INTRODUCTION

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PULMONARY EDEMA DEVELOPS as a result of either changes in the hydrostatic and/or oncotic pressure gradients across the pulmonary circulation or increased alveolocapillary permeability. As such, increased pulmonary capillary wedge pressures can result in pulmonary edema formation (13, 32). Several therapeutic options are available in clinical practice to reduce edema formation; however, approaches to increase alveolar fluid reabsorption (once edema has been formed) are less understood. Previous reports have suggested that vectorial Na⁺ transport and thus alveolar fluid reabsorption across the alveolocapillary barrier are important in keeping the air spaces free of edema (16, 23, 29). Alveolar fluid reabsorption is regulated in epithelial cells by the rate of sodium entry, via the apical Na⁺ channels, coupled to the rate of Na⁺ extrusion, via the basolateral Na⁺-K⁺-ATPase.

Dopamine (DA) induces natriuresis in the kidney by inhibiting the Na⁺-K⁺-ATPase. However, in the lungs, DA and isoproterenol (ISO) have been shown to increase active Na⁺ transport and thus alveolar fluid reabsorption in normal rats as well as in rats exposed to hyperoxia by regulating Na⁺-K⁺-ATPase function in the alveolar epithelium (3, 4, 27).

Recently, it has been shown that alveolar fluid reabsorption is impaired in the presence of elevated left atrial pressures (Pla) (Refs. 7 and 22, and Saldias FJ, Azzam ZS, Ridge KM, Yeldandi A, Rutschman DH, Schraufnagel D, Sznajder JI, unpublished observations). The purpose of this study was to determine whether DA and ISO would increase alveolar fluid reabsorption in a model of increased Pla and investigate mechanisms contributing to these effects.

	METHODS AND MATERIALS

TOP ABSTRACT INTRODUCTION METHODS AND MATERIALS RESULTS DISCUSSION REFERENCES

Isolated-perfused lung. The lungs and heart of rats anesthetized with 50 mg/kg body wt of intraperitoneal pentobarbital were removed en bloc after a 10-min ventilation with 100% O₂ and anticoagulation with heparin as previously described (23, 28). The pulmonary artery and left atrium were catheterized and perfused continuously with a solution of 3% bovine serum albumin (BSA) in buffered physiological salt solution (135.5 mM Na⁺, 119.1 mM Cl, 25 mM HCO₃, 4.1 mM K⁺, 2.8 mM Mg⁺, 2.5 mM Ca²⁺, 0.8 mM SO₄², 8.3 mM glucose). Trace amounts of fluorescein-labeled (FITC) albumin were also added to the perfusate. The recirculating volume of the constant-pressure perfusion system was 90 ml; arterial and venous pressures were set at 15 and 0 cmH₂O, respectively, in the group of rats with normal Pla and 20 and 15 cmH₂O, respectively, in the increased Pla study groups. The vascular pressures were recorded every 10 s with a multichannel recorder (Cyber Sense, Nicholasville, KY). The lungs were immersed in a "pleural" bath (100 ml) filled with the same BSA solution. The entire system was maintained at 37°C in a water bath. Perfusate pH was maintained at 7.40 by bubbling with a gas mixture of 95% O₂-5% CO₂. The lungs were then instilled via tracheal cannula in two sequential phases with a total of 5-ml volume of the BSA solution containing 0.1 mg/ml Evans blue dye-labeled (EBD; Sigma Chemical, St. Louis, MO) albumin, 0.02 µCi/ml of ²²Na⁺ (Du Pont-NEN, Boston, MA) and 0.12 µCi/ml of [³H]mannitol (Du Pont-NEN). Samples were taken from the instillate, perfusate, and bath solutions after an equilibration time of 10 min from the instillation and 60 min later. To ensure a homogeneous sampling of the instillate, a volume of 2 ml was aspirated and reintroduced into the air spaces three times before each sample was removed. All samples were centrifuged at 3,000 g for 10 min. Absorbance analysis of the supernatant or EBD albumin was performed at 620 nm in a Hitachi model U2000 spectrometer (Hitachi, San Jose, CA). Analysis of FITC-albumin (excitation 487 nm and emission 520 nm) was performed in a Perkin-Elmer fluorometer (model LS-3B, Perkin-Elmer, Oakbrook, IL). Scintillation counts (for ²²Na⁺ and [³H]mannitol) were measured in a Beckman beta counter (model LS 6500, Beckman Instruments, Fullerton, CA).

Calculations. The mathematical calculation of lung liquid clearance, the movement of sodium, and the flux of mannitol were described elsewhere (23). Briefly, the amount of instilled EBD-albumin remains constant during the experimental protocol; thus any change in its concentration at a given time (t) will reflect the change in the air space volume (V_t)

(1)

(2)

(3)

where V₀ is the initial known volume instilled instilled into rat air spaces containing a known concentration of Evans blue dye-albumin [EBD]₀; V_t and [EBD]_t are the alveolar fluid volume and EBD concentration in the instillate at time t, respectively; and J is the volume flux during a time period (t).

(4)

(5)

Study groups. One hundred nineteen specific pathogen-free male Sprague-Dawley rats (275-325 g) were acquired from Harlan Sprague Dawley (Indianapolis, IN). DA, ISO, SCH-23390, colchicine, and -lumicolchicine were purchased from Sigma Chemical. Ouabain and propranolol were purchased from RBI (Natick, MA), and fenoldopam was generously provided by Neurex (Menlo Park, CA).

Control group of rats studied at Pla 0 cmH₂O (n = 7) and Pla 15 cmH₂O (n = 7). Rat lungs were instilled with 10⁴ M DA into the air space at Pla 0 cmH₂O (n = 8) and Pla 15 cmH₂O (n = 7). Rat lungs were perfused with 10⁶ M ISO through the pulmonary circulation at Pla 0 cmH₂O (n = 6) and Pla 15 cmH₂O (n = 6).

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Statistical analysis. Data are presented as means ± SE; n is the number of animals in each study group. One-way analysis of variance was used when multiple comparisons were made followed by a multiple comparison test (Tukey's) when the F statistic indicated significance. Results were considered significant when P < 0.05.

	RESULTS

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Alveolar fluid reabsorption. As depicted in Fig. 1, alveolar fluid reabsorption in rat lungs exposed to Pla of 15 cmH₂O was decreased by ~45% compared with control rats with Pla of 0 cmH₂O (from 0.51 ± 0.02 to 0.27 ± 0.02 ml/h, P < 0.001). Treatment with DA or ISO restored alveolar fluid reabsorption to control levels in rat lungs exposed to elevated Pla (0.44 ± 0.03 and 0.52 ± 0.02 ml/h, respectively).

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Dopamine (DA) and isoproterenol (ISO) increased alveolar fluid reabsorption in rat lungs exposed to left atrial pressure (Pla) of 0 (left) and 15 cmH2O (right). Bars represent means ± SE. *P < 0.001 vs. control (CT) group with Pla 0 cmH2O; +P < 0.001 vs. control group with Pla 0 cmH2O.

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View larger version (20K): [in this window] [in a new window]   Fig. 2.   A: fenoldopam (Fen) increased alveolar fluid reabsorption in rat lungs exposed to Pla of 15 cmH2O compared with CT. SCH-23390 prevented the DA-mediated increase in lung liquid clearance in rats exposed to Pla 15 cmH2O. *P < 0.01 vs. other study groups. B: propranolol (PRO) inhibited the stimulatory effects of ISO; however, it did not affect lung liquid clearance in the CT group or when instilled together with DA. Bars represent means ± SE. *P < 0.001 vs. the other study groups.

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View larger version (11K): [in this window] [in a new window]   Fig. 3.   Alveolar fluid reabsorption was inhibited in rat lungs perfused with 5 × 104 M ouabain (OUAB) and/or instilled with 104 M amiloride (AMI). DA did not stimulate clearance in the presence of these antagonists (AMI vs. AMI + DA, OUAB vs. OUAB + DA, OUAB + AMI vs. OUAB + AMI + DA), P > 0.05. Bars represent means ± SE. *P < 0.05 vs. DA-treated rats and both CT groups; +P < 0.001 vs. CT group and DA groups exposed to Pla 15 cmH2O.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Effect of DA on lung liquid clearance was inhibited by colchicine (COL) in rats exposed to Pla 15 cmH2O. However, -lumicolchicine (LUM) did not block the stimulatory effects of DA. The difference between CT and COL groups in rat lungs with Pla of 15 cmH2O was not statistically significant (P > 0.05). Bars represent means ± SE. *P < 0.01 vs. CT group of Pla 0 cmH2O.

Epithelial permeability. As shown in Table 1, alveolar epithelial permeability to the small solutes, as measured by ²²Na⁺ and [³H⁺]mannitol flux, was significantly increased in the rat lungs exposed to Pla of 15 cmH₂O compared with control rats. The movement of FITC-albumin from the pulmonary circulation into the air spaces was slightly increased in all rat lungs exposed to Pla 15 cmH₂O compared with control rats (Pla 0 cmH₂O).

	DISCUSSION

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During congestive heart failure with increased Pla, patients can develop cardiogenic pulmonary edema. Recently, it has been shown that, in animal models of increased pulmonary vascular pressures, there is not only increased edema formation but also impairment of alveolar fluid reabsorption (7, 22, Saldias et al., unpublished observations). Additionally, in some patients with hydrostatic pulmonary edema, the alveolar fluid reabsorption was preserved, whereas in other patients, the ability to clear edema was impaired (35). We reason that, in patients suffering from chronic heart failure, the neurohumoral axis is activated, which results in increased endogenous catecholamines (8, 10, 30). Thus it is possible that, in patients with increased levels of endogenous catecholamines, alveolar fluid reabsorption is preserved (20, 21). We report here that, in an isolated rat lung model with acute increase in Pla, there was decreased alveolar fluid reabsorption. Our data are concordant with a previous report showing that the alveolar fluid clearance was normal in sheep exposed to moderate left atrial hypertension. In contrast, in adrenalectomized sheep where the secretion of endogenous catecholamines was abolished, the clearance was significantly reduced (7).

DA has been used clinically to induce natriuresis in patients with pulmonary edema by inhibiting the Na⁺-K⁺-ATPase activity in the renal tubular epithelium (17). In contrast, DA enhanced active Na⁺ transport and increased alveolar fluid reabsorption in normal rat lungs and in a model of lung injury induced by exposure of rats to 100% oxygen for 64 h (3, 27). In accord with these data, we are reporting that, in a model of increased Pla, DA, via the D₁ receptors, and ISO, via activation of -adrenergic receptors, increased alveolar fluid reabsorption. These effects were blocked by the D₁-receptor antagonist SCH-23390 and increased by the D₁ agonist fenoldopam (Fig. 2A). The -adrenergic-receptor antagonist propranolol did not inhibit the stimulatory effects of DA (Fig. 2B). These results suggest that the DA effects in this model are mediated by the dopaminergic (D₁) and not the -adrenergic pathway.

The effects of DA and ISO were inhibited by ouabain and amiloride, confirming that -adrenergic and dopaminergic agonists increase lung edema clearance by stimulating the alveolar epithelial Na⁺-K⁺-ATPase function and amiloride-sensitive Na⁺ pathways in rat lungs with increased Pla (Fig. 3), as has been previously reported in control rats (3, 4, 28). Upregulation of Na⁺-K⁺-ATPase function could be due to increased transcription, translation, protein assembly, recruitment, and translocation to the plasma membrane from intracellular pools and metabolic activation (5, 6).

Recent studies have suggested that the cell microtubular transport system and cytoskeleton proteins are involved in Na⁺ pump recruitment from intracellular pools to the plasma membrane (6). Therefore, we tested in physiological experiments whether the stimulatory effects of DA and ISO in rat lungs exposed to increased Pla occur by stimulation of preexisting membrane-bound Na⁺ pumps or by recruitment of Na⁺-K⁺- ATPase proteins from intracellular pools to the cell plasma membrane. We reasoned that cell microtubular transport disruption by colchicine could provide information about whether the stimulatory effects of DA and ISO on active Na⁺ transport and lung edema clearance in rat lungs with increased Pla could be due to Na⁺ pump recycling. We indeed observed that colchicine inhibited DA stimulation of edema clearance in rat lungs with increased Pla (Fig. 4). Meanwhile, the isomer -lumicolchicine, which shares many colchicine properties with the exception of inhibiting cell microtubular transport (27, 28), did not inhibit the DA modulation of lung edema clearance. These results suggest that, in a model of increased Pla, DA and ISO upregulation of lung edema clearance is mediated by recruitment of Na⁺ pumps from intracellular pools to the plasma membrane of alveolar epithelial cells.

The data presented in this report support the notion that increased pulmonary capillary hydrostatic pressures represent a model of lung injury that impairs alveolar fluid reabsorption. Our data agree with reports in sheep (7, 11) and rats (Saldias et al., unpublished observations) in which the rate of alveolar fluid reabsorption was impaired when hydrostatic pulmonary circulation pressures were increased.

The epithelial permeability to small solutes and to albumin was slightly increased compared with rat groups with normal Pla. These observations are similar to studies in which increased permeability of tracers across the alveolocapillary barrier was reported when Pla increased (19, 34, 36). We reason that this is possibly due to capillary stress failure or stretch pore phenomena that may transiently affect the permeability of the alveolocapillary barrier (2, 36).

This report demonstrates the beneficial effects of DA and ISO by improving alveolar fluid reabsorption in a model of increased hydrostatic pulmonary circulation pressures. Left heart failure with resultant decrease in effective arterial volume is characterized mainly by decreased cardiac output and left atrial enlargement (1, 14). Consequently, this may lead to increased levels of atrial natriuretic peptides, angiotensin II, endothelin, and possibly endogenous ouabain-like substances (12, 15, 24, 30). It has been reported that atrial natriuretic peptides instilled in the rat air spaces decreased the active Na⁺ transport and alveolar fluid reabsorption (18, 33). Also, endogenous ouabain-like substances may decrease alveolar fluid reabsorption by inhibiting alveolar Na⁺-K⁺-ATPase. We reason that DA and ISO can counteract these decreases in active Na⁺ transport and increase alveolar fluid reabsorption.

In summary, we report that DA and ISO increase alveolar fluid reabsorption in a model of increased Pla. The effects of DA are mediated by the activation of dopaminergic D₁ receptors, whereas the effects of ISO are mediated by the activation of -adrenergic receptors that cause an upregulation of the amiloride-sensitive Na⁺ pathways and Na⁺-K⁺-ATPase. This new information is of potential clinical relevance in the treatment of patients with cardiogenic pulmonary edema.