Lung Membrane Transport Group, Tayside Institute of Child Health, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, United Kingdom
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ABSTRACT |
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Distal lung epithelial
cells isolated from fetal rats were cultured (48 h) on permeable
supports so that transepithelial ion transport could be quantified
electrometrically. Unstimulated cells generated a short-circuit current
(Isc) that was inhibited (~80%) by apical
amiloride. The current is thus due, predominantly, to the absorption of
Na+ from the apical solution. Isoprenaline increased the
amiloride-sensitive Isc about twofold.
Experiments in which apical membrane Na+ currents were
monitored in basolaterally permeabilized cells showed that this was
accompanied by a rise in apical Na+ conductance
(GNa+). Isoprenaline also increased apical
Cl conductance (GCl
) by
activating an anion channel species sensitive to glibenclamide but
unaffected by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid
(DIDS). The isoprenaline-evoked changes in
GNa+ and GCl
could account for the changes in Isc observed in
intact cells. Glibenclamide had no effect upon the isoprenaline-evoked
stimulation of Isc or
GNa+ demonstrating that the rise in
GCl
is not essential to the stimulation
of Na+ transport.
alveolar ion transport; Ussing chambers; permeabilized epithelia
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INTRODUCTION |
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THROUGHOUT FETAL
LIFE the distal lung epithelia secrete fluid into the developing
air spaces (31) and establish a distending pressure that
is crucial to lung morphogenesis (11). However, this
liquid must be removed from the lungs if the newborn infant is to
breathe at birth. The absorption of this liquid occurs during the final
stages of gestation and is dependent upon the active withdrawal of
Na+ from the lung lumen, a process that can be controlled
via -adrenoceptors (23, 30, 36-38). The means by
which this control is achieved are not fully understood, but there is
evidence that the process involves a rise in apical Na+
conductance (GNa+) (15, 20).
It is also known that
-adrenoceptor agonists can increase apical
Cl
conductance (GCl
) in
these cells, and it has been suggested that this may facilitate
Na+ transport by increasing the driving force for
Na+ entry (16, 29). It is, however, difficult
to see how the ionic gradients that normally prevail in epithelial
cells could allow Na+ transport to be controlled in this
way (see Refs. 20, 44). To clarify the means by which
-adrenoceptor agonists can control Na+ absorption in the
distal lung epithelia, we now explore the effects of isoprenaline upon
the conductive properties of the apical membrane.
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METHODS |
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Isolation and culture of rat fetal distal lung epithelial cells.
Fetuses removed from anesthetized (3% halothane), 20-day pregnant
(term = 22 days) rats were immediately decapitated, and their lung
tissue was collected into ice-cold, Ca2+- and
Mg2+-free Hanks' balanced salt solution. The anesthetized
animals were then killed (cervical dislocation/exsanguination) before regaining consciousness. The fetal lung tissue was chopped into pieces
(<0.5 mm) and disaggregated using 0.2% trypsin/0.012% DNase (2 × 20 min, 37°C) followed by 0.1% collagenase/0.012% DNase [15 min, 37°C, both in Dulbecco's modified Eagle's medium (DMEM)]. Cells pelleted from the resultant digest were resuspended in DMEM containing 10% fetal calf serum and then incubated (1 h, 37°C) in a
75-cm2 culture flask. The supernatant was then gently
decanted to separate nonadherent epithelial cells from fibroblasts and
smooth muscle cells. After a second such fractionation, the nonadherent
cells were resuspended in culture medium, plated (1.5 × 106 cells/cm2) onto Transwell-Col membranes
(Costar, High Wycomb, UK) and incubated (37°C) in an atmosphere of
water-saturated room air containing 5% CO2 and sufficient
N2 to reduce partial pressure of oxygen (PO2) to the level found in the adult alveolar
region (100 mmHg). Cells were incubated in PC-1 medium unless
otherwise stated. This is a serum-free medium that contains
defined amounts of the hormones/growth factors found in fetal calf
serum, which is almost invariably added to media used to maintain
epithelial cells in primary culture. Its exact composition, however, is
regarded as commercially sensitive. After 24 h, we changed the
medium and removed nonviable cells by gently washing each culture. The
cells were then incubated for a further 24 h before being used in
experiments. By this time, the cells had almost invariably (>90% of
cell preparations) become integrated into epithelial sheets with
transepithelial resistances (Rt) >200
cm2 (see also Refs. 1, 37).
Although the cells are isolated from fetal animals, previously
published work (1, 33, 36) shows that maintaining these
cells in an atmosphere that mimics the PO2 of
the postnatal alveolar region (~100 mmHg) causes the development of a
Na+-absorbing phenotype typical of the neonatal, rather
than the fetal lung.
Measurement of Isc and the conductive properties of
the apical membrane.
Cultured epithelia were mounted in Ussing chambers where they were
bathed with bicarbonate-buffered physiological salt solution (composition given in Solutions and chemicals) that was
continually circulated with a gas mixture identical to that in which
the epithelia had been incubated (PO2 = 100 mmHg). Initially, the cells were maintained under
open-circuit conditions until the transepithelial potential difference
(Vt) had stabilized (30-40 min).
Vt was then clamped to 0 mV, and the current
required to hold this potential [short-circuit current
(Isc)] was digitized (4 Hz) and displayed on a
computer screen while simultaneously being recorded to computer disk
using a PowerLab computer interface and associated software (ADI
Instruments, Hastings, UK). In some experiments,
Rt was also monitored by observing the currents
flowing in response to repeated, 1-mV excursions in
Vt. The conductive properties of the apical membrane were explored by using polyene antibiotics (nystatin or
amphotericin B) to permeabilize the basolateral plasma membrane. These
compounds form pores in cholesterol-containing membranes that are
permeable to Na+, K+, and Cl but
not to divalent cations or higher-molecular-weight substances and thus
allow experimental control over the cytoplasmic [Na+],
[K+], and [Cl
], whereas intracellular
Ca2+ is regulated by the normal, physiological mechanisms.
Because the pores formed by amphotericin B have a higher
Cl
conductance than do those formed by nystatin
(13, 21), the former compound was used to make
measurements of GCl
.
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Quantification of Na+ pump capacity. The method used to determine the capacity of the basolateral Na+ pump is described elsewhere (21, 36), and so only brief details are presented here. Epithelial monolayers were mounted in Ussing chambers, bathed with the standard physiological solution (i.e., high Na+), and treated with apical amiloride (10 µM) to block the Na+ channels in this membrane, which was then permeabilized using nystatin (75 µM). This elicited a slowly developing rise in Isc attributed to the activity of the basolaterally located Na+ pump. The fall in Isc evoked by subsequently adding ouabain (1 mM) to the basolateral solution (Ipump) was then measured to provide an indicator of the Na+ extrusion capacity of this pump. Our previously published work (36) shows that 1 mM ouabain never entirely abolished the Isc recorded from nystatin-permeabilized cells, and it is therefore possible that the method may slightly underestimate the capacity of the basolateral pump. However, we are forced to accept this limitation as higher concentrations of ouabain caused loss of epithelial integrity.
Data analysis and experimental design. Data are presented as means ± SE, and values of n refer to the number of times a protocol was repeated using cells prepared from different litters. The control current was defined as that measured under basal conditions at the onset of the experiment, and control and experimental cells were age matched and derived from the same litters. The statistical significance of differences between mean values was assessed using Student's t-test. In studies of intact cells, positive Isc was defined as the current carried by cations moving from the apical to the basolateral compartments, whereas, in basolaterally permeabilized preparations, positive Iap is that carried by cations leaving the cytoplasm. These are standard electrophysiological conventions.
Solutions and chemicals.
The standard physiological salt solution contained (in mM) 117 NaCl, 25 NaHCO3, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 2.5 CaCl2, and 11 D-glucose, pH 7.3-7.4 when bubbled with 5%
CO2. The K+-gluconate solution was prepared by
isosmotically replacing Cl in this standard solution with
gluconate, whereas the K+-Cl
solution was
prepared by replacing Na+ with K+. Both ionic
substitutions were made in the K+-gluconate solution. The
amount of calcium gluconate added to gluconate-containing solutions was
raised to 11.5 mM to maintain Ca2+ activity despite
gluconate's capacity to bind this cation. All solutions were
bicarbonate buffered and continually bubbled with 5% CO2
to maintain pH. The minimal defined-composition serum-free (MDSF)
medium used in some experiments consisted of a mixture (1:1) of
DMEM/Ham's F-12 nutrient mix that contained bovine serum albumin (1.25 mg/ml), L-glutamine (2 mM), and nonessential amino acids
(0.1%). Amiloride and isoprenaline were freshly prepared (10 mM in
distilled water) on each experimental day, whereas stock solutions (10 mM) of ethylisopropyl amiloride (EIPA; RBI International, Gillingham,
UK) and glibenclamide (Tocris, St. Albans, UK) were prepared in
dimethyl sulfoxide, and benzamil (50 mM; Molecular Probes) was
dissolved in distilled water containing 20% (vol/vol) methanol. These
solutions were divided into aliquots and stored at
20°C.
Appropriate experiments showed that the solvent vehicles had no effect
upon the parameters under study. Cell culture reagents were purchased
from Paisley Life Technologies (Paisley, UK), and general laboratory
reagents were from Sigma Chemical (Poole, UK).
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RESULTS |
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Properties of intact epithelia.
Apical amiloride (10 µM) caused the spontaneous
Isc to fall rapidly (20-40 s) to a value
that was 23.1 ± 6.6% (n = 7) of the control
level, and the concentration required to exert half-maximal inhibition
(IC50) was 0.47 ± 0.03 µM (Fig.
1A). EIPA and benzamil also
caused ~80% inhibition of the basal Isc, and
the IC50 values for these compounds were 3.3 ± 0.8 µM and 10.8 ± 1.8 nM, respectively (Fig. 1A). The
rank order of potency amongst these Na+ channel antagonists
was thus benzamil > amiloride > EIPA, a profile suggesting
the involvement of selective Na+ channels in the generation
of the basal Isc (4). These
experiments also revealed an amiloride-resistant component to the
spontaneous Isc, and previous work has shown
that subsequent application of bumetanide causes very little further
fall in the spontaneous current; the ionic basis of this current is
thus unknown (34). Basolateral isoprenaline (10 µM)
caused a slowly developing rise in Isc, although
an initial, more rapid component to this response was usually evident
(Fig. 1B, see also Refs. 36, 37).
After 30-40 min stimulation with this drug,
Isc had risen to a value that was 173.2 ± 10.3% of the control level. Subsequent addition of apical amiloride
(10 µM) caused a rapid fall to a value that was only 34.1 ± 3.8% of the control current measured at the onset of the experiment
(Fig. 1B). Further experiments were undertaken in which
amiloride-pretreated cells were exposed to isoprenaline. Examination of
the currents recorded at the onset of these experiments confirmed that
amiloride caused ~80% inhibition of basal
Isc, and, as anticipated, the response to
isoprenaline was essentially abolished (94.7 ± 1.3% inhibition)
by this drug (Fig. 1B).
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Effects of basolateral Ba2+.
We studied the effects of basolateral Ba2+, a cation that
blocks epithelial K+ channels, in an attempt to explore the
role of these channels in the response to -adrenoceptor agonists.
The application of Ba2+ to unstimulated cells caused a
rapid fall in spontaneous Isc, but this effect
was transient, and Isc returned to its basal
value within 10 min (Fig. 2). Subsequent
application of isoprenaline also caused a small fall in
Isc, which contrasted with the initial rapid
rise seen under control conditions. Although the recorded current
returned to its basal value after ~5 min, Isc
failed to rise above this level during 30-40 min of exposure to
isoprenaline (Fig. 2). Ba2+ thus abolishes the
isoprenaline-evoked rise in Isc. Subsequent experiments in which we monitored Na+ pump capacity (see
METHODS) in control and Ba2+-treated cells
showed that this cation caused 40.2 ± 6.5% inhibition of
Ipump (control, 10.9 ± 1.4 µA/cm2, Ba2+-treated, 6.5 ± 1.1 µA/cm2, P < 0.01, Student's paired
t-test), indicating that Ba2+, a well-known
inhibitor of epithelial K+ channels, causes substantial
loss of Na+ pump capacity.
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Regulation of GCl.
Imposing an outwardly directed Cl
gradient upon
basolaterally permeabilized cells evoked only small (1-2
µA/cm2) currents, demonstrating that
GCl
is normally low (~30
µS/cm2). Application of DIDS or glibenclamide (both 100 µM) reduced Iap, establishing that this small
conductance is due to the efflux of Cl
through channels
sensitive to these compounds. Analysis of these data showed that the
DIDS-sensitive and glibenclamide-sensitive components of
GCl
were 10.7 ± 1.7 µS/cm2 (n = 4) and 11.8 ± 4.0 µS/cm2 (n = 4), respectively. Basolateral
isoprenaline (10 µM) evoked a rise in Iap that
occurred with no discernible latency, reaching a level that was
~15-fold greater than the basal value after ~20 s.
Iap then fell to a plateau value two- to
threefold above control (Fig.
3A). The application of
glibenclamide (100 µM) to isoprenaline-stimulated preparations caused
a rapid fall in Iap, and subsequent application of DIDS (100 µM) caused a further small fall (Fig. 3A).
Analysis of these data indicated that isoprenaline augmented the
glibenclamide-sensitive component of GCl
approximately fivefold (55.7 ± 7.2 µS/cm2,
P < 0.05 vs. control data presented above) but had no
significant effect upon the DIDS-sensitive component (17.1 ± 1.9 µS/cm2). Experiments in which cells were exposed to these
Cl
channel blockers (100 µM) before stimulation with
isoprenaline (10 µM) showed that glibenclamide caused ~80%
inhibition of the peak response (Fig. 3, B and C)
but that DIDS had no effect (Fig. 3C). Isoprenaline thus
increases GCl
by selectively activating
the glibenclamide-sensitive conductance. Further studies of
basolaterally permeabilized cells (n = 4) showed that
basolateral isoprenaline had no discernible effect upon
Iap when both apical and basolateral
[Cl
] were maintained at 10.3 mM. The
isoprenaline-evoked changes in Iap are thus
dependent upon the presence of the Cl
gradient and so
reflect an increase in GCl
that would
facilitate the flow of Cl
down its electrochemical
gradient. To establish the extent to which this response is maintained
during prolonged stimulation, cells were first exposed to isoprenaline
while bathed with standard physiological salt solution, and the
basolateral membrane was then permeabilized to allow the
glibenclamide-sensitive component of GCl
to be measured. These experiments revealed an approximately fourfold
stimulation of glibenclamide sensitive GCl
after 30-40 min of stimulation
(control: 8.5 ± 2.4 µS/cm2;
isoprenaline-stimulated: 34.3 ± 4.5 µS/cm2,
n = 4, P < 0.01). Forskolin (100 µM), a drug that directly activates adenylate cyclase
(40), caused changes in GCl
essentially identical to those seen during stimulation with
isoprenaline (n = 4), and this response, in common with
the response to isoprenaline, was inhibited by glibenclamide but
unaffected by DIDS (Fig. 3D).
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Regulation of GNa+.
Experiments in which Iap was recorded from
basolaterally permeabilized cells exposed to an inwardly directed
Na+ gradient showed that isoprenaline had no effect over a
10- to 15-min period (n = 4). However, the
isoprenaline-evoked rise in Isc develops over
30-40 min (Fig. 2), and so further experiments were undertaken in
which cells were permeabilized and GNa+ was
quantified once this response had become fully established. The data
derived from intact cells (Fig.
4B) confirmed that
isoprenaline evoked a slowly developing rise in
Isc, and the corresponding measurements of
GNa+ (Fig. 4, Aii and
Bii) showed that this was accompanied by a rise
(approximately twofold) in GNa+ (control:
60.0 ± 9.9 µS/cm2, n = 6;
isoprenaline-stimulated: 138 ± 12.0 µS/cm2,
n = 4, P < 0.01). We studied the
effects of glibenclamide upon this response to explore the role of the
cAMP-activated anion channels (16, 29). These studies
showed that apical glibenclamide had no significant effect upon basal
Isc (control Isc:
7.4 ± 1.4 µA/cm2; postglibenclamide
Isc: 7.4 ± 1.7 µA/cm2,
n = 4). Moreover, application of basolateral
isoprenaline evoked a rise in Isc (3.5 ± 1.5 µA/cm2, n = 4) in the
glibenclamide-treated cells that did not differ significantly from that
seen in age-matched control cells at identical passage (3.6 ± 0.94 µA/cm2, n = 5). Once these responses
to isoprenaline had become established, the control and
glibenclamide-treated cells were basolaterally permeabilized so that
GNa+ could be measured. This analysis
showed that glibenclamide had no statistically significant effect upon
this parameter (control: 138.4 ± 12.0 µS/cm2,
n = 5; glibenclamide-treated, 143.1 ± 26.4 µS/cm2, n = 4). Thus glibenclamide, at a
concentration that can cause >90% inhibition of the cAMP-evoked rise
in GNa+, has no significant effect upon the
overall response to isoprenaline.
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Responses to isoprenaline in cells maintained in MDSF medium.
Experiments in which the standard culture medium (PC-1) was replaced
with the MDSF medium (see METHODS) used in a previous study
of alveolar epithelial cells isolated from adult animals (16) showed that the cells still became incorporated into
epithelial layers (Rt = 510 ± 27 cm2) under these conditions. These cultured epithelia
generated a spontaneous Isc (10.4 ± 0.5 µA/cm2) similar to that recorded from cells maintained in
PC-1 medium. Apical amiloride (10 µM) elicited an immediate
inhibition of this spontaneous current (83.4 ± 1.5%
n = 6). Further studies of cells cultured under these
conditions (n = 5; spontaneous
Isc = 7.5 ± 0.6 µA/cm2;
Rt = 389 ± 81
cm2) showed
that basolateral isoprenaline (10 µM) elicited only a barely
discernible (
Isc < 0.5 µA/cm2), transient fall in Isc, so
that the current measured after 20-30 min exposure to this drug
(7.5 ± 1.4 µA/cm2) did not differ significantly
from the initial control value. Although cells maintained in MDSF
medium thus generate a spontaneous Isc that
appears to be due largely to electrogenic Na+ absorption,
this transport process does not appear to be subject to
-adrenoceptor-mediated control under these conditions. This contrasts markedly with the consistent stimulation of
Isc seen in cells maintained in medium PC-1
(Fig. 1).
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DISCUSSION |
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Properties of intact cells.
Data obtained from cells maintained in medium PC-1 accord with the view
that cultured distal lung epithelial cells spontaneously absorb
Na+ from the apical solution and show that cAMP-coupled
agonists elicit a slowly developing but substantial rise in the rate of transepithelial ion transport. This response was due to a stimulation of amiloride-sensitive Isc, and so these data
confirm that the Na+ transport process underlying the basal
Isc is subject to -adrenoceptor-mediated control (see also Refs. 3, 23,
26, 36-38). However, net Na+
absorption is not a feature of the fetal lung where the dominant ion
transport process is the secretion of anions into the lung lumen
(31). This discrepancy between the present observations and those made in the intact fetal lung almost certainly reflects the
fact that the present study was undertaken using cells cultured at
adult alveolar PO2 (100 mmHg) rather than at
fetal PO2 (~23 mmHg). We (1, 36)
and others (2, 33) have recently shown that this causes
the development of the Na+-absorbing phenotype
characteristic of the adult lung. Although the present study was
undertaken using cells isolated from fetal animals, we believe that
these cells expressed an adult phenotype when used in the experiments
but cannot formally exclude the possibility that they may have adopted
an intermediate phenotype.
Ba2+-evoked changes in
Isc.
It is well documented that basolateral K+ channels, which
can be blocked by Ba2+, make an important contribution to
the maintenance of membrane potential (Vm) in
epithelial cells (32), and such channels could thus play
an important role in epithelial Na+ transport by
maintaining the driving force for Na+ entry (25,
27). The present study, in common with earlier data
(27), showed clearly that Ba2+ abolishes the
response to isoprenaline, but, in our hands, this cation caused only
transient inhibition of basal Isc. This result is difficult to reconcile with the view that Ba2+ acts by
selectively inhibiting K+ channels, and subsequent
experiments showed that it also reduced the activity of the basolateral
Na+ pump. Although this provides an alternative explanation
for the inhibition of the response to isoprenaline, it cannot account for the transient inhibition of the basal Isc.
High concentrations of Ba2+, in common with other
alkaline earth ions, have the potential to interfere with the control
of the internal free Ca2+ concentration
([Ca2+]i) (18, 19), and it is
interesting, in this context, that Marunaka and colleagues
(22) attribute the isoprenaline-evoked increase in
Isc to a cAMP-evoked increase in intracellular
[Ca2+]i. The inhibitory effect of
Ba2+ that we now describe could, therefore, also involve a
direct interaction with the -adrenoceptor-activated signal
transduction pathway. The important point to emerge from these
experiments is that Ba2+ exerts a number of effects under
the present conditions, and so this cation cannot be used as a
selective inhibitor of the K+ channels found in
Na+-absorbing epithelia.
Conductive properties of the apical membrane.
The small GCl measured in unstimulated
cells appears to be due to the presence of anion channels sensitive to
DIDS and glibenclamide. Isoprenaline and forskolin elicited clear
increases in GCl
, and these responses were inhibited by glibenclamide but not DIDS (see also Ref.
16), suggesting (see for example Refs. 10,
24, 41) that the currents elicited by
cAMP-coupled agonists reflect the activation of anion channels formed
by the cystic fibrosis transmembrane conductance regulator (CFTR), the
gene product that is defective in cystic fibrosis (CF)
(43). Support for this view came from parallel studies
(O. G. Best, A. Collett, S. M. Wilson, and A. Mehta,
unpublished data) in which apical membrane Cl
currents
were characterized in two human airway epithelial cell lines. In
wild-type cells (16HBE14o
), forskolin evoked changes in
GCl
that were qualitatively similar to
those described here. However, this response was absent from cells
expressing a CF phenotype (CFBE41D
), which have only
minimal levels of functional CFTR in the apical membrane. Our findings
thus accord with the results of earlier, more detailed studies of cells
isolated from adult animals that identified CFTR-like anion channels in
distal lung epithelial cells (16, 29). However, in our
hands, the cAMP-evoked increase in GCl
consisted of an initial peak followed by a slower, more sustained
phase, whereas previous studies of anion currents flowing through
CFTR-like channels have reported that cAMP causes a much more sustained
response (12, 16). The reason for this discrepancy between
these two studies is unknown but may reflect the different origin of
the cells used in the two studies. However, CFTR can be subject to
relatively rapid "rundown" in many experimental situations, and it
has become clear that this process is controlled, at least in part, by
Ca2+-dependent protein kinases (45). The cells
used in the present study were cultured under conditions different to
those used by Jiang et al. (16), and so it is therefore
possible that the variations between the observed time courses may
reflect slight differences in the basal level of protein kinase C
activity. However, it is also possible that the increase in
GCl
that we report may involve the
activation of other anion channel species. It is interesting, in this
context, that activation of CFTR has been shown to trigger the
increased activity of other anion channels in epithelial cells and that
this downstream effect is abolished when CFTR is blocked (9,
39). It is therefore possible that the ionic movements
underlying the cAMP-evoked Cl
currents that we now
describe may not occur exclusively via CFTR.
Physiological effects of the changes in
GNa+ and GCl.
The present estimates of GCl
and
GNa+ were combined with previously reported
values of Vm and internal [Cl
]
and [Na+] to allow us to predict the Cl
and
Na+ currents flowing across the apical membrane under basal
conditions and after prolonged stimulation with isoprenaline. Although
there is a driving force for Cl
entry in unstimulated
cells, GCl
is so low that only a
negligible (<0.5 µA/cm2) Cl
current can
flow (Table 2). However, our analysis
suggests that this would be accompanied by a substantial (~7
µA/cm2) inward Na+ current (Table 2) and thus
predicts that Na+ absorption is the dominant electrogenic
transport process in unstimulated cells. Stimulation with isoprenaline
reverses the driving force for Cl
movement, suggesting
that this drug may evoke the secretion of this anion, and so our
analysis thus predicts a rapid but transient rise in
Isc due to the secretion of Cl
,
although it is difficult to anticipate the magnitude of this early
current as Vm and internal [Cl
]
are both subject to rapid change at this time (22, 42). During prolonged stimulation, however,
GCl
remains relatively low, and so the
total, inwardly directed Cl
current would amount to only
~1 µA cm
2 (Table 2). Most importantly, our model
predicts that this initial transient would be followed by a slowly
developing but persistent rise in Isc that is
due, very largely (>90%), to increased Na+ absorption.
There is thus excellent agreement between the observed and predicted
responses, and so the changes in GCl
and
GNa+ that we now report can explain the
well-documented effects of isoprenaline upon these cells (23, 30,
36-38). Moreover, in A6 cells, a Na+-absorbing
cell line derived from the amphibian kidney, cAMP-dependent agonists
have been reported to elicit a biphasic response similar to that
described above (7). This pattern of response may thus be
a generalized feature of salt-absorbing epithelia.
|
Comparison with previous work.
When stimulated with cAMP-coupled agonists, the adult cells used by
Jiang et al. (16) displayed a rapid fall in
Isc followed by a slow recovery toward the basal
level consistent with a rapid stimulation of Cl
absorption superimposed upon a slowly developing stimulation of
Na+ transport, a finding that accords with data from
earlier isotope flux studies of such cells (17). The
fetally derived cells used in the present study, however, responded to
isoprenaline with a clear stimulation of Na+ transport that
appeared to be accompanied by only a very small stimulation of
Cl
secretion. As anion secretion is an important
physiological feature of the fetal lung (31), it could be
argued that the presence of this small secretory response implies that
the cells have retained some features of the fetal lung.
Possible role of the CFTR-like channels.
The present study shows that prolonged stimulation with cAMP-coupled
agonists leads to a clear rise in GNa+, which implies increased Na+ entry despite a fall in
VNa+ (Table 2). Because Jiang et al.
(16) did not detect such control over
GNa+, they concluded that cAMP-dependent
agonists do not activate Na+ channels in alveolar
epithelia. Instead, they suggested that such drugs might stimulate
Na+ transport by activating the CFTR-like anion channels,
hyperpolarizing the cell and thus increasing
VNa+ (16). There are, however,
conceptual problems with this model, some of which have been discussed
by Widdicombe (44). For example, the analysis presented
here suggests that VCl is negative under
resting conditions, and so, rather than hyperpolarizing the cell, a
rise in GCl
would initially favor depolarization and a fall in VNa+ (Table
2).
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ACKNOWLEDGEMENTS |
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The authors are grateful to Helen Murphie for skilled technical help and to Sarah Inglis for many helpful comments and suggestions.
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FOOTNOTES |
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The authors are also grateful to the Wellcome Trust for the financial support (program grant no. 0548/Z/99/Z/JMW/CP/JF) that made this study possible.
Address for reprint requests and other correspondence: S. M. Wilson, Lung Membrane Transport Group, Tayside Institute of Child Health, Ninewells Hospital and Medical School, Univ. of Dundee, Dundee DD1 9SY, UK (E-mail: S.M.Wilson{at}Dundee.ac.uk).
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.00142.2001
Received 24 April 2001; accepted in final form 5 November 2001.
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REFERENCES |
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