolateral membrane of ATII cells. The data also suggest that activation of this receptor contributes to edema removal from the alveolar space. Our data further indicate that sodium transport in the lungs may be upregulated by administration of pharmacological agents that stimulate the D₁ receptor. Dopaminergic stimulation represents a novel and rapid mechanism to stimulate edema removal from the lungs and should be evaluated as a strategy in the treatment of patients with hypoxemic respiratory failure caused by pulmonary edema.