Chloride transport in rabbit esophageal epithelial cells

Solange Abdulnour-Nakhoul, Nazih L. Nakhoul, Canan Caymaz-Bor, and Roy C. Orlando

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<UP><SUB>i</SUB><SUP>Cl</SUP></UP>), basolateral or apical membrane potentials (VmBL or VmC), and the voltage divider ratio. When a basal cell was impaled, VmBL was -73 ± 4.3 mV and a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> was 16.4 ± 2.1 mM, which were similar in presence or absence of bicarbonate. Removal of serosal Cl- caused a transient depolarization of VmBL and a decrease in a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> of 6.5 ± 0.9 mM. The depolarization and the rate of decrease of a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> were inhibited by ~60% in the presence of the Cl--channel blocker flufenamate. Serosal bumetanide significantly decreased the rate of change of a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> on removal and readdition of serosal Cl-. When a luminal cell was impaled, VmC was -65 ± 3.6 mV and a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> was 16.3 ± 2.2 mM. Removal of luminal Cl- depolarized VmC and decreased a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> by only 2.5 ± 0.9 mM. Subsequent removal of Cl- from the serosal bath decreased a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> in the luminal cell by an additional 6.4 ± 1.0 mM. A plot of VmBL measurements vs. log a<UP><SUB>i</SUB><SUP>Cl</SUP></UP>/log a<UP><SUB>o</SUB><SUP>Cl</SUP></UP> (a<UP><SUB>o</SUB><SUP>Cl</SUP></UP> is the activity of Cl- in a luminal or serosal bath) yielded a straight line [slope (S) = 67.8 mV/decade of change in a<UP><SUB>i</SUB><SUP>Cl</SUP></UP>/a<UP><SUB>o</SUB><SUP>Cl</SUP></UP>]. In contrast, VmC correlated very poorly with log a<UP><SUB>i</SUB><SUP>Cl</SUP></UP>/a<UP><SUB>o</SUB><SUP>Cl</SUP></UP> (S = 18.9 mV/decade of change in a<UP><SUB>i</SUB><SUP>Cl</SUP></UP>/a<UP><SUB>o</SUB><SUP>Cl</SUP></UP>). These results indicate that 1) in rabbit EEC, a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> is higher than equilibrium across apical and basolateral membranes, and this process is independent of bicarbonate; 2) the basolateral cell membrane possesses a conductive Cl- 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<UP><SUB>i</SUB><SUP>Cl</SUP></UP> in this tissue is maintained above electrochemical equilibrium independently of the presence of bicarbonate. We have also identified a Ca2+-dependant conductive pathway for Cl- 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 (Delta VTE)] as follows
R<SUB>TE</SUB><IT>=&Dgr;V</IT><SUB>TE</SUB><IT>/</IT>I (1)
The esophagus possesses relatively large basal cells (20-40 µm) that allow stable impalements with double-barreled microelectrodes. Double-barreled microelectrodes were used in basal cells to measure simultaneously and in the same cell the cell membrane potential difference [basolateral membrane potential (VmBL)] and the intracellular activity of Cl- (a<UP><SUB>i</SUB><SUP>Cl</SUP></UP>). Luminal cells are exceptionally long (100-250 µm) and slender (5-10 µm) so that single-barreled microelectrode impalements proved more stable. Thus, 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.

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 MOmega , 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 MOmega , and the tip potentials were <5 mV.

The slope (S) of the Cl- microelectrode (or ion-sensitive barrel) was determined from the equation
S=<FR><NU>V<SUB>100 NaCl</SUB><IT>−V</IT><SUB>10 NaCl</SUB></NU><DE>0.94</DE></FR> (2)
where V100mM NaCl and V10mM NaCl denote the electrode potential in the solutions as noted, 0.94 equals the logarithm (base 10) of the Cl- 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 kCl-HCO3 for Cl- over HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> was calculated from the equation
k<SUB>Cl<IT>−</IT>HCO<SUB>3</SUB></SUB><IT>=</IT>10<SUP><IT>V</IT>100HCO<SUB>3</SUB><IT>−V</IT>100Cl<IT>/</IT>S</SUP> (3)
where V100HCO3 is the voltage of the Cl- electrode in 100 mM NaHCO3 and V100Cl is the voltage of the Cl- electrode in 100 mM NaCl.

The intracellular ionic activities (a<UP><SUB>i</SUB><SUP>Cl</SUP></UP>) were calculated from the potential readings of cellular impalements of the Cl- 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
a<SUP>Cl</SUP><SUB>i</SUB><IT>=</IT>a<SUP>Cl</SUP><SUB>o</SUB><IT>×</IT>10<SUP>(<IT>V</IT><SUP>Cl</SUP><SUB>i</SUB>−V<SUB>o</SUB><IT>−V</IT><SUP>Cl</SUP><SUB>Ringer</SUB>)<IT>/</IT>S</SUP><IT>−K</IT><SUB>Cl−HCO<SUB>3</SUB></SUB>(a<SUP>HCO<SUB>3</SUB></SUP><SUB>i</SUB>) (4)
where a<UP><SUB>o</SUB><SUP>Cl</SUP></UP> indicates the activity of Cl- 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<UP><SUB>o</SUB><SUP>Cl</SUP></UP> is the reading of the electrode in the bathing solution. In 50 Cl- electrodes used for the study, the mean Cl--to-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> selectivity ratio was 10:1. Because of the low estimated intracellular activity of bicarbonate in the cell and the good selectivity of our electrodes, the correction factor (KCl-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> · a<SUB>i</SUB><SUP>HCO<SUB>3</SUB><SUP>−</SUP></SUP>) for the selectivity was dropped from the calculations of a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> (2).

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 (Delta VmC) and VmBL (Delta VmBL), respectively] according to the equation
Ra<IT>/R</IT>b<IT>=&Dgr;V</IT><SUB>mC</SUB><IT>/&Dgr;V</IT><SUB>mBL</SUB> (5)
when a serosal cell was impaled, Delta VmBL produced by the transepithelial current pulse was measured directly and Delta VmC was calculated as Delta VTE - Delta VmBL. When a luminal cell was impaled, Delta VmC was measured directly and Delta VmBL was calculated as Delta VTE - Delta 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
E<SUB>Cl</SUB><IT>=</IT>−<FR><NU>RT</NU><DE>zF</DE></FR> ln <FR><NU>a<SUB>i</SUB>Cl</NU><DE>a<SUB>o</SUB>Cl</DE></FR> (6)
where R is the gas constant, T the absolute temperature, z is the valence, F is the Faraday constant, a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> is the intracellular Cl- activity, and a<UP><SUB>o</SUB><SUP>Cl</SUP></UP> indicates the activity of Cl- 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.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Composition of solutions

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 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> Ringer solution (Table 1, solution 1) yielded the following values: VmBL = -70 ± 4.2 mV, VTE = -12.9 ± 0.7 mV, a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> = 16.2 ± 2.0 mM, RTE = 2,807 ± 147 Omega  · cm2 and Ra/Rb= 4.74 ± 0.6 (n = 16).

On the basis of the measured a<UP><SUB>i</SUB><SUP>Cl</SUP></UP>, the electrochemical equilibrium for Cl- (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, a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> is higher than the a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> predicted by electrochemical equilibrium across the basolateral membrane.

Effect of removal of serosal Cl- in the presence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. When the serosal bath solution was switched to a Cl--free solution (solution 2, Table 1), a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> decreased from 14.3 ± 1.9 to 5.7 ± 1.1 mM (n = 12, P < 0.001), and this decrease was accompanied by a rapid (<1 min) initial depolarization of VmBL as shown in Fig. 1, segment ab, from -66 ± 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 Omega  · cm2 to 2,787 ± 170 Omega  · 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<UP><SUB>i</SUB><SUP>Cl</SUP></UP> monotonically increased to its control value over the course of 5-8 min.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 1.   Tracing from an experiment showing the effect of serosal bath removal of Cl- in a basal cell. In control bicarbonate Ringer, removal of Cl- from the serosal bath caused an initial transient depolarization (ab) of the basolateral membrane potential (VmBL) that was followed by a quick recovery (bc) to a value not significantly different from control. The transepithelial potential (VTE) also depolarized and partially recovered, whereas intracellular Cl- activity (a<UP><SUB>i</SUB><SUP>Cl</SUP></UP>) decreased substantially. The changes were reversible on restoring bath Cl-.



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 2.   Effect of Cl- removal from the serosal bath in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> Ringer in a basal cell, on VmBL (A), VTE (B), a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> (C), transepithelial resistance (RTE; D), and apical-to-basolateral membrane resistance ratio (Ra/Rb; E). Removal of serosal bath Cl- caused an initial depolarization in VmBL of 5.2 ± 0.8 mV, which then recovered to a value not significantly different from control (n = 21; P < 0.001). VTE depolarized initially by 7.9 ± 0.5 mV and recovered partially to a value 4.3 ± 0.5 mV more positive than control. a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> decreased by 8.6 ± 1.4 mM (n = 12; P < 0.001). RTE increased by 257 ± 35 Omega  · cm2 (n = 21; P < 0.001), and Ra/Rb decreased by 0.79 ± 0.14 (n = 10; P < 0.001). *Significantly different from control, paired data (P < 0.05).

Effect of SITS or DIDS on removal of serosal Cl- in CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> Ringer. Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange and other bicarbonate-dependent transporters are inhibited by the stilbene derivatives SITS and DIDS in a variety of tissues. To check the contribution of such a mechanism to Cl- 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<UP><SUB>i</SUB><SUP>Cl</SUP></UP> decreased from 20.8 ± 5.0 to 9.7 ± 2.0 mM. The changes in VmBL observed on removal of serosal Cl- 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<UP><SUB>i</SUB><SUP>Cl</SUP></UP> on removal and readdition of serosal Cl- were not different in the absence and presence of SITS (Table 2).

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Rates of change in a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> of basal cells caused by removal and readdition of bath Cl- under different conditions

In a different set of experiments, the esophageal tissue from 4 different rabbits were exposed to DIDS (0.1 mM) for 15 min before the removal of serosal Cl-. 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<UP><SUB>i</SUB><SUP>Cl</SUP></UP> by 7.4 ± 1.2 mM in the absence and 6.2 ± 3.05 mM (n = 4, P > 0.05) in the presence of DIDS. All the observed changes in cell membrane potential difference, resistance, and transepithelial parameters were not statistically different in the absence or presence of DIDS (n = 4, P > 0.05). The rates of change in a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> on removal and readdition of serosal Cl- 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<UP><SUB>i</SUB><SUP>Cl</SUP></UP>- 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/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. Tissues from 11 rabbits were therefore perfused with a bicarbonate-free HEPES Ringer solution (solution 3, Table 1), and the following values were obtained: VmBL was -73 ± 4.3 mV, VTE was -12.6 ± 1.3 mV, and a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> was 16.4 ± 2.1 mM. RTE was 2,357 ± 263 Omega  · 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<UP><SUB>i</SUB><SUP>Cl</SUP></UP> remains higher than the activity of Cl- 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<UP><SUB>3</SUB><SUP>−</SUP></UP>. On removal of bath Cl-, a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> decreased significantly from 16.4 ± 2.1 to 9.9 ± 1.5 mM (n = 13, P < 0.001). This was accompanied by a very rapid (<1 min) initial depolarization of VmBL (Fig. 3, segment ab) from -68 ± 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 Omega  · 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<UP><SUB>i</SUB><SUP>Cl</SUP></UP> monotonically increased to its control value over the course of 5-8 min. The rates of change of a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> on removal and restoration of serosal Cl- were not different in the presence and absence of bicarbonate (Table 2).


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 3.   Tracing from an experiment showing the effect of removal of serosal Cl- in a basal cell, on a<UP><SUB>i</SUB><SUP>Cl</SUP></UP>, VmBL, and VTE in the absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (HEPES Ringer). Removal of Cl- from the serosal bath caused an initial transient depolarization (ab) of VmBL that was followed by a quick hyperpolarization and recovery (bc) to a value slightly lower but not significantly different from control. VTE also quickly depolarized and partially recovered, whereas a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> decreased substantially. The changes were reversible on restoring bath Cl-.



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of removal of Cl- from the serosal bath in the absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>in a basal cell, on VmBL (A), VTE (B), a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> (C), RTE (D), and Ra/Rb (E). Removal of serosal Cl- caused an initial depolarization of VmBL of 5.0 ± 0.8 mV, which then recovered to a value not significantly different from control (n = 30; P < 0.001). VTE depolarized initially by 8.6 ± 0.6 mV and recovered partially to a value 6.3 ± 0.7 mV more positive than control (n = 32; P < 0.001). a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> decreased by 6.5 ± 0.94 mM (n = 13; P < 0.001). RTE increased by 283 ± 29 Omega  · cm2 (n = 26; P < 0.001), and Ra/Rb decreased by 1.91 ± 0.54 (n = 13; P < 0.001). *Significantly different from control, paired data (P < 0.05).

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 a<UP><SUB>i</SUB><SUP>Cl</SUP></UP>. The change in a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> (Delta a<UP><SUB>i</SUB><SUP>Cl</SUP></UP>) on removal of serosal Cl- was -4.6 ± 2.6 mM in the presence of bumetanide, which is slightly but not significantly different from Delta a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> of -7.4 ± 1.7 mM in the absence of bumetanide (n = 5, P > 0.05). However, the rate of decrease in a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> on removal of serosal Cl- 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<UP><SUB>i</SUB><SUP>Cl</SUP></UP> on restoration of serosal Cl- 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, Delta a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> was -9.0 ± 2.3 mM, a value not significantly different from Delta a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> of -9.1 ± 1.7 mM in the absence of DIOA (n = 5, P > 0.05). Also, the Delta VmBL and Delta VTE were similar in the absence and presence of bumetanide or DIOA (P > 0.05). The rate of change in a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> on removal and addition of serosal Cl- 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<UP><SUB>i</SUB><SUP>Cl</SUP></UP>, bumetanide decreased the rate of change of a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> on removal and addition of serosal Cl- 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 a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> in either bicarbonate- or HEPES-containing solutions. Delta VmBL, Delta VTE, Delta a<UP><SUB>i</SUB><SUP>Cl</SUP></UP>, and the rate of decrease in a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> on the removal of serosal Cl- were not significantly different in the absence and presence of each of these four inhibitors (Table 3).

                              
View this table:
[in this window]
[in a new window]
 
Table 3.   Effect of Cl- channel inhibitors on changes in VmBL, a<UP><SUB>i</SUB><SUP>Cl</SUP></UP>, and VTE upon removal of Cl- from the serosal bath

Flufenamic acid, at concentrations ranging from 0.1 to 0.5 mM, is an inhibitor of Cl- 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<UP><SUB>i</SUB><SUP>Cl</SUP></UP>. Moreover, in the presence of flufenamate, removal of serosal Cl- decreased a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> by 5.9 ± 0.74 mM, a value not significantly different from the decrease in a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> (6.8 ± 1.8 mM) in the absence of the inhibitor. However, in the presence of flufenamate, VmBL depolarized by only 2.9 ± 0.5 mV (Fig. 5, segment a'b') compared with 7.4 ± 1 mV in control (Fig. 5, segment ab), and the repolarization was only partial (Fig. 5, segment b'c'). Similarly, the initial depolarization of VTE was only 5.8 ± 0.6 mV compared with 9.0 ± 0.7 mV in control, thus the peak depolarizations of VmBL and VTE caused by Cl- removal in the presence of flufenamate were significantly smaller than those in its absence (P < 0.01, n = 14). The rate of decrease of a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> was markedly reduced from -2.9 ± 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.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 5.   Tracing from an experiment showing the effect of removal of serosal Cl- in a basal cell on a<UP><SUB>i</SUB><SUP>Cl</SUP></UP>, VmBL, and VTE in the absence and in the presence of flufenamate. Removal of Cl- from the serosal bath in the presence of flufenamate caused a marked reduction in the initial transient depolarization of VmBL (a'b' vs. ab) and a marked reduction in the depolarization of VTE. It can also be noted that the initial rate of decrease in a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> is reduced in the presence of flufenamate. When Cl- was given back to the bath, the hyperpolarization of VmBL (cd) observed under control conditions is also markedly reduced (c'd').

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<UP><SUB>i</SUB><SUP>Cl</SUP></UP>, RTE, and Ra/Rb did not change significantly. The removal of Cl- 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<UP><SUB>i</SUB><SUP>Cl</SUP></UP> decreased by 4.6 ± 1.8 mM in control, a value not significantly different from 7.8 ± 0.7 mM in the absence of Ca2+ (n = 3, P > 0.05). The inhibition of the depolarization on removal of Cl- 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.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 6.   Tracing from an experiment showing the effect of removal of serosal Cl- in a basal cell on VmBL and VTE in the presence and absence Ca2+. Removal of Cl- from the serosal bath in the absence of Ca2+ caused a marked reduction in the initial transient depolarization of VmBL (a'b' vs. ab). When Cl- was given back to the bath, the hyperpolarization of VmBL (cd) observed under control conditions is also markedly reduced (c'd').

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 (10-4 M), for several minutes. 8-bromocyclic AMP did not have an effect on the steady-state values of VmBL, VTE, or a<UP><SUB>i</SUB><SUP>Cl</SUP></UP>. When Cl- 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<UP><SUB>i</SUB><SUP>Cl</SUP></UP> decreased by 10.2 ± 1.5 mM, with a rate of change in a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> that was not different from control (Table 2). VTE depolarized transiently by 4.0 ± 2.3 mV and then repolarized to a value 2.4 ± 0.4 mV more positive than control. Those values were not significantly different from the values observed in the absence of 8-bromo-cAMP in the same experiments (n = 4, P > 0.05).

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).


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 7.   Tracing from an experiment in which 2 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. Cells 1 and 2 indicate 2 adjacent cells a few millimeters apart. The 2 cells have similar membrane potential differences (VmC), and the potential changes produced by the removal of Na+ or Cl- from the luminal bath are the same in the 2 cells.

Measurements of intracellular Cl- in the presence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> Ringer. In tissues from six rabbits perfused with control HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> Ringer (Table 1, solution 1), VmC was -53 ± 5.1 mV, VTE was -11.1 ± 2.5 mV, and a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> was 26.7 ± 3.3 mM. RTE was 2,237 Omega  · cm2, and Ra/Rb was 1.75 ± 0.37. On the basis of the measured value of a<UP><SUB>i</SUB><SUP>Cl</SUP></UP>, the calculated electrochemical equilibrium potential in luminal cells for Cl- (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<UP><SUB>3</SUB><SUP>−</SUP></UP>, intracellular Cl- 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<UP><SUB>i</SUB><SUP>Cl</SUP></UP> was 16.3 ± 2.2 mM. RTE was 2,413 ± 177 Omega  · cm2, and Ra/Rb was 2.27 ± 0.2 (n = 12). ECl, calculated from Eq. 6, was -49 mV in the absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, a value significantly smaller than the electrical potential difference of -64 mV observed across the apical cell membrane. These experiments indicate that a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> is higher than the activity of Cl- predicted by electrochemical equilibrium across both the apical and the basolateral membranes both in the presence or absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>.

Effect of removal of luminal and basolateral Cl- in the absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. To investigate Cl- 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/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, VmC depolarized from -65 ± 3.0 to -57 ± 2.7 mV (segment abc), VTE hyperpolarized from -14.5 ± 1.5 to -21.8 ± 1.1 mV, and a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> decreased from 16.3 ± 2.2 to 13.8 ± 1.9 mM. As shown in Fig. 8, the changes in VmC and VTE were not transient, which is different from the changes in VmBL and VTE when Cl- was removed from the basolateral bath. RTE increased from 2,413 ± 177 to 3,100 ± 247 Omega  · 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.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 8.   Tracing from an experiment showing the effect of removal of luminal Cl- in an apical cell on a<UP><SUB>i</SUB><SUP>Cl</SUP></UP>, VmC, and on VTE. When luminal Cl- was removed, there was a sustained depolarization of VmC (ab), a hyperpolarization of VTE, and a small decrease in a<UP><SUB>i</SUB><SUP>Cl</SUP></UP>. The changes were reversible when Cl- was returned to the luminal bath.

When Cl- 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 Omega  · cm2. The a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> in the luminal cell decreased from 15.2 ± 3.4 to 8.8 ± 2.4 mM (n = 6, P < 0.001) in response to basolateral Cl- removal. Thus the decrease in a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> in the apical cell is significantly larger (6.3 ± 1.0 mM) when Cl- 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.


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 9.   Effect of removal of Cl- from the luminal bath, and its subsequent removal from the serosal bath in an apical cell in the absence of CO2/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> on VmC (A), VTE (B), a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> (C), and RTE (D). The removal of Cl- from the lumen depolarized VmC by 8.3 ± 0.9 mV, decreased the a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> by 2.5 ± 0.9 mM, hyperpolarized VTE by 7.4 ± 0.4 mV, increased RTE by 687 ± 79 Omega  · cm2, (n = 12; P < 0.02). Subsequent removal of Cl- from the serosal bath in the continuous absence of luminal Cl- transiently hyperpolarized VmC by 8.0 ± 0.9 mV, decreased a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> by 6.4 ± 1.0 mM, and initially depolarized VTE by 10.3 mV, which recovered partially by 4.9 ± 1.6 mV (n = 6; P < 0.001). *Significantly different from control; §significantly different from 0 Cl- lumen, paired data (P < 0.05).

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 (10-4 M) for several minutes. 8-bromo-cAMP did not have an effect on the steady-state values of VmC, VTE, a<UP><SUB>i</SUB><SUP>Cl</SUP></UP>, Ra/Rb, or RTE. The removal of Cl- 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 Omega  · cm2. Those changes are not significantly different from the changes observed in the absence of cAMP (n = 4, P > 0.05).

The above experiment was repeated in the presence of bicarbonate to examine the possible presence of a cAMP-stimulated apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange. In the presence of bicarbonate Ringer, we exposed the luminal side of the tissue to 8-bromo-cAMP (10-4 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<UP><SUB>i</SUB><SUP>Cl</SUP></UP>, Ra/Rb, or RTE. The removal of Cl- from the luminal solution in the continuous presence of 8-bromo-cAMP caused VmC to depolarize by 5.5 ± 2.5 mV, and a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> decreased by 2.7 ± 1.9 mM at a rate of -0.2 ± 0.12 mM/min. VTE hyperpolarized by 7.45 ± 0.9 mV, Ra/Rb increased slightly but not significantly, and RTE increased by 353 ± 41Omega · 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 a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> (-0.2 ± 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<UP><SUB>i</SUB><SUP>Cl</SUP></UP> in the basal cell on removal of serosal Cl-, 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<UP><SUB>3</SUB><SUP>−</SUP></UP> (and, therefore, HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-dependent Cl- transports could be active), neither SITS nor DIDS changed the steady-state value of a<UP><SUB>i</SUB><SUP>Cl</SUP></UP>- 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 a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> appreciably.

Among 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<UP><SUB>i</SUB><SUP>Cl</SUP></UP> on removal and addition of serosal Cl- 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<UP><SUB>i</SUB><SUP>Cl</SUP></UP> is maintained above the value predicted by equilibrium potential in esophageal cells, undoubtedly, results from the action of several transporters including Na+/K/2Cl-, a leak pathway for Cl- must exist to counterbalance active Cl- loading to maintain a steady-state a<UP><SUB>i</SUB><SUP>Cl</SUP></UP>. A likely mechanism for such a leak is through a conductive pathway for Cl-.

Basolateral Cl- Channels

Our experiments support the presence of a conductive pathway for Cl-, 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<UP><SUB>i</SUB><SUP>Cl</SUP></UP> exists. When VmBL is plotted vs. the ratio of a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> to a<UP><SUB>o</SUB><SUP>Cl</SUP></UP> in tissues bathed in control HEPES Ringer, a straight line is generated (Fig. 10A), the S of which is 67.8 mV/decade of change in a<UP><SUB>i</SUB><SUP>Cl</SUP></UP>/<UP><SUB>q</SUB><SUP>Cl</SUP></UP> (R2 = 0.83, n = 13, P < 0.001). Third, serosal flufenamate, a Cl--channel blocker blocked the depolarization induced by the removal of Cl- by 60%. Fourth, the rate of decrease in a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> on removal of serosal Cl- 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.


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 10.   Plot of the logarithm of the ratio of intracellular to extracellular activity of Cl- (a<UP><SUB>i</SUB><SUP>Cl</SUP></UP>/a<UP><SUB>o</SUB><SUP>Cl</SUP></UP>) against VmBL (A) and VmC (B) when the tissues were perfused with control HEPES Ringer. The regression equation for VmC against a<UP><SUB>i</SUB><SUP>Cl</SUP></UP>/a<UP><SUB>o</SUB><SUP>Cl</SUP></UP> is y = 18.9x - 51.9 R2 = 0.24 (n = 14; P > 0.05), whereas the regression equation for VmBL against a<UP><SUB>i</SUB><SUP>Cl</SUP></UP>/a<UP><SUB>o</SUB><SUP>Cl</SUP></UP> is y = 67.8x - 17.0, R2 = 0.83, (n = 13; P < 0.001).

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 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and -61 ± 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 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>and -78 ± 2.6 mV, respectively, in HEPES, which was not statistically different from the average measured VmBL (-70 ± 4.2 mV in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and -73 ± 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

The conductance of the apical membrane of luminal cells to Cl- 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<UP><SUB>i</SUB><SUP>Cl</SUP></UP>. For example, when VmC is plotted vs. the ratio of a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> to a<UP><SUB>o</SUB><SUP>Cl</SUP></UP> in tissues bathed in control HEPES Ringer, a straight line (Fig. 10B) with poor correlation is generated. The S of this line is 18.9 mV/decade of change in a<UP><SUB>i</SUB><SUP>Cl</SUP></UP>/<UP><SUB>q</SUB><SUP>Cl</SUP></UP> (R2 = 0.24, n = 14, P = 0.07). Second, whereas removal of basolateral Cl- reduced a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> in the basal cell by 40%, luminal Cl- removal reduced a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> in the luminal cell by only 15%. In fact, basolateral Cl- deletion caused an even larger drop in luminal cell a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> than did luminal Cl- deletion (see RESULTS). Third, the rate of decrease in a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> in the apical cell on removal of luminal Cl- is severalfold smaller than the rate of decrease in a<UP><SUB>i</SUB><SUP>Cl</SUP></UP> in the serosal cell on removal of serosal Cl-. 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<UP><SUB>i</SUB><SUP>Cl</SUP></UP> and that there is little transport of Cl- 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<UP><SUB>i</SUB><SUP>Cl</SUP></UP> in the esophageal epithelium is maintained above electrochemical equilibrium is indicative of additional bicarbonate-independent secondary active pathways for Cl- 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[Abstract/Free Full Text].

2.   Abdulnour-Nakhoul, S, and Boulpaep EL. Transcellular chloride pathways in ambystoma proximal tubule. J Membr Biol 166: 15-35, 1998[ISI][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[Abstract].

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[ISI][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[Abstract/Free Full Text].

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[ISI][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[ISI][Medline].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

17.   Orlando, RC, Lacy ER, Tobey NA, and Cowart K. Barriers to paracellular permeability in rabbit esophageal epithelium. Gastroenterology 102: 910-923, 1992[ISI][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[Abstract/Free Full Text].

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[ISI][Medline].

20.   Powell, DW, Morris SM, and Boyd DD. Water and electrolyte transport by rabbit esophagus. Am J Physiol 229: 438-443, 1975[ISI][Medline].

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[ISI][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[Abstract].

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[Abstract].

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[ISI][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[ISI][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[ISI][Medline].

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[ISI][Medline].

29.   Zeiske, W, Atia F, and Van Driessche W. Apical Cl- channels in A6 cells. J Membr Biol 166: 169-178, 1998[ISI][Medline].


Am J Physiol Gastrointest Liver Physiol 282(4):G663-G675
0193-1857/02 $5.00 Copyright © 2002 the American Physiological Society




This Article
Abstract
Full Text (PDF)
All Versions of this Article:
282/4/G663    most recent
00085.2001v1
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Abdulnour-Nakhoul, S.
Articles by Orlando, R. C.
Articles citing this Article
PubMed
PubMed Citation
Articles by Abdulnour-Nakhoul, S.
Articles by Orlando, R. C.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online