SPECIAL TOPIC
Pre- and Postnatal Lung Development, Maturation, and Plasticity
Distal air space epithelial fluid clearance in near-term rat fetuses is fast and requires endogenous catecholamines

Hans G. Folkesson1, Michael A. Matthay2, Cheryl J. Chapin2, Nicolas F. M. Porta2,3, and Joseph A. Kitterman2,3

1 Department of Physiology, Northeastern Ohio Universities College of Medicine, Rootstown, Ohio 44272-0095; 2 Cardiovascular Research Institute, and 3 Department of Pediatrics, University of California, San Francisco, California 94143-0130


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Knowledge about the conversion of the epithelium in the distal air spaces of the lung from secretion to absorption is imperative to the understanding of postnatal lung development; little such information is available in rats. Distal air space fluid clearance was therefore measured in 21- to 22-day gestation rat fetuses and newborn (40 h) rats. Distal air space fluid clearance was measured from the increase in 131I-albumin concentration in an isosmolar, physiological solution instilled into the developing lungs. There was no net fluid movement across the distal air space epithelium in the lungs of 21-day gestation fetuses. Twenty-four hours later, distal air space fluid was cleared at a rapid rate in the 22-day gestation fetuses. Within the first 40 h after birth, the rate rapidly declined to adult levels. The high distal air space fluid clearance at 22 days gestation and at 40 h after birth was mediated by beta -adrenergic receptors as demonstrated by elevated plasma epinephrine levels and inhibition by propranolol. Interestingly, the elevated distal air space fluid clearance in the 22-day gestation fetuses was only minimally amiloride sensitive; however, amiloride sensitivity increased over the first 40 h after birth. In conclusion, these studies demonstrate that 1) rapid rates of net alveolar fluid clearance occur late in gestation in the rat and 2) this clearance is driven by elevations of endogenous epinephrine.

amiloride sensitivity; development; distal air space epithelium; epinephrine; Na+ transport; pulmonary edema; neonatal respiratory distress syndrome


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

CONVERSION OF THE EPITHELIUM in the distal air spaces of the lung from secretion to absorption is critical for survival of the newborn near term when the fetus has to switch from placental to pulmonary gas exchange. Recent as well as older data have identified a significant role for endogenous epinephrine in stimulating Na+ and fluid absorption near term (35, 49) and in newborn and adult lungs (14, 34), although some studies have suggested that it might not be the only factor of importance (1, 3, 40). It is known that active Na+ transport drives alveolar fluid across the normal healthy adult alveolar epithelium into the lung interstitium and vasculature (17, 30, 32, 34). Amiloride-sensitive epithelial Na+ channels, localized to alveolar epithelial type II cells, and a basolaterally located Na+-K+-ATPase constitute one molecular mechanism involved in apical Na+ uptake (20, 30, 31, 37, 48).

Although some investigators have studied epithelial fluid transport during the later periods of gestation in larger animals such as sheep and guinea pigs (6, 10, 14, 38), there have been no studies of distal air space fluid clearance in developing rat fetuses. Data on expression and regulation of the transporters implied in transepithelial Na+ transport, the sodium channel, and the Na+-K+-ATPase are available (21, 22, 47), although functional studies are lacking. Because the mature rat lung has fluid transport properties similar to the human lung in terms of both the rate of transport and the fractional sensitivity to amiloride (18, 41, 42), studies in rat fetuses may provide insights regarding fetal fluid transport that could be germane to the human lung. Therefore, the first objective of the current study was to study distal air space fluid clearance at different developmental stages near term in rats. The age groups studied were 20- to 22-day gestation rat fetuses and newborn (40 h) rats. We also measured extravascular lung water in the developing rat fetuses. Our goal was to determine the efficiency of distal air space fluid clearance immediately before and after birth. Because it has been shown that catecholamines may be important for regulating distal air space fluid clearance at birth (14, 49), our second aim was to investigate the contribution of endogenous beta -adrenergic stimulation on clearance of fetal lung fluid in late-gestation rat fetuses and at birth. We used the beta -adrenergic antagonist propranolol to investigate whether endogenous catecholamines were responsible for clearing the distal air spaces from fluid. Plasma samples were obtained from each developmental stage for measurements of endogenous epinephrine levels. Because epithelial Na+ transport via amiloride-sensitive Na+ channels provides much of the driving force for alveolar fluid reabsorption in adult and newborn lungs (14, 30, 38), the third aim was to determine whether the amiloride-sensitive fraction of distal air space fluid clearance in the fetal and newborn lungs.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals

Rats of the Sprague-Dawley strain (n = 138, divided in 16 litters, Charles River, Wilmington, MA) were used in the study. Both male and female rat fetuses were used in the study. The timed-pregnant rats were housed in separate cages in temperature- and humidity-controlled units (20 ± 2°C and 55 ± 10% relative humidity). Adult rats (n = 6) were used for determination of the normal dry lung extravascular lung water. All studies were approved by the University of California at San Francisco Committee on Animal Research.

Surgery

Timed-pregnant rats and their fetuses were anesthetized by maternal intramuscular injections of ketamine (50 mg/kg body wt; Ketalar, Parke-Davis, Warner-Lambert, Morris Plains, NJ) mixed with xylazine (5 mg/kg body wt; Rompun, Bayer, Pittsburgh, PA). After all fetuses had been delivered, the female rats were euthanized with an overdose intracardiac pentobarbital sodium (100 mg/kg body wt; Nembutal, Abbott Laboratories, Chicago, IL) and bilateral pneumothoraces.

The anesthetized timed-pregnant female rats were placed on a temperature-controlled pad heated to 38°C to maintain their body temperature. An abdominal hysterotomy was carried out by a midline laparotomy, and the fetuses were delivered one by one. Between deliveries of the fetuses, the uterus was kept closed by a noninjurious hemostat. Once the fetus was delivered, pithing the spinal cord with a needle immediately killed it. The fetus was fixed to a surgical board after death. The lower jaw was dislocated by bilateral cuts. The larynx was visualized, and a tracheal cannula (Angiocath no. 24, Becton-Dickinson, Sandy, UT), connected to a 1-ml syringe containing the fluid instillate, was inserted through the larynx into the trachea. The syringe was fixed to the surgical board with modeling clay. The rat fetuses were placed under a heating lamp to maintain normal body temperature during the experimental time period, and the heating lamp was cycled on and off to maintain an incubating temperature of 38-40°C in the incubation area. Temperature in the incubation area was measured by the use of a calibrated temperature probe.

General Experimental Protocol

The 5% albumin instillate solution with the 131I-labeled albumin as a distal air space protein tracer was instilled into both lungs over 2-3 s through the tracheal cannula. The fetuses were given 10 ml/kg body wt of fluid as instillate. The volume of instilled fluid does not affect the rate of alveolar fluid clearance (24, 32). After fluid instillation, the fetuses remained in the incubator area for the 30-min experiment. At the end of the experiment, remaining distal air space fluid was aspirated through the tracheal cannula. Radioactivities in instillates and air space samples of the 131I-labeled albumin were measured. Both lungs were excised and placed in a sample tube and counted for radioactivity as a confirmation of the location of the fluid instillate. Lungs that lacked significant radioactivity, i.e., when the radioactivity left in the lungs was below 70% of the instilled radioactivity, were discarded with their corresponding distal air space fluid samples because of uncertainty of location of the fluid instillate in those fetuses. Plasma epinephrine levels were measured in plasma samples from littermates to the ones used for the distal air space fluid clearance studies in all age groups by a commercially available ELISA method (IBL, Hamburg, Germany). The interassay and intra-assay coefficients of variation for the epinephrine ELISA were 6.2 and 15.2%, respectively.

Preparation of Instillates

The 131I-labeled 5% albumin instillate solution was prepared by dissolving 50 mg/ml bovine serum albumin (Sigma Biochemical, St. Louis, MO) in an isosmolar aqueous solution of 0.9% NaCl with 0.1 µCi 131I-labeled human serum albumin (Frosst Laboratories, Montreal, Canada). For studies of endogenous epinephrine dependence of distal air space fluid clearance, we added the general beta -adrenergic antagonist, propranolol (Sigma) at the concentration of 10-4 M to the 131I-labeled 5% albumin instillate solution. For determination of fractional amiloride inhibition of distal air space fluid clearance, we added amiloride (ICN Biochemicals, Costa Mesa, CA) at the concentration of 10-3 M to the 131I-labeled 5% albumin instillate solution. We used 10-3 M amiloride because ~50% of amiloride is protein bound and another significant fraction escapes from the air spaces, resulting in lower functional concentrations (38, 51).

Specific Experimental Protocols

The following experimental groups and specific experimental protocols were used. All rat fetuses used for distal air space fluid clearance studies were studied for 30 min and processed as described above in General Experimental Protocol. All experimental groups contained fetuses from at least two different dams. The n values of the groups represent individual fetuses. To verify that our method provided values of alveolar fluid clearance that did not differ between anesthetized rats and our in situ continuous positive airway pressure oxygenated fetuses, we compared the alveolar fluid clearance in the 40-h postnatal rats with the adult ventilated rat. In the 40-h-old rats, alveolar fluid clearance was not different from that in the adult rat when extrapolated to 1 h (16 ± 2%) in the 40-h-old rats and compared with that in adult rats (17 ± 2%, data from Ref. 16).

Group 1. Determination of preexisting distal air space fluid volume [21-day gestation, n = 6; 22-day gestation, n = 6; newborn (40 h), n = 6]. Because the lung is filled with a protein-free fluid in utero (6), we needed a method to account for the fraction of this fluid that would still be present in the air spaces of the lungs. The presence of fetal lung fluid in the air spaces would dilute the 131I-labeled albumin concentration in the instilled solution and, therefore, confound calculations of distal air space fluid clearance, depending on volume of fluid present in different age groups. Therefore, the volume of the preexisting fluid was used to correct the instilled 131I-labeled albumin concentrations for the dilution of the instillate that would occur if there were already fluid in the lung before instillation of the 131I-labeled 5% albumin solution in corresponding experimental groups. Littermates to rat fetuses used for distal air space fluid clearance experiments were instilled with 10 ml/kg body wt of the 131I-labeled 5% albumin instillate into the lungs. The fluid was immediately retrieved and reinstilled four times to ensure adequate mixing with preexisting lung fluid, as we have done before (14). After the last mixing, the fluid was retrieved and radioactivity counted. The whole procedure required ~1 min; during this time, it is unlikely that significant amounts of 131I-labeled albumin left or entered the air spaces or that significant volumes of fluid were reabsorbed from or secreted into the air spaces. Because protein would not cross the distal air space epithelium during this short time, any change in 131I-labeled albumin concentration would represent a dilution by preexisting protein-free fluid. We calculated preexisting fluid volume by the relationship
V<SUB>L</SUB><IT>=</IT>[(V<SUB>I</SUB><IT>·</IT>C<SUB>I</SUB>)<IT>/</IT>C<SUB>L</SUB>]<IT>−</IT>V<SUB>I</SUB> (1)
where VL is preexisting lung fluid volume (ml), VI is instilled fluid volume (ml), CI is 131I-labeled albumin concentration in instilled fluid [counts/min (cpm)/ml], and CL is 131I-labeled albumin concentration in aspirated fluid (cpm/ml).

Group 2. Control studies [21-day gestation, n = 8; 22-day gestation, n = 8; newborn (40 h), n = 6]. Rat fetuses were instilled with 10 ml/kg body wt of the 131I-labeled 5% albumin instillate into the lungs. In preliminary experiments, we also attempted to use 20-day gestation rat fetuses, but these fetuses were too small and fragile to carry out successful studies of distal air space fluid clearance.

Group 3. Endogenous epinephrine studies [21-day gestation, n = 6; 22-day gestation, n = 6; newborn (40 h), n = 6]. Rat fetuses were instilled with 10 ml/kg body wt of the 131I-labeled 5% albumin instillate containing 10-4 M propranolol into the lungs. Endogenous epinephrine levels were measured in plasma from littermates to the rat fetuses used for the distal air space fluid clearance studies in each group (n = 6-9 in each group, 21- to 22-day gestation and newborn, i.e., 40 h) and in 20-day gestation fetuses.

Group 4. Amiloride studies [21-day gestation, n = 6; 22-day gestation, n = 8; newborn (40 h), n = 6]. Rat fetuses were instilled with 10 ml/kg body wt of the 131I-labeled 5% albumin instillate containing 10-3 M amiloride into the lungs.

Group 5. Extravascular lung water determination [20-day gestation, n = 6; 21-day gestation, n = 5; 22-day gestation, n = 7; newborn (40 h), n = 6; adult, n = 6]. We measured extravascular lung water in 20-day gestation rat fetuses in addition to littermates of rat fetuses that were used for the studies of distal air space fluid clearance. In these groups, blood for hemoglobin measurement was obtained by decapitation of the rat fetuses. Then, the lungs were rapidly excised, the heart was removed, and the lungs were placed in preweighed sample tubes and immediately weighed. Distilled water (200 µl) was added to the tubes, the lungs were reweighed and then kept frozen at -20°C until they were homogenized and processed as described below. We used adult rats as a reference point for these studies. The adult rats were killed with 100 mg/kg body wt pentobarbital sodium intraperitoneally. A blood sample was obtained for hemoglobin measurement before the heart stopped beating. In adult rats, the lungs were excised through a midline sternotomy; the heart was removed, and the lungs were placed in tubes. Distilled water (4 ml) was added, and the lungs were homogenized and further processed as described below. This method for determination of excess lung water has been described previously in detail (4). In brief, extravascular lung water was determined by measuring extravascular water-to-dry weight ratio (g water/g dry lung) (43) and was calculated as
E<IT>=</IT>W<IT>/</IT>D (2)
where E is extravascular lung water, and W and D are wet and dry weights, respectively, of rat lungs corrected for blood volume. The lung blood volume was determined as previously described (4, 15) from measurements of hemoglobin concentrations in blood and lung homogenate supernatants.

Distal Air Space Fluid Clearance Calculation

Clearance of fluid from the lung distal air spaces was measured by the increase in distal air space protein concentration of the instilled solution over 30 min. The increase in protein concentration due to removal of water from the air spaces is a direct reflection of distal air space fluid clearance, as demonstrated before (4, 17, 32, 34). Data are presented as a ratio between final and instilled protein concentration or as distal air space fluid clearance in percent instilled volume. This method is accurate for measuring distal air space fluid clearance, since protein leaks due to a change in epithelial and endothelial permeability were likely to be negligible. The term distal air space fluid clearance does not imply that all fluid is reabsorbed at the alveolar level, since certainly some of the fluid may be reabsorbed by the distal airways. For example, Ballard and colleagues (2) have demonstrated that distal airways can transport sodium and thereby potentially absorb luminal fluid. Distal air space fluid clearance (AFC) was calculated from the equation
AFC<IT>=</IT>[(V<SUB>I</SUB><IT>−</IT>V<SUB>F</SUB>)<IT>/</IT>V<SUB>I</SUB>]<IT>·</IT>100 (<IT>%</IT>instilled volume) (3)
where VI is instilled volume (ml) corrected for preexisting lung fluid volume as per Eq. 1, and VF is final distal air space volume (ml) calculated from the 131I-labeled albumin concentrations in instilled and final distal air space fluids.

Statistics

All data are presented as means ± SD. Data were analyzed with one-way analysis of variance (ANOVA) with Tukey's test as post hoc. Two groups were compared with unpaired Student's t-test. Differences were considered significant when a P value of <0.05 was reached. The n for the different groups represents individual fetuses.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Distal Air Space Fluid Volume in Developing Fetal and Newborn Lungs

Because lungs are fluid filled in utero, the presence of preexisting lung fluid was assessed. As expected, the most premature lungs contained the highest volume of preexisting lung fluid (Fig. 1). The volume of preexisting distal air space fluid corresponded to a 38% dilution of the instilled fluid's 131I-labeled albumin concentration in the 21-day gestation rat fetuses, a dilution that decreased to 14% dilution in the 22-day gestation rat fetuses and declined to only 2% at 40 h after birth. This volume was used to correct the distal air space fluid clearance calculations.


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Fig. 1.   Preexisting distal air space fluid volume in developing fetal rat lungs. Dotted line indicates time of birth. Values are means ± SD. *P < 0.05 compared with 40-h postnatal rats; dagger P < 0.05 compared with 21-day gestation rat fetuses (ANOVA with Tukey's test as post hoc).

Distal Air Space Fluid Clearance During Development

The results are presented as the ratio between final to instilled 131I-labeled albumin concentrations and as the distal air space fluid clearance in percent instilled volume (Figs. 2, 4, and 5). There was no net distal air space fluid clearance in 21-day rat fetuses (Fig. 2). The final to instilled 131I-labeled albumin concentration was highest in 22-day gestation rat fetuses, showing that 18 ± 8% of the instilled volume was removed from the distal air spaces (Fig. 2). In the 40-h-old rats, alveolar fluid clearance was 8 ± 2%, a level not different from that in the adult rat when extrapolated to 1 h (Fig. 2) (16 ± 2%) in the 40-h-old rats and compared with that in adult rats (17 ± 2%, data from Ref. 16).


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Fig. 2.   Distal air space epithelial fluid clearance over 30 min in developing rat fetuses after instillation of 5% albumin solution. Values are means ± SD. *P < 0.05 compared with 21-day gestation rat fetuses; dagger P < 0.05 compared with 40-h postnatal rats (ANOVA with Tukey's test as post hoc).

Sensitivity to Endogenous beta -Adrenergic Stimulation During Development

Circulating epinephrine levels were low in 20-day (276 ± 110 pg/ml) and 21-day (358 ± 102 pg/ml) gestation rat fetuses but significantly elevated in 22-day gestation (23,241 ± 10,494 pg/ml) rat fetuses (Fig. 3). In 40-h-old rats, even though the distal air space fluid clearance had tapered toward adult levels, circulating epinephrine levels remained high (13,998 ± 4,418 pg/ml).


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Fig. 3.   Plasma epinephrine levels in developing rat fetuses.

Because endogenous plasma epinephrine levels in near-term rat fetuses (22-day gestation) and newborn rats (40 h old) were high (Fig. 3) and alveolar fluid clearance in adult rats can be stimulated by exogenous administration of beta -adrenergic agonists (24), we studied whether distal air space fluid clearance is sensitive to inhibition by the beta -adrenergic antagonist propranolol and whether such a sensitivity varies with development and plasma epinephrine levels. Addition of propranolol significantly decreased the elevated distal air space fluid clearance in 22-day gestation rat fetuses and completely abolished fluid clearance in rat lungs at 40 h after birth (Fig. 4). Propranolol blocked distal air space fluid clearance only when plasma epinephrine levels were elevated.


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Fig. 4.   Distal air space fluid clearance over 30 min in developing rat fetuses after beta -adrenergic receptor inhibition with 10-4 M propranolol. Distal air space fluid clearance <0% corresponds to net secretion. For n values, see Specific Experimental Protocols. Values are means ± SD. *P < 0.05 compared with age-matched control fetuses (t-test).

Involvement Of Amiloride-Sensitive Na+ Channels During Development

We used amiloride to determine the contribution of the amiloride-sensitive Na+ channels for distal air space fluid clearance during late gestation and early postnatal development. There was no inhibition by amiloride (10-3 M) in 21-day gestation rat fetuses (Fig. 5). Addition of amiloride to the 131I-labeled 5% albumin instillate demonstrated a small, but not significant, inhibition by amiloride of distal air space fluid clearance in 22-day gestation rat fetuses, an inhibition that significantly increased in 40-h-old postnatal rats (Fig. 5).


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Fig. 5.   Distal air space fluid clearance over 30 min in developing rat fetuses after Na+ channel inhibition with 10-3 M amiloride. Distal air space fluid clearance <0% corresponds to net secretion. For n values, see Specific Experimental Protocols. Values are means ± SD. *P < 0.05 compared with age-matched control fetuses (t-test).

Extravascular Lung Water

Extravascular lung water was highest in the most premature rat fetuses and steadily decreased as term approached (Fig. 6). A further decrease in extravascular lung water was observed after birth in 40-h rats. However, extravascular lung water was still significantly higher in 40-h rats than in adult rats.


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Fig. 6.   Extravascular lung water in fetal, neonatal, and adult rats. For n values, see Specific Experimental Protocols. Values are means ± SD. All extravascular lung water values were significantly different from each other (P < 0.05; ANOVA with Tukey's test as post hoc).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Because the majority of infants make the transition from intrauterine life to postnatal life without complications, it is easy to forget that, only hours before birth, the lungs are filled with an essentially protein-free saltwater solution that is actively secreted by the lung's epithelium. Were this fluid not removed rapidly and effectively at birth, adequate oxygenation would be difficult to achieve. We therefore determined the rate of distal air space fluid clearance in developing rat fetuses and whether it varied with development near term. There were significant differences in the ability of the distal air space epithelium to clear excess fluid from the air spaces during late fetal and early postnatal life in the rat. One day before birth, at 21 days gestation, the fetal lung still secreted fluid into the air spaces. Distal air space fluid clearance then increased significantly over the next 12-24 h to reach a very high rate in 22-day gestation rat fetuses, a rate that rapidly decreased after birth and already reached adult levels 40 h after delivery. High endogenous plasma epinephrine levels seemed to mediate the high distal air space fluid clearance in 22-day gestation rat fetuses. Even though the rate of distal air space fluid clearance decreased over the first 40 h after birth, plasma epinephrine levels remained elevated. We measured extravascular lung water in littermates to rat fetuses that were used for the distal air space fluid clearance experiments. Extravascular lung water decreased toward the end of gestation and birth but remained significantly elevated in newborn rats compared with adult levels, suggesting that significant volumes of fetal lung fluid still were present in the developing postnatal rat lungs. Furthermore, distal air space fluid clearance became amiloride sensitive first at birth, although it reached a higher sensitivity at 40 h after birth.

What regulates distal air space fluid clearance at birth? In adult animals, it has been demonstrated that addition of exogenous beta -adrenergic agonists can stimulate reabsorption of fluid from the air spaces (4, 5, 24, 34). Also, endogenous epinephrine can stimulate reabsorption of fetal lung fluid near term (6, 7, 13, 14, 49). Endogenous epinephrine, levels of which increase in the blood circulation during labor and delivery (7), may play an important role in lung fluid clearance at birth (14). However, even though some data exist to support this hypothesis, there is little functional evidence linking endogenous epinephrine levels and fluid absorption rates in fetal rats. Therefore, the involvement of endogenous epinephrine in regulating distal air space fluid clearance at birth was determined by two methods. First, endogenous epinephrine plasma levels were measured in the developing rat fetuses, and we correlated these levels to the ability to clear excess distal air space fluid. Second, functional inhibition of beta -adrenergic receptor stimulation by addition of propranolol to the instilled fluid determined the extent of receptor stimulation. Endogenous plasma epinephrine levels were partly correlated with an increased ability of the lung to clear excess distal air space fluid as term approached. Endogenous epinephrine levels were significantly elevated in 22-day gestation rat fetuses, at the time when distal air space fluid clearance was the highest (Figs. 3 and 4). However, distal air space fluid clearance decreased rapidly during the first 40 h after birth, whereas epinephrine levels remained elevated, suggesting that other factors may be involved or that there is another reason why epinephrine levels remained high. As confirmation of the importance of epinephrine and beta -adrenergic receptors for clearance of fetal lung fluid at birth, distal air space fluid absorption in 22-day gestation fetal rat lungs and in 40-h postnatal rats was highly sensitive to propranolol-inhibition (Fig. 4). Thus propranolol inhibited distal air space fluid clearance only when endogenous plasma epinephrine levels were elevated, indicating that beta -adrenergic receptor stimulation was a major mechanism for clearing the fetal lung fluid from the distal air spaces of the lung.

Why would epinephrine levels in fetal plasma remain elevated 40 h after birth in rats? In guinea pigs, we found a decrease in plasma epinephrine concentrations immediately after birth that paralleled the decline in alveolar fluid clearance (14). In contrast, in rats the distal air space fluid clearance decreased immediately after birth, whereas no apparent decrease in plasma epinephrine levels was observed. Why? Guinea pigs are born with mature, fully alveolarized lungs (27), whereas rats are born with saccular lungs, and alveolarization primarily occurs postnatally (8). Less mature lungs may be expected to contain more extravascular lung water. As such, newborn rats may experience transient periods of hypoxia, situations known to release epinephrine (25, 44). In fact, our data show that extravascular lung water in the 40-h postnatal rats was significantly elevated compared with that in adult animals (Fig. 6). Extravascular lung water of 5.8 ± 0.6 g water/g dry lung in newborn 40-h rats is comparable with excess water present in the lungs. Although not measured in this study, this could result in a hypoxic situation for newborn 40-h rats, which in turn may result in increased plasma epinephrine levels and propranolol sensitivity even though these animals are fully air breathing. Another possibility may relate to postnatal development of rat lungs and differences in response to beta -adrenergic stimulation between airways and alveoli. The rat is born with a saccular lung, which likely would have a larger proportion of airway surface area compared with alveolar surface area (8). In adult animals, it has been shown that beta -adrenergic stimulation results in fluid secretion in airways (23) and increased fluid absorption in distal alveolar regions of the lung (31). If the surface area of the airways constitutes a proportionally larger fraction of the transporting surface area in newborn rats, it is plausible that secretory forces may counteract the otherwise expected elevation of distal air space fluid clearance at birth from the increased plasma epinephrine levels. Our data suggest, however, that in rats, epinephrine may not be the only factor responsible for stimulation of distal air space fluid clearance at birth. In fact, other studies have suggested that epinephrine is not the only factor in, or even necessary for, the clearance of fluid from the lungs at birth (11, 33). Although not studied here, plasma cortisol, thyroid hormone, and change in postnatal O2 levels may contribute to development of distal air space fluid clearance at birth in addition to the apparent role of epinephrine as has been implied in other studies (1, 3, 40).

What is the mechanism for clearing fetal lung fluid near term and at birth? In adult lungs, active Na+ transport through amiloride-sensitive epithelial Na+ channels (ENaC) is a key mechanism for creating a driving force for alveolar epithelial water reabsorption (for review see Refs. 29 and 31). Amiloride has been demonstrated to impair reabsorption of fetal lung fluid in several species (14, 38, 39). Little if any functional data are available on possible differences in sensitivity to amiloride inhibition during late gestation and postnatal lung development in rats. Therefore, we investigated whether inhibition of distal air space fluid clearance by amiloride differed at different developmental stages. Interestingly, the results showed that a significantly larger fraction of distal air space fluid clearance was inhibited by amiloride in newborn 40-h rats than in 21-day gestation and 22-day gestation fetal rats (Fig. 5). The alpha -ENaC subunit is found earliest at gestational day 19, whereas neither the beta - nor gamma -subunit is significantly expressed until or after birth in isolated distal lung epithelial cells and in whole lung preparations (47). The fact that ENaC requires all three subunits to become fully functional as a Na+ channel may explain the low amiloride sensitivity on the day of birth (22-day gestation rat fetuses) seen in our study and the greatly increased amiloride sensitivity after birth in 40-h-old rats. However, it is also possible that amiloride affects other sodium transporting pathways, even the basolateral Na+-K+-ATPase. If a significant amiloride inhibition of the Na+-K+-ATPase occurs here, an inhibition of the alveolar fluid clearance as early as fetal day 21 would be expected because Na+-K+-ATPase is expressed then (21). In summary, the rat data largely confirm the conclusion that ENaC may be responsible for a large fraction of distal air space fluid clearance at birth, becoming fully active in rats just after birth, and being already active before birth in guinea pigs (14) and perhaps also in human lungs (19, 45).

Our results also indicate that amiloride-insensitive pathways may mediate a fraction of the alveolar fluid clearance at birth in the rat. It was recently demonstrated that amiloride-insensitive Na+ transport was responsible for a large fraction of the alveolar fluid clearance observed after beta -adrenergic stimulation in the adult rat (36). This fluid clearance was likely to be mediated by the amiloride-insensitive cyclic nucleotide-gated (CNG) sodium channels, which have been shown to be present in the rat lung (12). However, the role for these channels at birth is unknown. In fetal sheep, it has been demonstrated that CNG channels lacked significance when the lungs were to be cleared of fetal lung fluid at birth (26). It is, however, possible that there exist species differences and that such channels may be active at birth in the rat.

A role for the water-only pathways, the aquaporins, in clearing the alveolar spaces from fluid at birth has been postulated, and experiments have been carried out to test this. A developmental expression pattern exists that might suggest a role for the aquaporins at birth (9, 50). However, more recent studies using transgenic mice suggest that aquaporins are of limited importance for the absorption of the fetal lung fluid at birth because genetically modified animals lacking these channels survive birth without respiratory complications (28, 46).

In conclusion, we found functional evidence in late-gestation fetal rat lungs for a stimulated distal air space fluid clearance that rapidly decreased toward adult levels within hours after birth. The elevated distal air space fluid clearance depended on elevations in plasma levels of epinephrine near term.


    ACKNOWLEDGEMENTS

This study was supported by National Heart, Lung, and Blood Institute Grant RO#1 HL-51854 and Program Project Grant HL-24075, the Swedish Natural Science Research Council, the Crafoord Foundation, and the Magnus Bergwall Foundation.


    FOOTNOTES

Address for reprint requests and other correspondence: H. G. Folkesson, Dept. of Physiology, Northeastern Ohio Univs. College of Medicine, 4209 State Rte. 44, P.O. Box 95, Rootstown, OH 44272-0095 (E-mail: hgfolkes{at}neoucom.edu).

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.

10.1152/ajplung.00183.2001

Received 25 May 2001; accepted in final form 3 October 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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