(Received for publication, March 30, 1995; and in revised form, May 31, 1995)
From the
The mechanisms mediating transepithelial ion transport in
salivary ducts were characterized and localized by studying the
regulation of [Na]
,
[K
]
, and pH
in isolated intralobular ducts and perfused main ducts of
the submandibular salivary gland. A new procedure was developed for the
rapid preparation of intralobular ducts. Measurements of pH
revealed the presence of Na
/H
and Cl
/HCO
exchange activities in intralobular duct cells. We could not obtain
evidence for a coupled K
/H
exchange
activity which was postulated to exist in the luminal membrane of duct
cells. Rather, a K
-dependent pathway
which mediates the transport of
H
/HCO
and Na
was found. This pathway was absent from acinar cells of the same
gland and was active in unstimulated duct cells incubated in 5
mM K
. Accordingly, inhibition
of the Na
pump with ouabain resulted in rapid and
large Na
influx in duct but not acinar cells.
Perfusion experiments with the experimentally accessible main duct and
measurements of pH
were used to provide the first
direct localization of ion transporters in salivary ducts. The luminal
and basolateral membranes of the duct express separate
Na
/H
and
Cl
/HCO
exchangers.
Na
/H
exchange activity in both
membranes was similar, whereas the luminal
Cl
/HCO
exchange
activity was higher than that in the basolateral membrane. The perfused
main duct was also used to localize the newly discovered
K
-dependent
H
/HCO
and Na
transport pathway to the luminal membrane, which suggests that
this pathway may play an important role in Na
reabsorption and K
and
HCO
secretion by the salivary ductal
system.
Fluid and electrolyte secretion by the submandibular salivary
gland occurs in two steps(1, 2) . First, a plasma like
fluid is secreted by the acinar cells and cells in the
acinar-intercalated duct region. Subsequently, the electrolyte
composition of the primary fluid is modified by the ductal system. The
ducts reabsorb Na and Cl
, secrete
K
and HCO
, and reduce
the osmolarity of the fluid, apparently without a significant change in
the volume of the secreted fluid(3) .
The majority of the
studies to reveal the mechanism of vectorial ion transport in duct
cells was performed with the experimentally available perfused main
duct(3) . Ion substitution and the use of blockers led to a
model in which luminal K secretion is mediated by a
coupled, electroneutral K
/H
exchanger
in parallel with an epithelial type Na
/H
exchanger(3, 4) . Na
absorption
in the luminal membrane is mediated by the
Na
/H
exchanger and an
amiloride-sensitive Na
channel(5, 6) . Na
exit and
K
entry at the basolateral membrane are mediated by
the Na
pump. Salivary secretion is under autonomic
control. Both cholinergic and
-adrenergic stimulation inhibit
Na
reabsorption and reduce the transepithelial
potential (7, 8, 9, 10, 11, 12) .
-Adrenergic stimulation has variable effects depending on agonist
concentration(8, 10, 13) . Several
gastrointestinal hormones, including substance P, also modulate ductal
secretion similar to cholinergic stimulation(3, 14) .
In recent years techniques were developed to examine the mechanisms
by which agonists modulate salivary electrolyte secretion on the
cellular level. Patch clamp of intralobular duct cells was used to
demonstrate the presence of an amiloride-sensitive Na channel (6) and a Cl
channel in the
luminal membrane (15) . Measurement of pH
(
)by microspectrofluorometry demonstrated the
presence of a Na
/H
exchange in
intralobular striated duct cells(16) , but the membrane
localization of the exchanger has not been determined. No evidence for
Cl
/HCO
or coupled
K
/H
exchange was found(16) .
Measurements of [Ca
]
showed that cholinergic,
-adrenergic(17, 18) , and
-adrenergic (19) stimulation caused an increase in
[Ca
]
.
In the
present study we identified the major mechanisms of
Na, K
and
H
/HCO
transport in
submandibular duct cells. For the purpose of these studies we
simplified the technique of Dehaye and Turner (20) for
preparation of intralobular granular duct cells. We also used
measurements of pH
in the perfused main duct to
provide the first direct membrane localization of the transporters
participating in regulation of pH
,
[Na
]
, and
transepithelial electrolyte transport. The subsequent manuscript (38) describes the properties and regulation of this pathway
during agonist stimulation.
Figure 1:
Light micrographs of isolated acini,
intralobular ducts and the perfused main submandibular ducts. A shows the mixture of acini and ducts obtained after collagenase
digestion of submandibular glands (magnification 80). B shows the cannula perfusing a main duct (left side) held
in place by a polyethylene tubing (magnification
16).
SBFI fluorescence was recorded
at excitation wavelengths of 340 and 370 nm. The image ratios of
340/370 were calibrated essentially as described before(23) .
To clamp Na, the cells were perfused with
solutions containing several concentrations of NaCl (between 0 and 90
mM), 10 mM Hepes (pH 7.4), 5 µM
gramicidin, and 2.5 µM monensin. The dye behaved similarly
in duct and acinar cells, and similar changes in fluorescence ratio
were obtained by clamping Na
at different
concentrations. Therefore, the calibration curves from both cells were
pooled. The apparent K
for Na
in the cells was about 48 ± 6.3 mM (n = 4). The calibration curve and the ratios were used to
calculate [Na
]
.
Figure 2:
Cl/HCO
and Na
/H
exchange in
intralobular ducts. Ducts and acini loaded with BCECF were used to
measure pH
by image acquisition and analysis (a and b). Cells incubated in solution A (NaCl,
Hepes) were perfused in sequence with solutions D
(Cl
-free, Hepes), A, E (NaCl,
HCO
), F (Cl
-free,
HCO
), and E. After treatment with 0.5
mM DIDS for 8 min, the cells were perfused with solution F
containing 0.5 mM DIDS. The figure shows 1 of 4 experiments
with similar results. In c, ductal pH
was
measured by photon counting of BCECF fluorescence from 8 cells. The
duct was perfused with solution E in which 20 mM NH
Cl replaced 20 mM NaCl and then solution G
(Na
-free, HCO
). After
stabilization of pH
, the cells were perfused with
solution G in which 5 or 140 mM K
replaced
similar [NMG
] and then with solution E
containing 20 µM DMA before perfusion with solution E.
This experiment is similar to 2 others with the same conditions and to
6 others in Hepes-buffered solutions.
In an attempt to identify the
proposed(3, 4) K/H
exchange activity, we tested the effect of external K
on H
efflux from acid-loaded cells (Fig. 2c). Addition of 5 mM
K
to acidified cells caused a small
increase in pH
. However, increasing
K
up to 140 mM had no further
effect on pH
. Exposing these cells to 5 mM K
and 140 mM Na
in the presence of 20 µM dimethylamiloride (DMA)
increased pH
similar to that observed with
K
alone. Removal of DMA resulted in recovery of
resting pH
due to Na
/H
exchange. Hence, even in strongly acidified cells and in the
presence of HCO
, external K
had minimal effect on pH
.
Another
protocol to test for K/H
exchange is
shown in Fig. 3. Removal of external K
caused
cytosolic acidification at an initial rate of about 0.13 ± 0.02
pH unit/min (n = 12). Up to 20 µM amiloride, which is sufficient to block the luminal Na
channel(6) , had no effect on the rate or extent of the
acidification. Inhibition of the Na
/H
exchanger with 0.5 mM amiloride caused a further
reduction of pH
by about 0.16 ± 0.03 pH
unit (n = 4) (Fig. 3a). Inhibition of
the Na
/H
exchanger prior to removal
of K
showed that the maximal rate of
acidification due to removal of K
was
0.34 ± 0.02 pH unit/min (n = 5) with
stabilization of pH
within 128 ± 5.7 s (Fig. 3b). Fig. 3c shows that
HCO
somewhat reduced the rate and
significantly blunted the extent of the acidification. However, a
similar set point of pH
was attained under the
various conditions. In Hepes-buffered medium, pH
stabilized at 6.98 ± 0.03 (n = 4),
whereas in HCO
-buffered medium,
pH
stabilized at 6.93 ± 0.04 (n = 3).
Figure 3:
Effect of K on pH
in intralobular ducts. In experiment a, ducts loaded with BCECF were perfused with solution B
(K
-free, Na
containing, Hepes),
solution B containing 0.5 mM amiloride, solution B, and then
solution A (containing 5 mM K
). In experiment b, ducts were incubated in solution A containing 25 µM DMA before exposure to solution B containing DMA. In experiment c, the ducts were equilibrated with
HCO
-buffered solution E (NaCl,
HCO
) before exposure to 25 µM DMA and then solution E in which 5 mM NMG-Cl replaced 5
mM KCl (K
-free).
In the absence of amiloride or DMA, addition of
K rapidly increased pH
back to resting levels (Fig. 3a). DMA (Fig. 3, b and c) reduced, but did not
prevent, the rapid alkalinization observed on addition of
K
. Hence, the initial rapid increase in
pH
induced by K
did
not require functioning Na
/H
exchanger.
Fig. 4, a and b,
illustrates the dependence of H influx and efflux on
[K
]. Gradual reduction of
K
from 5 to 1 mM had a small
effect on pH
(Fig. 4a). The same
effect was observed if K
was reduced in
one step from 5 to 1 mM. Reducing K
from 1 to 0 mM resulted in the typical acidification. Fig. 4b shows that in duct cells acidified by reducing
K
from 5 to 0 mM, the addition
of 1 mM K
was sufficient to
rapidly restore normal pH
. In an attempt to
determine the specificity of the extracellular K
site,
we found that substituting Li
for K
had no effect on the K
-dependent
acidification (not shown). Fig. 4c shows that
Cs
at 5 mM largely prevented the
acidification. On the other hand, when 5 mM Cs
was added to acidified cells incubated in the absence of
K
, resting pH
was
recovered.
Figure 4:
Dependence of pH on
K
concentration. Ducts incubated in
solution A were perfused with the same solutions in which increasing
concentrations of NMG
substituted for K
(a and b). In trace c, the duct was
perfused with solution A in which 5 mM CsCl replaced 5 mM KCl before perfusion with solution B (K
-free) and
then solution A.
The characteristics of H fluxes
demonstrated in Fig. 2-4 are not compatible with a coupled
K
/H
exchange. Another possibility is
transport of the ions through conductive pathways as was suggested for
the striated duct(16) . However, K
and
H
fluxes did not require anion transport since
incubation of the cells in Cl
-free medium in the
presence or absence of HCO
, to deplete
intracellular and extracellular Cl
, had no effect on
H
fluxes due to K
removal or addition (not shown, but see Na
fluxes in accompanying manuscript (38) ). The
K
-dependent acidification was not
affected by up to 10 mM triethylammonium (not shown), but was
inhibited by Ba
. Fig. 5a shows that
incubating the cells with 2.5 mM Ba
increased steady-state pH
by 0.067 ±
0.015 (n = 3) unit and inhibited the acidification by
about 84 ± 7% (n = 3). The inhibition by
Ba
was reversible. A somewhat unexpected finding is
illustrated in Fig. 5b. Inhibition of the pathway
required for H
fluxes by Ba
did not
result in recovery of pH
due to
Na
/H
exchange. This did not occur
until K
was added to the incubation medium in the
absence (not shown) or presence (Fig. 5b) of
Ba
. Hence, Ba
inhibits H
efflux but not H
influx induced by changes in
K
.
Figure 5:
Inhibition by Ba of
K
-dependent acidification. In experiment a, the duct was perfused with solution A containing 2.5
mM Ba
, solution B (K
-free)
containing 2.5 mM Ba
, and then solution B.
In experiment b, the duct was perfused with solution B and
then solution B and A containing 2.5 mM
Ba
.
A clue to what might be limiting
the activity of the Na/H
exchanger to
set pH
at about 7.1-7.2 in the absence of
K
(Fig. 3-5) was obtained
by testing the effect of Na
on
pH
. Fig. 6a shows that removal of
Na
reduced pH
to
6.86 ± 0.03 (n = 3). Removal of
K
in the absence of
Na
increased, rather than
decreased, pH
to about 6.96 ± 0.04 (n = 3). On the other hand, removal of
Na
after incubation in the absence of
K
resulted in a rapid and large
reduction in pH
to about 6.34 ± 0.03 (n = 5) (Fig. 6b). The same behavior was
observed in the presence of HCO
(Fig. 6c).
Figure 6:
Effect of Na on pH
of ducts incubated in the presence or
absence of K
. In experiment a (dashed line), the duct was perfused with solution C
(Na
-free, Hepes), K
-free solution C
and finally with solution A. In experiment b (solid
line), the same duct was subsequently perfused with solution B
(K
-free, Hepes) and then with the Na
-
and K
-free solution. The same results were obtained
when only part b of the experiment was performed. In
experiment c, a duct incubated in solution E (NaCl,
HCO
) was perfused with solution E and
solution G (Na
-free,
HCO
) in which 5 mM NMG
replaced the K
and finally
with solution E.
Figure 7:
Effect of K on [Na
]
of
intralobular ducts and acini. SBFI-loaded submandibular ducts and acini
present in the same recording field were used to measure
[Na
]
by image
acquisition and analysis. Cells in solution A (NaCl, Hepes) were
perfused with solution B (K
-free, Hepes) and then
solution A. Subsequently, the cells were perfused sequentially with
solution E (NaCl, HCO
),
K
-free solution E, and then solution E. Note that
removal of K
in the presence or absence
of HCO
increased
Na
in ducts but initially reduced
Na
in acini.
To evaluate the Na permeability of resting
duct cells and demonstrate that removal of K
increased [Na
]
beyond that expected from Na
pump
inhibition, we tested the effect of ouabain on
[Na
]
. Fig. 8, a-d, shows that ouabain inhibited the Na
pump of duct cells at resting (before removal of
K
) and at high
[Na
]
with an apparent
affinity of about 100 mM, typical of the
1 isoform found
in rat tissue(25) . Further, 1 mM ouabain inhibited
the pump even in the presence of 40 mM
K
(Fig. 8e). Yet,
ouabain at 2 mM increased
[Na
]
less and slower
than removal of K
(Fig. 8a). Thus, removal of
K
increased Na
influx
through a K
-dependent pathway.
Figure 8:
Effect of ouabain on
[Na]
of duct cells.
SBFI-loaded ducts were perfused with solution A containing 0.02 (a), 0.1 (b), 0.5 (c), or 2 (d)
mM ouabain and then solution B (K
-free,
Hepes). After stabilization of
[Na
]
, the ducts were
perfused with solution B and solution A containing the above
concentrations of ouabain. Subsequent perfusion with solution A caused
rapid reversal of the inhibition by ouabain as shown in experiment d. Resting [Na
]
of all ducts was similar to that in trace d. Traces a-c were separated vertically to improve visualization
of the effect of ouabain. In experiment e, a duct incubated in
solution B was perfused with solution B containing 1 mM ouabain, solution A in which 35 mM K
replaced 35 mM Na
and also containing 1
mM ouabain, the high K
solution, and then solution A. Results similar to those in a-e were observed in at least 3 separate
experiments.
The
combined experiments in Fig. 7and Fig. 8show that at 5
mM K duct cells have
significant permeability to Na
which is countered by
the Na
pump. Removal of K
inhibits the Na
pump but also increases
Na
permeability by 4.2-fold (4.56 ± 0.6
mM/min (n = 4) in the presence of ouabain and
19.2 ± 0.8 mM/min (n = 33) due to
removal of K
). The accompanying
manuscript describes the characterization and regulation of this duct
cell specific ion transport pathway.
Fig. 9shows the presence of a
Na/H
exchange activity in the luminal
side and provides the first demonstration of similar activity in the
basolateral side of the duct. Fig. 8a shows that
removal of Na
from the lumen had a small effect on
pH
. Removal of Na
from the bath
in the absence of luminal Na
caused rapid
acidification. Addition of Na
to the lumen in the
absence of Na
in the bath rapidly increased
pH
. However, addition of Na
to
the bath was required to restore resting pH
. The
second part of Fig. 8a shows a reverse sequence of
Na
removal and addition. In this case, addition of
Na
to the bath in the absence of luminal Na
partially restored pH
, and addition of
Na
to the lumen further increased
pH
. Fig. 8b shows that DMA
inhibited the Na
-dependent H
efflux
in both membranes. Removal of DMA was followed by recovery of normal
pH
.
Figure 9:
Localization of
Na/H
exchangers in the luminal and
basolateral membranes of the main submandibular duct. The main duct was
cannulated, loaded with BCECF, and perfused with separate luminal and
bath solutions. In experiment a, the luminal and bath
perfusion solutions were switched from solution A (NaCl, Hepes) to
solution C (Na
-free, Hepes) as indicated. In
experiment b, the duct was perfused with
Na
-free solution C and acidified by an
NH
pulse before perfusing the lumen with
solution A (140 mM Na
) containing 20
µM DMA and then solution A. In the second part of the
experiment, the bath of the acidified duct was perfused with solution A
containing 20 µM DMA and then solution A. The experiments
were performed with separate ducts and represent 5 (a) and 3 (b) others with the same results whether both experiments were
performed with the same or separate ducts.
Fig. 10provides evidence for
separate luminal and basolateral
Cl/HCO
exchangers in
submandibular ducts. Removal of Cl
from the bath in
the presence of luminal Cl
caused a small, transient
(in 5 out of 5 experiments) increase in pH
(Fig. 10a). Removal of Cl
from the lumen caused a rapid, large, and sustained increase in
pH
. Addition of Cl
to the bath
in the absence of luminal Cl
partially reduced
pH
, and addition of Cl
to the
lumen restored normal pH
. The reverse experiment
is shown in Fig. 10b. In this case, removal of
Cl
from the lumen in the presence of Cl
in the bath resulted in a large and sustained increase in
pH
. Addition of Cl
to the lumen
in the absence of Cl
in the bath was nearly
sufficient to restore resting pH
. All effects of
Cl
removal were largely inhibited by incubation with
0.5 mM DIDS.
Figure 10:
Localization of
Cl/HCO
exchange
activity in the luminal and basolateral membranes of the main
submandibular duct. The lumen and bath of a BCECF-loaded duct in
solution E (NaCl, HCO
) were perfused with
solution F (Cl
-free,
HCO
) and then solution E (a).
The same duct was used to perform the experiment in trace b in
which the lumen and then the bath were perfused with solution F and
then solution E. Finally, the lumen and then the bath were perfused
with solution F containing 0.5 mM DIDS (c). All the
experiments were performed with the same duct and represent another two
with similar results. Similar results were also obtained in two
experiments in which part b was performed before part a.
Fig. 11shows the localization of the
K-sensitive pathway in the luminal
membrane. Removal of K
from the luminal solution
caused small acidification. Removal of K
from the bath
was required to obtain the maximal acidification (Fig. 10a) (n = 3). On the other hand,
removal of K
from the bath in the presence of luminal
K
had a small effect on pH
, and
removal of K
from the lumen caused significant
acidification (Fig. 10b) (n = 3).
Addition of K
to the lumen in the absence of
K
in the bath had a small effect on
pH
. Addition of K
to the bath was
required for the rapid H
efflux (Fig. 10a). The simplest explanation for the need to
remove bath K
to observe the maximal acidification is
the effect of bath K
on the Na
pump.
In the perfused duct, large intracellular Na
accumulation due to removal of luminal K
was
prevented because of Na
efflux across the basolateral
membrane by the Na
/K
pump. This
prevented partial inhibition of the Na
/H
exchangers which removed some of the H
entering
the cytosol. That this is likely the case is shown by the experiments
in the second part of Fig. 10, a and b. Fig. 10a shows that when K
was removed
from the lumen and then the bath to allow accumulation of intracellular
Na
as in Fig. 6b, removal of
Na
from the bath and then the lumen caused large and
rapid cytosolic acidification. On the other hand, Fig. 10b shows that maintaining luminal K
at 5 mM throughout the experiment reduced the acidification observed on
removal of Na
from both sides. Thus, removal of
luminal K
increased the accumulation of intracellular
Na
and the acidification. Unfortunately we were unable
to confirm this finding by measuring
[Na
]
with SBFI or
Na
-Green due to small signal/noise.
Figure 11:
Localization of the
K-dependent H
and
Na
influx pathway. In experiment a, the lumen
and bath of the duct were alternately perfused with solution B
(K
-free, Hepes) and then solution A. In the second
part of the experiment after perfusing the lumen and then bath with
solution B, the bath and then the lumen were perfused with a
Na
- and K
-free solution C. This
caused large acidification of the cytosol. In experiment b,
the reverse protocol was performed in which the bath and then the lumen
were perfused with solution B. In the second part of the experiment,
the lumen was continuously perfused with a solution containing 5 mM K
. When the lumen was not exposed to a
K
-free solution, removal of Na
from
the bath and then the lumen caused a small reduction in
pH
.
Ductal systems of various exocrine glands control the final
electrolyte composition of the secreted fluid. In the gastrointestinal
tract, the ducts alkalize the fluid by secreting
HCO and/or absorbing H
.
HCO
concentration in the secreted fluid
of some species can be as high as 140
mM(3, 26) . The submandibular salivary gland
has been extensively used as a model system to study electrolyte
secretion by duct cells(3) . Salivary duct cells reabsorb
Na
and Cl
and secrete K
and HCO
. The concentrations of
Na
and K
in the salivary fluid varies
depending on secretory rate but, in general, they change in a
reciprocal manner(3) . Cl
absorption
parallels that and accounts for about 25% of Na
absorption (4, 27) and appears to be only
partially coupled to HCO
secretion(3) . HCO
and
K
secretion partially correlates, but usually
K
secretion significantly exceeds
HCO
secretion(5, 7, 8, 11, 12) .
In the extreme cases of total removal of luminal Na
(4, 5, 28) or in the presence of
luminal amiloride(16) , when secretion is slow but measurable,
K
and HCO
secretion (or
H
absorption) become increasingly coupled(4) .
Based on these findings and their elegant studies on the dependence of
ion transport on the luminal concentrations of Na
and
K
, Knauf et al.(4) suggested that
luminal K
and HCO
secretion are mediated largely by separate, but functionally coupled,
Na
/H
and
K
/H
exchangers. With few
modifications, the model proposed by Knauf et al.(4) becomes the accepted mechanism of ion transport by the
salivary duct.
Partial validation of the model was obtained with the
findings of an amiloride-sensitive Na channel (6) and a Cl
conductance (15) in the
luminal membrane of salivary duct cells. Measurement of pH
in the present studies confirmed the presence of a
Na
/H
exchange activity in
intralobular duct cells(16, 24) . Unlike a previous
report using similar cells and techniques, we also found a
Cl
/HCO
exchange
activity in these cells (Fig. 2). The simplified cell
preparation technique used for the present studies may have allowed the
demonstration of this activity in intralobular ducts. As a control for
these experiments, we measured pH
in ducts and
acini in the same recording field since the acini were shown to have a
Cl
/HCO
exchanger(29) . In both cell types, removal of Cl
in the absence of HCO
acidified,
rather than alkalinized, the cytosol. These findings are similar to
those in pancreatic acini (30) in which the exchanger
transports HCO
but not
OH
. In the presence of
HCO
, removal of
Cl
caused a DIDS-sensitive,
Na
-independent alkalinization, all indicative of a
Cl
/HCO
exchange.
The
perfusion experiments with the main submandibular ducts provide the
first direct localization and the first evidence for the presence of
Na/H
and
Cl
/HCO
exchange
activities on both sides of the duct. Thus, the luminal and basolateral
sides of the ducts displayed similar rates of
Na
/H
exchange. The rate and extent of
pH
changes due to Cl
removal
suggests that luminal
Cl
/HCO
exchange
activity is much higher than the basolateral activity. The influence of
the ions in one side on the activity of the exchanger present in the
opposite side indicates that the exchangers share common pools of
intracellular Na
, Cl
, and
H
/HCO
. This would imply
that the luminal and basolateral exchangers are present in the same
cells or in cells coupled with respect to Na
,
Cl
, and
H
/HCO
. This is
particularly relevant since the submandibular duct contains at least
two morphologically distinct cell types(3) . In the present
studies, we were unable to address this question directly since the
thick layer of connective tissue of the main duct precluded
identification of single cells (see Fig. 1B). However,
pancreatic duct cells were shown to be electrically coupled through gap
junctions(31) , which are permeable to a variety of small
charged molecules(32) . It is thus possible that the exchangers
exist in the same or separate submandibular duct cells coupled by gap
junction.
The presence of more than one type of a similar
transporter in selective membranes of the same epithelial cell has been
extensively studied in the kidney(33, 34) . It is
believed that in these cases one of the transporters is involved in
transepithelial electrolyte transport, whereas the other maintains the
normal cytosolic concentration of the respective ion(34) . It
is likely that the basolateral Na/H
and Cl
/HCO
exchangers in the salivary duct regulate pH
. The
luminal Na
/H
exchanger probably
mediates some of the nonelectrogenic Na
reabsorption(3, 4) . On the other hand, the
luminal Cl
/HCO
exchanger is likely to play only a minor role in Na
absorption and K
and HCO
secretion since removal of luminal Cl
reduces
Na
reabsorption by only 25%(4, 27) .
The most significant finding of the present studies is a
K-dependent pathway permeable to
Na
and/or H
(HCO
) in submandibular duct, but
not acinar cells. We believe that this can be a major pathway of
K
and HCO
secretion in
the luminal membrane of the duct.
Our results do not support the
existence of a coupled K/H
exchange
or a K
/HCO
cotransport
mechanism in submandibular duct cells. The most compelling evidence is
the inability of K
to cause substantial
H
efflux from acid-loaded cells (Fig. 2). In
separate experiments we used the protocol in Fig. 6b to
first deplete the cells from K
and load them with
Na
and then acidify the cells by incubation in
Na
-free medium. Even in these acid-loaded and
K
-depleted cells K
was
not effective in causing H
efflux. In duct cells
incubated in the absence of Na
, removal
of K
alkalinized, rather than acidified,
the cytosol (Fig. 6a). All previously reported
K
/H
exchange mechanisms had markedly
different properties (35, 36, 37) from the
K
-dependent H
fluxes in
submandibular ducts. These include saturation at a 100-150 mM K
(35, 37) , H
transport only against the concentration gradient for
K
(35, 36, 37) insensitivity
to Ba
(36, 37) and transport of
Cs
and Li
(37) . Since
Ba
and 5 mM Cs
inhibited the K
-dependent
H
/HCO
and Na
(38) fluxes, it is possible that the pathway is sensitive
to the transepithelial potential and K
and
H
(HCO
) transport is
mediated by conductive pathways. Obviously, additional studies are
needed to identify the nature of this pathway(s) with certainty.
However, irrespective of the exact mechanism, this transporter is
likely to play a major role in ductal electrolyte secretion.
At this
point it is useful to indicate the similarities and inter-relationship
between the K-induced H
and Na
fluxes which are described in the
accompanying manuscript(38) . This suggests that
H
/HCO
and Na
and maybe K
are transported by the same pathway
or by separate but tightly coupled pathways. The physiological
significance of these observations is highlighted by our ability to
localize this pathway to the luminal membrane of submandibular duct
cells (Fig. 11). Thus, the luminal membrane of the submandibular
duct has an ion transport pathway(s) which is sensitive to the
extraluminal concentrations of K
and mediates the
concomitant transport of Na
and H
and/or HCO
into the cells and
K
out of the cells. Such a pathway is ideal in
mediating K
and HCO
efflux to the lumen and some of the Na
reabsorption
during salivary secretion.