| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Departments of Medicine and Physiology, Tulane University School of Medicine, and Veterans Administration Medical Center, New Orleans, Louisiana 70112-2699
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
We
investigated Cl
transport pathways in the apical and
basolateral membranes of rabbit esophageal epithelial cells (EEC) using
conventional and ion-selective microelectrodes. Intact sections of
esophageal epithelium were mounted serosal or luminal side up in a
modified Ussing chamber, where transepithelial potential difference and
transepithelial resistance could be determined. Microelectrodes were
used to measure intracellular Cl activity
(a
73 ± 4.3 mV and
a
caused a transient depolarization of VmBL and a
decrease in a-channel
blocker flufenamate. Serosal bumetanide significantly decreased the
rate of change of a. When a luminal cell was impaled,
VmC was 65 ± 3.6 mV and
a depolarized VmC and decreased
a from the serosal bath decreased a
in a luminal or serosal bath) yielded
a straight line [slope (S) = 67.8 mV/decade of change
in a pathway sensitive to
flufenamate; and 3) the apical membrane has limited
permeability to Cl, which is consistent with the limited
capacity for transepithelial Cl transport. Transport of
Cl at the basolateral membrane is likely the dominant
pathway for regulation of intracellular Cl.
microelectrodes; Ca2+-sensitive Cl
channels; flufenamate; anion transport blockers
| |
INTRODUCTION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
THE ESOPHAGUS IS LINED
BY a moist stratified squamous epithelium, which actively
transports Na+ from lumen to serosa. Active Na+
transport accounts for the main portion (85%) of the short-circuit current developed across this tissue (~10 µAmp/cm2),
whereas the majority of the remaining current is due to the transport
of Cl from serosa to mucosa (20). Although
the contribution of Cl to the short-circuit current is
very small, cellular transport of Cl, similar to other
epithelia, is fundamental for the maintenance of the intracellular
milieu. Regulation of cellular Cl transport is
particularly important in the esophageal epithelium, a tissue regularly
exposed to wide fluctuations in luminal content including high levels
of NaCl (salt) in food and HCl (gastric acid) due to the reflux of
gastric content (14, 16). Because very little information
is available about Cl transport in esophageal epithelial
cells, the purpose of this study was to examine its transport pathways
across both the basolateral and the apical cell membranes of this
epithelium. This was done using microelectrodes to measure
intracellular Cl activity and basolateral and apical
membrane potential of esophageal cells within the intact epithelium.
Our study indicates that the a
transport across the basolateral membrane that is sensitive to the
nonsteroidal anti-inflammatory aromatic Cl-channel
blocker flufenamate. A component of Cl transport in the
serosal cells is sensitive to bumetanide. Moreover, there is little
capacity for the transport of Cl across the apical
membrane, which likely accounts for the minimal contribution of
Cl to net active transport of ions across this epithelium.
| |
METHODS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Animal and Tissue Preparation
New Zealand rabbits were killed by administration of an intravenous overdose of pentobarbital sodium (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 were dissected free with a scalpel. The sheet of mucosa thus obtained was cut, and a section was mounted horizontally serosal side up (for basal cells impalements) or mucosal side up (for luminal cell impalements) in a modified Ussing chamber with an aperture of 1.13 cm2. The chamber allows independent and continuous perfusion of the apical and the serosal side of the tissue (1). 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) that allow one of six experimental solutions to flow to each side of the chamber. The volume of fluid in the upper and in the lower sides of the chamber was ~0.25 ml. The rate of flow of the solution to the upper chamber was 2 ml/min, which yielded a turnover rate (i.e., time for a solution to be washed out completely from the chamber) of ~8 s. Perfusion fluid to the lower part of the chamber was delivered at 2 ml/min; however, only part of this fluid (~1 ml/min) entered the chamber, the rest leaving through a drain that was kept at a fixed level. The turnover rate in the lower side of the chamber was ~15 s. All solutions were placed at the same height from the chamber. The solutions were prewarmed and delivered to the chamber at 37°C.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 luminal side and a similar electrode placed in the bath fluid of the serosal side (luminal electrode potential serosal electrode potential). Both
electrodes were fitted with an Ag-AgCl wire, and the leads were
connected to the amplifier of a voltage clamp (model VCC 600 Physiologic Instruments, San Diego, CA). The voltage clamp allowed
automatic compensation for voltage errors due to the resistance of the
fluid and was also used to deliver a direct-current (DC) pulse (I) 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 [change in
VTE (
VTE)] as follows
|
(1) |
(a-sensitive microelectrode for intracellular
Cl measurements.
The following is a brief description of microelectrode preparation:
double-barreled microelectrodes were prepared from two 1.2-mm OD
borosilicate glass fiber capillaries (A-M Systems, Everett, WA) that
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
100oC for 2 h. The Cl exchanger (WPI,
Sarasota, FL.) was then introduced into the tip of the silanized barrel
by means of very fine glass capillaries. The silanized barrel was
further backfilled with 0.5 M KCl, and 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). The resistance of the reference barrel ranged from 50 to 100 M
, and the tip potential was <5 mV. Single-barreled
Cl-selective microelectrodes were pulled from 1.2-mm-OD
borosilicate glass capillaries dried in the oven at 200°C for 2 h. Ten microliters of tri-n-butyl-chlorosilane were then
introduced in a closed vessel (300 ml) that contained the
microelectrodes, after which the silane fumes were vented and the
electrodes were left in the oven for an additional 30 min. The
electrodes were then filled and backfilled as described for the
ion-sensitive barrel above. Ling-Gerard microelectrodes were pulled
from 1.2-mm-OD borosilicate glass fiber capillaries and filled with 3 M
KCl. Their resistance ranged from 20 to 30 M, and the tip potentials
were <5 mV.
The slope (S) of the Cl microelectrode (or
ion-sensitive barrel) was determined from the equation
|
(2) |
activity ratio of pure 100 mM to pure 10 mM NaCl. The
S of the electrodes averaged 55 mV/decade of change in
activity of Cl. The selectivity coefficient
kClHCO3 for Cl over
HCO
|
(3) |
electrode in 100 mM NaHCO3 and
V100Cl is the voltage of the Cl electrode in
100 mM NaCl.
The intracellular ionic activities (a sensitive barrel of the double-barreled microelectrode
or of the single barreled Cl sensitive microelectrode.
The total potential (ViCl) of the ion-sensitive
electrode was measured as the voltage difference between the
ion-sensitive microelectrode and the free flowing reference electrode
in the bath. The pure ionic potential was obtained by subtracting
electronically VmBL or apical membrane potential
(VmC) from the total potential of the
ion-sensitive electrode.
The intracellular ionic activity was calculated according to the
equation
|
(4) |
in the bathing solution, the activity coefficient
being 0.76 at 37°C, Vcell is the cell membrane
potential difference (VmBL or
VmC), and V
electrodes used for the study, the mean
Cl-to-HCO
When a basal cell was impaled, the basolateral membrane potential (VmBL) was read as the voltage difference between the reference barrel of the double-barreled microelectrode and the reference electrode in the serosal bath. When a luminal cell was impaled, the VmC was read as the voltage difference between the single-barreled microelectrode (Ling-Gerard) and the reference electrode in the luminal bath.
The apparent ratio (23) of the apical to the basolateral
membrane resistance (Ra/Rb) was determined from
the ratio of the voltage deflections produced by the transepithelial DC
pulse across the apical and the basolateral membranes [change in
VmC (VmC) and VmBL (VmBL),
respectively] according to the equation
|
(5) |
VmBL
produced by the transepithelial current pulse was measured directly and
VmC was calculated as
VTE VmBL.
When a luminal cell was impaled, VmC was
measured directly and VmBL was calculated as
VTE VmC . This calculation relies on the assumption that
VTE = VmBL + VmC, which was verified only in simple tissues.
To validate that in this multilayered tissue this calculation is a
reasonable approximation we did the following. We used the signal from
the bath voltage electrode at either the serosal or the luminal side of
the tissue to drive the circuit ground of the intracellular measuring
electrometer, thus allowing the intracellular potential (from the
voltage microelectrode) to be referenced to either the serosal or the
mucosal side of the tissue. We used this approach to compare during
serosal impalements a calculated VmC (from
VmBL VTE) to a
presumed VmC measured as cell potential
referenced to the luminal electrode. On the other hand, during luminal
impalements, we compared a calculated VmBL (from
VmC VTE) with a
presumed VmBL measured as cell potential referenced to the serosal electrode. The differences between the calculated and measured values of VmBL or of
VmC were within an acceptable range close to the
standard error of the measurement.
Readings were recorded on a three-channel strip chart recorder (Kipp & Zonen, Bohemia, NY).
The Nernst equilibrium potential for Cl was calculated
from the equation
|
(6) |
activity, and a in the luminal or serosal bath.
Solutions
The composition of Ringer solutions is given in Table 1. We have measured ionized Ca2+ in gluconate-containing solutions and found that ionized Ca2+ concentration was reduced by almost 50% compared with control Ringer. Consequently, we have doubled the concentration of Ca2+ in all solutions containing gluconate. Chelation of Ca2+ in 0 Cl solution
was corrected in a similar manner by Boron and Boulpaep (3). Indanyloxyacetic acid 94 (R(+)-IAA-94) and
R(+)-butylindazone [R(+)-DIOA] were purchased from RBI-Sigma (St
Louis, MO), diphenylamine-2-carboxylate (DPC) was from Fluka
(Milwaukee, WI), 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB)
was from Calbiochem (La Jolla, CA), and DIDS, SITS, and all other
chemicals were from Sigma. Inhibitors insoluble in aqueous solutions
were dissolved in a small volume of dimethyl sulfoxide and added to the
solution. The concentrations used were based on the concentrations
required to achieve maximal inhibition of the transporters as reported
previously in other preparations (7, 18). The
concentration of dimethyl sulfoxide never exceeded 0.5% 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 unless otherwise indicated. n Is the number of observations.| |
RESULTS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Basolateral Membrane Studies in Basal Cells
Measurements in control bicarbonate Ringer.
Intracellular measurements of basal cells obtained in tissues from 10 rabbits, bathed bilaterally with control HCO70 ± 4.2 mV, VTE = 12.9 ± 0.7 mV, a · cm2 and Ra/Rb=
4.74 ± 0.6 (n = 16).
(ECl), as calculated from
Eq. 6, is 46 mV, a value significantly smaller than the
electrical potential difference of 70 mV observed across the cell
basolateral membrane. Thus, in the basal cells, aEffect of removal of serosal Cl in the presence of
CO2/HCO-free
solution (solution 2, Table 1), a66 ± 3.0 to 61 ± 3.5 mV (n = 21, P < 0.001). This initial depolarization was then
followed by a slower (~5-8 min) repolarization of
VmBL (segment bc) until the membrane
potential reached 67 ± 3.3 mV, a value not significantly
different from control. The switch to Cl-free solution
also resulted in the rapid depolarization of VTE from 12.8 ± 0.6 to 4.9 ± 0.6 mV and then partial
repolarization to 8.9 ± 0.8 mV. RTE
increased from 2,530 ± 162 · cm2 to
2,787 ± 170 · cm2, whereas
Ra/Rb decreased from 4.32 ± 0.85 to
3.53 ± 0.73 (n = 10, P < 0.001).
Data from this and similar experiments are summarized in Fig.
2. When Cl was returned to
the serosal solution, all changes were completely reversed.
VmBL and VTE
hyperpolarized rapidly (<1 min.) and transiently (Fig. 1,
segment cd) to 74 ± 5.8 and 16.1 ± 0.9 mV,
respectively, before going back to their control values.
RTE and Ra/Rb returned to
normal, and a
|
|
Effect of SITS or DIDS on removal of serosal Cl in
CO2/HCO/HCO exit on removal of
serosal Cl, we treated tissues from four different
animals with 0.5 mM SITS for 15 min before the removal of extracellular
Cl. SITS did not have an effect on the steady-state
parameters in the treated tissues. Moreover, when Cl was
removed in the presence of SITS, we observed a transient depolarization
of VmBL of 8.6 ± 0.6 mV, after which
VmBL hyperpolarized to a value not significantly
different from the control value in the presence of SITS. Also,
a in the presence of SITS are not significantly
different (n = 4, P > 0.05) from those
observed in its absence (see above). The rate of change in
a were not different in the absence and presence of SITS
(Table 2).
|
. DIDS had no significant effect on
the steady-state parameters. The initial depolarization of
VmBL on removal of serosal Cl was
4.3 ± 1.9 and 3.3 ± 1.9 mV in the absence and presence of DIDS, respectively. This initial depolarization was followed by a
hyperpolarization of VmBL and a reduction
in the a were not different in the absence and presence of
DIDS. (Table 2).
Measurements of intracellular Cl in the nominal
absence of bicarbonate.
The lack of inhibition by SITS or DIDS of the changes in
a and the depolarization of
VmBL associated with Cl removal
suggest that a conductive pathway for Cl exists across
the basolateral membrane of basal cells. If this is the case, then the
cellular changes induced by removal of serosal Cl are
expected to persist in the absence of
CO2/HCO73 ± 4.3 mV,
VTE was 12.6 ± 1.3 mV, and
a · cm2, and Ra/Rb was
5.47 ± 1.1 (n = 13). From these data, the
ECl, as calculated from the Nernst equation (Eq. 6), was 48 mV, which is significantly smaller than the
electrical potential difference of 73 mV observed across the
basolateral cell membrane. Thus the a predicted by electrochemical
equilibrium across the basolateral membrane even in the absence of
bicarbonate. This indicates that the mechanism for maintenance of
Cl above electrochemical equilibrium is essentially
bicarbonate independent.
Effect of removal of serosal Cl in the absence of
bicarbonate.
When the serosal bath HEPES solution was switched to a
Cl-free HEPES solution (solution 4, Table 1)
the observed changes in cellular and transepithelial parameters were
very similar to the changes observed on removal of Cl in
the presence of HCO, a68 ± 2.5 to 63 ± 2.8 mV (n = 30, P < 0.001) followed by a slow (~5-8 min)
repolarization (segment bc) until the membrane potential
reached 66 ± 2.5 mV, a value not significantly different from
control. VTE depolarized rapidly from
11.5 ± 1.0 to 2.9 ± 1.1 mV and then repolarized to
5.5 ± 1.2 mV (n = 32, P < 0.001). RTE increased from 1,995 ± 144 to
2,278 ± 158 · cm2 (n = 26, P < 0.001), whereas Ra/Rb
decreased from 5.47 ± 1.1 to 3.56 ± 0.6 (n = 13, P < 0.001). Data from this and similar experiments are summarized in Fig. 4. All
changes were fully reversed when Cl was restored to the
bath. There was a rapid (<1 min) and transient hyperpolarization of
VmBL and VTE (Fig. 3,
segment cd) by 6.1 ± 0.5 and 8.9 ± 0.3 mV,
respectively, before going back to their control values (segment
de). RTE and Ra/Rb
returned to normal values, whereas a were not different in the presence and absence
of bicarbonate (Table 2).
|
|
Removal of serosal Cl in the presence of anion
transport inhibitors.
To further investigate the ion transport processes involved in the
cellular responses to removal of serosal Cl, we tested
the effect of two known inhibitors of Cl cotransport. The
experiments were run in the absence of bicarbonate to minimize the
effect of putative bicarbonate transporting mechanisms. Bumetanide
blocks Na-K-2Cl cotransport in a large variety of tissues, and
DIOA blocks K-Cl cotransport (6). In these
experiments, esophageal tissues were exposed to the inhibitors for
~15 min, after which the solution was switched to a chloride-free
solution also containing the inhibitor. Application of bumetanide (0.1 mM) or DIOA (0.1 mM), each in three different rabbits, had no effect on
steady-state VmBL or aa was 4.6 ± 2.6 mM in the presence of
bumetanide, which is slightly but not significantly different from
a7.4 ± 1.7 mM in the absence of
bumetanide (n = 5, P > 0.05). However,
the rate of decrease in a was 0.96 ± 0.3 in the presence of bumetanide,
a value significantly smaller than 2.67 ± 0.79 mM/min
in its absence. Moreover, the rate of increase in a was 0.97 ± 0.3 mM/min
in the presence of bumetanide, a value significantly smaller than
1.93 ± 0.63 mM/min in its absence (n = 5, P < 0.05; Table 2). In the presence of DIOA,
a9.0 ± 2.3 mM, a value not
significantly different from a9.1 ± 1.7 mM in the absence of DIOA (n = 5, P > 0.05). Also, the VmBL and
VTE were similar in the absence and presence
of bumetanide or DIOA (P > 0.05). The rate of change
in a was 3.3 ± 0.28 and 2.02 ± 0.3 mM/min,
respectively, in the absence and 2.83 ± 0.44 and 1.09 ± 0.36 mM/min, respectively, in the presence of DIOA. Although the rates
were slightly reduced in the presence of the inhibitor, the values were
not statistically different. These data indicate that although under
steady-state conditions, bumetanide or DIOA do not alter
a without affecting the accompanying changes in cell
membrane potential difference. This finding is consistent with the
presence of an electroneutral transport mechanism for Cl
transport at the basolateral membrane, likely Na-K-2Cl.
Removal of serosal Cl in the presence of
Cl-channel blockers.
Because the depolarization of VmBL on removal of
serosal Cl indicates that a conductive pathway for
Cl exists at the basolateral membrane of basal cells, the
following four compounds reported to inhibit different types of
Cl channels in other preparations were investigated:
anthracene-9-carboxylate (A-9-COOH), DPC, NPPB, and R(+)-IAA 94 (7, 12, 13). Application of each of these compounds,
A-9-COOH (1 mM), DPC (0.1 mM), NPPB (0.1 mM), or R(+)-IAA94 (20 µM)
in at least three tissues from different animals had no effect on
steady-state VmBL or aVmBL, VTE, a were
not significantly different in the absence and presence of each of
these four inhibitors (Table 3).
|
conductance, particularly the
Ca+2-activated Cl channel (27,
29). Similar to A-9-COOH, DPC, NPPB, and R(+)-IAA 94, serosal
application of the Cl-channel inhibitor flufenamate (0.5 mM) did not cause significant changes in steady-state values of
VmBL or a
decreased a removal in the
presence of flufenamate were significantly smaller than those in its
absence (P < 0.01, n = 14). The rate
of decrease of a2.9 ± 0.8 to 0.9 ± 0.2 mM/min in the presence of
flufenamate (n = 5, P < 0.05). These
results are summarized in Table 3.
|
Effect of removal of serosal Cl in the absence
of Ca2+.
The effect of flufenamate is indicative of the presence of
Ca2+-activated Cl channels in the basolateral
membrane (5, 27). Prolonged exposure of the tissue to a
Ca2+-free solution in the presence of a Ca2+
chelator is likely to result in reduction of intracellular
Ca2+. Moreover, studies in other epithelial cells have
indicated that Cl channels can be regulated by external
and internal Ca2+ (24, 28). We, therefore,
investigated the effect of Cl removal in the absence of
Ca2+ from the bathing solutions. In seven esophageal
tissues from six different rabbits, Ca2+ removal was
achieved by perfusing the esophagus bilaterally for ~20 min with a
Ca2+-free HEPES solution to which 3 mM EDTA was added
(solution 5). Figure 6 is a
representative tracing showing the results of those experiments. The
removal of Ca2+ caused a slow depolarization of
VmBL from 57 ± 3.5 to 42 ± 1.9 mV and of VTE from 11.9 ± 0.6 to
8.6 ± 0.5 mV (n = 12, P < 0.001), whereas a in the absence of Ca2+ (solution
6) caused an initial small depolarization of
VmBL, which was followed by a repolarization
(segment a'b'c'). In the absence of Ca2+, the
initial depolarization caused by Cl removal
(segment a'b') averaged 2.9 ± 0.8 mV, which was
significantly smaller than the depolarization of 6.1 ± 0.6 mV in
control (segment ab; n = 10, P < 0.006). Subsequent to this depolarization, in the
absence of Ca2+, VmBL repolarized to
a value of 9.3 ± 1.0 mV (n = 10, P < 0.001), more negative than the initial value
(segment b'c') whereas in the presence of Ca2+,
it repolarized to a value not significantly different from control (segment bc). The transient hyperpolarization of
VmBL (segment cd; 5.8 ± 0.9 mV) on the return of Cl to the bath was also inhibited
(2.0 ± 0.4 mV) in the absence of Ca2+
(segment c'd'). VTE depolarized
initially by 8.6 ± 0.6 mV on removal of Cl in the
absence of Ca2+, a value slightly smaller than in control
(10.1 ± 0.6). The changes in RTE and
Ra/Rb were similar in the absence and presence of
Ca2+. a both in the presence of flufenamate and in
the absence of Ca2+ strongly supports the presence of
Ca2+-sensitive Cl channels on the basolateral
membrane.
|
Cyclic AMP.
To examine the effect of a Cl-channel activator on
Cl transport in the basal esophageal cell, we studied the
effect of cAMP on the changes induced by the removal of
Cl. We exposed the serosal side of the tissue to a Ringer
solution containing the permeable analog of cAMP, 8-bromo-cAMP
(104 M), for several minutes. 8-bromocyclic AMP did not
have an effect on the steady-state values of
VmBL, VTE, or
a was removed from the
serosal bath, VmBL depolarized transiently by
4.8 ± 3.0 mV, after which it repolarized to a value not
significantly different from control, whereas a
Apical Membrane Studies in Luminal Cells
For apical membrane studies, two adjacent luminal cells were impaled with two single-barreled microelectrodes, one a Ling-Gerard microelectrode for cell membrane potential measurement and the other a Cl-sensitive microelectrode for intracellular
Cl measurements. For these measurements to be valid, the
membrane potentials recorded by the two electrodes should be identical and ionic substitutions in the luminal bath should cause equal changes
in the two cell membrane potential differences. To validate the
measurements thus obtained, experiments were performed in which two
different luminal cells in the same tissue were impaled using a
Ling-Gerard microelectrode and a dummy microelectrode identical to the
ion-selective microelectrode but filled with 3 M KCl. As shown in Fig.
7, cells 1 and 2
have similar membrane potential differences, and the potential changes
produced by the removal of Na+ or Cl from the
luminal bath are the same in the two cells. This experiment was
repeated in tissues from three different rabbits, and the luminal cell
membrane potential difference was 62 ± 1.9 and 61 ± 2.1 mV in the simultaneously impaled cells, cells 1 and
2 (n = 9, P > 0.05). In the
absence of Cl from the luminal bath those values were
60 ± 3.3 and 59 ± 2.3, mV respectively
(n = 4, P > 0.05).
|
Measurements of intracellular Cl in the presence of
HCO53 ± 5.1 mV,
VTE was 11.1 ± 2.5 mV, and
a · cm2, and
Ra/Rb was 1.75 ± 0.37. On the basis of the
measured value of a
(ECl) was 34 mV, which is significantly smaller than the
luminal cell potential difference VmC of 53 mV
observed in these cells. Thus, in the presence of
CO2/HCO
is higher than predicted from electrochemical equilibrium.
Measurements of intracellular Cl in the nominal
absence of bicarbonate.
In tissues from five rabbits perfused with HEPES Ringer (solution
3, Table 1), VmC was 65 ± 3.6 mV,
VTE was 14.5 ± 1.5 mV, and
a · cm2, and Ra/Rb was
2.27 ± 0.2 (n = 12). ECl, calculated
from Eq. 6, was 49 mV in the absence of
CO2/HCO64 mV observed
across the apical cell membrane. These experiments indicate that
a
predicted by electrochemical equilibrium across both the apical and the
basolateral membranes both in the presence or absence of
CO2/HCO
Effect of removal of luminal and basolateral Cl in
the absence of CO2/HCO transport at the apical membrane of
luminal cells of EE, we conducted the experiment depicted in Fig.
8. When the luminal bath solution was
switched to a Cl-free solution in the nominal absence of
CO2/HCO65 ± 3.0 to
57 ± 2.7 mV (segment abc), VTE hyperpolarized from 14.5 ± 1.5 to
21.8 ± 1.1 mV, and a was removed from the basolateral bath.
RTE increased from 2,413 ± 177 to
3,100 ± 247 · cm2, and
Ra/Rb increased from 2.27 ± 0.2 to
2.63 ± 0.2 (n = 12, P < 0.02).
The hyperpolarization of VTE on luminal
Cl removal could be explained by a diffusion potential
for Cl across the paracellular shunt, a fact also
supported by the observed increase in RTE.
|
was subsequently removed from the basolateral
solution in the continuous absence of luminal Cl, there
was an initial transient hyperpolarization of
VmC from 55 ± 2.2 to 63 ± 2.4 mV
after which VmC depolarized to 51 ± 3.6 mV and VTE depolarized transiently from
23.6 ± 1.4 to 13.2 ± 1.4 mV to recover to 18.5 ± 2.2 mV. RTE increased from 3,292 ± 264 to 3,675 ± 323 · cm2. The
a removal. Thus the
decrease in a is removed from the
basolateral side than when removed from the luminal side (2.6 ± 0.9 mM; unpaired t-test, n = 15, P < 0.02). These data are summarized in Fig.
9.
|
Effect of cyclic AMP.
To investigate the possibility that a cystic fibrosis
transmembrane conductance regulator-like channel is expressed at the luminal cell membrane, we examined the effect of removal of luminal Cl in the presence of the permeable analog of cAMP,
8-bromo-cAMP. We exposed the luminal side of the tissue to a control
HEPES solution containing 8-bromo-cAMP (104 M) for
several minutes. 8-bromo-cAMP did not have an effect on the
steady-state values of VmC,
VTE, a from the luminal solution in the continuous
presence of 8-bromo-cAMP caused VmC to
depolarize by 6.3 ± 2.2 mV, and VTE
hyperpolarized by 7.8 ± 0.4 mV, Ra/Rb
remained unchanged, and RTE increased by 282 ± 3 · cm2. Those changes are not
significantly different from the changes observed in the absence of
cAMP (n = 4, P > 0.05).
/HCO4 M) for several minutes. Similar to the
experiment in the absence of bicarbonate, 8-bromo-cAMP did not have an
effect on the steady-state values of VmC,
VTE, a from the luminal solution in the continuous
presence of 8-bromo-cAMP caused VmC to
depolarize by 5.5 ± 2.5 mV, and a0.2 ± 0.12 mM/min.
VTE hyperpolarized by 7.45 ± 0.9 mV,
Ra/Rb increased slightly but not significantly,
and RTE increased by 353 ± 41 · cm2. Those changes are not significantly
different from the changes observed in the absence of cAMP
(n = 4, P > 0.05). It should be noted
that the rate of decrease in a0.2 ± 0.12 mM/min) in the apical cell on removal of luminal Cl was
about 10-fold smaller than the rate of decrease in a, a fact
consistent with the lower permeability of the luminal cell membrane to
Cl.
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Steady-State Measurements
The aim of this study was to investigate the mechanisms for Cl transport across the apical and basolateral membranes
of esophageal epithelial cells. Intracellular measurements were
obtained either in luminal cells (across the apical membrane) or basal
cells (across the basolateral membrane) of intact sections of rabbit
esophageal epithelium using conventional and ion-selective
microelectrodes. The results of these investigations establish for the
first time the level of intracellular Cl activity in the
luminal and basal cells of this stratified squamous epithelium.
Intracellular activity of Cl was higher than equilibrium
in both cell types even when the esophageal tissue was exposed to a
nominally bicarbonate-free solution, indicating that active transport
mechanism(s) for Cl entry exists in these cells and that
these mechanism(s) are not dependent on the presence of bicarbonate.
Moreover, in the presence of
CO2/HCO transports could be
active), neither SITS nor DIDS changed the steady-state value of
a nor the changes observed on removal
of serosal Cl. This finding is consistent with the fact
that removal of bicarbonate from the perfusing solutions did not alter
aAmong the possible bicarbonate-independent mechanisms that could be
responsible for Cl entry are Na-K-2Cl and/or K-Cl
cotransport. In our experiments, the rate of change in
a was significantly reduced in the presence of
bumetanide, which indicates that Na-K-2Cl is contributing to the
movement of Cl across the basolateral membrane at least
when Cl gradient is perturbed across this membrane. The
possibility exists that under physiological conditions, one or both
transporters are present but only activated in the face of an
osmotic challenge to the cell (15, 25). Other
transporters likely to drive Cl into the cells in the
absence of bicarbonate are Cl/OH, or other
Cl-base exchangers similar to those reported in the
kidney (10, 26).
Although the mechanism by which a, a leak pathway for
Cl must exist to counterbalance active Cl
loading to maintain a steady-state a.
Basolateral Cl Channels
, which is more prominent on the basolateral than on
the apical membrane of the cell. This finding is supported by several
pieces of evidence. First, the removal of external Cl
causes a fast depolarization of the basolateral membrane potential difference in the basal cell. Although VmBL
gradually recovers to its original value, this recovery is likely due
to compensatory secondary mechanisms. This behavior of the cell
membrane is consistent with the presence in the basolateral membrane of
two conductive pathways, one for K+ and the other for
Cl. The rapid depolarization due to Cl exit
causes K+ to leave the cell (9), which brings
back the cell membrane PD to its original value. In fact, the presence
of a conductive pathway for K+ in the basolateral
membrane of esophageal basal cells has already been established
(11). Second, a linear relationship between VmBL and a
-channel blocker blocked the depolarization induced by
the removal of Cl by 60%. Fourth, the rate of decrease
in a was
significantly inhibited in the presence of flufenamate. It is also of
importance to note that this conductive pathway for Cl
was less sensitive to treatment with other Cl-channel
inhibitors, such as SITS, DIDS, DPC, A-9-COOH, NPPB, or IAA-94, and
that 8-bromocyclic-AMP did not stimulate this Cl
conductance.
|
The removal of serosal Cl caused apparent
Ra/Rb to decrease substantially. Although we
cannot rule out a change in the shunt resistance contributing to the
observed change in the divider ratio, the observed decrease in
Ra/Rb on removal of serosal Cl
reflects a possible increase in basolateral membrane resistance, indicating that Cl may contribute significantly to the
conductance across this membrane. It should be noted that the
calculation of Ra/Rb (Eq. 5) from the
voltage divider ratio is an approximation that holds true only if
VTE = VmBL + VmC . This assumption was validated in
relatively simple epithelia (21, 22). Given the complexity
of the esophageal epithelium [~25 layers of cells
(17)], we relied on the following observations as an
indication that VTE approximately equals
VmBL + VmC. First,
our pooled data of measured VmBL and
VTE during basolateral impalements indicate that
the average calculated value of VmC in these
experiments (57 ± 4.1 mV in HCO61 ± 4.1 mV in HEPES Ringer) was not statistically different from the measured values of VmC (50 ± 4.8 and 66 ± 3.6 mV, respectively) obtained from experiments in
which luminal impalements were performed. Similarly, the data of
measured VmC and VTE
during luminal impalements yielded an average calculated value of
VmBL in these experiments of 64 ± 5.5 in
HCO78 ± 2.6 mV, respectively, in HEPES,
which was not statistically different from the average measured
VmBL (70 ± 4.2 mV in
HCO73 ± 4.3 mV in HEPES Ringer)
obtained from basolateral impalements. Second, as described in
METHODS, the calculated value of VmC
(as VTE VmBL;
56 ± 4.8 mV) obtained from serosal impalements and the measured
value of a presumed VmC (57 ± 5.0 mV)
obtained from readings of the intracellular voltage microelectrode
against the reference electrode in the luminal bath (rather than the
serosal bath) were also similar.
The inhibition by flufenamate suggests the presence of a
Ca2+-sensitive Cl channel at the basolateral
membrane of the esophageal cell. This is supported by the fact that in
the absence of extracellular Ca2+, the initial
depolarization induced by Cl removal was reduced by
~50%. Moreover, in the absence of Ca2+, the transient
hyperpolarization on readdition of basolateral Cl was
significantly inhibited. It remains to be determined whether this
Cl channel shares common characteristics with the
Ca2+-activated Cl channel family, which play
an important role in anion transport and cell volume regulation and may
have a role as cell adhesion molecules (4, 8, 19).
Apical Membrane Cl Transport
is very limited and could not be stimulated by cAMP.
Transport of Cl is more prominent on the basolateral
side. This is supported by several observations. First, the apical
membrane potential is not linearly proportional to changes in
a reduced a removal reduced a
deletion caused an even larger drop in luminal cell
a deletion (see
RESULTS). Third, the rate of decrease in
a is severalfold smaller than the rate of decrease in
a. It is also to be noted that the removal of
basolateral Cl caused Ra/Rb to
decrease by 35%, whereas luminal Cl removal caused an
increase in Ra/Rb of only 16%. These findings indicate that Cl transport at the basolateral membrane is
the major determinant of a across the apical membrane. Such findings
are also in agreement with the observation that Cl
transport from serosa to mucosa contributes only a small (~10%) component of net active ion transport (as reflected in the
short-circuit current) across rabbit esophageal epithelium. Such
limited conductance of the apical membrane of luminal cells, however,
is likely a reflection of its barrier properties, protecting the
surface cells from hydrochloric acid refluxing from the stomach and
from osmotic and concentration gradients in various ingested solutes.
In summary, our study indicates the presence of a conductive pathway
for Cl transport at the basolateral membrane of the cell,
which is sensitive to flufenamate and to the absence of
Ca2+. The fact that a entry into the cell. Our results also
indicate that there is little capacity for the transport of
Cl across the apical membrane, and this likely accounts
for the minimal contribution of Cl to net active
transport of ions across this epithelium.
| |
ACKNOWLEDGEMENTS |
|---|
This work was supported by a Veteran's Administration merit grant and a National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-36013.
| |
FOOTNOTES |
|---|
Address for reprint requests and other correspondence: S. Abdulnour-Nakhoul, Dept. of Medicine, Section of Gastroenterology SL 35, 1430 Tulane Ave., New Orleans, LA 70112-2699 (E-mail: solange{at}tulane.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published December 12, 2001;10.1152/ajpgi.00085.2001
Received 7 March 2001; accepted in final form 26 November 2001.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
1.
Abdulnour-Nakhoul, S,
Bor S,
Imeryuz N,
and
Orlando RC.
Mechanisms of basolateral Na+ transport in rabbit esophageal epithelial cells.
Am J Physiol Gastrointest Liver Physiol
276:
G507-G517,
1999
2.
Abdulnour-Nakhoul, S,
and
Boulpaep EL.
Transcellular chloride pathways in ambystoma proximal tubule.
J Membr Biol
166:
15-35,
1998[Web of Science][Medline].
3.
Boron, WF,
and
Boulpaep EL.
Intracellular pH regulation in the renal proximal tubule of the salamander. Na-H exchange.
J Gen Physiol
81:
29-52,
1983
4.
Fuller, CM,
and
Benos DJ.
Electrophysiological characteristics of the Ca2+-activated Cl channel family of anion transport proteins.
Clin Exp Pharmacol Physiol
27:
906-910,
2000[Web of Science][Medline].
5.
Gandhi, R,
Elble RC,
Gruber AD,
Schreur KD,
Ji HL,
Fuller CM,
and
Pauli BU.
Molecular and functional characterization of a calcium-sensitive chloride channel from mouse lung.
J Biol Chem
273:
32096-32101,
1998
6.
Garay, RP,
Nazaret C,
Hannaert PA,
and
Cragoe EJ, Jr.
Demonstration of a [K+,Cl]-cotransport system in human red cells by its sensitivity to [(dihydroindenyl)oxy]alkanoic acids: regulation of cell swelling and distinction from the bumetanide-sensitive [Na+,K+,Cl]-cotransport system.
Mol Pharmacol
33:
696-701,
1988[Abstract].
7.
Greger, R.
Chloride channel blockers.
Methods Enzymol
191:
793-810,
1990[Medline].
8.
Gruber, AD,
Gandhi R,
and
Pauli BU.
The murine calcium-sensitive chloride channel (mCaCC) is widely expressed in secretory epithelia and in other select tissues.
Histochem Cell Biol
110:
43-49,
1998[Web of Science][Medline].
9.
Hodgkin, AL,
and
Horowicz P.
The influence of potassium and chloride ions in the membrane potential of single muscle fibres.
J Physiol (Lond)
148:
127-160,
1959.
10.
Karniski, LP,
and
Aronson PS.
Anion exchange pathways for Cl transport in rabbit renal microvillus membranes.
Am J Physiol Renal Fluid Electrolyte Physiol
253:
F513-F521,
1987
11.
Khalbuss, WE,
Alkiek R,
Marousis CG,
and
Orlando RC.
Potassium conductance in rabbit esophageal epithelium.
Am J Physiol Gastrointest Liver Physiol
265:
G28-G34,
1993
12.
Landry, DW,
Akabas MA,
Redhead C,
and
al-Awqati Q.
Purification and reconstitution of epithelial chloride channels.
Methods Enzymol
191:
572-583,
1990[Medline].
13.
Landry, DW,
Reitman M,
Cragoe EJ, Jr,
and
Al-Awqati Q.
Epithelial chloride channel. Development of inhibitory ligands.
J Gen Physiol
90:
779-798,
1987
14.
Long, JD,
Marten E,
Tobey NA,
and
Orlando RC.
Luminal hypertonicity and the susceptibility of rabbit esophagus to acid injury.
Dis Esophagus
11:
94-100,
1998[Medline].
15.
Lytle, C,
and
Forbush d B.
Na-K-Cl cotransport in the shark rectal gland. II Regulation in isolated tubules.
Am J Physiol Cell Physiol
262:
C1009-C1017,
1992
16.
Orlando, RC,
Bryson JC,
and
Powell DW.
Mechanisms of H+ injury in rabbit esophageal epithelium.
Am J Physiol Gastrointest Liver Physiol
246:
G718-G724,
1984
17.
Orlando, RC,
Lacy ER,
Tobey NA,
and
Cowart K.
Barriers to paracellular permeability in rabbit esophageal epithelium.
Gastroenterology
102:
910-923,
1992[Web of Science][Medline].
18.
Palade, PT,
and
Barchi RL.
On the inhibition of muscle membrane chloride conductance by aromatic carboxylic acids.
J Gen Physiol
69:
879-896,
1977
19.
Pauli, BU,
Abdel-Ghany M,
Cheng HC,
Gruber AD,
Archibald HA,
and
Elble RC.
Molecular characteristics and functional diversity of CLCA family members.
Clin Exp Pharmacol Physiol
27:
901-905,
2000[Web of Science][Medline].
20.
Powell, DW,
Morris SM,
and
Boyd DD.
Water and electrolyte transport by rabbit esophagus.
Am J Physiol
229:
438-443,
1975.
21.
Reuss, L,
and
Finn AL.
Electrical properties of the cellular transepithelial pathway in Necturus gallbladder. I Circuit analysis and steady-state effects of mucosal solution ionic substitutions.
J Membr Biol
25:
115-139,
1975[Web of Science][Medline].
22.
Reuss, L,
and
Finn AL.
Passive electrical properties of toad urinary bladder epithelium. Intercellular electrical coupling and transepithelial cellular and shunt conductances.
J Gen Physiol
64:
1-25,
1974
23.
Reuss, L,
Reinach P,
Weinman SA,
and
Grady TP.
Intracellular ion activities and Cl-transport mechanisms in bullfrog corneal epithelium.
Am J Physiol Cell Physiol
244:
C336-C347,
1983
24.
Stewart, GS,
Glanville M,
Aziz O,
Simmons NL,
and
Gray MA.
Regulation of an outwardly rectifying chloride conductance in renal epithelial cells by external and internal calcium.
J Membr Biol
180:
49-64,
2001[Web of Science][Medline].
25.
Tobey, NA,
Cragoe EJ, Jr,
and
Orlando RC.
HCl-induced cell edema in rabbit esophageal epithelium: a bumetanide-sensitive process.
Gastroenterology
109:
414-421,
1995[Web of Science][Medline].
26.
Warnock, DG,
and
Yee VJ.
Chloride uptake by brush border membrane vesicles isolated from rabbit renal cortex. Coupling to proton gradients and K+ diffusion potentials.
J Clin Invest
67:
103-115,
1981.
27.
White, MM,
and
Aylwin M.
Niflumic and flufenamic acids are potent reversible blockers of Ca2(+)-activated Cl channels in Xenopus oocytes.
Mol Pharmacol
37:
720-724,
1990[Abstract].
28.
Wladkowski, SL,
Lin W,
McPheeters M,
Kinnamon SC,
and
Mierson S.
A basolateral chloride conductance in rat lingual epithelium.
J Membr Biol
164:
91-101,
1998[Web of Science][Medline].
29.
Zeiske, W,
Atia F,
and
Van Driessche W.
Apical Cl- channels in A6 cells.
J Membr Biol
166:
169-178,
1998[Web of Science][Medline].
This article has been cited by other articles:
|
|
 |
|
  N. A. Tobey, C. M. Argote, X. C. Vanegas, W. Barlow, and R. C. Orlando Electrical parameters and ion species for active transport in human esophageal stratified squamous epithelium and Barrett's specialized columnar epithelium Am J Physiol Gastrointest Liver Physiol, July 1, 2007; 293(1): G264 - G270. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
