American Journal of Physiology-, Lung Cellular and Molecular Physiology, February 2000, Volume 278 (22)
AMILORIDE-SENSITIVE ACTIVE TRANSPORT of Na across the
alveolar epithelium removes lung liquid in the neonate and protects against edema in the adult. Several lines of evidence have established that this process is stimulated by Active Na absorption across the alveolar epithelium presumably occurs
by the standard mechanism (8): diffusion of Na across the apical
membrane down its electrochemical gradient via amiloride-sensitive channels followed by active extrusion across the basolateral membrane by Na-K-ATPase. The inward Na current across the apical membrane (INa) is given by the product of the membrane
conductance to Na (GNa) and the driving force for
net Na entry (Va The overall Na conductance of the alveolar apical membrane (in
mS/cm2) is given by GNa = g · Po · N,
where g is the conductance of individual channels,
Po is their average probability of being open, and
N is their frequency per unit area of membrane. Patch-clamp studies on cultured type II cells (9) have shown that the
predominant Na channel is amiloride sensitive and has a G of
~27 pS. Terbutaline approximately doubles the Po
of this channel without affecting G (9). The percent increase
in Po of this channel is approximately the same as
the percent increase in active Na absorption measured as
amiloride-sensitive Isc in Ussing chambers.
However, these experiments were done on single isolated cells, not
confluent cell sheets with tight junctions and a measurable transepithelial resistance. It is argued (9) that "ATII cells in
primary culture orient themselves so that their basal membranes are
attached to the substratum and the apical membranes are pointing upward." But without tight junctions, one cannot be
sure that the upper membrane has the same composition as a true apical
membrane. In fact, it almost certainly doesn't. Structural
specializations on the apical membranes of confluent polarized cells
(e.g., microvilli and glycocalyx) make them notoriously hard to patch
clamp, which is why most studies are performed on single cells or small
clumps of cells. Extrapolation of the results from single cells to the function of the intact epithelium should be done with caution; absence
of tight junctions may alter the types of ion transport present. In
airway epithelium, for instance, the Na-K-2Cl cotransporter of intact
epithelium becomes a Na-Cl cotransporter on cell dispersion (5), and
nonconfluent cells contain a form of Ca-activated Cl conductance absent
from the apical membrane of intact epithelia (1).
In contrast to a direct opening of Na channels, O'Grady et al. (11)
suggest an indirect role for Cl in modulating active absorption of Na.
Specifically, they propose that the apical membrane of alveolar type II
cells has a cAMP-dependent Cl conductance. Also, the electrochemical
gradient for Cl is inward across the apical membrane (i.e.,
ECl is more negative than Va).
Therefore, the opening of Cl channels by cAMP results in
hyperpolarization of the apical membrane and an increased driving force
for entry of Na.
This is plausible, but close inspection of the original article by
Jiang et al. (6) shows that terbutaline did not in fact increase
overall Isc nor are data presented that show that
terbutaline increases amiloride-sensitive Isc. In
fact, representative traces suggest that terbutaline did not increase
active Na absorption (see Fig. 1 in Ref. 11). Nevertheless, others
using slightly different alveolar type II cell cultures have reported
increases of 40 (4) or 110% (10) in active Na absorption with
terbutaline, and it would be worth using such cells to test the
mechanism proposed by O'Grady et al. (11). The first step of O'Grady
et al.'s hypothesis has been established: cultures of alveolar cells
do indeed have an apical membrane Cl conductance. Thus Jiang et al. (6)
permeated the basolateral membrane of confluent cell sheets with
amphotericin and imposed a Cl gradient across the remaining apical
membrane. The addition of terbutaline or cAMP to such preparations
increased the Isc. The authors used the same
permeabilized preparation to provide evidence against a cAMP-activated
apical GNa. With a Na gradient imposed across the
isolated apical membrane, cAMP failed to increase amiloride-sensitive
transepithelial currents. But given that nonpermeabilized cultures
showed no increase in amiloride-sensitive Isc in
response to terbutaline, this result was to be expected.
The other assumptions in the scheme proposed by O'Grady et al. (11)
are that the electrochemical gradient for Cl is directed inward across
the apical membrane and that the opening of Cl channels by cAMP results
in hyperpolarization of Va. However, there is a
difficulty with this part of the proposed mechanism: the driving force
for Na entry is already comparatively large and would need to be
increased substantially by opening the Cl channels. The driving force
for Na entry is ENa In addition to a possible role in regulating active Na absorption,
O'Grady et al. (11) suggest that liquid absorption across the alveolar
epithelium can be stimulated by raising transcellular Cl permeability
or by increasing active absorption of Cl. The first possibility has
recently been demonstrated in primary cultures of bovine airway
epithelium (13). The second suggestion is based on the observation by
Kim et al. (7) that addition of terbutaline to short-circuited cultures
induced a significant net absorption of Cl. This interesting finding
can be explained by the downhill movement of Cl through apical membrane
Cl channels followed by extrusion across the basolateral membrane by
cotransport with K. This would constitute a form of secondary active
transport driven by the concentration gradient for K across the
basolateral membrane.
In summary, more work needs to be done to test both hypotheses. The
apical GNa needs to be studied in confluent cell
sheets as well as in single isolated cells. A good approach is to
measure Va with microelectrodes and alter the Na
concentration in the mucosal bath in the presence and absence of
amiloride. Preparations in which the basolateral membrane has been
permeated could also be used in Ussing chambers, provided it is first
established that the intact tissue does indeed show increases in
amiloride-sensitive Isc in response to
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REFERENCES
-adrenergic agents via elevation of cAMP. First, when the lungs are filled with saline in vivo, then
-adrenergic agents or exogenous cAMP stimulates removal of this
liquid (2, 3, 12). Furthermore, the increase in liquid clearance
induced by terbutaline is inhibited by amiloride (3, 12). Second, in
cultured cells, the amiloride-sensitive short-circuit current
(Isc) is stimulated by terbutaline (4, 10).
Finally, the terbutaline-induced increase in Isc is
mirrored by an increase in net Na absorption as determined from the
transepithelial fluxes of 22Na (7).
ENa),
where Va is the apical membrane potential
difference and ENa is the equilibrium potential for
Na. In this issue of the American Journal of Physiology-Lung
Cellular and Molecular Physiology, Lazrak et al. (9) argue that
-adrenergic agents stimulate Na absorption by an effect on
GNa, whereas O'Grady et al. (11) believe that the
stimulation is due to a change in Va brought about
by an increase in Cl conductance.
Va.
ENa is likely to be approximately +50 mV, and
Va in short-circuited tissues (the kind used to
measure active Na transport) is probably negative [it is
60 mV in short-circuited airway epithelium (14)]. Thus the
driving force for Na entry should be between 50 and 100 mV. Therefore,
to increase active Na absorption by 40-110% solely by altering
the driving force will require changes in Va of at
least 20 mV and possibly much more. Thus ECl would
have to be considerably more negative than Va
(i.e., intracellular Cl activity would be much lower than
the equilibrium level predicted for passive distribution according to
Va). Clearly, microelectrode measurements of
Va and intracellular Cl activity are needed to test
these predictions.
-adrenergic agents or cAMP. As to the idea that opening Cl channels
increases Na transport by increasing the driving force for Na across
the apical membrane, microelectrodes (both ion sensitive and
conventional) need to be used to determine the direction of the
electrochemical driving force for Cl and to see whether the opening of
Cl channels does indeed hyperpolarize Va by the
required amount. Finally, it must be noted that stimulating Na
absorption by increasing the GNa or the
electrochemical driving force for Na entry are not mutually exclusive
possibilities, and both sets of workers might be partially right.
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