INTRODUCTION

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The study of pulmonary edema and its pathologic effects has for some time centered on determining mechanisms affecting the formation of alveolar edema, and consequently devising potential ways to prevent accumulation of fluid within the alveolar space. A more likely clinical scenario, however, involves treatment of patients who have already developed pulmonary edema. It has been demonstrated recently that the alveolar epithelium is capable of actively reabsorbing fluid from the alveolar space, and that this occurs following actively generated sodium gradients by means involving entry of sodium via apical sodium channels and its extrusion through active basolateral transport (1). The latter is principally driven by the activity of the sodium pump, Na,K-ATPase, which transports three Na⁺ ions out of cells and two K⁺ ions into cells at the expense of energy derived from ATP (5). Importantly, this ability of the lungs to clear fluid appears to be regulated by a number of mechanisms, whose elucidation might lead to improved clinical management of respiratory failure due to pulmonary edema.

A large body of information has accumulated regarding regulation of sodium transport in the kidney (6), but much less is known about other transporting epithelia, notably the alveolar epithelium of the lung. A recent focus of our laboratory and others has been the short-term regulation of the sodium pump in the alveolar epithelium (10) and specifically, the ability of dopamine to regulate lung liquid clearance.

Catecholamines, both exogenous (in particular -adrenergic agonists such as terbutaline or isoproterenol) and endogenous, have previously been shown to increase sodium channel and Na,K-ATPase activity in pulmonary alveolar epithelium and to stimulate lung edema clearance (14). We therefore hypothesized that dopamine could accelerate sodium transport and lung edema clearance in the isolated perfused rat lung model, and report herein the results of this investigation.

	METHODS

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Isolated Perfused Lungs

The isolated perfused lung preparation used in our laboratory has been described in detail (3, 4, 11). Briefly, lungs are isolated from anesthetized rats following a 10-minute ventilation with 100% O₂. The diffusive absorption of oxygen facilitates the filling of alveoli with instillate fluid. The pulmonary artery and left atrial appendage are cannulated and perfused with a solution of 3% bovine serum albumin (BSA) in buffered physiological salt solution. Fluorescein-labeled (FITC-) albumin is 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 is 90 ml; arterial and venous pressures are set at 12 and 0 cm H₂O, respectively. The lungs are excised from the thoracic cavity and placed in a "pleural" bath (100 ml) filled with the same BSA solution. The entire system is maintained at 37° C in a water bath. The lungs are then instilled via the tracheal catheter with 5 ml BSA containing Evans blue dye-labeled (EBD-) albumin, ²²Na⁺, and ³H-mannitol. Samples are taken from the instillate, perfusate, and bath solutions following a ten-minute equilibration period and 60 minutes later. Absorbance at 620 nm (for EBD-albumin), fluorescence (excitation 487 nm; emission 520 nm; for FITC-albumin), and scintillation counting (for ²²Na⁺ and ³H-mannitol) are measured in centrifuged samples from each compartment. The EBD-albumin remains in the airspace and is used to determine the fluid volume remaining in the isolated lung (3). The decrease in ²²Na⁺ and ³H-mannitol in the instillate is used to calculate the total and passive small solute and fluid movement.

Experimental Protocols

In isolated lungs where drugs were added to the alveolar instillate, the compounds were present throughout the course of the experiment. When substances were perfused through the pulmonary vasculature, the compounds were added to the perfusate at the start of the experiment and were present throughout the instillation procedure, including the 10-min stabilization period. Dopamine (Sigma Chemical Co., St. Louis, MO) was freshly prepared before each experiment as a 1,000× stock in normal saline. Amiloride (Sigma) was prepared as a 25 mM stock and added to the instillate fluid to obtain the final concentration used. Ouabain (ICN Biochemicals, Aurora, OH) was dissolved in 200 µl DMSO before addition to the perfusate.

A total of 78 isolated perfused rat lungs were studied. The experimental groups were as follows, with the number of animals in each group in parentheses: controls (n = 12); dopamine administered via the airway instillate at concentrations of 10¹⁰ M (n = 6), 10⁹ M (n = 4), 10⁸ M (n = 6), 10⁶ M (n = 6), and 10⁴ M (n = 8); dopamine administered via the perfusate at concentrations of 10⁸ M (n = 5), 10⁶ M (n = 6), and 10⁴ M (n = 4); treatment with amiloride via the airway instillate to inhibit epithelial apical sodium channels, using concentrations of 10⁶ M amiloride (n = 4), 10⁶ M amiloride + 10⁴ M instilled dopamine (n = 5), 10⁴ M amiloride (n = 4), and 10⁴ M amiloride + 10⁴ M instilled dopamine (n = 5); and treatment with ouabain via the perfusate to inhibit basolateral Na,K-ATPase, with concentrations of 5 × 10⁴ M ouabain (n = 4) and 5 × 10⁴ M ouabain + 10⁴ M instilled dopamine (n = 3).

Calculations

The derivation of all equations involved in the mathematical model of edema clearance has been previously described in detail (3). Concentration of EBD-albumin was used to estimate airspace volume. As virtually all EDB-albumin remains in the airspace, we may calculate instillate volume (V) at a given time t as:

where V₀ is the initial volume instilled and [EBD]₀ and [EBD]_t are the concentrations of EBD-albumin at times 0 and t, respectively. The removal of sodium from the alveolar space during a defined period of time is probably accompanied by isotonic water flux and is given by:

where J_Na,net is the net or active Na⁺ transport, J_Na,out is the total or unidirectional Na⁺ outflux and J_Na,in is the back flux of Na⁺ into the alveolar fluid by passive bidirectional movement. Since Na⁺ concentration [Na⁺] remains constant in all compartments, the net Na⁺ flux (which we refer to as active Na⁺ transport) from the airspace is:

The unidirectional outflux of Na⁺ (J_Na,out) from the alveolar space, a result of active transport and passive movement, was calculated as:

Similarly, the (unidirectional) volume flux of ³H-mannitol (typically expressed as PA, permeability of surface area) was calculated as:

Albumin flux from the pulmonary circulation into the alveolar space was determined from the fraction of FITC-albumin that appears in the alveolar space during the experimental protocol. For reasons of comparison fluxes are reported as volume fluxes (volume/time) by using the appropriate solute concentrations.

Measurement of cAMP

Levels of cAMP were measured in lung homogenates prepared following physiologic determinations in control lungs and lungs instilled with 10⁶ M dopamine. Crude homogenates were passed over anion exchange columns (Amprep SAX; Amersham, Arlington Heights, IL) and the eluted cAMP preparations were lyophilized and redissolved in assay buffer. Amounts of cAMP were determined by a commercial ELISA (Amersham) and were expressed as pg/lungs.

Statistical Analysis

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

	RESULTS

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In isolated perfused lungs, instilled dopamine increased lung liquid clearance (Figure 1) out of the alveoli. The increased clearance was dose-dependent, with 10⁸ M and greater concentrations of dopamine increasing clearance to 27 to 33% above controls (p < 0.05). Dopamine at any of the concentrations utilized did not affect passive sodium or manitol flux (Table 1, p = 0.75 for Na⁺, p = 0.93 for mannitol) or increase epithelial permeability, as monitored by FITC-albumin flux (Table 2). Perfusate flow did not change with the administration of dopamine (Table 2). In most experiments, dopamine was placed in the instillate to maximize the concentration at the epithelial cell level. When added to the perfusate, dopamine increased clearance although the effect was not consistent at 10⁶ M (Figure 2).

View larger version (13K): [in this window] [in a new window]   Figure 1.   Dose-response of increased lung liquid clearance in response to increasing concentrations of dopamine in the isolated perfused lung. Dopamine at the indicated concentrations was added to the instillate. *p < 0.05 as compared with controls.

View larger version (11K): [in this window] [in a new window]   Figure 2.   Dose-response of increased lung liquid clearance seen when dopamine was perfused through the pulmonary circulation at the indicated concentrations. *p < 0.05 as compared with controls.

To determine the cellular pathway for sodium transport affected by dopamine, we used both amiloride to block sodium channels and ouabain to specifically block Na,K-ATPase in the absence and presence of dopamine.

Two different doses of amiloride were used in our studies. As shown in Figure 3, amiloride inhibited active sodium transport and lung liquid clearance. When lungs were instilled with 10⁶ M amiloride, clearance decreased to 66% of control values, while 10⁴ M amiloride decreased clearance to 51% of controls. When amiloride was coinstilled with 10⁴ M dopamine, 10⁴ M amiloride completely inhibited the dopamine-stimulated clearance (89% of 10⁴ M amiloride alone), while dopamine was still able to elicit an increase in clearance when coinstilled with 10⁶ M amiloride (154% of 10⁶ M amiloride alone). Passive sodium and mannitol flux were not affected by either dose of amiloride in the presence or absence of dopamine (Table 1, p = 0.47 for Na⁺, p = 0.78 for mannitol); permeability to albumin was also not changed (Table 2). Perfusate flow was not significantly different in any group (Table 2).

View larger version (10K): [in this window] [in a new window]   Figure 3.   Amiloride instilled into the alveolar space inhibited alveolar liquid clearance. The effect of amiloride on dopamine-stimulated clearance was dependent upon the dose of amiloride used. Dopamine concentration was 104 M. Amil: Amiloride at the indicated concentrations. *p < 0.05 as compared with control. +p < 0.05 as compared with low amiloride alone or with high amiloride plus dopamine. There was no significant difference from dopamine alone.

We then determined whether ouabain, the specific inhibitor of Na,K-ATPase, inhibited sodium transport and lung liquid clearance (Figure 4). Ouabain (5 × 10⁴ M) decreased clearance to 45% of controls, and also eliminated the stimulation of clearance by dopamine (ouabain with 10⁴ M dopamine was 86% of ouabain alone). Passive sodium and mannitol fluxes (Table 1, p = 0.46 for Na⁺, p = 0.67 for mannitol) and albumin flux (Table 2) were not significantly different. Perfusate flow was not different (Table 2).

View larger version (11K): [in this window] [in a new window]   Figure 4.   Ouabain (5 × 104 M) perfused through the pulmonary circulation inhibited lung liquid clearance in both control lungs and in lungs instilled with 104 M dopamine. *p < 0.05 as compared with controls. +p < 0.05 compared with dopamine alone. Dopamine did not increase clearance in ouabain-perfused lungs.

Cyclic AMP measurements were performed in lung homogenates from controls (n = 5) and from lungs instilled with 10⁶ M dopamine (n = 4). The amount of cAMP calculated per set of lungs did not increase significantly following incubation with dopamine (30.7 ± 15.6 pmol versus 25.8 ± 6.1 pmol for control lungs).

	DISCUSSION

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Regulation of sodium transport and consequently edema clearance by the alveolar epithelium might potentially provide new approaches to clinical management of patients with pulmonary edema. In the present studies we have demonstrated that dopamine, which is already widely used clinically, stimulates clearance of alveolar edema in the isolated perfused rat lung in a dose-dependent manner (Figures 1 and 2). We evaluated mechanisms contributing to the increased lung liquid clearance by selectively inhibiting components of the sodium pathway in the cell, using both amiloride and ouabain, inhibitors of sodium channels and Na,K-ATPase, respectively. We focused on the mechanisms that contribute significantly to vectorial sodium flux, although we recognize that other mechanisms outside the scope of this manuscript (Na-glucose exchange and Na-H exchange, for example) could also contribute to transpulmonary sodium flux (14).

Because -adrenergic agonists, another class of catecholamines, stimulate clearance and increase the activity of epithelial sodium channels (16, 18, 19), we instilled lungs with amiloride to inhibit the sodium channels (Figure 3). At 10⁶ M, which specifically inhibits sodium channels, the stimulatory effect of dopamine on clearance was preserved, albeit at a lower total clearance. At 10⁴ M amiloride, the ability of dopamine to stimulate sodium transport and liquid clearance was fully inhibited. As amiloride at higher concentrations is known to inhibit several sodium transport mechanisms in addition to sodium channels, including Na,K-ATPase (20), we hypothesized that when the residual stimulatory effect of dopamine seen in the presence of 10⁶ M amiloride was abolished by 10⁴ M amiloride, we could be inhibiting the sodium pump as well. Even if the sodium channels are not 100% inhibited (and we agree that at 10⁶ M, they are probably not), the proportion of fluid transport that is amiloride-sensitive should increase if an increase in sodium channel activity is necessary to increase overall clearance. We did not see an increase in this proportion, leading us to conclude that the increased clearance produced by dopamine was probably not mediated by an increase in sodium channel activity, but rather by other mechanisms.

We then perfused lungs with 5 × 10⁴ M ouabain to specifically inhibit Na,K-ATPase. In these lungs, however, not only was basal liquid clearance decreased, the dopamine-stimulated increased in lung fluid clearance was abolished (Figure 4). These data suggest that dopamine increases lung liquid clearance by stimulating active Na⁺ transport. Although this study does not allow us to deduce the exact mechanism responsible for the increase in Na⁺ transport, the time course of this stimulation suggests a short-term regulation of the transport mechanisms (for example, translocation or recruitment of new Na⁺ pumps to the cell membrane), because it is unlikely that synthesis of new Na,K-ATPase protein occurred within the time frame involved.

Most of the previous work concerning regulation of Na,K-ATPase by dopamine and the mechanisms of regulation (second messenger systems, protein phosphorylation, cytoskeletal effects, etc.) has concentrated on the transporting epithelial cells of the kidney and dopamine-sensitive cells of the central nervous system (21, 22). While these studies define the short-term regulation of Na,K-ATPase in the tissues mentioned, the behavior of other cell types may very well be different, as pump regulation is likely determined by the different functions of the cells. For example, in the small intestine, which shares its embryological origins with the lungs, dopamine has been found to stimulate sodium absorption (23), while in renal tubules, the effects of dopamine on sodium transport are generally inhibitory (7, 22). Clearly, even within different types of epithelial cells regulation of Na,K-ATPase may vary from one cell type to another.

We are not aware of any previous publications on the effect of dopamine as a possible short-term modulator of lung Na,K-ATPase activity and lung liquid clearance. Recently, an abstract was presented in a different model in which dobutamine, but not dopamine, increased lung edema clearance (24); other laboratories have begun to explore the possible cellular mechanisms of sodium transport or lung edema clearance by other mediators.

Regulation of Na,K-ATPase activity and consequently sodium movement in transporting epithelia may have important physiologic consequences. Regulation of Na,K-ATPase varies in different organs and cell lines (6, 25), with evidence that mechanisms mediated by cAMP-dependent protein kinase (PKA) or protein kinase C (PKC) may play important intracellular signaling roles. Activation of these cellular pathways has also been shown to affect other transporters involved in cellular sodium homeostasis, notably the activation of sodium channels by cAMP (19, 29). Although the mechanism of regulation cannot be deduced from the current study, it has provided us with a model of short-term sodium transport and edema clearance upregulation to study in the alveolar epithelium.

It has been demonstrated by several groups of investigators that administration of cyclic AMP analogues (30) or agents that increase intracellular cAMP (15, 17, 30, 31) increases basal lung liquid clearance and sodium transport across the alveolar epithelium. It has also been shown that this is likely an important physiological means of regulating these processes, as shown by increased catecholamine release, which mediates enhanced lung liquid clearance in septic shock (16), and by the finding that increased intracellular cAMP can stimulate edema clearance following ischemia-reperfusion lung injury (32). The effects of dopamine, however, appear to be distinct from those of -adrenergic agonists, as isoproterenol or terbutaline have been reported to produce clearances which are approximately twice controls (31, 33, 34), and are greater than the increase we saw with dopamine (33%).

In many cell types, dopamine activates cAMP via D₁ (or DA₁) type dopamine receptors (7, 21, 22). We therefore postulated that the effects of dopamine in our isolated perfused lung model could have been mediated through this pathway. Our findings were not definitive, as we were able to measure only a small (nonsignificant) increase in cAMP levels in lung homogenates following incubation with dopamine. These data agree with studies in renal cortical plasma membranes in which D₁ receptor occupation activated signal transduction pathways independent of cAMP (35). Unfortunately, we cannot rule out the possibility that there was an increase in cAMP which we were unable to detect, either because of dilution of epithelial cAMP by other cells present in lung tissue, or by a degradation of any initial increase in cAMP over the course of the experiment. Further experiments at the cellular level will be necessary to more definitively resolve both which receptor type is activated and which second messenger system is responsible for the observed increase in clearance.

In summary, our data show that dopamine stimulates lung liquid clearance in a dose-dependent manner. As this stimulation is inhibited by ouabain or amiloride, it is likely that dopamine exerts its effects by increasing active Na⁺ transport. Furthermore, the ability of ouabain but not low concentrations of amiloride to inhibit the dopamine-stimulated Na⁺ transport and lung liquid clearance, leads us to reason that this occurs via a modulation of the lung Na,K-ATPase. While it is beyond the scope of this manuscript to comment on the cellular pathways which are involved in mediating the response to dopamine, further studies are warranted to define the cellular mechanisms in detail.