(Received for publication, March 30, 1995; and in revised form, May 31, 1995)
From the
In the preceding manuscript (Zhao, H., Xu, X., Diaz, J., and
Muallem, S.(1995) J. Biol. Chem. 270, 19599-19605), we
described a K-dependent
H
/HCO
and Na
influx pathway in the luminal membrane of salivary duct cells. In
the present studies, we further characterized this pathway to show that
the K
-dependent Na
influx was not mediated by the luminal amiloride-sensitive
Na
channel, the Na
/H
exchangers, or any electroneutral or conductive
Cl
-dependent transport pathway. Thus, K
efflux probably maintained electroneutrality during Na
influx induced by removal of K
.
Accordingly, Na
influx was largely inhibited by 2.5
mM external Ba
. The K
site
of the K
-dependent Na
influx showed the selectivity sequence Cs
>
K
> NH
>
Li
which is different from that of several known
K
channels. More importantly, Na
influx is 50% inhibited at about 20 mM
K
, and significant Na
influx occurred even at 80 mM
K
. This is a critical property for the
pathway to play a role in Na
reabsorption and
K
secretion by the duct. The large Na
influx in resting duct cells is matched by high activity of the
ductal Na
pump which is about 8-fold faster than that
of acinar cells. Stimulation of submandibular ducts with various
agonists increased [Na
]
in an agonist-specific manner. The parasympathetic agonist
epinephrine was more effective than isoproterenol and the sympathetic
agonist carbachol. The use of various inhibitors of Na
and K
transporters suggests that different
pathways mediate Na
influx in stimulated acinar and
duct cells of the gland. In duct cells, Na
influx was
inhibited only by extracellular Cs
and
Ba
. The overall findings support a significant role
for the K
-dependent pathway(s) in
Na
reabsorption and K
and
HCO
secretion and explain several
features of transepithelial electrolyte transport by salivary ducts.
The main function of the salivary gland ductal system is the
reabsorption of Na and Cl
and the
secretion of K
and HCO
(1) . Cl
absorption is coupled in part to
HCO
secretion and in part to
Na
absorption(2, 3, 4) . A
significant portion of HCO
secretion is
coupled to K
secretion(2, 5) .
However, the bulk of K
secretion is coupled to
Na
absorption(5, 6, 7) .
These findings together with measurements of inter-relationships among
ions transported during transepithelial electrolyte transport (2) have led to a model in which most Na
reabsorption and K
and
HCO
secretion are mediated by a luminal
Na
/H
and
K
/H
exchangers(1, 2) .
In the preceding
manuscript (8) we measured pH(
)and [Na
]
in isolated submandibular ducts and the perfused main duct
to show the localization and inter-relationships between several
anticipated (1, 2) and several newly discovered (8) ion transporters. We confirmed the existence of luminal
Na
/H
and
Cl
/HCO
exchangers and
demonstrated the presence of similar transporters in the basolateral
membrane of duct cells. However, we could not obtain evidence for a
coupled K
/H
exchanger, similar to
that described in other epithelial
cells(9, 10, 11) . Instead, we identified a
luminal, duct-specific, K
-sensitive ion
transport pathway(s) which transports
H
/HCO
and
Na
. We postulate that the
K
-sensitive pathway(s) mediates most of
the K
secretion and some of the Na
reabsorption by salivary ducts. To perform these tasks, the
pathway should be regulated by ductal secretagogues and allow the
transport of H
/HCO
and
Na
in the presence of high
K
. Thus, the concentration of
K
in the primary saliva produced by acinar cells is
about 5 mM whereas that measured in the secondary saliva is
about 70-80
mM(3, 7, 12, 13, 14, 15) .
In the present studies we show that Na transport
initiated by changes in K
is not
mediated by any of the previously described Na
transporters of duct cells and that the pathway can mediate
Na
influx in the presence of 40-60 mM
K
. Stimulation of duct cells with
various ductal secretagogues increases
[Na
]
, which may be
mediated by the K
-dependent pathway.
All materials and experimental procedures were the same as in
the preceding manuscript(8) . In the present studies we used
SBFI or Na-Green to estimate
[Na
]
. It was necessary
to use Na
-Green to evaluate the effect of amiloride on
ductal Na
transport because amiloride and all
analogues tested have strong fluorescence when excited at the
340-380 nm range. A disadvantage of using
Na
-Green is that this dye has no isosbestic point and
in many experiments a large fraction of the dye behaved as if it were
extracellular. This reduces the signal/noise ratio, prevents proper
calibration, and precludes testing the effect of various
Na
concentrations on Na
transport.
Na-Green was loaded by incubating
the cells with 2.5 µM Na
-Green/AM for 15
min at room temperature. The cells were then washed once, resuspended
in about 5 ml of PSA, and kept on ice until plating on coverslips.
Na
-Green fluorescence was measured by photon counting
at an excitation wavelength of 490 nm. The emitted light was passed
through a dichroic mirror and filter set DM510 from Nikon and directed
to a photomultiplier tube. The operational, storage, and data analysis
programs were from PTI.
Figure 1:
Effect of amiloride on
K-dependent Na
influx.
Intralobular ducts from the submandibular salivary gland loaded with
Na
-Green were perfused with solution A (NaCl, 5 mM K
) or solution B (K
-free) (a and b). As indicated, 0.5 mM amiloride (a) or 25 µM DMA (b) was included in the
perfusate.
These cells have a luminal Cl channel (18) which may affect electrogenic cation transport. The
ubiquitous Na
-K
-2Cl
cotransporter (19) can potentially mediate some of the
Na
influx. Fig. 2a shows that removal
of Cl
slightly reduced the rate of
Na
influx (32 ± 8%, n = 4).
However, this may be secondary to the acidification of the cytosol
caused by Cl
removal (see Fig. 2in the
preceding manuscript(8) ). Indeed, in the presence of
HCO
, Na
influx was not
affected by Cl
removal (within 8 ± 11% of
control) (Fig. 2c). Inhibition of the cotransporter
with bumetanide had no apparent effect on the rates of Na
influx or efflux induced by changes in K
(Fig. 2b). We therefore conclude that
Cl
fluxes are not required for Na
influx and/or K
efflux induced by removal of
K
.
Figure 2:
Na influx is independent
of Cl
transport in duct cells. In experiment a, SBFI-loaded ducts were perfused with solution A (NaCl)
containing 5 mM K
, solution B
(K
-free), and then solution A (control period), before
perfusion with solution D (Cl
-free, 5 mM K
), K
-free solution D, and then
solution D. In experiment b, ducts were perfused with solution
A or solution B and with or without 0.1 mM bumetanide as
indicated. In experiment c, the ducts were equilibrated in
solution E (NaCl, HCO
) before perfusion
with solution F (Cl
-free,
HCO
) and K
-free solution
F.
To test the possible involvement
of ion channels in Na influx, we measured the effect
of known inhibitors of K
and nonselective cation
channels on Na
influx and the ionic selectivity of the
K
site. Fig. 3a shows
that 10 mM tetraethylammonium had no effect on Na
fluxes. Similarly, Na
influx was not inhibited
by tetrabutylammonium, quinine, quinidine, and apamin (not shown). On
the other hand, 2.5 mM Ba
reduced the
steady-state [Na
]
of
resting duct cells by about 4.6 ± 0.6 mM (n = 5) (Fig. 3b). Ba
also
inhibited the Na
influx induced by
K
removal in a reversible manner. Fig. 3c shows that addition of Ba
to
cells incubated in K
-free medium caused only a small
reduction in [Na
]
.
However, when K
was added to the medium, the cells
rapidly reduced [Na
]
,
in the presence (Fig. 2c) or absence (not shown) of
Ba
. The effects of Ba
on
Na
influx and efflux are identical with those on
H
fluxes shown in the preceding manuscript (8) .
Figure 3:
Effect of
channel blockers on Na influx. In all experiments (a-c), the ducts were alternately perfused with solution
A (NaCl, 5 mM K
) or solution B
(K
-free). Where indicated, these solutions also
contained 10 mM tetraethylammonium (a) or 2.5 mM Ba
(b and c).
The ionic selectivity of the
K site is shown in Fig. 4. Fig. 4a shows that Li
up to 10 mM could not substitute K
either in
preventing Na
influx or in activating the
Na
pump-mediated Na
efflux from
Na
-loaded cells. NH
at 5
mM partially substituted K
in
inhibiting Na
influx but was a poor substitute for the
Na
pump (Fig. 4b). Higher
concentrations of NH
were not tested
because of its effect on pH
. Fig. 4c shows that Cs
substituted well for
K
in inhibiting Na
influx and as a substrate for the Na
pump.
Interestingly, unlike K
, the second
removal of Cs
from the medium failed to cause rapid
and large Na
influx. This is probably because
Cs
cannot be transported by the
K
-dependent pathway to mediate
Cs
- Na
exchange. Fig. 4d shows the apparent affinity for
Cs
in inhibiting Na
influx. In this
experiment, Na
influx was inhibited by Cs
and the Na
pump was inhibited by ouabain.
Stepwise reduction in Cs
caused a
stepwise increase in
[Na
]
. Cs
inhibited the K
-dependent
Na
influx pathway with an apparent IC
of
about 2.5 mM.
Figure 4:
Effect of K congeners on
Na
influx in duct cells. In experiment a,
ducts in solution A (NaCl, 5 mM K
) were
perfused with solution B (K
-free) in which 10 mM Li
replaced 10 mM Na
before perfusion with solution B, solution B containing 10 mM Li
, and then solution A. In experiments b and c, the same protocol was followed except that 5
mM NH
(b) or 10 mM Cs
(c) replaced equivalent amounts of
Na
in solution B. In experiment d, the duct
was perfused with solution B containing 10 mM Cs
and then the same solution containing 0.5 mM ouabain.
Subsequently, the concentration of Cs
was reduced by
replacement with an appropriate concentration of
Na
.
Fig. 5shows the
K dependence of Na
influx. In the absence of ouabain, reducing
K
from 5 to 2 mM had a small
effect on [Na
]
(Fig. 5a). Further reduction in
K
to 1, 0.5, 0.25, and 0 mM resulted in a stepwise increase and stabilization of
[Na
]
at different
levels. A subsequent stepwise increase in K
caused a proportional reduction in
[Na
]
. Notably,
[Na
]
stabilized at
different levels at the same K
concentration during Na
influx and efflux. This
was observed in all experiments tested (n = 7). This
probably reflects the effect of
[Na
]
on the apparent
affinity of the pump for K
(20) .
The behavior of Na
influx in Fig. 5a shows the marked impact and activity of the Na
pump in intralobular ducts.
Figure 5:
Dependence of Na influx
on K
concentration. In experiment a, ducts were perfused with solution A containing the
indicated K
concentrations. In experiment b
, the duct was perfused with solution A in which
55 mM Na
was replaced with 55 mM K
and then with the high K
solution containing 1 mM ouabain. Trace b
shows the control experiment, in which NMG
replaced Na
. The experiment in c shows
the protocol used to measure the effect of K
on Na
influx independent of the Na
pump. The ducts were perfused with solution A in which different
concentrations of K
replaced equivalent concentrations
of Na
. All solutions also contained 1 mM ouabain to inhibit the Na
pump. Na
influx independent of the
K
-dependent channel and due to
inhibition of the Na
pump was estimated from
Na
influx in the presence of ouabain and 60
mM K
(broken line,
taken from experiment b
). The contribution of the
K
-dependent channel to Na
influx was calculated as the differences between the
[Na
]
in experiments c and b
(panel
d).
Fig. 5b shows how
the contribution of the K-sensitive
pathway and the Na
pump to Na
influx
were separated. Fig. 5b
shows that
increasing K
from 5 to 60 mM reduced [Na
]
by
about 6.7 ± 1.2 mM (n = 3). This was
due to inhibition of Na
influx and not stimulation of
the Na
pump, since pump activity was maximal at 5
mM K
. Fig. 5b
provides a control experiment
showing that the reduced Na
influx at 60 mM
K
was not due to the reduction in
Na
. Hence, at 90 mM
Na
and 5 mM
K
, ouabain caused substantial
Na
influx.
The conditions of Fig. 5b were used to determine the dependence of Na influx on K
, and the results are
illustrated in Fig. 5c. Na
influx was
inhibited by 60 mM K
, and the
Na
pump was inhibited by 1 mM ouabain.
Remarkably, reducing K
from 60 to 40
mM was sufficient to cause Na
influx, and the
influx was almost maximal at 5 mM
K
. Fig. 5d displays the
summary of 4 similar experiments and shows that the dependence of
Na
influx on K
followed
simple saturation kinetics with an apparent affinity for
K
of about 18.2 ± 3.4 mM (n = 4). Furthermore, extrapolation of the results
to higher K
indicates that the pathway
will allow some Na
influx even at 80 mM
K
. This property is a key feature of the
luminal K
-dependent pathway with respect
to its physiological role.
Fig. 6a shows that
stimulation of duct cells with Epi caused a rapid (27.8 ± 1.6
mM/min) and sustained increase in
[Na]
by about 7.4
± 0.4 mM (n = 21). Carbachol
stimulation increased [Na
]
of duct cells at a rate of 12.5 ± 0.8 mM/min
to an average of 4.7 ± 0.3 mM (n = 11)
above resting (Fig. 6b). In the same experiments,
carbachol rapidly and strongly increased
[Na
]
of acinar cells by
62 ± 4.7 mM (not shown). Isoproterenol had the smallest
effect on [Na
]
.
Isoproterenol stimulation increased ductal
[Na
]
(Fig. 6c) at a rate of 5.6 ± 0.4
mM/min, which stabilized at 2.8 ± 0.55 mM (n = 6) above control.
Figure 6:
Effect of agonist stimulation on
[Na]
of submandibular
duct cells. The cells were stimulated by perfusion with solution A
containing 10 µM Epi (a), 100 µM carbachol (b), or 1 µM isoproterenol (c). In experiment b, cell stimulation was terminated
by perfusion with solution A containing 10 µM atropine.
In an attempt to
evaluate the contribution of the different Na influx
pathways to the agonist-dependent
[Na
]
increase, we
tested the effect of several transport inhibitors on Na
influx. Results with the submandibular acinar cells are presented
as a positive control and to contrast the behaviors of the two cell
types. Carbachol was the most active agonist in acinar cells, and,
therefore, results obtained with this agonist are shown in Fig. 7, a-d. Inhibition of the
Na
-K
-2Cl
cotransporter with bumetanide reduced the rate of Na
influx into carbachol-stimulated acinar cells by about 24
± 9% (n = 4), but had a minimal effect on the
extent of [Na
]
increase. Removal of bumetanide slightly increased
[Na
]
(Fig. 7b). Ba
reduced the
rate of Na
influx by 46 ± 11% (n = 3) (Fig. 7c). However, most of the effect
of Ba
is probably indirect since Ba
is likely to interfere with the initial KCl efflux and cell
shrinkage, which is critical for activation of Na
influx in stimulated acinar cells(22) . DMA inhibited
Na
influx rate by about 72 ± 17% (n = 4) (Fig. 7a). Since Epi is the most
active agonist in granular intralobular ducts, the results obtained
with this agonist are shown in Fig. 7, e-h. Fig. 7f shows that bumetanide had no apparent effect on
the Epi-stimulated [Na
]
increase (n = 6). Notably, 25 µM DMA had no apparent effect on the agonist-mediated
[Na
]
increase in the
presence or absence of K
(Fig. 7g), excluding both the amiloride-sensitive
Na
channel and the Na
/H
exchangers as major contributors to this Na
influx. Fig. 7h shows that 2.5 mM Ba
nearly abolished the Epi-stimulated
[Na
]
increase and the
large Na
influx observed after removal of
K
.
Figure 7:
Effect of various inhibitors on
agonist-induced [Na]
increase in duct and acinar cells. In experiments a-c, SBFI-loaded acinar cells were stimulated with 100
µM carbachol after perfusion in the absence (a)
or presence of 0.1 mM bumetanide (b) or 2.5 mM Ba
(c). In experiment d, a
Na
-Green-loaded acinus was perfused with solution A
containing 25 µM DMA before stimulation with 100
µM carbachol. In experiments e, f, and h, SBFI-loaded ducts were stimulated with 10 µM Epi in the absence (e) or presence of 0.1 mM
bumetanide (f) or 2.5 mM Ba
(h). Where indicated in experiment h, the
stimulated duct was also perfused with solution B containing 2.5 mM Ba
and then solution B. In experiment g, a Na
-Green-loaded submandibular duct was
incubated with 25 µM DMA before stimulation with 10
µM Epi. Removal of K
in the
continuous presence of DMA rapidly increased
[Na
]
.
Additional evidence that different
pathways mediate the major portion of the Na influx in
duct and acinar cells is presented in Fig. 8. Fig. 8a shows that replacing K
with 5
mM Cs
inhibited the Epi-evoked
[Na
]
increase in duct
cells. Removal of Cs
from the incubation medium
resulted in a rapid increase in
[Na
]
. Replacing
K
with 5 mM Cs
had a minimal effect on the carbachol-induced
[Na
]
increase in acinar
cells (not shown). Fig. 8, b and c, shows that
increasing K
from 5 to 40 mM reduced [Na
]
in
Epi-stimulated ducts by 4.7 ± 0.6 mM (n = 3), whereas it increased acinar
[Na
]
by 6.2 ±
0.8 mM (n = 3).
Figure 8:
Inhibition of agonist-induced
[Na]
increase by
Cs
and high K
in duct
but not acinar cells. In experiment a, the duct was incubated
in solution B (K
-free) containing 5 mM Cs
before stimulation with 10 µM Epi
and then with solution B and again with solution B containing 5 mM Cs
. In experiments b and c,
acinar and duct cells present in the same recording field were
stimulated with 10 µM Epi before exposure to solution A in
which 35 mM K
replaced 35 mM Na
.
The lack of effect of DMA
and the inhibition by Cs and Ba
of
the agonist-dependent [Na
]
increase in duct cells suggests that the
K
-dependent pathway may mediate a
significant part of Na
influx in stimulated duct
cells. To further test this possibility, we measured the effect of
agonist stimulation on Na
influx evoked by removal of
K
. Fig. 9a shows that
Epi stimulation increased the rate of Na
influx due to
K
removal by 1.84 ± 0.06-fold,
and [Na
]
stabilized at
about 67 ± 5.1 mM (n = 8). In addition,
Epi stimulation of cells incubated in the absence of
K
further increased
[Na
]
by about 9.6
± 1.7 mM (Fig. 9b).
Figure 9:
Stimulation by Epi of
K-dependent Na
influx
in duct cells. In experiment a, the duct was perfused with
solution A (NaCl, 5 mM K
), solution B
(K
-free), and then solution A (control period), before
stimulation with 10 µM Epi and perfusion with solution B.
In experiment b, the duct was perfused with solution B before
stimulation with 10 µM Epi and perfusion with solution
A.
To evaluate the
effect of cell stimulation on Na influx independent of
Na
pump activity, we measured the effect of ouabain on
Na
influx in stimulated cells. Fig. 10a shows that stimulation of cells incubated with ouabain increased
the rate of Na
influx from 9.8 ± 0.6 to 34.7
± 4.2 mM/min, and
[Na
]
stabilized at
about 58 ± 4 mM (n = 3), which was
about 10-12 mM lower than that caused by removal of
K
. Removal of K
increased [Na
]
in
duct cells treated with ouabain and Epi (Fig. 10b) by
about 13 mM. The reciprocal protocol is shown in Fig. 10b. Addition of ouabain to Epi-stimulated cells
caused [Na
]
to increase
at a rate of 28.4 ± 2.6 mM/min, which was about
2.9-fold faster than in unstimulated cells (Fig. 10a).
Figure 10:
Effect
of ouabain on [Na]
of
Epi-stimulated duct cells. Ducts were treated with 0.5 mM ouabain before (a) or after (b) stimulation with
10 µM Epi. In experiment b, where indicated, the
duct was exposed to solution B (K
-free) containing 0.5
mM ouabain.
The major function of the salivary ductal system is the
modification of electrolyte composition of the primary
saliva(1) . This is achieved by HCO and K
secretion and Na
reabsorption, processes that are coupled to each
other(1, 2) . It has been difficult to fully explain
the coupling of these activities under various physiological and
experimental conditions(2) . The most commonly accepted model
assumes that luminal Na
/H
and
K
/H
exchangers are responsible for
the cotransport of these ions in duct cells(1, 2) .
The model, however, does not account well for
HCO
secretion and does not explain
several properties of ductal electrolyte transport. For example it was
shown that 10 µM amiloride, which blocks the luminal
Na
channel (16) but is not sufficient to
inhibit the epithelial type Na
/H
exchanger(8, 17) , largely blocked Na
reabsorption(5, 23, 24, 25) .
It is not clear why Na
concentration of the luminal
solution has to be reduced well below that needed for Na
reabsorption (2, 5) in order to stimulate
HCO
secretion(2, 15) .
In addition, a K
/H
exchange activity
cannot be demonstrated in mildly (8, 17) or strongly
acidified duct cells(8) . Hence, alternative mechanisms have to
be invoked for HCO
and K
fluxes at the luminal membrane.
In the preceding
manuscript(8) , we reported the presence of a luminal,
K-dependent pathway which allows the
transport of H
/HCO
and
Na
(8) . In the present studies, we show that
this pathway is different from any amiloride-sensitive Na
pathway present in duct cells. Neither low nor high
concentrations of amiloride inhibited Na
influx
induced by removal of K
. Low
concentrations of amiloride (up to 20 µM) had no effect on
[Na
]
. Amiloride reduced
resting [Na
]
only at
high concentrations. This indicates that the
K
-dependent Na
influx
is not mediated by the amiloride-sensitive Na
channel
and that this channel has minimal contribution to Na
influx in resting duct cells.
Another critical implication of
the experiments with amiloride is that a coupling between
K/H
and
Na
/H
exchangers cannot possibly
mediate Na
influx and K
efflux from
duct cells. Coupling between the exchangers requires that when
Na
influx is initiated by removal of
K
, H
influx induced by
K
/H
exchange is matched by
Na
/H
exchange to maintain relatively stable pH
and
mediate large Na
influx. In this case, a high
concentration of amiloride was expected to completely inhibit
Na
influx. In fact, as judged from measurements with
Na
-Green, amiloride reduced Na
influx
by only about 13%. This corresponds to about 5.7 mM Na
influx based on measurements of
[Na
]
with SBFI. This
value agrees reasonably well with measurement of pH
shown in the preceding manuscript (8) in which
amiloride increased the K
-dependent
acidification by about 0.16 pH unit. Measurement of H
buffer capacity of duct cells using established
procedures(26, 27) showed it to be about 35
mM/pH unit at a pH
of 7.1. This
translates to the transport of 5.4 mM H
, and,
therefore Na
, by the Na
/H
exchanger. Hence, Na
/H
exchange
mediates a small fraction of Na
influx in duct cells
and thus coupled Na
/H
and
K
/H
exchange cannot account for
K
and HCO
secretion and
Na
reabsorption by the duct.
Other pathways that do
not contribute to, or are essential for, the Na influx
induced by removal of K
are the
Cl
-dependent pathways. These include
Na
-K
-2Cl
and NaCl
cotransport and the luminal and basolateral Cl
channels present in duct cells from various
species(1, 18, 28) . An important implication
of this conclusion is that another ion must be transported together
with Na
to maintain electroneutrality. This ion must
be K
. Therefore, although we did not measure
K
transport directly, K
efflux must
occur and account for the bulk of Na
influx induced by
removal of K
.
A potential transporter
that can mediate the K-dependent
Na
influx is the nonselective cation channel found in
many epithelial cells, including pancreatic and salivary acinar
cells(29) . We could not obtain evidence for the involvement of
this channel in Na
influx into resting or stimulated
submandibular duct cells. Although the
K
-dependent Na
influx
was completely inhibited by Ba
, it was not sensitive
to other channel blockers such as tetraethylammonium, quinidine, or
apamin. Furthermore, the K
-dependent
Na
influx was active at resting levels of
[Ca
]
in duct cells,
whereas no such activity was found in submandibular acinar cells
present in the same recording field. Finally, replacing
K
with Cs
inhibited
Na
influx even when the Na
pump was
inhibited, indicated that Cs
directly inhibited the
K
-dependent pathway. Cs
should not inhibit Na
influx by the nonselective
channel, as replacing K
with
Cs
in the absence of Cs
influx should
have hyperpolarized the cells and augmented Na
influx
mediated by a nonselective channel. Although these observations do not
completely exclude that the K
-dependent
Na
influx is mediated by the nonselective cation
channel, they suggest that such is unlikely.
An important property
of the K-dependent pathway is that it
allows Na
influx in the presence of very high luminal
K
(60-80 mM). Of course, the high
concentration of K
in saliva(3, 7, 12, 13, 14, 15) dictates
that if the pathway is to play a role in salivary electrolyte transport
it must allow Na
influx at high luminal
K
. Nevertheless finding such a property was important
in establishing a physiological role for this transporter. Since the
K
-sensitive pathway dominates
Na
influx in submandibular duct cells, it is possible
that the main function of the amiloride-sensitive Na
channel is to regulate the potential across the luminal membrane
and thus control fluxes through the
K
-dependent pathway. In this case, the
activity of both transporters is required for electrolyte transport by
the duct. The major role of the luminal
Na
/H
exchanger may be to prevent
accumulations of H
next to the apical membrane during
HCO
secretion and thus facilitate
Cl
reabsorption by the luminal
Cl
/HCO
exchanger.
Indeed, 100 µM amiloride inhibits the ductal
Na
/H
exchanger by less than 50% (17) , whereas 10 µM amiloride inhibits
Na
absorption by more than
90%(5, 23, 25) . If a major role of the
luminal Na
/H
exchanger was
Na
reabsorption, then 10 µM amiloride
should have reduced the reabsorption by about 10-15%.
It is
clear that the K-dependent pathway is
active in resting cells when luminal K
is 5
mM. This is concluded from the unmasking of significant
Na
influx by ouabain. The effect of ouabain on
[Na
]
also indicates a
tight coupling between Na
influx by the luminal
K
-dependent pathway and the basolateral
Na
pump. This finding was also demonstrated in the
main perfused duct when the luminal and basolateral transporters and
solutions are separated (Fig. 10, (8) ). Therefore,
unlike most cells, in salivary duct cells the major Na
influx mechanism that fuels the Na
pump is the
K
-dependent pathway and not the
Na
/H
exchanger. The physiological
consequence of this coupling is the maintenance of low
[Na
]
during
Na
reabsorption. Low
[Na
]
prevents
inhibition of the Na
/H
exchangers,
which probably function to remove the excess cytosolic H
accumulating during HCO
secretion.
Another interesting finding of the present studies was the
properties of the agonist-dependent
[Na]
increase in
submandibular duct cells. Bumetanide and amiloride had no effect on the
agonist-dependent [Na
]
increase. On the other hand, this activity was blocked by
Cs
and Ba
, and agonist stimulation
increased the rate of K
-dependent
Na
influx by 2-3-fold. This would suggest that
most Na
influx in stimulated submandibular duct cells
is mediated by the K
-dependent pathway.
Hence, independent of its identity, it is quite remarkable that the
K
-dependent pathway dominates
Na
influx in resting and stimulated submandibular duct
cells, whereas the amiloride-sensitive pathways make minor
contributions to Na
influx. It is therefore possible
that the K
-dependent transporter is the
major Na
influx pathway responsible for Na
reabsorption. If it can also mediate K
efflux
during Na
influx, it might also be responsible for the
secretion of K
into the duct lumen.
The findings
presented in the preceding (8) and present manuscripts explain
several features of electrolyte transport by duct cells which could not
be explained by previous models. The
K-dependent pathway provides a suitable
mechanism for ductal secretion of HCO
and
for the coupling between HCO
and
K
secretion. Our results also explain why the
secretion of K
and HCO
is augmented and becomes increasingly coupled in the absence of
luminal
Na
(2, 3, 5, 6) .
Under these conditions, the K
-dependent
pathway is expected to mostly extrude HCO
rather than absorb Na
. In the absence of luminal
Cl
, Na
reabsorption is reduced by
25%, and Na
influx becomes equal to K
efflux(2, 3) . Under these conditions,
Na
reabsorption can occur through the
K
-sensitive pathway in exchange for
intracellular K
. HCO
secretion by the rat submandibular duct is low compared to the
active transport of Na
and
K
(1, 2) . In the presence of 5 mM luminal K
, removal of luminal Na
increases HCO
secretion about
3-fold(2) . The lack of luminal K
/H
exchange (8) requires alternative pathways that allow
the K
and HCO
uptake
under these conditions. The K
-dependent
pathway(s) has the required properties to mediate such fluxes. Finally,
our results explain why agonist stimulation reduces the transepithelial
potential difference of the ducts(14, 15) . The
agonists activate the luminal
K
-dependent, Na
- and
K
-permeable pathways and increase
[Na
]
and probably
reduce [K
]
. Both of
these effects reduce the transepithelial potential difference. The
maintained high [Na
]
in
stimulated cells (Fig. 6) is expected to reduce the rate of
Na
influx at the luminal membrane and at least
partially inhibit the Na
/H
exchangers(30, 31) . As a consequence, agonist
stimulation is expected to reduce the rate of Na
reabsorption. This was indeed found in many studies using the
perfused main duct(1) .