1 Department of Animal Physiology, Lund University, SE-223 62 Lund, Sweden; and 2 Department of Physiology, Northeastern Ohio Universities College of Medicine, Rootstown, Ohio 44272-0095
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ABSTRACT |
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We tested the hypothesis
that labor-induced epinephrine release would stimulate alveolar fluid
clearance in preterm fetuses. Preterm fetuses were obtained by cesarean
section from timed-pregnant guinea pigs at 61-69 days
postconception. Fetal guinea pigs were euthanized and placed on
continuous positive airway pressure oxygenation, and an isosmolar 5%
albumin solution was instilled. Alveolar fluid clearance was measured
over 1 h. The fetal lung began to absorb fluid at 64-66 days
postconception, and at birth, alveolar fluid clearance quadrupled.
Baseline alveolar fluid clearance when present was sensitive to
propranolol inhibition and depended on -adrenergic stimulation.
Measurements of plasma epinephrine in fetal animals confirmed high
epinephrine levels in 66- to 69-day postconception fetuses.
Prenatal alveolar fluid clearance when present was highly amiloride
sensitive, suggesting that amiloride-sensitive Na+ channels
were critical. Oxytocin-induced labor initiated an amiloride- and
propranolol-sensitive net alveolar fluid clearance in 61-day-gestation animals. Moreover, oxytocin induced significant epinephrine release in
all fetuses. These results have clinical implications for infants delivered by cesarean section before the onset of labor. Use of pharmacological agents to induce labor may reduce the occurrence and
severity of perinatal respiratory distress.
alveolar epithelium; -adrenergic stimulation; infant respiratory
distress syndrome; prenatal development; epinephrine; sodium
transport
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INTRODUCTION |
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NORMAL ALVEOLAR
DEVELOPMENT requires fluid-filled fetal lungs, and gas exchange
occurs through the placental cord (30). The lung fluid is
produced and secreted by the pulmonary epithelium by
Na+-coupled Cl secretion (41).
This fluid must be removed to allow gas exchange across the alveolar
epithelial-endothelial barrier at the transition from placental to
pulmonary gas exchange. Absorption of alveolar fluid is driven by
Na+ absorption in newborn (16) and adult
(32) guinea pig lungs. Transepithelial Na+
absorption is driven by the basolateral
Na+-K+-ATPase (15, 23) and is
dependent on apical Na+ channels (17, 22, 28,
29). Studies have suggested that fluid absorption occurs in
near-term lamb (6, 40), guinea pig (16, 34),
and rabbit (7) lungs. Na+ transport pathways
have been demonstrated to be developmentally regulated in other rodent
models of lung development (12, 33), whereas no functional
physiological studies of fluid transport in small rodents have been
carried out. Regulation of lung fluid transport during late prenatal
and early postnatal development is poorly understood, but several
hormones and other factors are proposed to be involved. Cortisol and
triiodothyronine (T3) stimulate lung fluid clearance in
lambs (3) and adult rats (18). Epinephrine rapidly increases alveolar fluid clearance in newborn guinea pig (16) and lamb (40) lungs.
Little is known about the signals that initiate fluid absorption in
fetal lungs. Thus our first aim was to investigate normal late-gestational development of alveolar fluid clearance in preterm guinea pigs. Fetal guinea pigs of gestational ages 61-69 days (term = 69 days) were obtained by cesarean section from
timed-pregnant guinea pigs, and alveolar fluid clearance was studied by
measuring the change in the concentration of an instilled albumin
solution over 1 h. Because epinephrine stimulates alveolar fluid
clearance at birth in guinea pigs (16), our second aim was
to investigate the time point when alveolar fluid clearance becomes
sensitive to endogenous -adrenergic stimulation as well as to
measure plasma epinephrine levels. Because labor releases
catecholamines from the adrenal glands (11) and
catecholamines are involved in the stimulation of alveolar fluid
clearance (16), our third aim was to investigate whether
preterm labor induced fluid absorption in fetal lungs. Preterm labor
was induced by injecting oxytocin into the timed-pregnant guinea pigs
followed by measurement of alveolar fluid clearance in the fetuses.
Plasma epinephrine levels were also measured after labor induction. The
acute effects on alveolar fluid clearance from oxytocin instilled into
the lungs were also investigated. A fourth aim was to study the
development of the fractional contribution of amiloride-sensitive
transport to alveolar fluid clearance under normal conditions and after premature induction of labor with oxytocin.
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MATERIALS AND METHODS |
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Animals and Oxytocin Pretreatment
Preterm and newborn Dunkin-Hartley guinea pigs (n = 251 from 60 litters; Sahlins Försöksdjursfarm, Malmö, Sweden) were used. The timed-pregnant guinea pigs were maintained on 12:12-h day-night rhythm and had free access to food (standard guinea pig chow, Special Diet Services, Witham, UK) and tap water. The Committee on Animal Experiments at Lund University (Lund, Sweden) approved this study.Preterm labor was induced by subcutaneous oxytocin injections (1 mg/kg body weight; Ferring, Malmö, Sweden) every 15 min for 45 min. The fetuses were delivered by cesarean section (see Surgical Preparation) after 45 min if a normal vaginal delivery did not occur. Timed-pregnant guinea pigs with 1 and 3 days gestation remaining usually delivered their fetuses vaginally within 45 min after oxytocin (68 days gestation, 100% vaginal delivery; 66 days gestation, 98% vaginal delivery), whereas fetuses of timed-pregnant guinea pigs with 5 and 8 days gestation remaining were delivered by cesarean section (64 days gestation, 98% cesarean section; 61 days gestation, 100% cesarean section). The oxytocin dose was adapted from the clinical dose for labor induction in humans (9). Separate sets of timed-pregnant guinea pigs (gestational ages 61 and 66 days) were injected with the vehicle (0.9% NaCl) used to dissolve oxytocin to control for effects from the injections per se. Timed-pregnant guinea pigs that were injected with vehicle or were not injected were all delivered by cesarean section at all gestational ages.
Solutions
A 5% albumin solution was prepared by dissolving 50 mg/ml of bovine serum albumin (Sigma, St. Louis, MO) in 0.9% NaCl. In some studies, the Na+ channel inhibitor amiloride (1 mM; Sigma) or the generalSurgical Preparation
The timed-pregnant guinea pigs were anesthetized by intraperitoneal injections of pentobarbital sodium (120 mg/kg body weight; Apoteksbolaget, Umeå, Sweden) and euthanized by intracardiac injections of 60 mg of pentobarbital sodium. A laparotomy was rapidly done, and the fetuses were carefully delivered. The umbilical cord was ligated to prevent bleeding. The fetuses were immediately euthanized with intraperitoneal pentobarbital sodium (12 mg) with 500 IU of heparin (Lövens, Ballerup, Denmark). Newborn guinea pigs were euthanized by intraperitoneal pentobarbital sodium (18 mg) with 500 IU of heparin.After euthanasia, an endotracheal tube (PE-190, Clay Adams, Becton Dickinson, Parsippany, NJ) was inserted through a tracheostomy. The animals were immediately connected to a constant oxygen flow (oxygen fraction 1.0; AGA, Lidingö, Sweden), and the lungs were expanded by adjusting the oxygen flow to a constant positive airway pressure (CPAP) of 5 cmH2O. The whole surgical procedure after euthanasia required 5 min. The animals were placed in-between heating pads to maintain body temperature during the experiments. A rectal temperature probe measured body temperature, and heating was adjusted to maintain the temperature at 37-38°C. Airway pressure was continuously monitored by calibrated pressure transducers (UFI model 1050B or TSD104A, BioPac Systems, Goleta, CA) and analog-to-digital converters and amplifiers (UIM100 and MP100, BioPac Systems).
Alveolar Fluid Clearance Experiments
After surgery and connection to the CPAP circuit, the albumin solution (10 ml/kg body weight) was instilled into the lungs through the endotracheal tube as follows. First, the animals were briefly disconnected from the CPAP circuit, and the lungs were deflated by gently aspirating residual air with the instillation syringe. The instillation solution was then instilled into the lungs and withdrawn again. This procedure was repeated four times to allow thorough and adequate mixing of instillate and preexisting fetal lung fluid, and the fluid was finally instilled. Then the animals were reconnected to the CPAP circuit and maintained on CPAP for the 1-h study period. A 0.1-ml sample of the instillation solution-lung fluid mixture (initial solution) remained in the syringe for protein measurement. After 1 h, the lungs and heart were carefully removed en bloc through a midline sternotomy, and a sample of the remaining alveolar fluid was collected. Total protein concentrations in the instilled, initial, and final solutions were determined spectrophotometrically (iEMS Reader MF, Labsystems, Helsinki, Finland) by the Lowry (27) method adapted for microtiter plates.Alveolar fluid clearance or alveolar fluid secretion was calculated
from the change in protein concentration over 1 h. This is
possible because the alveolar epithelium is relatively impermeable to
large molecules such as albumin (mol wt 67,000). Therefore, water
movement (absorption or secretion) will result in a change in airspace
protein concentration. Because the fetal lung is fluid filled in utero
(6, 7, 17), we expected that a certain fraction of fluid
would still be present in the lungs at the time of experiment. This
fluid is virtually free of protein and will not add protein to the
instilled albumin concentration. In contrast, it will dilute the
protein concentration in the instillates and thereby influence the
calculation of alveolar fluid clearance differently depending on the
volume of fluid present at the different developmental stages. To
control for this factor, we instilled the guinea pig fetuses as
described above with 10 ml/kg body weight of the 5% albumin
instillate, and the fluid was aspirated and reintroduced four times
before a final 0.1-ml sample was taken, the rest was instilled, and the
animal was studied for 1 h. The whole procedure required
~1-2 min. During this time, it was unlikely that a significant
quantity of protein left or entered the airspaces or that significant
volumes of fluid were reabsorbed from or secreted into the airspaces.
Therefore, any change in protein concentration would represent dilution
by preexisting fluid. The preexisting fluid volume calculated from
Eqs. 1 and 2 was used to correct the instilled
protein concentrations by the dilution of the instillate that would
occur if there was fluid already in the lung before instillation of the
5% albumin solution. The preexisting fluid volume (Vpre)
was corrected for body weight of the fetus and is expressed as
milligrams per kilogram of body weight in RESULTS. Alveolar
fluid clearance (AFC) or alveolar fluid secretion (AFS) was then
calculated from Eqs. 3 and 4
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(1) |
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(2) |
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(3) |
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(4) |
To be certain that alveolar fluid clearance proceeded at similar rates in our in situ lungs from the euthanized animals as in the in vivo lungs, we ventilated anesthetized newborn and adult guinea pigs according to previous protocols (16, 32) in parallel to the in situ CPAP studies. Alveolar fluid clearance in the ventilated and the in situ CPAP animals was not significantly different (data not shown).
Specific Protocols
Guinea pig fetuses of gestational ages 61, 64, 66, 68, and 69 (term) days were studied. Adult animals were studied for reference. The day of conception was set to the day when the timed-pregnant guinea pigs gave birth to their earlier litter because guinea pigs enter estrus immediately after birth. All groups contained fetuses from at least two litters.Control studies. Preterm guinea pig fetuses and newborn guinea pigs of 61 (n = 8), 64 (n = 7), 66 (n = 7), 68 (n = 8), and 69 (n = 11) days gestation were delivered from the timed-pregnant guinea pigs and surgically prepared as described in Surgical Preparation. The 5% albumin solution was instilled, and the fetuses were maintained on 5 cmH2O CPAP for 1 h. The lungs were removed, and a sample of the remaining alveolar fluid was collected. Protein concentrations were measured, and alveolar fluid clearance was calculated. A group of adult animals (n = 3) was studied for reference and to verify the CPAP method.
Oxytocin studies. Guinea pig fetuses of 61 (n = 6), 64 (n = 9), 66 (n = 6), and 68 (n = 9) days gestation were delivered from the oxytocin-injected timed-pregnant guinea pigs and were surgically prepared as described in Surgical Preparation. Labor was induced by giving the timed-pregnant guinea pigs oxytocin (1.0 mg/kg body weight) every 15 min for 45 min. The fetuses were delivered vaginally or surgically after a maximum of 45 min of oxytocin treatment. Separate sets of guinea pigs at gestational ages of 61 (n = 4) and 66 (n = 7) days were injected with vehicle (0.9% NaCl) alone every 15 min for 45 min to control for possible stress from the injections per se. The 5% albumin solution was instilled, and the fetuses were maintained on 5 cmH2O CPAP for 1 h. The lungs were removed, and a sample of the remaining alveolar fluid was collected. Protein concentrations were measured, and alveolar fluid clearance was calculated.
Acute effects of oxytocin.
Guinea pig fetuses of 61 (n = 3), 64 (n = 4), 66 (n = 5), and 68 (n = 4) days
gestation were delivered from the NaCl-injected timed-pregnant guinea
pigs and surgically prepared as described in Surgical
Preparation. The 5% albumin solution containing 104
M oxytocin was instilled, and the fetuses were maintained on 5 cmH2O CPAP for 1 h. The lungs were removed, and a
sample of the remaining alveolar fluid was collected. Protein
concentrations were measured, and alveolar fluid clearance was calculated.
Propranolol studies.
Guinea pig fetuses of 61 (n = 5), 64 (n = 5), 66 (n = 6), 68 (n = 4), and 69 (term; n = 4) days gestation were delivered from the
timed-pregnant guinea pigs and surgically prepared as described in
Surgical Preparation. Another set of guinea pig fetuses of 61 (n = 5), 64 (n = 6), 66 (n = 5), and 68 (n = 7) days gestation from the oxytocin-injected timed-pregnant guinea pigs was also prepared. Labor was induced by giving the timed-pregnant guinea pigs
oxytocin (1.0 mg/kg body weight) every 15 min for 45 min. The fetuses
were delivered vaginally or surgically after a maximum of 45 min of
oxytocin treatment. The 5% albumin solution containing 0.1 mM
propranolol (the general -adrenergic antagonist) was instilled, and
the fetuses were maintained on 5 cmH2O CPAP for 1 h.
The lungs were removed, and a sample of the remaining alveolar fluid
was collected. Protein concentrations were measured, and alveolar fluid
clearance was calculated.
Amiloride studies. Guinea pig fetuses of 61 (n = 6), 64 (n = 6), 66 (n = 6), 68 (n = 9), and 69 (term; n = 10) days gestation were delivered from the timed-pregnant guinea pigs and surgically prepared as described in Surgical Preparation. Another set of guinea pig fetuses of 61 (n = 5), 64 (n = 6), 66 (n = 6), and 68 (n = 6) days gestation from the oxytocin-injected timed-pregnant guinea pigs was also prepared. Labor was induced by giving the timed-pregnant guinea pigs oxytocin (1.0 mg/kg body weight) every 15 min for 45 min. The fetuses were delivered vaginally or surgically after a maximum of 45 min of oxytocin treatment. The 5% albumin solution containing 1 mM amiloride (Na+ channel inhibitor) was instilled, and the fetuses were maintained on 5 cmH2O CPAP for 1 h. Amiloride at a concentration of 1 mM was used because a large fraction becomes protein bound and another significant fraction rapidly leaves the airspaces due to its low molecular weight (34, 42); therefore, the active concentration in the alveoli was probably lower. Also, the same amiloride concentration has been used in several earlier studies of alveolar fluid clearance (16, 25, 32, 38). The lungs were removed, and a sample of the remaining alveolar fluid was collected. Protein concentrations were measured, and alveolar fluid clearance was calculated.
Measurement of Plasma Epinephrine
Plasma was collected from parallel groups of guinea pigs at all gestational ages (n = 4-5 in each experimental group) with and without oxytocin induction of premature labor. The plasma was immediately frozen in liquid nitrogen after centrifugation (3,000 g for 5 min) and then stored atStatistics
Values are presented as means ± SD. Statistical analysis was carried out with one-way analysis of variance (ANOVA) with Tukey's test post hoc or Student's t-test when appropriate. Differences were considered significant with P < 0.05. ![]() |
RESULTS |
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Alveolar Fluid Volume in Developing Fetal Lungs
Because the lungs are fluid filled in utero, the presence of preexisting fluid in the airspaces of the lungs had to be taken into account in these experiments. This was done by measuring the dilution of the instilled 5% albumin solution after the mixing and immediate retrieval of the instillate-lung fluid mixture. Because the preexisting lung fluid would be protein free (6, 7, 17), any dilution would represent a volume of preexisting alveolar fluid (16). We found, as expected, that the most immature lungs (61 days gestation) contained the highest volume of preexisting lung fluid (Fig. 1). There was no detectable preexisting fluid in the adult lungs. In the subsequent experiments, each individual animal served as its own internal control with regard to the preexisting alveolar fluid volume.
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Late-Gestational Development
Guinea pig fetuses of several gestational ages (61-69 days) were instilled with the 5% albumin solution, and alveolar fluid clearance was studied over 1 h. A fluid sample from the distal airspaces was collected by aspiration of the remaining alveolar fluid at the end of the 1-h experiment, and the alveolar fluid clearance or secretion was calculated from the increase or decrease, respectively, in the alveolar protein concentration. At 61 days from conception, the lungs were secreting fluid into the airspaces (Fig. 2). On gestational day 64, there was no net secretion or net absorption of lung fluid. From gestational day 64 to day 68, the fluid absorption rate slowly increased, and there was a marked increase between the last gestational day and the day of birth.
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Oxytocin-Induced Premature Labor
Timed-pregnant guinea pigs at different times postconception had labor induced prematurely by sequential oxytocin injections. As in the control studies, the guinea pig fetuses (61-68 days gestation) from the oxytocin-injected timed-pregnant guinea pigs were instilled with the 5% albumin solution, and alveolar fluid clearance was studied over 1 h. Premature onset of labor induced significant alveolar fluid clearance in guinea pig fetuses on gestational days 61 and 64 (Fig. 3). The onset of premature labor on gestational days 66 and 68 (when the lung already absorbed alveolar fluid) did not further stimulate alveolar fluid clearance, although there was a tendency for an increase in alveolar fluid clearance. A separate set of timed-pregnant control guinea pigs (gestational ages 61 and 66 days) were injected with the vehicle (0.9% NaCl) for oxytocin alone to study whether the injections per se induced stress hormone release and thereby affected the results from the oxytocin injections. There were no differences in alveolar fluid clearance between the fetuses from vehicle-injected guinea pigs and the fetuses from noninjected control animals (Table 1).
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Acute Effects From Oxytocin Instillation
Timed-pregnant guinea pigs at different times postconception were obtained, the guinea pig fetuses (61-68 days gestation) from the timed-pregnant guinea pigs were instilled with the 5% albumin solution containing 10
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Endogenous -Adrenergic Stimulation
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Alveolar fluid clearance after the oxytocin-induced labor was also
investigated by the use of propranolol inhibition. Induction of
premature labor at 61 and 64 days gestation with oxytocin induced propranolol-sensitive alveolar fluid clearance in those fetuses (Fig.
6). At both time points, propranolol
reversed alveolar fluid clearance to secretion after the
oxytocin-induced labor. One day before birth (day 68),
propranolol inhibited the oxytocin-induced alveolar fluid clearance by
76 ± 47%, in contrast to control conditions when propranolol
reversed alveolar fluid clearance to secretion.
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Plasma Epinephrine Levels
Plasma epinephrine levels were measured in guinea pigs of all ages with and without maternal oxytocin-induced premature labor. In normal fetuses, plasma epinephrine levels were low at 61 and 64 days gestation and began to increase at 66 days gestation, reaching maximal levels at the time of birth (69 days gestation; Fig. 7A). Concomitantly with the increased plasma epinephrine levels, there was an increase in alveolar fluid clearance (Fig. 7B). Maternal oxytocin treatment increased plasma epinephrine levels significantly in all guinea pig fetuses, although it was most evident in fetuses of 61 and 64 days gestation (Fig. 7A), paralleling the induction of alveolar fluid clearance in these age groups. In the 66- and the 68-day-gestation age groups, maternal oxytocin treatment did not significantly increase alveolar fluid clearance.
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Plasma epinephrine levels were also measured in the maternal blood with and without oxytocin injections. The plasma epinephrine levels were low in all control timed-pregnant guinea pigs. Induction of premature labor by oxytocin significantly increased plasma epinephrine levels in the maternal blood at all age groups studied (data not shown).
Fractional Amiloride Inhibition
The fractional amiloride inhibition of alveolar fluid clearance was investigated in the developing fetal guinea pigs. The guinea pig fetuses (61-69 days gestation) from the timed-pregnant guinea pigs were instilled with the 5% albumin solution containing 1 mM amiloride, and alveolar fluid clearance was studied over 1 h. At 61 days gestation, amiloride had no effect on the fluid secretion. However, from gestational days 64 to 68, amiloride completely inhibited the alveolar fluid clearance and even reversed net fluid absorption to net secretion in the fetuses (Fig. 8). In newborn guinea pigs, amiloride sensitivity decreased, and amiloride now inhibited the alveolar fluid clearance by 71 ± 13%.
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Fractional amiloride inhibition was also investigated after
oxytocin-induced labor in the timed-pregnant guinea pigs. The guinea
pig fetuses (61-68 days gestation) from the oxytocin-injected timed-pregnant guinea pigs were instilled with the 5% albumin solution
containing 1 mM amiloride, and alveolar fluid clearance was studied
over 1 h. Under these conditions, amiloride inhibited the
oxytocin-induced alveolar fluid clearance in the 61-day fetuses completely (Fig. 9). Amiloride also
inhibited alveolar fluid clearance in the fetuses at all other
gestational ages after maternal oxytocin-induced labor, in contrast to
control fetuses in which amiloride was only effective from 64 days
gestation (Fig. 9).
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DISCUSSION |
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We found remarkable differences in the capacity of the alveolar
epithelium to remove excess fluid from the airspaces during late-gestational development in guinea pig fetuses. What mechanisms initiate and regulate alveolar fluid clearance near term? In fetal and
adult animals, the addition of exogenous -adrenergic agonists stimulates alveolar fluid reabsorption (4, 17, 19, 29, 32). Plasma catecholamine levels increase during labor and
delivery (11, 16) and may be important for stimulating
lung fluid clearance at birth. We found the same plasma epinephrine
levels in the newborn animals in this study as in the earlier in vivo
study on ventilated newborn guinea pigs (16). However,
although some data are available, there is a lack of conclusive
functional evidence that links endogenous
-adrenergic stimulation
and alveolar fluid absorption in the preterm animals. In these studies,
we found propranolol-sensitive alveolar fluid clearance in animals of
64 days gestation and older. These results indicated that this level of
alveolar fluid clearance was maintained by endogenous
-adrenergic
stimulation, probably from epinephrine. This finding was also confirmed
by plasma epinephrine measurements where plasma epinephrine levels were
clearly detectable in fetuses older than 64 gestational days. Also,
plasma epinephrine levels increased sharply toward term and peaked at
birth. Could this have been due to an increase in receptor number
during late gestation or to increased
-adrenergic stimulation? The
small increase in the number of
-adrenergic receptors in developing guinea pig lungs can be explained by the increased alveolar
surface area (20), and thus our results cannot solely be
explained by changes in receptor number. Instead, it is more likely
that alveolar fluid clearance was elevated by increased receptor stimulation.
Can preterm endogenous epinephrine release and -adrenergic
stimulation induce the conversion from chloride and fluid secretion to
sodium and fluid absorption? Enhorning et al. (14)
discovered that injection of
-adrenergic agonists into pregnant
rabbits reduced fetal lung water. Intravenous epinephrine decreased
lung fluid secretion in late-gestational fetal sheep, an effect that was inhibited by
-adrenergic antagonists (40). A
positive correlation between plasma epinephrine levels and reabsorption
of fetal lung fluid has been demonstrated in the near-term lamb
(10) and the newborn guinea pig (16). In this
study, endogenous
-adrenergic stimulation increased as term
approached simultaneously with the appearance of amiloride-sensitive
alveolar fluid clearance. Paralleling the appearance of
-adrenergic
stimulation of alveolar fluid clearance, plasma epinephrine levels
increased, suggesting that this catecholamine was important for the
conversion of the alveolar epithelium from secretion to absorption in
the late-gestational lungs. The Na+ channel inhibitor
amiloride blocked a large fraction of the epinephrine-stimulated alveolar fluid reabsorption in the fetal lungs in both these studies and other investigations (16, 36), an inhibition that was reduced later in life (16, 32). This result suggested that epinephrine stimulated Na+ transport via
amiloride-sensitive Na+ channels primarily in the near-term
fetal but also in the newborn lungs.
Oxytocin-induced premature labor initiated alveolar fluid clearance in
preterm lungs at gestational ages in which secretion dominated. In
these studies, the oxytocin-induced alveolar fluid clearance was
completely inhibited by the addition of the -adrenergic inhibitor
propranolol, providing evidence that the oxytocin-induced alveolar
fluid clearance occurred by endogenous
-receptor stimulation. The
measurements of plasma epinephrine in the fetuses confirmed and
extended the interpretation that endogenous plasma epinephrine was the
main mechanism after oxytocin induction of alveolar fluid clearance.
However, oxytocin induction of labor had no effect on alveolar fluid
clearance at gestational days 66 and 68. At that
time, endogenous plasma epinephrine levels already had started to
increase and stimulate fluid absorption, and a further increase in
epinephrine plasma concentration appeared to have little effect on the
epinephrine-stimulated alveolar fluid clearance in these animals.
Similar results were observed in the earlier study by Finley et al.
(16) on newborn guinea pigs where exogenous
epinephrine failed to stimulate alveolar fluid clearance when there was
endogenous epinephrine stimulation. Another possible explanation is
that other stress hormones, i.e., cortisol, were released at these gestational ages and contributed to the endogenous stimulation of fluid
absorption in those animals. Plasma cortisol levels have been shown to
begin to rise 3 days before term in the guinea pig (1).
Because labor releases epinephrine (11) and rabbits born
after the onset of labor had less lung water than rabbits delivered by
cesarean section before the onset of labor (5), it is
likely that endogenous epinephrine released during the induced premature labor mediated the induction of alveolar fluid clearance at
gestational ages when fluid secretion predominated. Secretion of fluid
into the tracheae of fetal lambs and guinea pigs decreases before birth
(13, 26, 37) simultaneously with the appearance of
endogenous catecholamines and stress hormones.
During labor, not only is epinephrine released, but other hormones are released that could have potential effects on fluid movements across the alveolar epithelium. These hormones include cortisol, thyroid hormones, and prostaglandins. It has been shown that simultaneous pretreatment with thyroid hormones and glucocorticoid hormones synergistically promotes maturation of epinephrine-induced stimulation of amiloride-sensitive fluid absorption in the near-term fetal sheep lung (2). In the adult rat lung, pretreatment with dexamethasone and thyroid hormones upregulates alveolar fluid clearance via additive pathways (18). Also, in the adult guinea pig, endogenous cortisol maintains normal alveolar fluid clearance (31), and it was suggested that late-gestational expression of the epithelial Na+ channel was controlled by endogenous cortisol (1). These hormones might thus be involved in the normal maturation of fluid absorption as gestation proceeds to term. However, most of the effects from cortisol are mediated through protein synthesis, and the time frames of induction or stimulation (45 min) of alveolar fluid clearance in this study after oxytocin-induced labor are too short to be explained by regulation with cortisol alone.
Could oxytocin itself induce alveolar fluid clearance in the fetal guinea pigs? To answer this question, we carried out experiments where oxytocin was instilled directly into the fetal lungs. We found no induction of alveolar fluid clearance at any developmental stage. In contrast, oxytocin seemed to lower alveolar fluid clearance in the animals at the developmental stages where the lung could already clear alveolar fluid. One way of interpreting these data is that injected oxytocin increases alveolar fluid clearance via epinephrine secretion, whereas, although not studied here, instilled oxytocin has a direct inhibitory effect on clearance. Also, the concentration of oxytocin instilled was likely severalfold higher than that which could be possible if any oxytocin could cross the placenta to the fetuses. Thus it is unlikely that a direct effect of oxytocin is mediating the induction of alveolar fluid clearance seen after oxytocin injections at the gestational ages of 61 and 64 days.
To control for the effect of the injections alone, which may cause stress and discomfort to the animal, we injected control animals with the vehicle (0.9% NaCl) for oxytocin on the same schedule as oxytocin. There were no effects from the injections per se compared with noninjected control animals. Thus the induction of alveolar fluid clearance seen after the oxytocin injections must have been due to endogenous epinephrine release caused by oxytocin-induced labor.
There were different degrees of preexisting alveolar fluid in the lungs. The highest preexisting fluid volume was, as expected, observed in the least developed fetuses, i.e., the 61-day fetuses. The fluid volume then significantly decreased between gestational days 61 and 64, likely due to a decreased secretory rate of fluid into the alveolar spaces. Fluid secretion has been shown to decrease before birth in other animal species (26). Then alveolar fluid volume again started to decrease between gestational days 66 and 69 (term), concomitantly with the appearance of amiloride-sensitive Na+ transport and fluid absorptive capacities.
What is the driving force for the clearance of fetal lung fluid as term approaches? In adult lungs, Na+ transport through amiloride-sensitive and amiloride-insensitive Na+ channels is a key mechanism driving alveolar epithelial fluid reabsorption (for reviews, see Refs. 28, 29). In fetal and newborn guinea pig lungs, amiloride impairs reabsorption of fetal lung fluid to different degrees (16, 34, 36). However, few, if any, functional data link the possible differences in amiloride sensitivity to prenatal lung development. We expected that an amiloride-sensitive Na+ transport across the alveolar epithelium would be present whenever alveolar fluid clearance was evident in the developing fetal lungs. Our results indicate, in fact, that amiloride-sensitive pathways driving water reabsorption in the developing fetal guinea pig lungs first appeared around 64 days gestation, the time when secretion and absorption matched each other, so no net fluid movement across the alveolar epithelium occurred. Amiloride-sensitive fluid transport then increased as term approached.
Amiloride does not inhibit the same fraction of alveolar fluid
clearance in adult lungs after -adrenergic agonist stimulation or
stimulation by other factors (19, 32, 39) as that in the
fetal and developing newborn guinea pig lungs (16). Thus the increased amiloride sensitivity as term approaches cannot solely be
a result of the stimulation of alveolar fluid clearance from the
elevated endogenous epinephrine levels but also a result from more
Na+ transporting pathways in the alveolar epithelial cell
membranes and thus an increased amiloride-sensitive Na+
transport capacity. An increased expression and/or function of the
amiloride-sensitive epithelial Na+ channels could be
responsible for the elevated alveolar fluid clearance as term
approaches. In fact, Finley et al. (16) recently reported
that the
-subunit of the epithelial Na+ channel (ENaC)
mRNA expression was increased in the newborn guinea pig lung. Isolated
rat fetal distal lung epithelial cell studies suggested that
terbutaline promoted the trafficking of amiloride-sensitive cation
channels to the apical cell membranes (24). In a study set
up to investigate whether premature delivery by cesarean section would
affect expression and function of ENaC, Baines et al. (1) delivered guinea pigs before term and measured
-ENaC and
-ENaC expression postnatally. The expression data were well correlated with
the ability of the lung to reabsorb fluid that had been instilled into
the distal airspaces of prematurely delivered animals. In conclusion,
although terbutaline and other
-adrenergic agonists, e.g.,
epinephrine, will increase channel activity, the increased amiloride
sensitivity in the developing lungs is more likely due to an increased
synthesis and insertion of Na+ channels into the epithelial
cell membranes.
Vectorial transport of ions requires an entry step at the apical cell membrane and an exit step at the basolateral cell membrane. In the alveolar epithelium, the entry step is ENaCs and the exit step is basolateral Na+-K+-ATPase. In this study, we focused our attention on the apical amiloride-sensitive ENaCs and their role in driving alveolar fluid clearance in the fetal lung during normal development and after labor induction. It is likely that both the apical entry step (Na+ channels) and the basolateral exit step (Na+-K+-ATPase) are regulated in parallel to be able to accommodate the observed changes in fluid transport across the alveolar epithelium. Although not studied here, developmental changes in lung epithelial Na+-K+-ATPase levels and activity have been investigated in the chase for the mechanism responsible for clearing the developing airspaces from fluid at birth. Several studies (8, 12, 23) suggest that events associated with labor may stimulate the basolaterally located Na+-K+-ATPase in lung epithelial cells, contributing to the alveolar fluid volume decline seen at birth. The main findings suggest that pump activity increases at birth, whereas the number of Na+-K+-ATPase pumps increases after birth. Those findings go well with our findings of the changes in Na+ transport and fluid absorption rate in the late-gestation guinea pigs.
Did the induction of premature labor affect amiloride sensitivity?
Oxytocin already induced alveolar fluid clearance in fetuses at 61 days
gestation when there was net fluid secretion in age-matched control
fetuses. This induction of clearance resulted from endogenous -adrenergic stimulation by labor-induced epinephrine release because
the induced alveolar fluid clearance was completely inhibited by
propranolol and plasma epinephrine levels were significantly increased.
Moreover, the oxytocin-induced alveolar fluid clearance was also
completely blocked by amiloride, an inhibition that was not present in
normal age-matched control fetuses. Also, to functionally upregulate
fluid absorption across the alveolar epithelium, both the
Na+ channel number or activity and the
Na+-K+-ATPase number or activity have to be
stimulated. Thus oxytocin-induced labor initiated an insertion or an
activation or opening of functional Na+ channels and/or
Na+-K+-ATPases in the fetal epithelial cell
membranes, giving the fetal alveolar epithelium absorptive
characteristics. It is unlikely that significant new protein synthesis
took place because the time after oxytocin induction and the alveolar
fluid clearance experiments was too short (<90 min). Thus oxytocin
induction of labor induced the fetal lung to acquire adult
fluid-absorptive characteristics and become better prepared for the
air-breathing postnatal life.
The conversion by oxytocin from secretion to absorption in the fetal lungs may have significant clinical implications because babies that are prematurely delivered by cesarean section before the onset of labor may develop respiratory distress (21). This distress may result, in part, from an immature alveolar epithelium with a limited fluid absorption capacity in conjunction with a deficient surfactant secretion (35). In a clinical study of the influence of labor and route of delivery on respiratory morbidity in the neonate, it was demonstrated that a cesarean delivery after labor had started drastically reduced the respiratory morbidity in the neonate (21). Although no direct mechanism was proposed in that study, it is tempting to speculate that factors released during labor matured the lung fluid-absorptive capacity and perhaps surfactant release and production. If this alveolar fluid-absorptive capacity can be induced either permanently or transiently, respiratory distress may be less and discomfort for the newborn less severe. Also, because the alveolar epithelium acquired the capacity of fluid absorption after oxytocin-induced labor, the fetal lung seems to be more mature and ready for the postnatal life. Fetuses born or delivered by cesarean section from the oxytocin-injected timed-pregnant guinea pigs always seemed to be stronger and more alert in their general appearance compared with age-matched control fetuses.
Functional evidence was found for stimulated alveolar fluid clearance
in late-gestational guinea pig fetal lungs that rapidly increased
during the last gestational day. This elevated alveolar fluid clearance
depended on endogenous -adrenergic stimulation from labor-released
epinephrine. The ability to clear fluid from distal lung airspaces
correlated with the appearance of amiloride sensitivity.
Oxytocin-induced labor resulted in the induction of both propranolol-
and amiloride-sensitive fluid absorption in the fetal lungs at
gestational ages (61 days) when fluid secretion predominated in the
age-matched control fetuses. It was also demonstrated that oxytocin
induction of alveolar fluid clearance in the fetal animals relied on
endogenous epinephrine release. These results suggest that the
induction of premature labor may better prepare the prematurely
delivered babies for the air-breathing life and prevent neonatal
respiratory distress.
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ACKNOWLEDGEMENTS |
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We thank Dr. Michael A. Matthay (Department of Medicine and Cardiovascular Research Institute, University of California, San Francisco) for carefully reviewing the manuscript.
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FOOTNOTES |
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This work was supported by grants from the Swedish Natural Science Research Council, the Crafoord Foundation, the Royal Physiographic Society in Lund, the Magnus Bergwall Foundation, and the Hierta Retzius Foundation.
Address for reprint requests and other correspondence: H. G. Folkesson, Dept. of Physiology, Northeastern Ohio Universities College of Medicine, 4209 State Route 44, PO 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.
Received 12 January 2000; accepted in final form 25 July 2000.
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