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J Appl Physiol 90: 10-16, 2001;
8750-7587/01 $5.00
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Vol. 90, Issue 1, 10-16, January 2001

Denopamine, a beta 1-adrenergic agonist, increases alveolar fluid clearance in ex vivo rat and guinea pig lungs

Tsutomu Sakuma1, Chiharu Tuchihara1, Masanobu Ishigaki1, Kazuhiro Osanai1, Yoshihiro Nambu1, Hirohisa Toga1, Keiji Takahashi1, Nobuo Ohya1, Takayuki Kurihara2, and Michael A. Matthay3

Departments of 1 Pulmonary Medicine and 2 Basic Medical Science, Kanazawa Medical University, Uchinade, Ishikawa 920-0293, Japan; and 3 Departments of Medicine and Anesthesia, Cardiovascular Research Institute, University of California, San Francisco, California 94143


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The effect of denopamine, a selective beta 1-adrenergic agonist, on alveolar fluid clearance was determined in both ex vivo rat and guinea pig lungs. Alveolar fluid clearance was measured by the progressive increase in the concentration of Evans blue-labeled albumin over 1 h at 37°C. Denopamine (10-6 to 10-3 M) increased alveolar fluid clearance in a dose-dependent manner in ex vivo rat lungs. Denopamine also stimulated alveolar fluid clearance in guinea pig lungs. Atenolol, a selective beta 1-adrenergic antagonist, and amiloride, a sodium channel inhibitor, inhibited denopamine-stimulated alveolar fluid clearance. The potency of denopamine was similar to that of similar doses of isoproterenol or terbutaline. Short-term hypoxia (100% nitrogen for 1-2 h) did not alter the stimulatory effect of denopamine. Denopamine (10-4, 10-3 M) increased intracellular adenosine 3',5'-cyclic monophosphate levels in cultured rat alveolar type II cells. In summary, denopamine, a selective beta 1-adrenergic agonist, stimulates alveolar fluid clearance in both ex vivo rat and guinea pig lungs.

pulmonary edema; sodium transport; alveolar epithelium


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE EFFECTS OF beta -ADRENERGIC agonists on transepithelial ion transport and alveolar fluid clearance are different among experimental animal species. beta 2-Adrenergic agonists increase alveolar fluid clearance in dog (4, 13), sheep (5), mouse (1), and human lungs (25, 26). In rat lungs, terbutaline, isoproterenol, and salmeterol have been reported as potent stimulators of beta 2-adrenoceptors in alveolar fluid clearance (9, 11, 16, 25, 29, 30). Recently, a clinically relevant beta -adrenergic agonist, dobutamine, stimulated alveolar fluid clearance in in vivo rat lungs, and the stimulation was inhibited by ICI-118551, a selective beta 2-adrenergic antagonist (35). Furthermore, endogenous catecholamines stimulate alveolar fluid clearance in rats with septic shock (21) and with hemorrhagic shock (19). However, a beta 2-adrenergic agonist failed to increase alveolar fluid clearance in rabbits (31) and guinea pigs (20). In guinea pig lungs, isoproterenol increased alveolar fluid clearance, an effect that was inhibited by atenolol, a selective beta 1-adrenergic antagonist (20). However, it is unknown whether a selective beta 1-adrenergic agonist increases alveolar fluid clearance in rat and guinea pig lungs. Interestingly, the beta 1-adrenoceptor is present on alveolar type II cells (2).

The first objective in this study was to determine whether a selective beta 1-adrenergic agonist would stimulate alveolar fluid clearance in ex vivo rat and guinea pig lungs. We used denopamine, a selective beta 1-adrenergic agonist that has been used in patients with heart failure (7, 14). The second objective was to determine whether there was a difference in the magnitude of alveolar fluid clearance stimulated by denopamine, terbutaline, and isoproterenol. The same doses of terbutaline and isoproterenol were instilled in ex vivo rat lungs, and then the rates of alveolar fluid clearance were compared. The third objective was to determine whether denopamine-stimulated alveolar fluid clearance was preserved in rat lungs exposed to short-term hypoxia (2 h). Alveolar fluid clearance was measured in rat lungs exposed to 100% nitrogen for 1-2 h. The final objective was to determine whether denopamine increased cAMP levels in rat alveolar type II epithelial cells because prior studies indicated that cAMP may function as a second messenger in alveolar type II cells or lung tissues that were exposed to beta 2-adrenergic agonists (18, 32, 36).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Materials

Materials were obtained as follows: denopamine from Tanabe Pharmaceutical (Tokyo, Japan); atenolol, amiloride, terbutaline, and isoproterenol from Sigma Chemical (St. Louis, MO); ICI-118551 from Tocris Cookson (Bristol, UK); and Evans blue from Tokyo Kasei (Tokyo, Japan).

General Protocol

Ex vivo rat and guinea pig studies. As previously reported (25, 28), we isolated rat lungs and guinea pig lungs and measured alveolar fluid clearance in the absence of either pulmonary perfusion or ventilation. Briefly, male Sprague-Dawley rats (200-250 g) and male guinea pigs (300-350 g) were anesthetized by an intraperitoneal administration of pentobarbital sodium (50 mg/kg body wt). An endotracheal tube was inserted through a tracheostomy. The animals were exsanguinated through the abdominal aorta. Through a median sternotomy, the trachea, both lungs, and the heart were excised en bloc. The lungs were wrapped by Saran wrap to prevent dehydration and were placed in the humid incubator at 37°C. Warmed Ringer lactate solution (6 ml/kg body wt) containing 5% albumin and 0.15 mg/ml of Evans blue was instilled into both lungs followed by 4 ml of oxygen to deliver all of the instilled fluid into the alveolar spaces. The lungs were inflated with 100% oxygen at an airway pressure of 8 cmH2O. Alveolar fluid was aspirated 1 h after instillation. To estimate alveolar fluid clearance for 1 h, the concentrations of Evans blue-labeled albumin in the instilled and aspirated solutions were measured by a spectrophotometer at a wavelength of 621 nm (model BioSpec-1600, Shimadzu, Kyoto, Japan).

Isolation of rat alveolar type II epithelial cells. Alveolar type II epithelial cells were isolated from pathogen-free Sprague-Dawley rats (200-250 g) by elastase digestion followed by centrifugation over a discontinuous metrizamide density gradient. The purity of freshly isolated type II cell preparations was 72-84% and the purity of type II cells was >95% after 24 h after plating. The amount of [3H]thymidine uptake was maintained for 72 h after plating. There was not a significant release of lactate dehydrogenase in cultured medium. The isolated type II cells were seeded at a density of 2 × 106 cells (35-mm-diameter cell culture cluster dish, Costar, Cambridge, MA) in 2 ml DMEM containing 10% fetal bovine serum (FBS) for 42 h before the studies.

Measurement of alveolar fluid clearance. Alveolar fluid clearance was estimated by measuring the progressive increase in the concentrations of alveolar Evans blue-labeled albumin (25, 28). Alveolar fluid clearance (AFC) was calculated as follows
AFC = [(Vi − Vf)&cjs0823;  Vi] × 100
where Vi is the volume of instilled albumin solution, and Vf is the volume of the final alveolar fluid.
Vf = (Vi × EBi)&cjs0823;  EBf
where EBi is the concentration of Evans blue in the instilled albumin solution and EBf is the concentration in the final alveolar fluid.

Measurement of intracellular cAMP. Intracellular cAMP levels were measured by an enzyme immunoassay kit (Amersham, Little Chalfont, UK). The assays were done using a nonacetylation assay.

Measurement of extravascular lung water. The water content of left lung was measured by drying the lungs to a constant weight at 70°C for 48 h. Lung water-to-dry lung weight ratio (LW/DL) was calculated as LW/DL = (wet lung weight - dry lung weight)/(dry lung weight).

Specific Protocol

Group 1: Effects of denopamine on alveolar fluid clearance in ex vivo rat lungs (n = 36). To determine the dose-dependent effect of denopamine on alveolar fluid clearance in ex vivo rat lungs, an isosmolar albumin solution in the presence of denopamine was instilled into the alveolar spaces immediately after isolation of the rat lungs. The concentrations of denopamine were 10-8 M (n = 5), 10-7 M (n = 5), 10-6 M (n = 4), 10-5 M (n = 4), 10-4 M (n = 4), and 10-3 M (n = 4). As controls, an isosmolar 5% albumin solution in the absence of denopamine was instilled into the alveolar spaces (n = 10).

Group 2: Effects of beta 1-adrenergic antagonist and sodium channel inhibitor on denopamine-stimulated alveolar fluid clearance in ex vivo rat lungs (n = 16). To determine whether denopamine-stimulated alveolar fluid clearance was mediated by beta 1-adrenoceptors or beta 2-adrenoceptors, an isosmolar albumin solution in the presence of 10-3 M atenolol, a selective beta 1-adrenergic antagonist, and 10-5 M denopamine (n = 4) or in the presence of 10-4 M ICI-118551, a selective beta 2-adrenergic antagonist, and 10-5 M denopamine (n = 4) was instilled into the alveolar spaces immediately after isolation of the rat lungs. To determine the effect of atenolol alone on alveolar fluid clearance, an isosmolar albumin solution in the presence of 10-3 M atenolol was instilled into the alveolar spaces (n = 4). Furthermore, to determine whether denopamine-stimulated alveolar fluid clearance was mediated by an amiloride-sensitive sodium channel in ex vivo rat lungs, an isosmolar albumin solution in the presence of 10-5 M denopamine and 10-4 M amiloride, a sodium channel inhibitor, was instilled into the alveolar spaces (n = 4).

Group 3: Effects of denopamine on alveolar fluid clearance in ex vivo guinea pig lungs (n = 12). To determine whether denopamine increased alveolar fluid clearance in ex vivo guinea pig lungs, an isosmolar albumin solution in the presence of 10-5 M denopamine was instilled into the alveolar spaces immediately after isolation of the guinea pig lungs (n = 4). As controls, an isosmolar albumin solution in the absence of denopamine was instilled (n = 4). To determine whether denopamine-stimulated alveolar fluid clearance was mediated by an amiloride-sensitive sodium channel, an isosmolar albumin solution in the presence of 10-5 M denopamine and 10-4 M amiloride was instilled into the alveolar spaces (n = 4).

Group 4: Comparison with terbutaline and isoproterenol in ex vivo rat lungs (n = 20). To compare the potency of denopamine with that of terbutaline and isoproterenol, the magnitude of augmented alveolar fluid clearance was measured with the same dose (10-5 M) of terbutaline and isoproterenol in ex vivo rat lungs. First, an albumin solution in the presence of 10-5 M terbutaline was instilled into the alveolar spaces immediately after isolation of the rat lungs (n = 4). To determine whether terbutaline-stimulated alveolar fluid clearance was mediated by beta 2-adrenoceptors, an isosmolar albumin solution in the presence of 10-5 M terbutaline and 10-4 M ICI-118551 was instilled (n = 4). To determine whether terbutaline could increase alveolar fluid clearance in addition to denopamine, an isosmolar albumin solution in the presence of 10-5 M denopamine and 10-5 M terbutaline was instilled (n = 4). Second, an albumin solution in the presence of 10-5 M isoproterenol was instilled immediately after isolation of the rat lungs (n = 4). To determine whether isoproterenol-stimulated alveolar fluid clearance was mediated by a beta 2-adrenoceptor, an isosmolar albumin solution in the presence of 10-5 M isoproterenol and 10-4 M ICI-118551 was instilled (n = 4).

Group 5: Effects of denopamine on alveolar fluid clearance in acutely hypoxic rat lungs (n = 15). To expose rat lungs to hypoxia, oxygen in the lungs was replaced by inflation (5 cycles) with 4 ml of 100% nitrogen immediately after isolation of the lungs. Then, an albumin solution in the presence of 10-5 M denopamine was instilled into the alveolar spaces (n = 4). In addition, to expose rat lungs to hypoxia for 1 h before instillation of albumin solution, isolated rat lungs were inflated with 100% nitrogen at an airway pressure of 8 cmH2O for 1 h after the replacement of oxygen with nitrogen; thereafter, an albumin solution in the presence of 10-5 M denopamine was instilled into the alveolar spaces (n = 4). As controls, an albumin solution in the absence of denopamine was instilled into the alveolar spaces after the replacement of oxygen with nitrogen (n = 7). After instillation of albumin solution, the lungs were inflated with 100% nitrogen for 1 h at an airway pressure of 8 cmH2O.

Group 6: Effects of denopamine on cAMP levels in rat alveolar type II cells (n = 28). To determine whether intracellular cAMP levels played a role in denopamine-stimulated alveolar fluid clearance, the effects of denopamine on intracellular cAMP levels were determined in cultured rat alveolar type II cells. The confluent alveolar type II epithelial cells 48 h after plating were washed with cell culture medium, DMEM without 10% FBS, to remove dead cells, and the medium was replaced with fresh DMEM. Then, the cells were exposed to denopamine at the concentrations ranging from 10-8 to 10-3 M (n = 4 each) in DMEM or to DMEM medium alone as control (n = 4) for 15 min. Thereafter, after removal of the medium, 65% (vol/vol) of ice-cold ethanol was added to the wells that were placed on ice for 30 min. The supernatant was drawn off into the test tubes. The remaining precipitate was washed with ice-cold 65% (vol/vol) ethanol, and the washings were added to the tubes. The extracts were centrifuged at 2,000 g for 15 min at 4°C, and the supernatant was transferred to fresh tubes. The combined extracts were dried in a vacuum oven at 60°C and stored at -27°C until the assay was conducted.

Statistics

The data are summarized as means ± SD. The data were analyzed by one-way ANOVA with Student-Newman-Keuls post hoc test when multiple comparisons were needed. When comparisons were made between two experimental groups, an unpaired Student's t-test was used. We regarded as significant those differences with a P value of <0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Group 1: Effects of Denopamine on Alveolar Fluid Clearance in Ex Vivo Rat Lungs

Denopamine increased alveolar fluid clearance in a dose-dependent fashion. Although 10-8 and 10-7 M denopamine did not significantly increase alveolar fluid clearance, doses of denopamine ranging from 10-6 to 10-3 M significantly increased alveolar fluid clearance (Fig. 1).


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  		Fig. 1.   A dose-dependent effect of denopamine on alveolar fluid clearance in ex vivo rat lungs. Values are means ± SD. * P < 0.05 vs. control values (ANOVA).

Group 2: Effects of beta 1-Adrenergic Antagonist and Sodium Channel Inhibitor on Denopamine-Stimulated Alveolar Fluid Clearance in Ex Vivo Rat Lungs

Atenolol inhibited denopamine-stimulated alveolar fluid clearance (Fig. 2). Atenolol alone did not affect alveolar fluid clearance (Fig. 2). ICI-118551 did not inhibit denopamine-stimulated alveolar fluid clearance. Amiloride (10-4 M) inhibited denopamine-stimulated alveolar fluid clearance by 30%.


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  		Fig. 2.   Denopamine-stimulated alveolar fluid clearance mediated by beta 1-adrenoceptor and sodium channel in ex vivo rat lungs. Atenolol, a selective beta 1-adrenergic antagonist, inhibited denopamine-stimulated alveolar fluid clearance. Values are means ± SD. * P < 0.05 vs. control values (ANOVA). dagger  P < 0.05 vs. 10-5 M denopamine (ANOVA).

Group 3: Effects of Denopamine on Alveolar Fluid Clearance in Ex Vivo Guinea Pig Lungs

Basal alveolar fluid clearance was greater by 30% in ex vivo guinea pig lungs than in ex vivo rat lungs (Fig. 3). Denopamine (10-5 M) significantly increased alveolar fluid clearance by 30%. Amiloride (10-4 M) inhibited denopamine-stimulated alveolar fluid clearance.


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  		Fig. 3.   Effect of denopamine on alveolar fluid clearance in ex vivo guinea pig lungs. Denopamine increased alveolar fluid clearance to the level observed in rat lungs. Values are means ± SD. * P < 0.05 vs. control values (ANOVA).

Group 4: Comparison With Terbutaline and Isoproterenol in Ex Vivo Rat Lungs

Terbutaline (10-5 M) increased alveolar fluid clearance to the same extent as denopamine (Fig. 4). ICI-118551 inhibited the terbutaline-stimulated alveolar fluid clearance. There was no additional increase in the presence of 10-5 M denopamine plus 10-5 M terbutaline (Fig. 4). Isoproterenol (10-5 M) increased alveolar fluid clearance to the same degree as the same dose of denopamine and terbutaline (Fig. 4). ICI-118551 inhibited isoproterenol-stimulated alveolar fluid clearance (Fig. 4).


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  		Fig. 4.   Magnitude of denopamine effect compared with those of terbutaline and isoproterenol. The rate of alveolar fluid clearance increased by denopamine was similar to the rates of clearance with terbutaline and isoproterenol. There was no additional increase when denopamine was administered with terbutaline. Values are means ± SD.* P < 0.05 vs. 10-5 M terbutaline (ANOVA). dagger  P < 0.05 vs. 10-5 M isoproterenol (unpaired Student's t-test).

Group 5: Effects of Denopamine on Alveolar Fluid Clearance in Acutely Hypoxic Rat Lungs

Inflation with 100% nitrogen during measurement of alveolar fluid clearance and inflation with 100% nitrogen for 1 h before instillation of albumin solution did not alter basal alveolar fluid clearance and the effect of denopamine on alveolar fluid clearance (Fig. 5).


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  		Fig. 5.   Effect of denopamine on alveolar fluid clearance in hypoxic rat lungs. Short-term hypoxia (100% nitrogen for 1-2 h) did not alter basal alveolar fluid clearance. The effect of denopamine on alveolar fluid clearance was preserved in hypoxic rat lungs. Values are means ± SD. * P < 0.05 vs. oxygen values (unpaired Student's t-test). dagger  P < 0.05 vs. oxygen and nitrogen values (ANOVA).

Group 6: Effect of Denopamine on cAMP levels in Rat Alveolar Type II Cells

Denopamine (10-4 and 10-3 M) significantly increased intracellular cAMP levels in cultured alveolar type II cells (Fig. 6). However, denopamine at the concentrations ranging from 10-8 M to 10-5 M did not increase cAMP levels.


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  		Fig. 6.   Effect of denopamine on intracellular cAMP levels in cultured alveolar type II cells from rat lungs. Values are means ± SD. * P < 0.05 vs. control values (ANOVA).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The first objective in this study was to determine whether a selective beta 1-adrenergic agonist would stimulate alveolar fluid clearance in ex vivo rat and guinea pig lungs. Although the distribution of the beta -adrenoceptor subtypes measured on cell membranes obtained from whole rat lungs is 25% for the beta 1-adrenoceptor and 75% for the beta 2-adrenoceptor (24), with a similar distribution in the human alveolar wall (8), the functions of beta 1-adrenoceptors are uncertain. The first finding in this study was that denopamine, a selective beta 1-adrenergic agonist, increased alveolar fluid clearance (Fig. 1). Our laboratory previously reported dose-dependent effects of beta 2-adrenergic agonists in the resected human and rat lungs (25). Similar stimulatory effects of beta 2-adrenergic agonists on alveolar fluid clearance have also been reported from several laboratories (11, 16, 29, 30). However, to our knowledge, this is the first report that demonstrates a stimulatory effect of a selective beta 1-adrenergic agonist on alveolar fluid clearance.

To determine whether the denopamine effect was mediated by beta 1-adrenoceptors and amiloride-sensitive sodium pathways, atenolol, a selective beta 1-adrenergic antagonist, and amiloride, a sodium channel inhibitor, were administered in the presence of denopamine. Because atenolol inhibited denopamine-stimulated alveolar fluid clearance, the stimulation was mediated by beta 1-adrenoceptors. In addition, because a selective beta 2-adrenergic antagonist, ICI-118551, in the concentration that could inhibit terbutaline-stimulated alveolar fluid clearance, did not inhibit denopamine-stimulated alveolar fluid clearance, the selectivity of denopamine has shown to be a beta 1-adrenergic agonist. Also, the denopamine effect in guinea pig lungs is consistent with the report that isoproterenol-stimulated alveolar fluid clearance was inhibited by atenolol in the guinea pig lungs (20) and mouse lungs (12). Second, both amiloride-sensitive and -insensitive mechanisms play an important role in alveolar fluid clearance because the percentage of amiloride-sensitive alveolar fluid clearance ranges from 30 to 75% of basal alveolar fluid clearance (3, 15, 19, 26, 30, 31). However, it has been suggested that alveolar fluid clearance increased by the beta -adrenergic agonists isoproterenol, terbutaline, and epinephrine is primarily amiloride sensitive (15, 26, 33). Amiloride-sensitive sodium channel stimulation is also dominant in the upregulated alveolar fluid clearance stimulated by release of endogenous catecholamines (19). However, because amiloride inhibited only 30% of denopamine-increased alveolar fluid clearance in the present study, the degree of inhibition was not different from the basal amiloride-sensitive alveolar fluid clearance. Therefore, it is likely that denopamine may stimulate both amiloride-sensitive and amiloride-insensitive alveolar fluid clearance.

We tested the effect of denopamine in guinea pig lungs because species differences in the effect of beta -adrenergic agonists on alveolar fluid clearance have been reported (17) and because the beta 1-adrenoceptor was recently reported to play a role in alveolar fluid clearance in guinea pig lungs (20). In the present study, because denopamine increased alveolar fluid clearance and amiloride inhibited alveolar fluid clearance in guinea pig lungs, the data suggest that a selective beta 1-adrenergic agonist increases alveolar fluid clearance in guinea pig lungs as well as in rat lungs. This result is consistent with the report that isoproterenol-stimulated alveolar fluid clearance was inhibited by atenolol, a selective beta 1-adrenergic antagonist (20).

Our laboratory previously reported that there are significant species differences in the basal rates of alveolar fluid clearance (17, 25). First, Sakuma et al. (25) compared the rate of alveolar fluid clearance between rat and human lungs. The rate of alveolar fluid clearance was faster in ex vivo rat lungs than in ex vivo human lungs. Second, summarizing the previous reports, Matthay et al. (17) classified the rates of alveolar fluid clearance in different species. The highest clearance rates were measured in rabbits, rat, and mouse lungs. In the present study, because the rates of alveolar fluid clearance were independent of the instilled volume, ranging from 2 to 6 ml/kg (4, 17), the instilled volume (3 ml/kg body wt) was adjusted to be the same in the guinea pig lungs as in the rat lungs. We found that guinea pig has comparatively higher basal alveolar fluid clearance than rat (Figs. 1 and 3). The results in this study are consistent with the previous in vivo study in guinea pigs (20).

The second objective was to compare the potency of denopamine with that of terbutaline or isoproterenol in stimulating alveolar fluid clearance. To determine potency, the same dose (10-5 M) of terbutaline and isoproterenol was instilled. We used 10-5 M because this dose was at the plateau of the dose response for the effect of denopamine on alveolar fluid clearance (Fig. 1). The magnitude of the denopamine effect on alveolar fluid clearance was similar to that of isoproterenol or terbutaline. Also, the terbutaline- and isoproterenol-stimulated increase in alveolar fluid clearance was inhibited by ICI-118551, a selective beta 2-adrenergic antagonist (Fig. 4). In addition, the magnitude of alveolar fluid clearance stimulated by denopamine was probably at the plateau of the dose-response curve because the addition of 10-5 M terbutaline to 10-5 M denopamine did not produce further increase in alveolar fluid clearance. However, there may be a limitation in recycling of cAMP or beta -adrenoceptors because the alveolar fluid clearance was measured in the absence of pulmonary perfusion.

The third objective of these studies was to determine whether the effect of denopamine was preserved in hypoxic rat lungs. Recently, our laboratory reported that inflation of the alveoli regardless of the gas concentration was important to maintain the transport function of alveolar epithelial cells (28). For example, although lung deflation induced a decrease in alveolar fluid transport, the decrease was not observed when the lungs were inflated with 100% nitrogen in en vivo rat lungs (28). Not only basal alveolar fluid clearance but also alveolar fluid clearance stimulated by a beta 2-adrenergic agonist were preserved for 2 h when the lungs were inflated with 100% nitrogen (28). In the present study, short-term hypoxia (2 h) did not alter the stimulatory effect of denopamine on alveolar fluid clearance. Therefore, it is likely that the beta 1-adrenoceptor is resistant to short-term severe hypoxia. The resistance may be beneficial in the resolution of alveolar edema when alveolar epithelial cells are acutely exposed to hypoxia.

Recently, the effect of hypoxia on alveolar ion transport and fluid clearance has been reported in isolated alveolar epithelial cell studies. Hypoxia (0 and 3% oxygen) induced a downregulation of expression and activity of sodium channels and sodium pump activity in cultured alveolar type II cells from rat lungs (22, 23). Although sodium uptake was not altered in cultured type II cells exposed to 5% oxygen (22), alveolar fluid clearance and sodium pump activity were decreased in rats exposed to 10% oxygen for 48-72 h (33). In this study, basal alveolar fluid clearance was not altered by hypoxia for 2 h. The results are consistent with the previous results in cultured type II cells in which sodium transport was not altered within 3 h under 0% oxygen (22). Therefore, it is likely that both the concentration of oxygen and the term of exposure to hypoxia are important factors that play a role in the regulation of alveolar fluid clearance.

The final objective was to determine whether denopamine-stimulated alveolar fluid clearance was matched by an increase in intracellular cAMP. Although 10-4 and 10-3 M denopamine increased intracellular cAMP levels in cultured alveolar type II cells, denopamine at the concentrations ranging from 10-8 to 10-5 M did not. There was a discrepancy between the effect of 10-5 M denopamine on alveolar fluid clearance in ex vivo rat lungs and on cAMP levels in cultured type II cells. The discrepancy can be explained in several ways. First, the higher concentration of denopamine might be needed to stimulate cAMP production in isolated cells, although 10-5 to 10-6 M denopamine increased alveolar fluid clearance in ex vivo lungs, as shown in Fig. 1. It is possible that the discrepancy was caused by the differences in the experiments. Studies of isolated, cultured type II cells 48-72 h after isolation from the lung may only partially reflect their in vivo function because the alveolar type II cells progressively lose some of their phenotypic characteristics after isolation (10). Second, it is possible that the high stimulation of alveolar fluid clearance filled the interstitium with fluid and that hydrostatic forces counteracted any further alveolar fluid clearance. However, as previously reported in sheep (27), it is probable that interstitial fluid could move into pulmonary circulation because the pressure in pulmonary circulation is zero and the plasma protein concentration is higher than interstitial protein concentration. Third, it was reported that terbutaline caused an early rise in cellular cAMP that peaked within 5 min and then returned to basal level by 60 min (18). Inasmuch as we measured cAMP levels in cultured type II cells 15 min after exposure to denopamine, the time we measured cAMP may not be the point when cAMP increased maximally. Fourth, the effect of denopamine might not be mediated by cAMP in stimulating alveolar fluid clearance. However, further studies are needed to conclude that the effect of beta 1-adrenergic agonist on alveolar fluid clearance is not mediated by cAMP.

Recently, it was reported that isoproterenol increased Na+-K+ ATPase activity by membrane insertion of alpha -subunits in lung alveolar cells (6). However, it was not determined in this study whether denopamine played the same role as isoproterenol.

Some water clearance probably occurs through alveolar type I cells because type I cells cover >95% of the surface of the alveolar spaces and water channels were present on type I cells (17). However, because the study in which the effect of beta -adrenergic agonist was determined in freshly cultured type I cells is not available, the role of type I cells in fluid clearance from the alveolar spaces needs future study.

What are the clinical implications of this study? Because denopamine has been administered to patients with congestive heart failure (7), if denopamine could accelerate the resolution of clinical alveolar edema, this vasoactive agent may be beneficial for hastening the resolution of pulmonary edema and well as improving cardiac function.

In summary, denopamine, a beta 1-adrenergic agonist, increased alveolar fluid clearance in a dose-dependent manner in both ex vivo rat and guinea pig lungs. The potency of denopamine was similar to isoproterenol or terbutaline. Short-term hypoxia (100% nitrogen for 2 h) did not alter the stimulatory effect of denopamine. These findings may have clinical significance because short-term upregulation of alveolar fluid clearance could be achieved either with beta 1- or beta 2-adrenoceptor stimulation.


    ACKNOWLEDGEMENTS

This study was supported by Grants for Collaborative Research C98-6 and C99-3 from Kanazawa Medical University; a Grant-in Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan; and National Heart, Lung, and Blood Institute Grant HL-51854.


    FOOTNOTES

Address for reprint requests and other correspondence: T. Sakuma, Dept. of Pulmonary Medicine, Kanazawa Medical University, Uchinade, Ishikawa 920-0293, Japan (E-mail: sakuma-t{at}kanazawa-med.ac.jp).

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.

Received 24 February 2000; accepted in final form 28 July 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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4.   Berthiaume, Y, Broaddus VC, Gropper MA, Tanita T, and Matthay MA. Alveolar liquid and protein clearance from normal dog lungs. J Appl Physiol 65: 585-593, 1988[Abstract/Free Full Text].

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J APPL PHYSIOL 90(1):10-16
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