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
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We
investigated the importance of changes in intracellular
Ca2+ concentration ([Ca2+]i) for
amiloride-sensitive alveolar fluid clearance (AFC) in late-gestational
guinea pigs. Fetal guinea pigs of 61, 68, and 69 days (term) gestation
were investigated under normal conditions and after oxytocin-induced
preterm labor. AFC or alveolar fluid secretion was measured using an
impermeable tracer technique. At 61 days gestation there was net
secretion of fluid into the lungs, and at birth the lungs cleared
49 ± 7% of the instilled fluid volume over 1 h. Induction
of preterm labor with oxytocin induced AFC at 61 days gestation. When
present, AFC was inhibited or reversed to net fluid secretion by
amiloride (103 M). Inhibition of membrane
Ca2+ channels by verapamil (10
4 M) or
depletion of intracellular Ca2+ by thapsigargin
(10
5 M) reduced AFC when net AFC was evident. Amiloride
lacked an inhibitory effect on AFC when instilled with verapamil or
thapsigargin. The results indicate that AFC via amiloride-sensitive
pathways develops during late gestation, and that inducing preterm
labor precociously may activate such pathways. Our results suggest that Ca2+ may act as a second messenger in mediating
catecholamine-stimulated AFC.
amiloride; birth; sodium transport; oxytocin-induced preterm labor
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
IN UTERO, THE LUNGS SECRETE FLUID into the developing air spaces. This fluid secretion is necessary for development of future alveolar spaces (3, 24). At birth, the alveolar fluid must be removed to allow pulmonary gas exchange. Consequently, lung fluid secretion is reduced as full term approaches (2, 19), and at birth the alveolar fluid clearance (AFC) is rapid (4, 5, 19). The increased AFC rate after spontaneous labor (4, 19) or oxytocin-induced preterm labor (19) is stimulated by labor-released epinephrine. Epinephrine stimulation of AFC is at least partly mediated by cAMP, which acts as an intracellular second messenger (5, 17, 30). It has also been demonstrated that intracellular Ca2+ concentration ([Ca2+]i) is increased after terbutaline stimulation (14, 17, 28). In addition, a significant fraction of stimulated transepithelial Na+ transport may depend on increased [Ca2+]i (12, 13, 28).
AFC is secondary to Na+ absorption across the alveolar epithelium. Na+ enters through apical Na+ channels in the alveolar epithelial type II cells and is extruded basolaterally by the Na+-K+-ATPase (for reviews, see Refs. 15 and 16). A significant fraction of AFC is mediated through amiloride-sensitive pathways in fetal (19, 23) and newborn (5) animals. The epithelial Na+ channel (ENaC), which is amiloride sensitive, has been proposed as one pathway for Na+ absorption in near-term or newborn animals (1, 5, 32). Upregulation of the basolateral Na+-K+-ATPase may be involved in emptying the fetal air spaces of fluid (6).
Because evidence is present that epinephrine stimulation of AFC may
depend on changes in [Ca2+]i (14, 17,
28), we hypothesized that increased
[Ca2+]i would lead to an increased AFC in
near-term fetal guinea pigs. We therefore attempted to manipulate
[Ca2+]i by blocking Ca2+ channels
that could regulate [Ca2+]i by changing the
exchange of Ca2+ with the external microenvironment.
The first aim of this study was to investigate the role of
extracellular Ca2+ influx for -adrenergic stimulation of
AFC by inhibiting membrane Ca2+ channels in normal fetuses
and in age-matched fetuses after oxytocin-induced preterm labor. The
second purpose was to investigate the effect of
Ca2+-channel inhibition on the amiloride-sensitive fraction
of AFC during normal conditions and after oxytocin-induced preterm
labor. The third goal was to investigate the effects of disrupting the intracellular Ca2+ balance by inhibiting Ca2+
reuptake by the sarcoplasmatic Ca2+-ATPase (SERCA)
using thapsigargin. This eventually results in a depletion of
intracellular Ca2+ thus lowering the
[Ca2+]i. We investigated thapsigargin
inhibition during normal conditions and after oxytocin-induced preterm
labor with and without amiloride inhibition of the AFC.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals and Oxytocin Pretreatment
Fetuses obtained from timed-pregnant Dunkin-Hartley guinea pigs (Sahlins Försöksdjursfarm, Malmö, Sweden) were used for the experiments (n = 165 animals from 52 litters). The timed-pregnant guinea pigs were maintained on a 12:12-h day-night rhythm and had free access to food (standard guinea pig chow; SDS, Witham, Essex, UK) and tap water. The Committee on Animal Experiments at Lund University approved this study.Preterm labor was induced as we have done before (19) by subcutaneous injection of oxytocin (1 mg/kg body wt, which is equal to 1 IU/kg body wt; Ferring, Malmö, Sweden) every 15 min for 45 min. Fetuses were delivered by abdominal hysterectomy (see Surgical Preparations) after 45 min if normal vaginal delivery did not occur within that time. Timed-pregnant guinea pigs with 1 day of gestation remaining delivered their fetuses vaginally within 45 min after oxytocin administration, whereas fetuses of timed-pregnant guinea pigs with 8 days gestation remaining were delivered by abdominal hysterectomy.
Solutions
An isosmolar 5% albumin solution was prepared by dissolving 50 mg/ml bovine serum albumin (Sigma, St. Louis, MO) in 0.9% NaCl. In the studies of the role of extracellular Ca2+ influx, the L-type Ca2+-channel inhibitor verapamil hydrochloride (10Surgical Preparations
The timed-pregnant guinea pigs were deeply anesthetized by injections of pentobarbital sodium (120 mg/kg body wt ip; Apoteksbolaget, Umeå, Sweden) and euthanized by intracardiac injections of 60 mg of pentobarbital sodium. A laparotomy was performed, and fetuses were carefully delivered. The fetal umbilical cord was ligated to prevent bleeding. The fetuses were immediately euthanized by 12 mg ip of pentobarbital sodium with 500 IU of heparin (Lövens, Ballerup, Denmark). Newborn guinea pigs were euthanized by 18 mg ip of pentobarbital sodium with 500 IU of heparin.After euthanasia, an endotracheal tube (PE-190; Clay Adams, Becton Dickinson, Parsippany, NJ) was inserted through a tracheotomy. The fetuses and newborn animals were immediately connected to a constant oxygen-flow device (oxygen fraction, 1.0; AGA, Lidingö, Sweden), and the lungs were expanded by adjusting the oxygen flow to provide a constant positive airway pressure (CPAP) of 5 cmH2O. The entire surgical procedure after euthanasia required 5 min. Fetuses and newborn animals were placed 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 5% albumin instillation solution (10 ml/kg body wt) was instilled into the lungs through the endotracheal tube: first, the fetuses and newborn 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 then the fluid was finally instilled. The fetuses and newborn animals were reconnected to the CPAP circuit, and they remained on the CPAP device 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 remaining alveolar fluid was collected by gently advancing the PE-50 sampling tube to a wedged position and aspirating the fluid. Total protein concentrations in the instillates and in the initial and final solutions were determined spectrophotometrically (iEMS Reader MF, Labsystems, Helsinki, Finland) by the Lowry method (11) adapted for microtiter plates.AFC or alveolar fluid secretion (AFS) was calculated from the change in
alveolar protein concentration over 1 h. This was possible because
the alveolar epithelium is relatively impermeable to large molecules
such as albumin (mol wt 67,000). Therefore, water movement (absorption
or secretion) results in a changed air-space protein concentration.
Because the fetal lung is fluid filled in utero (3, 19,
24), we expected that a certain fraction of fluid would still be
present in the lungs at the time of the experiments. This fluid is
virtually protein free and will not add protein to the instilled
albumin concentration. In contrast, it will dilute the protein
concentration in the instillates and influence the calculations of AFC
differently depending on the volume of fluid that is present in the
lungs at the different developmental stages. To control for this
volume, we instilled guinea pig fetuses with 10 ml/kg body wt of the
5% albumin instillation solution. The fluid was aspirated and
reintroduced four times before a final 0.1-ml sample was taken and the
rest was instilled, and the fetus was studied for 1 h. The entire
procedure required ~1-2 min. During this time, it was unlikely
that a significant quantity of protein either left or entered the air
spaces or that significant volumes of fluid were reabsorbed from or
secreted into the air spaces. Therefore, changes in protein
concentration represent a dilution by preexisting fluid. The
preexisting fluid volume (Vpre) calculated from Eqs.
1 and 2 was used to correct the instilled protein
concentrations by the dilution of the instillate that would occur if
fluid were already in the lung before the 5% albumin instillation. AFC
or AFS was calculated from Eqs. 3 and 4
![]() |
(1) |
![]() |
(2) |
![]() |
(3) |
![]() |
(4) |
Specific Protocols
Guinea pig fetuses of 61 and 68 days postconception gestational age and newborn animals (69 days; term) were studied. Day of conception was set as the day when the timed-pregnant guinea pigs gave birth to their earlier litter, because guinea pigs enter estrus immediately after birth. All groups contain fetuses from at least two litters. All fetuses were studied for 1 h after which the lungs were removed and a sample of remaining alveolar fluid was collected. Protein concentrations were measured and AFC was calculated.Control studies. Preterm guinea pig fetuses of 61 (n = 6) and 68 (n = 6) days gestation and newborn guinea pigs (69 days gestation, term; n = 6) were surgically delivered from timed-pregnant guinea pigs, prepared as described (see Surgical Preparations), and instilled with the 5% albumin solution.
Oxytocin studies. Guinea pig fetuses from oxytocin-injected timed-pregnant guinea pigs of 61 (n = 6) and 68 (n = 6) days gestation were surgically prepared as described and instilled with the 5% albumin solution.
Amiloride studies.
Guinea pig fetuses of 61 (n = 6) and 68 (n = 6) days gestation and newborn guinea pigs (69 days
gestation, term; n = 6) were delivered from
timed-pregnant guinea pigs, surgically prepared as described, and
instilled with the 5% albumin solution containing 103 M
amiloride. Another set of guinea pig fetuses from oxytocin-injected timed-pregnant guinea pigs of 61 (n = 5) and 68 (n = 4) days gestation was also prepared and instilled
with the 5% albumin solution containing 10
3 M amiloride.
The 10
3 M concentration of amiloride was used because a
large fraction of amiloride becomes protein bound, and a significant
fraction rapidly leaves the air spaces due to its low molecular weight (21, 33); therefore, the effective alveolar concentration was probably lower. Also, the same amiloride concentration has been
used in other studies of AFC in both developing and adult animals
(5, 8, 18).
Extracellular Ca2+ influx
(verapamil) studies.
Guinea pig fetuses of 61 (n = 6) and 68 (n = 6) days gestation and newborn guinea pigs (69 days
gestation, term; n = 5) were delivered from
timed-pregnant guinea pigs, surgically prepared as described, and
instilled with the 5% albumin solution containing 104 M
of the L-type Ca2+ channel inhibitor verapamil. Another set
of guinea pig fetuses from oxytocin-injected timed-pregnant guinea pigs
of 61 (n = 7) and 68 (n = 6) days
gestation was also prepared and instilled with the 5% albumin solution
containing 10
4 M verapamil.
Extracellular Ca2+ influx and
Na+ channel (verapamil plus amiloride)
studies.
Guinea pig fetuses of 61 (n = 4) and 68 (n = 6) days gestation and newborn guinea pigs (69 days
gestation, term; n = 5) were delivered from
timed-pregnant guinea pigs, surgically prepared as described, and
instilled with the 5% albumin solution containing 104 M
verapamil and 10
3 M amiloride. Another set of guinea pig
fetuses from oxytocin-injected timed-pregnant guinea pigs of 61 (n = 4) and 68 (n = 6) days gestation was also prepared and instilled with the 5% albumin solution
containing 10
4 M verapamil and 10
3 M amiloride.
Intracellular Ca2+ mobilization
(thapsigargin) studies.
Guinea pig fetuses of 61 (n = 6) and 68 (n = 6) days gestation and newborn guinea pigs (69 days
gestation, term; n = 5) were delivered from
timed-pregnant guinea pigs, surgically prepared as described, and
instilled with the 5% albumin solution containing 105 M
of the intracellular Ca2+-ATPase inhibitor thapsigargin.
Another set of guinea pig fetuses from oxytocin-injected timed-pregnant
guinea pigs of 61 (n = 7) and 68 (n = 6) days gestation was also prepared and instilled with the 5% albumin
solution containing 10
5 M thapsigargin.
Intracellular Ca2+ mobilization and
Na+ channel (thapsigargin plus amiloride)
studies.
Guinea pig fetuses of 61 (n = 4) and 68 (n = 6) days gestation and newborn guinea pigs (69 days
gestation, term; n = 5) were delivered from
timed-pregnant guinea pigs, surgically prepared as described, and
instilled with the 5% albumin solution containing 105 M
thapsigargin and 10
3 M amiloride. Another set of guinea
pig fetuses from oxytocin-injected timed-pregnant guinea pigs of 61 (n = 4) and 68 (n = 4) days gestation was also prepared and instilled with the 5% albumin solution
containing 10
5 M thapsigargin and 10
3 M amiloride.
Statistics
Values are presented as means ± SD. Statistical analysis was carried out with one-way ANOVA with Tukey's test post hoc. Differences were considered statistically significant when P < 0.05. ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We instilled an isosmolar 5% albumin solution into the lungs of fetal guinea pigs and calculated AFC or AFS from the change in protein concentration over 1 h. Calculations were always corrected for the initial dilution of the instilled protein solution by endogenous fetal lung fluid.
AFC or AFS
At 61 days gestation, fetal guinea pig lungs secreted fluid into the lung lumen (Fig. 1). When 1 day remained until delivery (at 68 days gestation), the lungs had begun to absorb fluid; during the last gestational day, the fluid absorption rate rapidly increased and reached very high rates at birth. When preterm labor was induced by repeated oxytocin injections (four 1 mg/kg injections over 45 min), the fluid secretion observed in the 61-day gestation control fetuses was converted to fluid absorption. Also, oxytocin-induced preterm labor tended to increase the AFC 1 day before birth (at 68 days gestation), but this increase did not reach significance.
|
Effect of Amiloride on AFC or AFS
AFC depends on water absorption secondary to Na+ absorption through amiloride-sensitive and -insensitive pathways (5, 15, 16, 19). To investigate the fraction of AFC that was mediated through amiloride-sensitive Na+ absorption, we used the Na+ channel inhibitor amiloride (10
|
|
|
|
|
Effect of Extracellular Ca2+ Influx (Verapamil) on AFC and AFS
In these studies, verapamil (10Effect of Intracellular Ca2+ Mobilization (Thapsigargin) on AFC and AFS
The intracellular Ca2+-ATPase inhibitor thapsigargin (10 ![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previous studies have shown that the fetal lung fluid volume
decreases before delivery (2, 19) and that absorption of fluid starts during or before labor (4, 5, 10, 19). It has
been demonstrated that labor releases fetal epinephrine that can
stimulate AFC (4, 5, 19). Catecholamines (e.g., epinephrine) stimulate AFC at least partly via cAMP as an intracellular second messenger in fetal, newborn, and adult animals (5, 16, 18). There is also evidence that catecholamines may act through alternative or complementary intracellular second-messenger systems; terbutaline has been demonstrated to increase
[Ca2+]i and to increase
Ca2+-sensitive Na+ transport in rat fetal
distal lung epithelial (FDLE) cells (13, 28). Because AFC
depends on transepithelial Na+ transport (16)
and this Na+ transport can be stimulated by the
-adrenergic system (16), we studied basal and
epinephrine-stimulated alveolar fluid transport after inhibition of
Ca2+ influx in developing guinea pig lungs.
AFC was measured in situ using a method whereby the fetal guinea pig lungs were kept expanded with a CPAP. It has previously been demonstrated that although the animals do not have pulmonary circulation and are not ventilated, the calculated AFC does not differ significantly from the results obtained in experiments where the animals have been ventilated (9, 19, 26). Therefore, we believe that the results we have obtained using this method are reliable and are not influenced by the fact that the study is carried out on postmortem lungs.
At 61 days gestation (i.e., 8 days before normal birth), the fetal
guinea pig lung displayed a net secretion of fluid, but when 1 day
remained to birth (at 68 days gestation), there was a small but
significant net fluid absorption. In newborn animals, AFC was
stimulated and ~50% of the instilled solution was absorbed over
1 h (see Fig. 1). These findings were well in accordance with our
previous studies (5, 19), which showed that lung fluid
production ceases 3-5 days before birth and the lung slowly begins
to absorb fetal lung fluid. Our results also confirm our earlier study
(19) where we demonstrated that induction of preterm labor
by oxytocin stimulated AFC at 61 days gestation (when there normally
was net fluid secretion). The earlier study demonstrated that oxytocin
stimulated AFC by release of fetal epinephrine, which in turn
stimulated AFC through -adrenergic stimulation (19).
Studies of several animal species have demonstrated that a significant fraction of AFC (5, 16, 19, 23) or Na+ transport at a cellular level (12, 14, 28) can be inhibited by amiloride. We observed in this study that amiloride inhibited net fluid absorption once AFC became stimulated, which is similar to what we have demonstrated before (19). These findings suggest that amiloride-sensitive pathways are major pathways for AFC in preterm guinea pigs. At birth, amiloride inhibits a large fraction of AFC, and within a few days after birth, the amiloride-inhibited fraction of AFC has decreased to 40-50% (5), which is similar to the adult inhibition (18). This developmental pattern of amiloride sensitivity largely conforms to the development suggested for ENaC in rats (20, 32) and guinea pigs (1).
Several in vitro studies (14, 28) have suggested that
Ca2+ may act as a second messenger for terbutaline (a
-adrenergic agonist)-stimulated Na+ transport in the
lung, which consequently suggests that stimulated AFC may depend on
changes in [Ca2+]i.
[Ca2+]i can be increased by mobilization from
two sources: intracellular stores (endoplasmic reticulum) and/or the
extracellular spaces (29). In this study, we investigated
the role of extracellular Ca2+ influx for stimulation of
AFC by inhibiting L-type voltage-gated Ca2+ channels with
verapamil. Generally, this drug is used to inhibit Ca2+
channels in cardiac or smooth muscles, but there is strong evidence that this type of channel is also located in the alveolar epithelium, because verapamil and the verapamil analog nifedipine can block phosphatidylcholine secretion from rat alveolar type II pneumocytes (27, 31). In all animals where AFC was stimulated (i.e.,
in newborn guinea pigs at 68 days gestation and after oxytocin-induced preterm labor), verapamil very effectively inhibited AFC (see Figs.
3-6). We know from our earlier studies that endogenous plasma epinephrine is significantly elevated at these gestational ages (19). These findings thus support the hypothesis that
-adrenergic stimulation of AFC may be associated with changes in
[Ca2+]i. When we had established that
verapamil could inhibit epinephrine-stimulated AFC, and because AFC is
at least partially mediated through amiloride-sensitive pathways,
we investigated whether the amiloride-sensitive fraction was
affected by Ca2+ channel inhibition by verapamil. The
combined inhibitory effect of amiloride and verapamil never differed
from that when any of the drugs were given alone. This indicates that
verapamil affects the same fraction of AFC as amiloride; i.e.,
stimulation of amiloride-sensitive pathways for AFC may be associated
with influx of Ca2+ through verapamil-sensitive
Ca2+ channels in the plasma membrane of the alveolar
epithelial cells. Marunaka and co-workers (14) reported
that a nonspecific cation channel (NSC) can be stimulated by increased
[Ca2+]i. Several reports suggest that ENaC is
expressed in the lung epithelium near birth (1, 5, 20,
32), but no reports are present on its dependence or relation to
changes in [Ca2+]i in fetal lungs. However,
because the amiloride sensitivity was completely abolished after
verapamil administration, it appears as if
-adrenergic stimulation
of ENaC depends on Ca2+ from extracellular spaces in the in
vivo situation. There is also evidence that ENaC in fact might be
stimulated at low [Ca2+]i (25),
and as such our result may suggest that the observed fluid clearance in
the fetal guinea pig may be mediated by the NSC-type channels, as these
results also do not exclude involvement of the NSC channels because
these channels probably are amiloride sensitive at the concentrations
of amiloride used in here.
Would changes in [Ca2+]i from intracellular stores yield the same result? Thapsigargin, an inhibitor of Ca2+-ATPase (which is responsible for Ca2+ reuptake into the endoplasmic reticulum and restoration of a low resting [Ca2+]i), produced similar results as when verapamil was added to the instillate (see Figs. 3-6). This suggests that stimulated amiloride-sensitive AFC in fetal or newborn guinea pigs depends on changes in [Ca2+]i independently of its origin. A model for the role of Ca2+ for activation of NSC in rat FDLE cells has been suggested (21) where mobilization of Ca2+ occurs in two steps. First, terbutaline or dibutyryl cAMP produces a transient [Ca2+]i elevation caused by Ca2+ release from the endoplasmic reticulum. Second, the continuous stimulation is caused by a subsequent influx of Ca2+ through membrane Ca2+ channels. Consequently, by blocking either of the pathways for altering [Ca2+]i, we may expect a reduced AFC rate.
Thapsigargin per se might be expected to stimulate AFC, because the immediate result of thapsigargin administration would be increased [Ca2+]i owing to a constant leak of Ca2+ from the endoplasmic reticulum (6). However, this was not the case in our studies. This deviation from the expected result can be explained in several possible ways. First, the thapsigargin concentrations used together with the length of the experiment would more likely cause an overall reduction in [Ca2+]i, because virtually all cytosolic Ca2+ buffering would be lost; i.e., there would be no Ca2+ release from the endoplasmatic reticulum. Second, because terbutaline also increases intracellular cAMP concentration in addition to increasing [Ca2+]i, it may be that Ca2+ cannot act without a concomitant activation of cAMP. Third, several studies have demonstrated that continuous (long-term) conditions where [Ca2+]i is mediating a signal to an effector protein occur through [Ca2+]i oscillations (33). It is possible that we inhibited the Ca2+ oscillations in our study. Fourth, thapsigargin may result in a new [Ca2+]i level, which the cell must readjust to maintain normal cell functions. The ability to adjust to altered [Ca2+]i depends on cell type, and if a readjustment is not possible, transport systems in the cell that depend on [Ca2+]i may cease functioning. Accordingly, transepithelial Na+ transport would be expected to decrease, and as a consequence, AFC would decrease.
The effect from verapamil may be compromised by the fact that verapamil
also can inhibit Ca2+-gated K+ channels
(7). The effect from this may indirectly lead to an
inhibited AFC (22), because when K+ channels
are inhibited, less K+ will leave the cell. The normal
continuous flux of K+ out of the cell is connected to a
decreased intracellular Cl concentration
([Cl
]i). A decreased
[Cl
]i increases the sensitivity of NSC to
Ca2+ (28). Consequently, if or when
K+ channels are inhibited, the
[Cl
]i will remain relatively high or even
increase, which may prevent the NSC from responding to the increased
Ca2+. However, the hypothesis that verapamil acts directly
on the L-type Ca2+ channels is more likely because it also
has been shown to affect surfactant release (31), but a
limited effect of verapamil on K+ channels cannot be excluded.
In summary, AFC is rapidly increased during the last day of gestation and can be stimulated 8 days before birth after oxytocin-induced preterm labor. Preterm AFC (either control or oxytocin induced) was always amiloride sensitive. Verapamil instillation as well as thapsigargin instillation inhibited AFC when stimulated by preterm or near-term labor induction and in newborn animals. These results, together with the findings that terbutaline increases the open probability of ENaC (15) and amiloride-sensitive NSC channels (14, 28), support the hypothesis that [Ca2+]i may be involved in stimulation of amiloride-sensitive AFC in preterm animals. Stimulation of AFC depends on Ca2+ mobilization from both extracellular compartments (via membrane Ca2+ channels) and intracellular compartments (such as the endoplasmatic reticulum). The exact mechanism for Ca2+ stimulation of AFC was not investigated in this study, but we speculate that intracellular Ca2+ may act as a second messenger, probably by increasing the open probability of amiloride-sensitive Na+ channels in the alveolar epithelium.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported by grants from NEOUCOM Start-up Funds and the Swedish Natural Science Research Council, the Crafoord Foundation, the Royal Physiographic Society in Lund, and the Magnus Bergwall Foundation.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: H. G. Folkesson, Dept. of Physiology, Northeastern Ohio Universities College of Medicine, 4209 State Route 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.00417.2000
Received 22 November 2000; accepted in final form 18 September 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Baines, DL,
Folkesson HG,
Norlin A,
Bingle CD,
Yuan HT,
and
Olver RE.
The influence of mode of delivery, hormonal status, and postnatal O2 environment on epithelial sodium channel (ENaC) expression in perinatal guinea-pig lung.
J Physiol (Lond)
522:
147-157,
2000
2.
Berger, PJ,
Kyriakides MA,
Smolich JJ,
Ramsden CA,
and
Walker AM.
Massive decline in lung liquid before vaginal delivery at term in the fetal lamb.
Am J Obstet Gynecol
178:
223-227,
1998[ISI][Medline].
3.
Bland, RD,
McMillan DD,
Bresack MA,
and
Dong LA.
Clearance of liquid from lungs of newborn rabbits.
J Appl Physiol
49:
171-177,
1980
4.
Brown, MJ,
Olver RE,
Ramsden CA,
Strang LB,
and
Walters DV.
Effects of adrenaline and of spontaneous labour on the secretion and absorption of lung liquid in fetal lamb.
J Physiol (Lond)
344:
137-152,
1983[Abstract].
5.
Finley, N,
Norlin A,
Baines DL,
and
Folkesson HG.
Alveolar epithelial fluid clearance is mediated by endogenous catecholamines at birth in guinea pigs.
J Clin Invest
101:
972-981,
1998
6.
Ingbar, DH,
Burns Weeks C,
Gilmore-Herbert M,
Jacobsen E,
Duvick S,
Dowin R,
Savik SK,
and
Jamieson JD.
Developmental regulation of Na,K-ATPase in rat lung.
Am J Physiol Lung Cell Mol Physiol
270:
L619-L629,
1996
7.
Jacobs, ER,
and
DeCoursey TE.
Mechanisms of potassium channel block in rat alveolar epithelial cells.
J Pharmacol Exp Ther
255:
459-472,
1990[Abstract].
8.
Jayr, C,
Garat C,
Meignan M,
Pittet JF,
Zelter M,
and
Matthay MA.
Alveolar liquid and protein clearance in anesthetized ventilated rats.
J Appl Physiol
76:
2636-2642,
1994
9.
Jayr, C,
and
Matthay MA.
Alveolar and lung liquid clearance in the absence of pulmonary blood flow in sheep.
J Appl Physiol
71:
1679-1687,
1991
10.
Kitterman, JA,
Ballard PL,
Clements JA,
Mescher EJ,
and
Tooley WH.
Tracheal fluid in fetal lambs: spontaneous decrease before birth.
J Appl Physiol
47:
985-989,
1979
11.
Lowry, OH,
Rosebrough NJ,
Farr A,
and
Randall RJ.
Protein measurement with the folin phenol reagent.
J Biol Chem
193:
265-275,
1951
12.
Marunaka, Y.
Amiloride-blockable Ca2+-activated Na+-permeant channels in the fetal distal lung epithelium.
Pflügers Arch
431:
748-756,
1996[ISI][Medline].
13.
Marunaka, Y,
Niisato N,
O'Brodovich H,
and
Eaton DC.
Regulation of an amiloride-sensitive Na+-permeable channel by a 2-adrenergic agonist, cytosolic Ca2+, and Cl
in fetal rat alveolar epithelium.
J Physiol (Lond)
515:
669-683,
1999
14.
Marunaka, Y,
Tohda H,
Hagiwara N,
and
O'Brodovich H.
Cytosolic Ca2+-induced modulation of ion selectivity and amiloride sensitivity of a cation channel and -agonist action in fetal lung epithelium.
Biochem Biophys Res Commun
187:
648-656,
1992[ISI][Medline].
15.
Matalon, S,
Benos DJ,
and
Jackson RM.
Biophysical and molecular properties of amiloride-inhibitable Na+ channels in alveolar epithelial cells.
Am J Physiol Lung Cell Mol Physiol
271:
L1-L22,
1996
16.
Matthay, MA,
Folkesson HG,
and
Verkman AS.
Salt and water transport across alveolar and distal airway epithelia in the adult lung.
Am J Physiol Lung Cell Mol Physiol
270:
L487-L503,
1996
17.
Niisato, N,
Ito Y,
and
Marunaka Y.
cAMP stimulates Na+ transport in rat fetal pneumocyte: involvement of a PTK- but not a PKA-dependent pathway.
Am J Physiol Lung Cell Mol Physiol
277:
L727-L736,
1999
18.
Norlin, A,
Finley N,
Abedinpour P,
and
Folkesson HG.
Alveolar liquid clearance in the anesthetized ventilated guinea pig.
Am J Physiol Lung Cell Mol Physiol
274:
L235-L243,
1998
19.
Norlin, A,
and
Folkesson HG.
Alveolar liquid clearance in fetal guinea pigs after induced labor: mechanisms and regulation.
Am J Physiol Lung Cell Mol Physiol
280:
L606-L616,
2001
20.
O'Brodovich, H,
Canessa C,
Ueda J,
Rafii B,
Rossier C,
and
Edelson J.
Expression of the epithelial Na+ channel in the developing rat lung.
Am J Physiol Cell Physiol
265:
C491-C496,
1993
21.
O'Brodovich, H,
Hannam V,
Seear M,
and
Mullen JBM
Amiloride impairs lung water clearance in newborn guinea pigs.
J Appl Physiol
68:
1758-1762,
1990
22.
O'Brodovich, H,
and
Rafii B.
Effect of K channel blockers on basal and -agonist stimulated ion transport by fetal distal lung epithelium.
Can J Physiol Pharmacol
71:
54-57,
1993[ISI][Medline].
23.
Olver, RE,
Ramsden CA,
Strang LB,
and
Walters DV.
The role of amiloride-blockable sodium transport in adrenaline-induced lung liquid reabsorption in the fetal lamb.
J Physiol (Lond)
376:
321-340,
1986[Abstract].
24.
Olver, RE,
and
Strang LB.
Ion fluxes across the pulmonary epithelium and the secretion of lung liquid in the foetal lamb.
J Physiol (Lond)
241:
327-357,
1974[ISI][Medline].
25.
Palmer, LG,
and
Frindt G.
Effects of cell Ca and pH on Na channels from rat cortical collecting tubule.
Am J Physiol Renal Fluid Electrolyte Physiol
253:
F333-F339,
1987
26.
Sakuma, T,
Pittet JF,
Jayr C,
and
Matthay MA.
Alveolar liquid and protein clearance in the absence of blood flow or ventilation in sheep.
J Appl Physiol
74:
176-185,
1993[Abstract].
27.
Sen, N,
Grunstein MM,
and
Chander A.
Stimulation of lung surfactant secretion by endothelin-1 from rat alveolar type II cells.
Am J Physiol Lung Cell Mol Physiol
266:
L255-L262,
1994
28.
Tohda, H,
Foskett JK,
O'Brodovich H,
and
Marunaka Y.
Cl regulation of a Ca2+-activated nonselective cation channel in
-agonist-treated fetal distal lung epithelium.
Am J Physiol Cell Physiol
266:
C104-C109,
1994
29.
Tsien, RW,
and
Tsien RY.
Calcium channels, stores, and oscillations.
Annu Rev Cell Biol
6:
715-760,
1990[ISI].
30.
Walters, DV,
Ramsden CA,
and
Olver RE.
Dibutyryl cAMP induces a gestation-dependent absorption of fetal lung liquid.
J Appl Physiol
68:
2054-2059,
1990
31.
Warburton, D,
Parton L,
Buckley S,
and
Cosico L.
Verapamil: a novel probe of surfactant secretion from rat type II pneumocytes.
J Appl Physiol
66:
1304-1308,
1989
32.
Watanabe, S,
Matsushita K,
Stokes JB,
and
McCray PB, Jr.
Developmental regulation of epithelial sodium channel subunit mRNA expression in rat colon and lung.
Am J Physiol Gastrointest Liver Physiol
275:
G1227-G1235,
1998
33.
Yue, G,
and
Matalon S.
Mechanisms and sequelae of increased AFC in hyperoxic rats.
Am J Physiol Lung Cell Mol Physiol
272:
L407-L412,
1997