Vol. 276, Issue 2, G507-G517, February 1999
Mechanisms of basolateral Na+ transport in rabbit
esophageal epithelial cells
Solange
Abdulnour-Nakhoul,
Serhat
Bor,
Nese
Imeryuz, and
Roy C.
Orlando
Departments of Medicine and Physiology, Tulane University School of
Medicine and Veterans Affairs Medical Center, New Orleans,
Louisiana 70112-2699
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ABSTRACT |
We examined the mechanisms of cellular
Na+ transport, both
Cl
dependent and
Cl independent, in the
mammalian esophageal epithelium. Rabbit esophageal epithelium was
dissected from its muscular layers and mounted in a modified Ussing
chamber for impalement with ion-selective microelectrodes. In
bicarbonate Ringer, transepithelial potential difference was
14.9 ± 0.9 mV, the transepithelial resistance (RTE) was 1,879 ± 142 
· cm2, the
basolateral membrane potential difference
(VmBL) was
53 ± 1.5 mV, and the intracellular activity of
Na+
(aNai) was 24.6 ± 2.1 mM. Removal of
Na+ and
Cl from the serosal and
luminal baths decreased aNai to 6.6 ± 0.6 mM. Readdition of Na+ to the
serosal bath in the absence of
Cl increased
aNai by 21.8 ± 3.0 mM, whereas
VmBL and RTE remained
unchanged. When serosal Na+ was
readded in the presence of amiloride the increase in
aNai and the rate of
Na+ entry were decreased by
~50%.
5-(N-ethyl-N-isopropyl)amiloride mimicked the effect of amiloride, whereas phenamil did not. Subsequent readdition of Cl to the
serosal bath further increased aNai by
4.4 ± 1.9 mM. When the cells were acid loaded by pretreatment with NH+4 in nominally
HCO3-free Ringer, intracellular pH
measurements showed a pHi recovery
that is dependent on the presence of
Na+ in the serosal bath and that
can be blocked by amiloride. These data indicate that esophageal
epithelial cells possess a
Na+-dependent, amiloride-sensitive
electroneutral mechanism for Na+
entry consistent with the presence of a basolateral
Na+/H+ exchanger. The ability of
Cl to further enhance
Na+ entry supports the existence
of at least one additional
Cl-dependent component of
basolateral Na+ entry.
sodium/hydrogen exchange; amiloride; intracellular sodium; intracellular pH
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INTRODUCTION |
THE ESOPHAGUS IS LINED by a stratified squamous
epithelium that actively transports
Na+ from lumen to blood. According
to the Koefoed-Johnson and Ussing (10) two membrane model,
Na+ enters the cell passively
across the luminal membrane through Na+ channels and then exits across
the basolateral membrane and into the intercellular space via the
energy-requiring action of the Na+
pump. In addition, other mechanisms that can transport
Na+ across cell membranes have
been reported in esophageal cells. These include the
Na+/H+ exchanger,
Na+-dependent
Cl/HCO3
exchanger and the Na+-K+-2Cl
cotransporter. However, these mechanisms were described in either a
nonpolarized primary culture of esophageal epithelial cells or isolated
esophageal cells so that their localization to apical and/or
basolateral membrane remains uncertain (11, 23). Moreover, the
conditions under which these or other yet unidentified transporters contribute to Na+ transport in
esophageal epithelium are unknown. In addition to transcellular
Na+ uptake, the cellular pathways
for Na+ transport are responsible
for the maintenance of intracellular Na+ and pH homeostasis. This is
particularly important in the esophageal epithelium, which is
constantly exposed to various noxious luminal agents ranging from high
acidity, especially during episodes of gastroesophageal reflux, to
hypertonicity of ingested food and beverages (15).
The aim of this study was to define in the esophageal epithelium the
specific pathways contributing to
Na+ transport across the
basolateral membrane of the cells located within the stratum
germinativum. These cells reside on the basement membrane of this 30 or
more cell-layered epithelium and are accessible to impalement and study
by microelectrodes. Microelectrodes were utilized to measure
intracellular Na+ activity
(aNai), intracellular pH
(pHi), and basolateral membrane
potential
(VmBL) of
esophageal cells within the intact epithelium. Our study indicates that
a major fraction of Na+ is
transported across the basolateral side via an electroneutral mechanism
independent of Cl. The
sensitivity of this pathway to inhibitors is consistent with
Na+/H+ exchange.
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METHODS |
Animal and Tissue Preparation
New Zealand rabbits were killed by administration of an intravenous
overdose of pentobarbital (60 mg/ml). The esophagus was excised, opened
longitudinally, and pinned mucosal side down in a paraffin tray
containing ice-cold oxygenated Ringer. The muscle layers were lifted up
with forceps, and the underlying mucosa was dissected free with a
scalpel. The sheet of mucosa thus obtained was cut, and a section was
mounted horizontally in a modified Ussing chamber with an aperture of
1.13 cm2. A diagram of the
preparation is shown in Fig 1. The chamber allows independent and continuous perfusion of the apical and the
serosal side of the tissue. The fluid for the perfusion of the tissue
is delivered by gravity. The perfusion solutions can be switched
quickly and with minimal dead space by means of a combination of rotary
and slider valves (Rainin, Emeryville, CA), which allow one of six
experimental solutions to flow to each side of the chamber. The
solutions were prewarmed and delivered to the chamber at 37°C.
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Fig. 1.
Continuously perfused preparation of esophageal epithelium for
electrophysiological measurements. Esophageal epithelium is mounted on
a grid between 2 halves of a modified Ussing chamber.
VTE, potential
difference measured between a reference electrode in serosal side and
another electrode in luminal side of esophageal epithelium.
VmBL, potential
difference between reference barrel of double-barreled microelectrode
and electrode in serosal bath.
VNa, total
potential of Na+-sensitive barrel
of double-barreled microelectrode. Pure ionic potential of
Na+ electrode is obtained by
subtracting electronically
VmBL from
VNa.
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Electrodes
Transepithelial potential
(VTE) was
measured as the voltage difference between a free-flowing KCl (tip
<10 µm) electrode placed in the bath fluid of the serosal side and
a similar electrode placed in the bath fluid of the apical side. Both
electrodes were fitted with an Ag-AgCl wire, and the leads were
connected to the amplifier of a voltage clamp (Physiologic Instruments,
San Diego, CA). The voltage clamp was also used to deliver a DC pulse
of 5-15 µA via platinum wires located in each side of the
chamber. This allowed us to determine the transepithelial resistance
(RTE) from the
voltage deflection
VTE as follows
(where I is current)
The
esophagus possesses relatively large basal cells (20-40 µm),
which allow stable impalements with microelectrodes. Double-barreled microelectrodes were used to measure simultaneously and in the same
cell VmBL and the
aNai or
pHi. The following is a brief
description for the preparation of the double-barreled microelectrodes.
Two borosilicate glass fiber capillaries (A-M Systems, Everett, WA)
were held together with shrinkable tubing and twisted 360° over an
open flame. The electrodes were then pulled on a vertical
microelectrode puller (David Kopf, Tujunga, CA) to a tip smaller than
0.2 µm. One barrel was exposed to hexamethyldisilazane (Sigma, St.
Louis, MO) vapor for 30 min after which the electrodes were baked at
100°C for 2 h. The Na+ or the
H+ exchanger (Fluka, Ronkonkoma,
NY) was then introduced into the tip of the silanized barrel by means
of very fine glass capillaries. The silanized barrel was further
backfilled with 150 mM NaCl for the
Na+ microelectrode or with a
buffer solution containing 0.04 M
KH2PO4, 0.023 M NaOH, and 0.015 M NaCl, pH 7.0 (2a) for the pH
electrode. The other barrel or the reference barrel was
filled with 1 M KCl. Each barrel was connected to an Ag-AgCl half cell
and connected to one of the probes of a high input impedance
electrometer (WPI, Sarasota, FL). The resistance of the reference
barrel ranged from 50 to 100 M, and the tip potential was <5 mV.
The slope (S) of the Na+ electrode
(ion-sensitive barrel) was determined from the equation
where
V100 mM NaCl and
V10 mM NaCl are
the electrodes potential in the solutions as noted and 0.94 is the
logarithm (base 10) of the Na+
activity ratio of pure 100 mM over pure 10 mM NaCl. The slope of the
electrodes averaged 58 mV per decade change in activity of
Na+. The selectivity for
Na+ over
K+ was calculated from the
equation
In
40 Na+ electrodes used for the
study, the mean
Na+-to-K+
selectivity ratio was 40:1.
The pH electrodes were calibrated in HEPES buffer solutions of pH 6, 7, and 8. The average slope was 55 ± 1.1 mV/pH unit
(n = 8).
When a serosal cell was impaled the
VmBL was read as
the difference between the reference barrel of the double-barreled
microelectrode and the reference electrode in the serosal side. The
apical membrane potential could then be calculated as the difference
between VmBL and
VTE.
The ratio of the apical to basolateral membrane resistance
(Ra/Rb)
was determined from the ratio of the voltage deflections produced by
the transepithelial DC current pulse across the apical and the
basolateral membranes (Va and
Vb, respectively) according to the
equation
Vb
was measured directly, and
Va was calculated by
subtracting Vb from
VTE.
The intracellular ionic activities were calculated from the potential
readings of cellular impalements of the specific ion-sensitive barrel
of the double-barreled microelectrode. The total potential of the
ion-sensitive electrode was measured as the voltage difference between
the ion-sensitive microelectrode and the free-flowing reference
electrode in the serosal bath. The pure ionic potential was obtained by
subtracting electronically
VmBL from the
total potential of the ion-sensitive electrode. Readings were recorded on a three-channel strip chart recorder (Kipp & Zonen, Bohemia, NY).
Solutions
The composition of Ringer solutions is given in Table
1. The chemicals were obtained from Sigma.
5-(N-ethyl-N-isopropyl) amiloride (EIPA) was purchased from RBI (Natick, MA). Amiloride, EIPA,
phenamil, and bumetanide were dissolved in a small volume of DMSO and
added to the solution. The concentrations of amiloride, EIPA, and
phenamil used were based on the concentrations required to achieve
maximal inhibition of the transporters as reported previously in other
preparations (9) and in the esophageal cells (16, 22). The
concentration of DMSO never exceeded 0.1% of the final solution.
Statistical Analysis
The results are presented as means ± SE. Data were analyzed using
the two-tailed paired Student's
t-test;
n is the number of observations.
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RESULTS |
Steady-State Measurements in
HCO3 Ringer
With the use of esophagi from 30 rabbits, sections of esophageal
epithelium were mounted in modified Ussing chambers and superfused on
the luminal and serosal side with standard
HCO3 Ringer (solution
1, Table 1). From these experiments baseline mean
values for esophageal epithelium were
VTE = 14.9 ± 0.9 mV and
RTE = 1,879 ± 142 · cm2,
and for individual impaled basal cells
VmBL = 53 ± 1.5 mV, Ra/Rb = 1.6 ± 0.48 and aNai = 24.6 ± 2.1 mM.
To study the mechanisms by which
Na+ is transported across the
basolateral membrane, Na+ and
Cl were removed from both
serosal and luminal bathing solutions to inhibit
Na+ entry into cells and to
deplete intracellular Na+ stores.
Subsequent readdition of Na+ to
the serosal bathing solution, in the absence of
Cl, would drive
Na+ into the cell via
Cl-independent mechanisms
of Na+ entry.
Cl was then returned to the
serosal bath to determine if additional Cl-dependent mechanisms of
Na+ transport were also present.
Effect of Removal of Serosal and Luminal
Na+ and
Cl
As shown in Fig. 2, after the serosal bath
solution was switched from standard
HCO3 Ringer to a
Na+-free,
Cl-free solution
(Na+ replaced with
N-methyl-D-glucamine
and Cl replaced with
gluconate; Table 1, solution 2),
aNai decreased sharply
(segment a-b), whereas
VTE and
VmBL
hyperpolarized over a period of ~10 min. The mean decrease in
aNai was 17.8 ± 1.9 mM, and the mean
hyperpolarizations of
VTE and VmBL were
2.7 ± 0.6 mV and 8.7 ± 3.8 mV,
respectively (n = 17, P < 0.05). In addition,
RTE increased by
344 ± 34 · cm2. The
data are summarized in Fig. 3,
A and
B (2nd bars).
Ra/Rb remained unchanged (1.57 ± 0.5 vs. 1.69 ± 0.6, P > 0.05).
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Fig. 2.
Tracing of experiment in rabbit esophageal epithelium using
double-barreled Na+-sensitive
microelectrode. This experiment shows effect of bilateral removal of
Na+ and
Cl and subsequent
readdition of Na+ and then
Cl to serosal bath on
intracellular activity of Na+
(aNai; top
trace), basolateral membrane potential
(VmBL,
middle trace), and transepithelial
potential (VTE,
bottom trace). Data from this and
similar experiments are summarized in Fig. 3,
A and
B, and Fig. 4,
A and
B. NMDG,
N-methyl-D-glucamine.
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Fig. 3.
A: effect of removal of luminal (L)
and serosal (S) Na+ and
Cl on
VmBL and
aNai. When
Na+ and
Cl were deleted from
serosal bath (2nd bar),
VmBL
hyperpolarized by 8.7 ± 3.8 mV and
aNai decreased substantially by
17.8 ± 1.9 mM (n = 17, P < 0.05). Subsequent removal of
Na+ and
Cl from luminal bath
(3rd bar) hyperpolarized
VmBL further by
5.0 ± 2.2 mV and aNai decreased by
4.7 ± 1.8 mM (n = 13, P < 0.05). * Significantly
different from previous condition, paired
t-test.
B: effect of removal of luminal and
serosal Na+ and
Cl on
VTE and
transepithelial resistance
(RTE). Removal
of Na+ and
Cl from the serosal bath
(2nd bar) hyperpolarized
VTE by 2.7 ± 0.6 mV and increased
RTE by 344 ± 34 · cm2.
Subsequent removal of Na+ and
Cl from luminal bath
(3rd bar) caused
VTE to depolarize
by 9.9 ± 2.0 mV and
RTE to increase
by 785 ± 102 · cm2
(n = 13, P < 0.05). * Significantly
different from previous condition, paired
t-test.
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To further ensure that Na+ and
Cl entry would not occur
across the basolateral membrane as a result of luminal ions diffusing across the junctions and through the paracellular pathway, perfusion of
the serosal side with Na+-free,
Cl-free solution was
followed by perfusion of the luminal bath with Na+-free,
Cl-free solution (Table 1,
solution 2). As shown in Fig. 2
(segment b-c),
aNai decreased further,
VmBL
hyperpolarized, and
VTE depolarized.
The mean decline in aNai was 4.7 ± 1.8 mM, the mean hyperpolarization of
VmBL was
5.0 ± 2.2 mV, and the mean depolarization of
VTE was 9.9 ± 2.0 mV, whereas
RTE increased
further by 785 ± 105 · cm2
(n = 13, P < 0.05 for all values). These data
are summarized in Fig. 3, A and
B (3rd bars).
Ra/Rb
increased slightly but not significantly (1.52 ± 0.49 vs. 1.88 ± 0.64).
Effect of Readdition of Serosal
Na+ in Absence
of Cl
After intracellular Na+ was
depleted and baseline readings in
Na+-free,
Cl-free solution were
established, the pathways for cellular
Na+ entry independent of
Cl were examined by adding
Na+ to the serosal bath in the
absence of Cl (Table 1,
solution 3). As shown in Fig. 2
(segment c-d), readdition of serosal
Na+ increased
aNai and depolarized
VTE but had no
significant effect on
VmBL. As
summarized in Fig. 4,
A and
B (2nd bars),
aNai increased by 21.8 ± 3.0 mM,
VTE depolarized
by 5.6 ± 1.3 mV (n = 16, P < 0.001), and there was no effect
on RTE or
Ra/Rb
(1.98 ± 0.57 vs. 2.09 ± 0.55). It is noteworthy that
the increase in aNai was substantial,
restoring the aNai entirely to the
pretreatment baseline aNai value.
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Fig. 4.
A: effect of readdition of
Na+ and then
Cl to serosal bath on
aNai and
VmBL in
continuous absence of Na+ and
Cl in the lumen. Readdition
of Na+ to serosal bath did not
cause any significant change in
VmBL, whereas
there was a very substantial increase in
aNai of 21.8 ± 3.0 mM
(n = 16, P < 0.001). When
Cl was subsequently added
back to the serosal bath in the continuous absence of
Na+ and
Cl in the lumen,
VmBL depolarized
by 19.5 ± 5.3 mV and there was a small but significant increase in
aNai of 4.4 ± 1.2 mM
(n = 8, P < 0.01). * Significantly
different from previous condition, paired
t-test.
B: effect of readdition of
Na+ and then
Cl to serosal bath on
VTE and
RTE. In absence
of luminal Na+ and
Cl readdition of
Na+ to serosal bath (2nd
bar) depolarized
VTE by 5.6 ± 1.3 mV (n = 16, P < 0.001) but did not cause any
significant change in
RTE. When
Cl was given back to
serosal bath in continuous absence of
Na+ and
Cl in the lumen (3rd
bar), VTE
hyperpolarized by 6.8 ± 1.5 mV
(n = 11, P < 0.005) and
RTE remained
unchanged. * Significantly different from previous condition,
paired t-test.
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Effect of Readdition of Serosal Cl
The final step of the experiments depicted in Fig. 2 was the addition
of Cl to check for
Cl-linked
Na+ transport. When
Cl was given back to the
serosal bath (segment d-e) in the
continuous absence of Na+ and
Cl in the lumen
(solution 1 in serosal bath,
solution 2 in lumen), aNai increased,
VmBL depolarized,
and VTE
hyperpolarized. The mean increase in aNai
was 4.4 ± 1.2 mM (n = 8, P < 0.01), the mean depolarization
of VmBL was 19.5 ± 5.3 mV, and the mean hyperpolarization of
VTE was
6.8 ± 1.5 mV (n = 11, P < 0.005), whereas
RTE remained
unchanged. These results are summarized in Fig. 4,
A and
B (3rd bars).
Ra/Rb
increased significantly from a value of 1.6 ± 0.4 to 2.6 ± 0.4 (P < 0.001). Although significantly different from values for aNai before
addition of Cl, the overall
increase was small (~16% of the total increase in Na+ uptake).
Effect of Amiloride on
Na+ Entry
The above experiments showed that readdition of serosal
Na+ caused a marked increase in
aNai, which was not accompanied by a
significant change in
VmBL. This
suggests that Na+ entered the cell
via an electroneutral mechanism for
Na+ transport, a likely candidate
of which is the amiloride-sensitive Na+/H+
exchanger (14). To assess this possibility, we removed serosal and
luminal Na+ and
Cl and pretreated the
tissue with 103 M amiloride
in the serosal bath for ~10 min before the readdition of serosal
Na+, which was also done in the
presence of amiloride. A typical experiment is shown in Fig.
5. The results of this maneuver was that
Na+ entry was inhibited as
evidenced by an increase of only 10.0 ± 3.1 mM in the
presence of amiloride (segment
a'-b') compared with an increase of 19.8 ± 3.8 mM in its absence (n = 7, P < 0.04, Table
2). In addition, the initial rate of
Na+ entry into the cell was slower
in the presence of amiloride at 2.2 ± 0.8 vs. 5.9 ± 1.7 mM/min
in the absence of amiloride (P < 0.02). These data are summarized in Fig. 6,
A and
B. The changes observed in
VmBL,
VTE, and
Ra/Rb
on Na+ readdition were not
different in the presence or absence of amiloride (P > 0.05 for each comparison, Table
2).
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Fig. 5.
Tracing of experiment showing effect of readdition of serosal
Na+ in absence and in presence of
amiloride (103 M) in
serosal bath. Readdition of serosal
Na+ increased
aNai (top trace, segment
a-b). Amiloride decreased rate of
Na+ entry and also the increase in
aNai (segment
a'-b') on readdition of
Na+. Changes in
VmBL
(middle trace) and
VTE
(bottom trace) are not different in
absence and in presence of amiloride.
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Table 2.
Effect of amiloride on changes in VmBL,
aNai, VTE,
RTE, and Ra/Rb on readdition of
serosal Na+ in continued absence of
Cl
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Fig. 6.
Data summary of increase in aNai
(A) and initial rate of
Na+ entry
(B) induced by readdition of serosal
Na+ in absence and in presence of
amiloride (103 M). Initial
rate of Na+ entry and increase in
aNai were inhibited in the presence of
amiloride (n = 7).
* P < 0.04.
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Effect of EIPA and Phenamil on
Na+ Entry
The above experiments showed that
Na+ entry was inhibited by
amiloride at 103 M. Although this observation is consistent with
Na+ entry via the electroneutral
Na+/H+ exchanger, amiloride at this
concentration is not very specific, being capable of inhibiting
Na+ entry via both the
Na+/H+ exchanger and the
Na+ channel. For this reason we
repeated the experiment described above but substituted for amiloride
the more selective Na+/H+ exchange inhibitor
EIPA (5 × 105 M)
(24). As summarized in Fig. 7,
A and
B, EIPA mimicked the effect of
amiloride, reducing both the rate of
Na+ entry from 5.6 ± 1.5 to 2.2 ± 0.3 mM/min and the increase in aNai on readdition of serosal
Na+ from 18.03 ± 4.6 to
9.1 ± 1.3 mM (n = 5, P < 0.05). As in
the presence of amiloride the changes observed in
VmBL,
VTE, and
Ra/Rb on readdition of Na+ were not
different in the presence or absence of EIPA
(n = 5, P > 0.05 for each comparison; Table
3).
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Fig. 7.
Data summary of increase in aNai
(A) and rate of
Na+ entry
(B) in absence and in presence of
5-(N-ethyl-N-isopropyl)amiloride
(EIPA; 5 × 105 M).
Similar to amiloride EIPA reduced increase in
aNai on readdition of serosal
Na+ and inhibited initial rate of
Na+ entry
(n = 5).
* P < 0.05.
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Table 3.
Effect of EIPA on changes in VmBL,
aNai, VTE,
RTE, and Ra/Rb on readdition of
serosal Na+ in continued absence of
Cl
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To investigate whether Na+ entry
could have occurred through Na+
channels, we examined the effect of the specific
Na+ channel inhibitor phenamil. As
can be seen in Fig. 8 in the presence of
phenamil (105 M),
readdition of serosal Na+ still
increased aNai by 18.8 ± 4.7 mM
(segment a'-b'), did not
alter VmBL
significantly, depolarized
VTE by 8.4 ± 0.8, and RTE
remained unchanged. Those changes are not significantly different from
the changes observed on readdition of
Na+ in the absence of the
inhibitor (segment a-b;
n = 3, P > 0.05).
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Fig. 8.
Tracing of experiment showing effect of readdition of
Na+ to serosal bath in absence and
in presence of Na+ channel blocker
phenamil. Readdition of serosal
Na+ increased
aNai (top trace, segment
a-b). Phenamil had no effect on rate of
Na+ entry nor increase in
aNai (top trace, segment
a'-b'). Changes in
VmBL
(middle trace) or
VTE
(bottom trace) caused by readdition
of Na+ were also not affected by
phenamil.
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Effect of Bumetanide on
Na+ Entry
As noted above in Fig. 2, segment d-e,
the readdition of serosal
Cl caused an increase in
Na+ uptake beyond that caused by
addition of serosal Na+ alone,
suggesting the presence of at least one additional
Cl-dependent entry pathway
for Na+. One possible pathway
likely to be present in esophageal cells is the bumetanide-sensitive,
Na+-K+-2Cl cotransporter (21).
Therefore, we investigated whether this transporter contributed to the
Cl-dependent
Na+ entry by monitoring
aNai in tissues pretreated with bumetanide (104 M) before
and during readdition of Cl
to the serosal bath. In our experiments bumetanide had no effect on the
increase in aNai, and the changes in
VTE,
RTE, and
VmBL were not
significantly different from those obtained in the absence of
bumetanide (Table 4).
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Table 4.
Changes in VmBL, aNai,
VTE, and RTE on readdition of serosal
Cl in absence and presence of bumetanide
(104 M)
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Measurements of pHi in Absence of
CO2/HCO3.
To confirm the presence of Na+/H+ exchange as
an acid extruder at the basolateral membrane, we used pH-sensitive
microelectrodes to monitor pHi
during acid loading and recovery from an acid load. Acute intracellular
acid loading was obtained by the
NH3/NH+4 prepulse technique, which was originally described in the squid axons
(5, 6). The experiments were run in the nominal absence of
HCO3 (HEPES Ringer, Table 1,
solution 4) to minimize the effect
of HCO3 transporters on
pHi regulation. Under these
conditions baseline mean values for esophageal epithelium were
VTE = 11.8 ± 1.0 mV, RTE = 2,026 ± 184 · cm2, and
for individual impaled basal cells
pHi = 7.10 ± 0.06, VmBL = 53 ± 2.7 mV, and
Ra/Rb = 5.18 ± 0.95 (n = 10).
Intracellular acid loading with
NH3/NH+4.
After baseline values in HEPES Ringer were determined, the cells were
exposed to
NH3/NH+4
(20 mM NH4Cl, Table 1,
solution 5). A typical experiment is
shown in Fig. 9. The exposure to
NH3/NH+4
leads to the initial entry of NH3
(usually the more permeant component) into the cell where it gets
protonated, thereby causing a rapid alkalinization of pHi (Fig. 9,
segment a-b). The mean increase in
pHi was 0.19 ± 0.02 pH unit
(n = 10, P < 0.001).
VmBL depolarized
by 5.6 ± 1.3 mV, whereas
VTE,
RTE, and
Ra/Rb
remained unchanged. The alkalinization was followed by a small and slow
acidification (plateau acidification, segment
b-c) due to the slower NH+4
entry and to NH3 exit.
NH3/NH+4
was then removed from the bathing solution, and the tissue was now
exposed to a Na+-free HEPES
solution (Table 1, solution 6).
NH+4 (as well as
NH3) exits the cell as
NH3, leaving behind its
H+ and thus causing a significant
fall in pHi
(segment c-d). The average fall in
pHi on removal of
NH+4 was 0.69 ± 0.07 pH unit
(n = 10, P < 0.001). The changes in
VmBL were
transient and not statistically significant (46 ± 2.83 mV
vs. 48 ± 2.5 mV) as were the changes in
Ra/Rb,
whereas VTE
hyperpolarized by 5.97 ± 0.62 mV and
RTE increased by
250 ± 29 · cm2
(n = 11, P < 0.001). It is
important to note that, in the absence of external
Na+,
pHi remained low and there was
complete inhibition of pHi
recovery.
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|
Fig. 9.
Tracing of experiment showing intracellular pH
(pHi; top
trace),
VmBL
(middle trace), and
VTE
(bottom trace) during
NH+4 acid loading and recovery in
HCO3-free solution. Exposure to
NH3/NH+4 caused initial
alkalinization (segment a-b),
followed by slight acidification (segment
b-c). On removal of
NH3/NH+4 in
Na+-free solution,
pHi fell (segment
c-d). In absence of
Na+ no
pHi recovery was observed. When
Na+ was restored in bathing
solution, pHi recovered to its
initial value (segment d-e).
|
|
Na+-dependent
recovery and effect of amiloride.
After the cells were acid loaded by removal of
NH3/NH+4
in a Na+-free solution,
Na+ was given back to the bath
(HEPES Ringer, Table 1, solution 4).
The restoration of Na+ caused full
recovery of pHi from a value of
6.60 ± 0.07 to a value of 7.14 ± 0.14, at an initial rate of
0.15 ± 0.04 pH units/min (Fig. 9, segment
d-e), whereas
VmBL,
VTE, and
RTE recovered to their initial value (n = 5, P < 0.001). These experiments
confirm the presence of a
Na+-dependent acid-extruding
mechanism on the basolateral side of the cell, a finding consistent
with the presence of Na+/H+ exchange.
To check the sensitivity of this transporter to amiloride the same
experiment as the one described previously was repeated (Fig.
10). After acid loading, the tissue was
pretreated with 103 M
amiloride before and during the readdition of
Na+. In three different tissues,
amiloride inhibited almost completely the
Na+-dependent recovery of
pHi from the imposed acid load
(segment e-f). When amiloride
was then removed from the bathing solution, pHi recovered by ~90%
(segment f-g;
n = 3, P < 0.02).
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|
Fig. 10.
Tracing of experiment showing inhibition of
pHi recovery from acid load by
amiloride (103 M).
pHi,
VmBL, and
VTE are shown
during NH+4 acid loading. Amiloride was added
to bathing solution in absence of
Na+. When
Na+ was restored in continuous
presence of amiloride, recovery of
pHi was inhibited
(segment e-f).
pHi recovered only when amiloride
was removed from bathing solution (segment
f-g).
|
|
| |
DISCUSSION |
Steady-State Measurements
The present study was designed to identify the mechanisms by which
Na+ is transported across the
basolateral membrane of esophageal epithelial cells. To do so, we
impaled the basal cells of this multilayered epithelium by
intracellular microelectrodes and measured aNai,
VmBL,
VTE, and
RTE. Under
steady-state conditions in HCO3
Ringer, our measurements of
VTE and RTE are
comparable to those reported in the Ussing chamber (18) and are
consistent with a tight epithelium. Measurements of
aNai in esophageal cells have not been
reported before, and our value of 24.6 ± 2.2 mM is within
reasonable range of intracellular
Na+ values reported earlier in
other Na+ transporting epithelia
(2, 3, 19, 25).
Bilateral removal of
Na+ and
Cl and readdition of serosal
Na+.
To characterize the cellular mechanisms of basolateral
Na+ transport, we first removed
external Na+ and
Cl, which would lead to
depletion of intracellular stores of these ions. Indeed the basal cell
aNai was reduced by this maneuver almost
75% below resting levels in HCO3 Ringer, thereby creating on readdition of
Na+ to the serosal bath a maximal
driving force for Na+ entry across
the basolateral membrane. The removal of serosal and luminal
Na+ and
Cl causes a decrease in
active transport, thus depolarizing
VTE and causing
an increase in
RTE.
Cl-independent
Na+ transport was examined by
adding basolateral Na+ in the
absence of external Cl. The
readdition of Na+ in the absence
of Cl resulted in a marked
increase in aNai, an increase that
effectively approached the prior resting baseline value. Moreover, this
increase in cell Na+ was
unaccompanied by a change in
VmBL, indicating
that the route of Na+ entry was a
Cl-independent
electroneutral pathway. One pathway that fits this description is the
Na+/H+ exchanger. Rabbit esophageal epithelial
cells isolated or in culture possess a Na+/H+
exchanger (11, 22), and this exchanger by analysis of isolated mRNA was
reported to be of the NHE1-type isoform (20). However, the
Na+/H+ exchanger has never functionally been
localized to the basolateral membrane in esophageal cells.
Evidence for Na+/H+ exchange
In our experiments the evidence for Na+/H+
exchange at the basolateral membrane is that readdition of serosal
Na+ in the absence of luminal and
serosal Cl caused a
substantial increase in aNai with no
significant sustained changes in
VmBL. Moreover,
our experiments indicate that both the amount of change in
aNai and the rate of increase in
aNai were significantly blocked by
amiloride (103 M). Given
the limited specificity of amiloride (9) the same experiments were
carried out using EIPA, a selective and more potent inhibitor of
Na+/H+ exchange (24), and the results were
essentially the same as with amiloride. The substantial decrease in
Na+ influx in the presence of
amiloride strongly supports the presence of a
Na+/H+ exchanger on the basolateral membrane of
basal cells. Further evidence is provided by measurements of
pHi.
Our experiments using pHi
measurements show that the basal cells of the esophagus, acid-loaded by
exposure to
NH3/NH+4 in HCO3-free Ringer, can recover from
their acid load only when Na+ is
present in the serosal bath. This acid extrusion mechanism could be
blocked by amiloride and is therefore compatible with Na+/H+ exchange. The experiments were conducted
in the absence of
CO2/HCO3 to minimize the contribution of possible
HCO3-dependent pHi regulating mechanisms. Such
mechanisms are known to exist in the esophageal epithelial cells (12,
23) but have not yet been fully characterized in the intact epithelium.
A significant role of Na+/H+ in transport of
Na+ at the basolateral membrane
agrees with the results obtained by intracellular measurements of
Na+. Inhibition of
Na+/H+ exchange by amiloride was evident by
inhibition of pHi recovery when
experiments were conducted in the absence of
CO2/HCO3 and after acid loading the cells using
NH4Cl. However, in the presence of
HCO3 inhibition of
Na+ entry by amiloride was not
complete. This underscores the possibility that other transport
mechanisms for Na+ entry in
addition to Na+/H+ exchange are present.
Although the concomitant presence of a Na+/H+
exchanger on the luminal membrane of the basal cells cannot be ruled
out, our results indicate that Na+/H+ exchange
is present at the basolateral membrane of the intact esophageal epithelium.
The results of our study do not support the existence of sizable
Na+ transport through basolateral
Na+ channels. The evidence for
this observation is based on the fact that even when the
Na+ gradient is largely set in
favor of passive Na+ entry as it
is after removal of Na+ from the
bathing solutions, the readdition of
Na+ did not cause any significant
change in VmBL or
Ra/Rb.
Transport of Na+ across the
basolateral membrane through channels would be expected to cause a
depolarization of
VmBL and a
decrease in basolateral membrane resistance that would result in an
increase in
Ra/Rb. Both parameters did not change significantly on readdition of serosal
Na+. Moreover phenamil, a specific
inhibitor of Na+ channels, did not
have any effect on Na+ transport
across the basolateral membrane. This lack of
Na+ channels on the basolateral
membrane is consistent with studies in other
Na+-transporting epithelia (17),
including the prototypical stratified squamous epithelium, the frog
skin, in which Na+ channels are
localized to the apical membrane.
Na+/H+ exchange extrudes
H+ from the cell in exchange for
external Na+. Basolateral
Na+/H+ exchange in the esophageal epithelium,
like that of the kidney proximal tubule basolateral membrane (4) and
that of the frog skin (8), does not contribute directly to
transcellular (lumen to serosa)
Na+ uptake but nevertheless is
primarily active in regulation of pHi. In this capacity
Na+/H+ exchange may play a very important role
in regulating transport and maintaining cell homeostasis. For example,
luminal Na+ channels in a variety
of epithelia, are reported to be inhibited at low
pHi (13); therefore, luminal
Na+ influx can be blocked during
periods of low luminal pH, as frequently occurs in the esophagus. In
these cases, basolateral Na+
exchange not only will protect against intracellular acidosis (in face
of external low pH) but also may play a role in maintaining intracellular Na+ when luminal
Na+ entry is blocked. This is
probably very important in the esophageal cells, which are regularly
exposed to refluxed acidic gastric contents and through ingestion of
acidic beverages to acid loads of a wide range.
Readdition of Cl in the Presence
of Na+
Our results also indicate a small
Cl-dependent component of
basolateral Na+ transport. Our
evidence for this pathway is that addition of serosal
Cl was able to drive
Na+ uptake into the cell beyond
that caused by the addition of serosal Na+. This increase in
aNai cannot result from the action of a
Na+-dependent
Cl/HCO3
exchanger because this transporter would drive
Na+ out of the cell when
Cl is driven into the cell.
It is also unlikely that
Na+-K+-2Cl is responsible for
the increase in aNai because it was not
blocked by bumetanide. Although
Na+-K+-2Cl cotransport was found
to be present in the rabbit esophagus (21), this transporter may only
be activated in face of an osmotic challenge to the cell. Another
possibility is that the entry of
Na+ on readdition of
Cl is mediated by a
Na+-Cl transporter that is not sensitive to
bumetanide. However the depolarization of
VmBL on
readdition of Cl cannot be
explained on the basis of a Na+-Cl
transporter because this transporter is known to be electroneutral. The
observed depolarization of
VmBL can possibly
result from a finite permeability of the cell membrane to gluconate
that would leave the cell on replacement of gluconate with
Cl, as suggested by Guggino
et al. (7) in the kidney proximal tubule. It should be noted that, in
the presence of Na+ and
Cl in the lumen, readdition
of basolateral Cl causes an
initial hyperpolarization of
VmBL followed by
a depolarization, consistent with the presence of a basolateral
conductance to Cl (2). The
observed increase in
Ra/Rb
in our experiments on readdition of serosal
Cl is consistent with this finding.
In summary we have localized a Na+/H+ exchange
mechanism on the basolateral membrane of the esophageal cells. This
exchanger, sensitive to amiloride and to one of its analog, EIPA,
constitutes a major pathway for basolateral
Na+ entry and likely plays an
important role in the pHi
regulation and homeostasis of the esophageal epithelial cells.
| |
ACKNOWLEDGEMENTS |
We thank Dr. Nazih L. Nakhoul for helpful discussions and for
carefully reading the manuscript.
| |
FOOTNOTES |
This work was supported by the National Institute of Diabetes and
Digestive and Kidney Diseases Grant RO1-DK-36013.
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. §1734 solely to indicate this fact.
Address for reprint requests: S. Abdulnour-Nakhoul, Dept. of Medicine,
Section of Gastroenterology, SL 35, 1430 Tulane Ave., New Orleans, LA
70112-2699.
Received 19 February 1998; accepted in final form 19 October 1998.
| |
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Am J Physiol Gastroint Liver Physiol 276(2):G507-G517
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Abstract
Introduction
![<IT>k</IT><SUB>Na − K</SUB> = <FR><NU>a<SUP>100 mM NaCl</SUP><SUB>Na</SUB></NU><DE>a<SUP>100 mM KCl</SUP><SUB>K</SUB></DE></FR> × 10<SUP>−[<IT>V</IT><SUB>100 mM NaCl</SUB> − <IT>V</IT><SUB>100 mM KCl</SUB>]/S</SUP>](/content/vol276/issue2/fulltext/G507/img003.gif)
