Mechanisms of basolateral Na+ transport in rabbit esophageal epithelial cells

Solange Abdulnour-Nakhoul, Serhat Bor, Nese Imeryuz, and Roy C. Orlando

Departments of Medicine and Physiology, Tulane University School of Medicine and Veterans Affairs Medical Center, New Orleans, Louisiana 70112-2699


    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

We examined the mechanisms of cellular Na+ transport, both Cl- dependent and Cl- independent, in the mammalian esophageal epithelium. Rabbit esophageal epithelium was dissected from its muscular layers and mounted in a modified Ussing chamber for impalement with ion-selective microelectrodes. In bicarbonate Ringer, transepithelial potential difference was -14.9 ± 0.9 mV, the transepithelial resistance (RTE) was 1,879 ± 142 Omega  · cm2, the basolateral membrane potential difference (VmBL) was -53 ± 1.5 mV, and the intracellular activity of Na+ (aNai) was 24.6 ± 2.1 mM. Removal of Na+ and Cl- from the serosal and luminal baths decreased aNai to 6.6 ± 0.6 mM. Readdition of Na+ to the serosal bath in the absence of Cl- increased aNai by 21.8 ± 3.0 mM, whereas VmBL and RTE remained unchanged. When serosal Na+ was readded in the presence of amiloride the increase in aNai and the rate of Na+ entry were decreased by ~50%. 5-(N-ethyl-N-isopropyl)amiloride mimicked the effect of amiloride, whereas phenamil did not. Subsequent readdition of Cl- to the serosal bath further increased aNai by 4.4 ± 1.9 mM. When the cells were acid loaded by pretreatment with NH+4 in nominally HCO-3-free Ringer, intracellular pH measurements showed a pHi recovery that is dependent on the presence of Na+ in the serosal bath and that can be blocked by amiloride. These data indicate that esophageal epithelial cells possess a Na+-dependent, amiloride-sensitive electroneutral mechanism for Na+ entry consistent with the presence of a basolateral Na+/H+ exchanger. The ability of Cl- to further enhance Na+ entry supports the existence of at least one additional Cl--dependent component of basolateral Na+ entry.

sodium/hydrogen exchange; amiloride; intracellular sodium; intracellular pH


    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

THE ESOPHAGUS IS LINED by a stratified squamous epithelium that actively transports Na+ from lumen to blood. According to the Koefoed-Johnson and Ussing (10) two membrane model, Na+ enters the cell passively across the luminal membrane through Na+ channels and then exits across the basolateral membrane and into the intercellular space via the energy-requiring action of the Na+ pump. In addition, other mechanisms that can transport Na+ across cell membranes have been reported in esophageal cells. These include the Na+/H+ exchanger, Na+-dependent Cl-/HCO-3 exchanger and the Na+-K+-2Cl- cotransporter. However, these mechanisms were described in either a nonpolarized primary culture of esophageal epithelial cells or isolated esophageal cells so that their localization to apical and/or basolateral membrane remains uncertain (11, 23). Moreover, the conditions under which these or other yet unidentified transporters contribute to Na+ transport in esophageal epithelium are unknown. In addition to transcellular Na+ uptake, the cellular pathways for Na+ transport are responsible for the maintenance of intracellular Na+ and pH homeostasis. This is particularly important in the esophageal epithelium, which is constantly exposed to various noxious luminal agents ranging from high acidity, especially during episodes of gastroesophageal reflux, to hypertonicity of ingested food and beverages (15).

The aim of this study was to define in the esophageal epithelium the specific pathways contributing to Na+ transport across the basolateral membrane of the cells located within the stratum germinativum. These cells reside on the basement membrane of this 30 or more cell-layered epithelium and are accessible to impalement and study by microelectrodes. Microelectrodes were utilized to measure intracellular Na+ activity (aNai), intracellular pH (pHi), and basolateral membrane potential (VmBL) of esophageal cells within the intact epithelium. Our study indicates that a major fraction of Na+ is transported across the basolateral side via an electroneutral mechanism independent of Cl-. The sensitivity of this pathway to inhibitors is consistent with Na+/H+ exchange.


    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Animal and Tissue Preparation

New Zealand rabbits were killed by administration of an intravenous overdose of pentobarbital (60 mg/ml). The esophagus was excised, opened longitudinally, and pinned mucosal side down in a paraffin tray containing ice-cold oxygenated Ringer. The muscle layers were lifted up with forceps, and the underlying mucosa was dissected free with a scalpel. The sheet of mucosa thus obtained was cut, and a section was mounted horizontally in a modified Ussing chamber with an aperture of 1.13 cm2. A diagram of the preparation is shown in Fig 1. The chamber allows independent and continuous perfusion of the apical and the serosal side of the tissue. The fluid for the perfusion of the tissue is delivered by gravity. The perfusion solutions can be switched quickly and with minimal dead space by means of a combination of rotary and slider valves (Rainin, Emeryville, CA), which allow one of six experimental solutions to flow to each side of the chamber. The solutions were prewarmed and delivered to the chamber at 37°C.


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Fig. 1.   Continuously perfused preparation of esophageal epithelium for electrophysiological measurements. Esophageal epithelium is mounted on a grid between 2 halves of a modified Ussing chamber. VTE, potential difference measured between a reference electrode in serosal side and another electrode in luminal side of esophageal epithelium. VmBL, potential difference between reference barrel of double-barreled microelectrode and electrode in serosal bath. VNa, total potential of Na+-sensitive barrel of double-barreled microelectrode. Pure ionic potential of Na+ electrode is obtained by subtracting electronically VmBL from VNa.

Electrodes

Transepithelial potential (VTE) was measured as the voltage difference between a free-flowing KCl (tip <10 µm) electrode placed in the bath fluid of the serosal side and a similar electrode placed in the bath fluid of the apical side. Both electrodes were fitted with an Ag-AgCl wire, and the leads were connected to the amplifier of a voltage clamp (Physiologic Instruments, San Diego, CA). The voltage clamp was also used to deliver a DC pulse of 5-15 µA via platinum wires located in each side of the chamber. This allowed us to determine the transepithelial resistance (RTE) from the voltage deflection VTE as follows (where I is current)
<IT>R</IT><SUB>TE</SUB> = &Dgr;<IT>V</IT><SUB>TE</SUB>/<IT>I</IT>
The esophagus possesses relatively large basal cells (20-40 µm), which allow stable impalements with microelectrodes. Double-barreled microelectrodes were used to measure simultaneously and in the same cell VmBL and the aNai or pHi. The following is a brief description for the preparation of the double-barreled microelectrodes. Two borosilicate glass fiber capillaries (A-M Systems, Everett, WA) were held together with shrinkable tubing and twisted 360° over an open flame. The electrodes were then pulled on a vertical microelectrode puller (David Kopf, Tujunga, CA) to a tip smaller than 0.2 µm. One barrel was exposed to hexamethyldisilazane (Sigma, St. Louis, MO) vapor for 30 min after which the electrodes were baked at 100°C for 2 h. The Na+ or the H+ exchanger (Fluka, Ronkonkoma, NY) was then introduced into the tip of the silanized barrel by means of very fine glass capillaries. The silanized barrel was further backfilled with 150 mM NaCl for the Na+ microelectrode or with a buffer solution containing 0.04 M KH2PO4, 0.023 M NaOH, and 0.015 M NaCl, pH 7.0 (2a) for the pH electrode. The other barrel or the reference barrel was filled with 1 M KCl. Each barrel was connected to an Ag-AgCl half cell and connected to one of the probes of a high input impedance electrometer (WPI, Sarasota, FL). The resistance of the reference barrel ranged from 50 to 100 MOmega , and the tip potential was <5 mV.

The slope (S) of the Na+ electrode (ion-sensitive barrel) was determined from the equation
S = <FR><NU><IT>V</IT><SUB>100 mM NaCl</SUB> − <IT>V</IT><SUB>10 mM NaCl</SUB></NU><DE>0.94</DE></FR>
where V100 mM NaCl and V10 mM NaCl are the electrodes potential in the solutions as noted and 0.94 is the logarithm (base 10) of the Na+ activity ratio of pure 100 mM over pure 10 mM NaCl. The slope of the electrodes averaged 58 mV per decade change in activity of Na+. The selectivity for Na+ over K+ was calculated from the equation
<IT>k</IT><SUB>Na − K</SUB> = <FR><NU>a<SUP>100 mM NaCl</SUP><SUB>Na</SUB></NU><DE>a<SUP>100 mM KCl</SUP><SUB>K</SUB></DE></FR> × 10<SUP>−[<IT>V</IT><SUB>100 mM NaCl</SUB> − <IT>V</IT><SUB>100 mM KCl</SUB>]/S</SUP>
In 40 Na+ electrodes used for the study, the mean Na+-to-K+ selectivity ratio was 40:1.

The pH electrodes were calibrated in HEPES buffer solutions of pH 6, 7, and 8. The average slope was 55 ± 1.1 mV/pH unit (n = 8).

When a serosal cell was impaled the VmBL was read as the difference between the reference barrel of the double-barreled microelectrode and the reference electrode in the serosal side. The apical membrane potential could then be calculated as the difference between VmBL and VTE.

The ratio of the apical to basolateral membrane resistance (Ra/Rb) was determined from the ratio of the voltage deflections produced by the transepithelial DC current pulse across the apical and the basolateral membranes (Va and Vb, respectively) according to the equation
<IT>R</IT><SUB>a</SUB>/<IT>R</IT><SUB>b</SUB> = &Dgr;<IT>V</IT><SUB>a</SUB>/&Dgr;<IT>V</IT><SUB>b</SUB>
Delta Vb was measured directly, and Delta Va was calculated by subtracting Delta Vb from Delta VTE.

The intracellular ionic activities were calculated from the potential readings of cellular impalements of the specific ion-sensitive barrel of the double-barreled microelectrode. The total potential of the ion-sensitive electrode was measured as the voltage difference between the ion-sensitive microelectrode and the free-flowing reference electrode in the serosal bath. The pure ionic potential was obtained by subtracting electronically VmBL from the total potential of the ion-sensitive electrode. Readings were recorded on a three-channel strip chart recorder (Kipp & Zonen, Bohemia, NY).

Solutions

The composition of Ringer solutions is given in Table 1. The chemicals were obtained from Sigma. 5-(N-ethyl-N-isopropyl) amiloride (EIPA) was purchased from RBI (Natick, MA). Amiloride, EIPA, phenamil, and bumetanide were dissolved in a small volume of DMSO and added to the solution. The concentrations of amiloride, EIPA, and phenamil used were based on the concentrations required to achieve maximal inhibition of the transporters as reported previously in other preparations (9) and in the esophageal cells (16, 22). The concentration of DMSO never exceeded 0.1% of the final solution.

                              
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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; n is the number of observations.


    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Steady-State Measurements in HCO-3 Ringer

With the use of esophagi from 30 rabbits, sections of esophageal epithelium were mounted in modified Ussing chambers and superfused on the luminal and serosal side with standard HCO-3 Ringer (solution 1, Table 1). From these experiments baseline mean values for esophageal epithelium were VTE = -14.9 ± 0.9 mV and RTE = 1,879 ± 142 Omega  · cm2, and for individual impaled basal cells VmBL = -53 ± 1.5 mV, Ra/Rb = 1.6 ± 0.48 and aNai = 24.6 ± 2.1 mM.

To study the mechanisms by which Na+ is transported across the basolateral membrane, Na+ and Cl- were removed from both serosal and luminal bathing solutions to inhibit Na+ entry into cells and to deplete intracellular Na+ stores. Subsequent readdition of Na+ to the serosal bathing solution, in the absence of Cl-, would drive Na+ into the cell via Cl--independent mechanisms of Na+ entry. Cl- was then returned to the serosal bath to determine if additional Cl--dependent mechanisms of Na+ transport were also present.

Effect of Removal of Serosal and Luminal Na+ and Cl-

As shown in Fig. 2, after the serosal bath solution was switched from standard HCO-3 Ringer to a Na+-free, Cl--free solution (Na+ replaced with N-methyl-D-glucamine and Cl- replaced with gluconate; Table 1, solution 2), aNai decreased sharply (segment a-b), whereas VTE and VmBL hyperpolarized over a period of ~10 min. The mean decrease in aNai was 17.8 ± 1.9 mM, and the mean hyperpolarizations of VTE and VmBL were -2.7 ± 0.6 mV and -8.7 ± 3.8 mV, respectively (n = 17, P < 0.05). In addition, RTE increased by 344 ± 34 Omega  · cm2. The data are summarized in Fig. 3, A and B (2nd bars). Ra/Rb remained unchanged (1.57 ± 0.5 vs. 1.69 ± 0.6, P > 0.05).


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Fig. 2.   Tracing of experiment in rabbit esophageal epithelium using double-barreled Na+-sensitive microelectrode. This experiment shows effect of bilateral removal of Na+ and Cl- and subsequent readdition of Na+ and then Cl- to serosal bath on intracellular activity of Na+ (aNai; top trace), basolateral membrane potential (VmBL, middle trace), and transepithelial potential (VTE, bottom trace). Data from this and similar experiments are summarized in Fig. 3, A and B, and Fig. 4, A and B. NMDG, N-methyl-D-glucamine.


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Fig. 3.   A: effect of removal of luminal (L) and serosal (S) Na+ and Cl- on VmBL and aNai. When Na+ and Cl- were deleted from serosal bath (2nd bar), VmBL hyperpolarized by 8.7 ± 3.8 mV and aNai decreased substantially by 17.8 ± 1.9 mM (n = 17, P < 0.05). Subsequent removal of Na+ and Cl- from luminal bath (3rd bar) hyperpolarized VmBL further by 5.0 ± 2.2 mV and aNai decreased by 4.7 ± 1.8 mM (n = 13, P < 0.05). * Significantly different from previous condition, paired t-test. B: effect of removal of luminal and serosal Na+ and Cl- on VTE and transepithelial resistance (RTE). Removal of Na+ and Cl- from the serosal bath (2nd bar) hyperpolarized VTE by -2.7 ± 0.6 mV and increased RTE by 344 ± 34 Omega  · cm2. Subsequent removal of Na+ and Cl- from luminal bath (3rd bar) caused VTE to depolarize by 9.9 ± 2.0 mV and RTE to increase by 785 ± 102 Omega  · cm2 (n = 13, P < 0.05). * Significantly different from previous condition, paired t-test.

To further ensure that Na+ and Cl- entry would not occur across the basolateral membrane as a result of luminal ions diffusing across the junctions and through the paracellular pathway, perfusion of the serosal side with Na+-free, Cl--free solution was followed by perfusion of the luminal bath with Na+-free, Cl--free solution (Table 1, solution 2). As shown in Fig. 2 (segment b-c), aNai decreased further, VmBL hyperpolarized, and VTE depolarized. The mean decline in aNai was 4.7 ± 1.8 mM, the mean hyperpolarization of VmBL was -5.0 ± 2.2 mV, and the mean depolarization of VTE was 9.9 ± 2.0 mV, whereas RTE increased further by 785 ± 105 Omega  · cm2 (n = 13, P < 0.05 for all values). These data are summarized in Fig. 3, A and B (3rd bars). Ra/Rb increased slightly but not significantly (1.52 ± 0.49 vs. 1.88 ± 0.64).

Effect of Readdition of Serosal Na+ in Absence of Cl-

After intracellular Na+ was depleted and baseline readings in Na+-free, Cl--free solution were established, the pathways for cellular Na+ entry independent of Cl- were examined by adding Na+ to the serosal bath in the absence of Cl- (Table 1, solution 3). As shown in Fig. 2 (segment c-d), readdition of serosal Na+ increased aNai and depolarized VTE but had no significant effect on VmBL. As summarized in Fig. 4, A and B (2nd bars), aNai increased by 21.8 ± 3.0 mM, VTE depolarized by 5.6 ± 1.3 mV (n = 16, P < 0.001), and there was no effect on RTE or Ra/Rb (1.98 ± 0.57 vs. 2.09 ± 0.55). It is noteworthy that the increase in aNai was substantial, restoring the aNai entirely to the pretreatment baseline aNai value.


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Fig. 4.   A: effect of readdition of Na+ and then Cl- to serosal bath on aNai and VmBL in continuous absence of Na+ and Cl- in the lumen. Readdition of Na+ to serosal bath did not cause any significant change in VmBL, whereas there was a very substantial increase in aNai of 21.8 ± 3.0 mM (n = 16, P < 0.001). When Cl- was subsequently added back to the serosal bath in the continuous absence of Na+ and Cl- in the lumen, VmBL depolarized by 19.5 ± 5.3 mV and there was a small but significant increase in aNai of 4.4 ± 1.2 mM (n = 8, P < 0.01). * Significantly different from previous condition, paired t-test. B: effect of readdition of Na+ and then Cl- to serosal bath on VTE and RTE. In absence of luminal Na+ and Cl- readdition of Na+ to serosal bath (2nd bar) depolarized VTE by 5.6 ± 1.3 mV (n = 16, P < 0.001) but did not cause any significant change in RTE. When Cl- was given back to serosal bath in continuous absence of Na+ and Cl- in the lumen (3rd bar), VTE hyperpolarized by -6.8 ± 1.5 mV (n = 11, P < 0.005) and RTE remained unchanged. * Significantly different from previous condition, paired t-test.

Effect of Readdition of Serosal Cl-

The final step of the experiments depicted in Fig. 2 was the addition of Cl- to check for Cl--linked Na+ transport. When Cl- was given back to the serosal bath (segment d-e) in the continuous absence of Na+ and Cl- in the lumen (solution 1 in serosal bath, solution 2 in lumen), aNai increased, VmBL depolarized, and VTE hyperpolarized. The mean increase in aNai was 4.4 ± 1.2 mM (n = 8, P < 0.01), the mean depolarization of VmBL was 19.5 ± 5.3 mV, and the mean hyperpolarization of VTE was -6.8 ± 1.5 mV (n = 11, P < 0.005), whereas RTE remained unchanged. These results are summarized in Fig. 4, A and B (3rd bars). Ra/Rb increased significantly from a value of 1.6 ± 0.4 to 2.6 ± 0.4 (P < 0.001). Although significantly different from values for aNai before addition of Cl-, the overall increase was small (~16% of the total increase in Na+ uptake).

Effect of Amiloride on Na+ Entry

The above experiments showed that readdition of serosal Na+ caused a marked increase in aNai, which was not accompanied by a significant change in VmBL. This suggests that Na+ entered the cell via an electroneutral mechanism for Na+ transport, a likely candidate of which is the amiloride-sensitive Na+/H+ exchanger (14). To assess this possibility, we removed serosal and luminal Na+ and Cl- and pretreated the tissue with 10-3 M amiloride in the serosal bath for ~10 min before the readdition of serosal Na+, which was also done in the presence of amiloride. A typical experiment is shown in Fig. 5. The results of this maneuver was that Na+ entry was inhibited as evidenced by an increase of only 10.0 ± 3.1 mM in the presence of amiloride (segment a'-b') compared with an increase of 19.8 ± 3.8 mM in its absence (n = 7, P < 0.04, Table 2). In addition, the initial rate of Na+ entry into the cell was slower in the presence of amiloride at 2.2 ± 0.8 vs. 5.9 ± 1.7 mM/min in the absence of amiloride (P < 0.02). These data are summarized in Fig. 6, A and B. The changes observed in VmBL, VTE, and Ra/Rb on Na+ readdition were not different in the presence or absence of amiloride (P > 0.05 for each comparison, Table 2).


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Fig. 5.   Tracing of experiment showing effect of readdition of serosal Na+ in absence and in presence of amiloride (10-3 M) in serosal bath. Readdition of serosal Na+ increased aNai (top trace, segment a-b). Amiloride decreased rate of Na+ entry and also the increase in aNai (segment a'-b') on readdition of Na+. Changes in VmBL (middle trace) and VTE (bottom trace) are not different in absence and in presence of amiloride.

                              
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Table 2.   Effect of amiloride on changes in VmBL, aNai, VTE, RTE, and Ra/Rb on readdition of serosal Na+ in continued absence of Cl-



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Fig. 6.   Data summary of increase in aNai (A) and initial rate of Na+ entry (B) induced by readdition of serosal Na+ in absence and in presence of amiloride (10-3 M). Initial rate of Na+ entry and increase in aNai were inhibited in the presence of amiloride (n = 7). * P < 0.04.

Effect of EIPA and Phenamil on Na+ Entry

The above experiments showed that Na+ entry was inhibited by amiloride at 10-3 M. Although this observation is consistent with Na+ entry via the electroneutral Na+/H+ exchanger, amiloride at this concentration is not very specific, being capable of inhibiting Na+ entry via both the Na+/H+ exchanger and the Na+ channel. For this reason we repeated the experiment described above but substituted for amiloride the more selective Na+/H+ exchange inhibitor EIPA (5 × 10-5 M) (24). As summarized in Fig. 7, A and B, EIPA mimicked the effect of amiloride, reducing both the rate of Na+ entry from 5.6 ± 1.5 to 2.2 ± 0.3 mM/min and the increase in aNai on readdition of serosal Na+ from 18.03 ± 4.6 to 9.1 ± 1.3 mM (n = 5, P < 0.05). As in the presence of amiloride the changes observed in VmBL, VTE, and Ra/Rb on readdition of Na+ were not different in the presence or absence of EIPA (n = 5, P > 0.05 for each comparison; Table 3).


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Fig. 7.   Data summary of increase in aNai (A) and rate of Na+ entry (B) in absence and in presence of 5-(N-ethyl-N-isopropyl)amiloride (EIPA; 5 × 10-5 M). Similar to amiloride EIPA reduced increase in aNai on readdition of serosal Na+ and inhibited initial rate of Na+ entry (n = 5). * P < 0.05.

                              
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Table 3.   Effect of EIPA on changes in VmBL, aNai, VTE, RTE, and Ra/Rb on readdition of serosal Na+ in continued absence of Cl-

To investigate whether Na+ entry could have occurred through Na+ channels, we examined the effect of the specific Na+ channel inhibitor phenamil. As can be seen in Fig. 8 in the presence of phenamil (10-5 M), readdition of serosal Na+ still increased aNai by 18.8 ± 4.7 mM (segment a'-b'), did not alter VmBL significantly, depolarized VTE by 8.4 ± 0.8, and RTE remained unchanged. Those changes are not significantly different from the changes observed on readdition of Na+ in the absence of the inhibitor (segment a-b; n = 3, P > 0.05).


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Fig. 8.   Tracing of experiment showing effect of readdition of Na+ to serosal bath in absence and in presence of Na+ channel blocker phenamil. Readdition of serosal Na+ increased aNai (top trace, segment a-b). Phenamil had no effect on rate of Na+ entry nor increase in aNai (top trace, segment a'-b'). Changes in VmBL (middle trace) or VTE (bottom trace) caused by readdition of Na+ were also not affected by phenamil.

Effect of Bumetanide on Na+ Entry

As noted above in Fig. 2, segment d-e, the readdition of serosal Cl- caused an increase in Na+ uptake beyond that caused by addition of serosal Na+ alone, suggesting the presence of at least one additional Cl--dependent entry pathway for Na+. One possible pathway likely to be present in esophageal cells is the bumetanide-sensitive, Na+-K+-2Cl- cotransporter (21). Therefore, we investigated whether this transporter contributed to the Cl--dependent Na+ entry by monitoring aNai in tissues pretreated with bumetanide (10-4 M) before and during readdition of Cl- to the serosal bath. In our experiments bumetanide had no effect on the increase in aNai, and the changes in VTE, RTE, and VmBL were not significantly different from those obtained in the absence of bumetanide (Table 4).

                              
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Table 4.   Changes in VmBL, aNai, VTE, and RTE on readdition of serosal Cl- in absence and presence of bumetanide (10-4 M)

Measurements of pHi in Absence of CO2/HCO-3.

To confirm the presence of Na+/H+ exchange as an acid extruder at the basolateral membrane, we used pH-sensitive microelectrodes to monitor pHi during acid loading and recovery from an acid load. Acute intracellular acid loading was obtained by the NH3/NH+4 prepulse technique, which was originally described in the squid axons (5, 6). The experiments were run in the nominal absence of HCO-3 (HEPES Ringer, Table 1, solution 4) to minimize the effect of HCO-3 transporters on pHi regulation. Under these conditions baseline mean values for esophageal epithelium were VTE = -11.8 ± 1.0 mV, RTE = 2,026 ± 184 Omega  · cm2, and for individual impaled basal cells pHi = 7.10 ± 0.06, VmBL -53 ± 2.7 mV, and Ra/Rb = 5.18 ± 0.95 (n = 10).

Intracellular acid loading with NH3/NH+4. After baseline values in HEPES Ringer were determined, the cells were exposed to NH3/NH+4 (20 mM NH4Cl, Table 1, solution 5). A typical experiment is shown in Fig. 9. The exposure to NH3/NH+4 leads to the initial entry of NH3 (usually the more permeant component) into the cell where it gets protonated, thereby causing a rapid alkalinization of pHi (Fig. 9, segment a-b). The mean increase in pHi was 0.19 ± 0.02 pH unit (n = 10, P < 0.001). VmBL depolarized by 5.6 ± 1.3 mV, whereas VTE, RTE, and Ra/Rb remained unchanged. The alkalinization was followed by a small and slow acidification (plateau acidification, segment b-c) due to the slower NH+4 entry and to NH3 exit. NH3/NH+4 was then removed from the bathing solution, and the tissue was now exposed to a Na+-free HEPES solution (Table 1, solution 6). NH+4 (as well as NH3) exits the cell as NH3, leaving behind its H+ and thus causing a significant fall in pHi (segment c-d). The average fall in pHi on removal of NH+4 was 0.69 ± 0.07 pH unit (n = 10, P < 0.001). The changes in VmBL were transient and not statistically significant (-46 ± 2.83 mV vs. -48 ± 2.5 mV) as were the changes in Ra/Rb, whereas VTE hyperpolarized by -5.97 ± 0.62 mV and RTE increased by 250 ± 29 Omega  · cm2 (n = 11, P < 0.001). It is important to note that, in the absence of external Na+, pHi remained low and there was complete inhibition of pHi recovery.


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Fig. 9.   Tracing of experiment showing intracellular pH (pHi; top trace), VmBL (middle trace), and VTE (bottom trace) during NH+4 acid loading and recovery in HCO-3-free solution. Exposure to NH3/NH+4 caused initial alkalinization (segment a-b), followed by slight acidification (segment b-c). On removal of NH3/NH+4 in Na+-free solution, pHi fell (segment c-d). In absence of Na+ no pHi recovery was observed. When Na+ was restored in bathing solution, pHi recovered to its initial value (segment d-e).

Na+-dependent recovery and effect of amiloride. After the cells were acid loaded by removal of NH3/NH+4 in a Na+-free solution, Na+ was given back to the bath (HEPES Ringer, Table 1, solution 4). The restoration of Na+ caused full recovery of pHi from a value of 6.60 ± 0.07 to a value of 7.14 ± 0.14, at an initial rate of 0.15 ± 0.04 pH units/min (Fig. 9, segment d-e), whereas VmBL, VTE, and RTE recovered to their initial value (n = 5, P < 0.001). These experiments confirm the presence of a Na+-dependent acid-extruding mechanism on the basolateral side of the cell, a finding consistent with the presence of Na+/H+ exchange.

To check the sensitivity of this transporter to amiloride the same experiment as the one described previously was repeated (Fig. 10). After acid loading, the tissue was pretreated with 10-3 M amiloride before and during the readdition of Na+. In three different tissues, amiloride inhibited almost completely the Na+-dependent recovery of pHi from the imposed acid load (segment e-f). When amiloride was then removed from the bathing solution, pHi recovered by ~90% (segment f-g; n = 3, P < 0.02).


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Fig. 10.   Tracing of experiment showing inhibition of pHi recovery from acid load by amiloride (10-3 M). pHi, VmBL, and VTE are shown during NH+4 acid loading. Amiloride was added to bathing solution in absence of Na+. When Na+ was restored in continuous presence of amiloride, recovery of pHi was inhibited (segment e-f). pHi recovered only when amiloride was removed from bathing solution (segment f-g).


    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

Steady-State Measurements

The present study was designed to identify the mechanisms by which Na+ is transported across the basolateral membrane of esophageal epithelial cells. To do so, we impaled the basal cells of this multilayered epithelium by intracellular microelectrodes and measured aNai, VmBL, VTE, and RTE. Under steady-state conditions in HCO-3 Ringer, our measurements of VTE and RTE are comparable to those reported in the Ussing chamber (18) and are consistent with a tight epithelium. Measurements of aNai in esophageal cells have not been reported before, and our value of 24.6 ± 2.2 mM is within reasonable range of intracellular Na+ values reported earlier in other Na+ transporting epithelia (2, 3, 19, 25).

Bilateral removal of Na+ and Cl- and readdition of serosal Na+. To characterize the cellular mechanisms of basolateral Na+ transport, we first removed external Na+ and Cl-, which would lead to depletion of intracellular stores of these ions. Indeed the basal cell aNai was reduced by this maneuver almost 75% below resting levels in HCO-3 Ringer, thereby creating on readdition of Na+ to the serosal bath a maximal driving force for Na+ entry across the basolateral membrane. The removal of serosal and luminal Na+ and Cl- causes a decrease in active transport, thus depolarizing VTE and causing an increase in RTE.

Cl--independent Na+ transport was examined by adding basolateral Na+ in the absence of external Cl-. The readdition of Na+ in the absence of Cl- resulted in a marked increase in aNai, an increase that effectively approached the prior resting baseline value. Moreover, this increase in cell Na+ was unaccompanied by a change in VmBL, indicating that the route of Na+ entry was a Cl--independent electroneutral pathway. One pathway that fits this description is the Na+/H+ exchanger. Rabbit esophageal epithelial cells isolated or in culture possess a Na+/H+ exchanger (11, 22), and this exchanger by analysis of isolated mRNA was reported to be of the NHE1-type isoform (20). However, the Na+/H+ exchanger has never functionally been localized to the basolateral membrane in esophageal cells.

Evidence for Na+/H+ exchange

In our experiments the evidence for Na+/H+ exchange at the basolateral membrane is that readdition of serosal Na+ in the absence of luminal and serosal Cl- caused a substantial increase in aNai with no significant sustained changes in VmBL. Moreover, our experiments indicate that both the amount of change in aNai and the rate of increase in aNai were significantly blocked by amiloride (10-3 M). Given the limited specificity of amiloride (9) the same experiments were carried out using EIPA, a selective and more potent inhibitor of Na+/H+ exchange (24), and the results were essentially the same as with amiloride. The substantial decrease in Na+ influx in the presence of amiloride strongly supports the presence of a Na+/H+ exchanger on the basolateral membrane of basal cells. Further evidence is provided by measurements of pHi.

Our experiments using pHi measurements show that the basal cells of the esophagus, acid-loaded by exposure to NH3/NH+4 in HCO-3-free Ringer, can recover from their acid load only when Na+ is present in the serosal bath. This acid extrusion mechanism could be blocked by amiloride and is therefore compatible with Na+/H+ exchange. The experiments were conducted in the absence of CO2/HCO-3 to minimize the contribution of possible HCO-3-dependent pHi regulating mechanisms. Such mechanisms are known to exist in the esophageal epithelial cells (12, 23) but have not yet been fully characterized in the intact epithelium.

A significant role of Na+/H+ in transport of Na+ at the basolateral membrane agrees with the results obtained by intracellular measurements of Na+. Inhibition of Na+/H+ exchange by amiloride was evident by inhibition of pHi recovery when experiments were conducted in the absence of CO2/HCO-3 and after acid loading the cells using NH4Cl. However, in the presence of HCO-3 inhibition of Na+ entry by amiloride was not complete. This underscores the possibility that other transport mechanisms for Na+ entry in addition to Na+/H+ exchange are present. Although the concomitant presence of a Na+/H+ exchanger on the luminal membrane of the basal cells cannot be ruled out, our results indicate that Na+/H+ exchange is present at the basolateral membrane of the intact esophageal epithelium.

The results of our study do not support the existence of sizable Na+ transport through basolateral Na+ channels. The evidence for this observation is based on the fact that even when the Na+ gradient is largely set in favor of passive Na+ entry as it is after removal of Na+ from the bathing solutions, the readdition of Na+ did not cause any significant change in VmBL or Ra/Rb. Transport of Na+ across the basolateral membrane through channels would be expected to cause a depolarization of VmBL and a decrease in basolateral membrane resistance that would result in an increase in Ra/Rb. Both parameters did not change significantly on readdition of serosal Na+. Moreover phenamil, a specific inhibitor of Na+ channels, did not have any effect on Na+ transport across the basolateral membrane. This lack of Na+ channels on the basolateral membrane is consistent with studies in other Na+-transporting epithelia (17), including the prototypical stratified squamous epithelium, the frog skin, in which Na+ channels are localized to the apical membrane.

Na+/H+ exchange extrudes H+ from the cell in exchange for external Na+. Basolateral Na+/H+ exchange in the esophageal epithelium, like that of the kidney proximal tubule basolateral membrane (4) and that of the frog skin (8), does not contribute directly to transcellular (lumen to serosa) Na+ uptake but nevertheless is primarily active in regulation of pHi. In this capacity Na+/H+ exchange may play a very important role in regulating transport and maintaining cell homeostasis. For example, luminal Na+ channels in a variety of epithelia, are reported to be inhibited at low pHi (13); therefore, luminal Na+ influx can be blocked during periods of low luminal pH, as frequently occurs in the esophagus. In these cases, basolateral Na+ exchange not only will protect against intracellular acidosis (in face of external low pH) but also may play a role in maintaining intracellular Na+ when luminal Na+ entry is blocked. This is probably very important in the esophageal cells, which are regularly exposed to refluxed acidic gastric contents and through ingestion of acidic beverages to acid loads of a wide range.

Readdition of Cl- in the Presence of Na+

Our results also indicate a small Cl--dependent component of basolateral Na+ transport. Our evidence for this pathway is that addition of serosal Cl- was able to drive Na+ uptake into the cell beyond that caused by the addition of serosal Na+. This increase in aNai cannot result from the action of a Na+-dependent Cl-/HCO3- exchanger because this transporter would drive Na+ out of the cell when Cl- is driven into the cell. It is also unlikely that Na+-K+-2Cl- is responsible for the increase in aNai because it was not blocked by bumetanide. Although Na+-K+-2Cl- cotransport was found to be present in the rabbit esophagus (21), this transporter may only be activated in face of an osmotic challenge to the cell. Another possibility is that the entry of Na+ on readdition of Cl- is mediated by a Na+-Cl- transporter that is not sensitive to bumetanide. However the depolarization of VmBL on readdition of Cl- cannot be explained on the basis of a Na+-Cl- transporter because this transporter is known to be electroneutral. The observed depolarization of VmBL can possibly result from a finite permeability of the cell membrane to gluconate that would leave the cell on replacement of gluconate with Cl-, as suggested by Guggino et al. (7) in the kidney proximal tubule. It should be noted that, in the presence of Na+ and Cl- in the lumen, readdition of basolateral Cl- causes an initial hyperpolarization of VmBL followed by a depolarization, consistent with the presence of a basolateral conductance to Cl- (2). The observed increase in Ra/Rb in our experiments on readdition of serosal Cl- is consistent with this finding.

In summary we have localized a Na+/H+ exchange mechanism on the basolateral membrane of the esophageal cells. This exchanger, sensitive to amiloride and to one of its analog, EIPA, constitutes a major pathway for basolateral Na+ entry and likely plays an important role in the pHi regulation and homeostasis of the esophageal epithelial cells.


    ACKNOWLEDGEMENTS

We thank Dr. Nazih L. Nakhoul for helpful discussions and for carefully reading the manuscript.


    FOOTNOTES

This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-36013.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: S. Abdulnour-Nakhoul, Dept. of Medicine, Section of Gastroenterology, SL 35, 1430 Tulane Ave., New Orleans, LA 70112-2699.

Received 19 February 1998; accepted in final form 19 October 1998.


    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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