Regulatory volume decrease in human esophageal epithelial cells

Geraldine S. Orlando, Nelia A. Tobey, Paul Wang, Solange Abdulnour-Nakhoul, 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
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In vivo human esophageal epithelial cells are regularly exposed to hyposmolal stress. This stress, however, only becomes destructive when the surface epithelial cell (barrier) layers are breached and there is contact of the hyposmolal solution with the basolateral cell membranes. The present investigation was designed to examine the effects of hyposmolal stress in the latter circumstance using as a model for human esophageal epithelial cells the noncancer-derived HET-1A cell line. Cell volume and the response to hyposmolal stress in suspensions of HET-1A cells were determined by cell passage through a Coulter Counter Multisizer II. HET-1A cells behaved as osmometers over the range of 280 to 118 mosmol/kgH2O with rapid increases in cell volume <= 15-20% above baseline. Following swelling, the cells exhibited regulatory volume decrease (RVD), restoring baseline volume within 30 min, despite continued hyposmolal stress. With the use of pharmacologic agents and ion substitutions, RVD appeared to result from rapid activation of parallel K+ and Cl- conductance pathways and this was subsequently joined by activation of a KCl cotransporter. Exposure to hyposmolal stress in an acidic environment, pH 6.6, inhibited, but did not abolish, RVD. These data indicate that human esophageal epithelial cells can protect against hyposmolal stress by RVD and that the redundancy in mechanisms may, to some extent, serve as added protection in patients with reflux disease when hyposmolal stress may occur in an acidic environment.

hyposmolality; HET-1A cells; ion channels; KCl cotransport; Coulter Counter


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE HUMAN ESOPHAGUS IS LINED by a moist stratified squamous epithelium that through eating, drinking, and gastroesophageal reflux is frequently exposed to liquid media whose physicochemical properties are potentially noxious. Among the noxious environments to which these epithelial cells are exposed is that of hyposmolality, ie., severe hyposmolality generated by the simple act of drinking tap water being capable of inducing cell swelling and lysis. Yet, the esophageal epithelium in most people remains intact and healthy despite exposure to hyposmolal solutions because of protection afforded by its surface cell layers. The surface cell layers are composed of individual pancake-shaped cells whose apical cell membranes and intercellular junctional complexes combine to produce an effective permeability barrier against the influx of luminal content (9). In particular, the barrier created by these structures limits exposure of the surface cells' basolateral cell membranes and entire membrane of cells of the deeper layers to the wide swings in osmolality occurring regularly within the esophageal lumen. This capacity of the surface cell layers for protecting deeper cell layers has been previously observed by Goldstein et al. (4) when they reported no effect of luminal hyposmolality on the short-circuit current of Ussing-chambered rabbit esophageal epithelium, whereas short circuit current, a reflection of net transepithelial ion transport, was significantly increased by serosal hyposmolality.

Gastroesophageal reflux disease results from repeated contact of the esophageal epithelium with refluxed gastric acid and pepsin. Based on the prevalence of its characteristic symptom, heartburn, it represents one of the most common modern disorders of adult Americans. Moreover, there is electrical and morphologic evidence that patients with heartburn, even with nonerosive disease, have a breakdown in barrier function of the esophageal epithelium (9-11, 15) and that in a significant percentage of these individuals, breakdown of the epithelial barrier can progress to erosions and ulceration that are grossly visible on upper endoscopy. Indeed, although acid and pepsin may, in fact, have been the initiators of this injury, destruction of the (permeability) barrier, both microscopically and macroscopically, provide luminal content greater access to cells of the lower layers, access that is maximized in the damaged esophagus by the lack of a protective surface mucous layer as exists in stomach and duodenum (1, 2). The consequence of this is that the cells of the lower layers are now exposed and so vulnerable to damage from exposure to luminal environments, including hyposmolality from ingestion of tap water.

In this study, we utilized HET-1A cells, a noncancer-derived esophageal epithelial cell line, to examine the effects of hyposmolal stress on human esophageal epithelial cells and to establish the nature of the membrane transport mechanisms responsible for regulatory volume decrease (RVD). Our results indicate that HET-1A cells behave like osmometers under hyposmolal stress and exhibit RVD as a means for protection against hyposmolal-induced cell death. Furthermore, RVD in HET-1A cells appears to occur via two distinct mechanisms: 1) rapidly activated parallel K+ and Cl- conductance pathways and 2) a slowly activated KCl cotransporter. The need for such a combination of mechanisms may indicate the importance of RVD for cell survival in this epithelial cell type.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

HET-1A cell culture. HET-1A cells, a SV-40 immortalized human esophageal epithelial cell line, were a generous gift from Dr. Gary Stoner (Medicine and Public Health Administration, Ohio State University, Columbus, OH). Given the fact that these cells have the capacity to replicate, they are more representative of cells within the basal layers of esophagus (stratum basalis) than the more mature nonreplicating cells of the upper strata (stratum spinosum and stratum corneum). The cells were grown in 250-ml flasks in DMEM with high glucose (4.5 g/l), supplemented with 2% FCS, 1 mM Na pyruvate, 2 mM/l L-glutamine, 5 µg/ml insulin, 5 µg/ml transferrin, 0.01 µg/ml hydrocortisone and 0.01 µg/ml cholera toxin. Cultured cells were incubated at 37°C in 5% CO2-95% O2 in high humidity.

Cells were harvested after 3 days of growth by removing the medium and incubation for 3 min at 37°C with 3 ml of 0.1% trypsin containing 5 mM EDTA in PBS whose composition (in mM/l) was 137 Na+, 2.7 K+, 4.3 HPO<UP><SUB>4</SUB><SUP>2−</SUP></UP>, and 1.4 mM H2PO<UP><SUB>4</SUB><SUP>−</SUP></UP>. Cells were then suspended in solution with gentle tapping of the flask, and the enzymatic reaction was stopped by the addition of 5 ml of DMEM containing 2% FCS. Cells were centrifuged at 2,000 rpm for 5 min, washed twice with PBS, and resuspended in 5 ml of an isotonic HEPES buffer composed of (in mM/l): 141 Na+, 5 K+, 1 Ca2+, 1 Mg2+, 159 Cl-, 10 HEPES, 5 D-glucose, and 290 mosmol/kgH2O, pH 7.4 after being adjusted with 1 mol/l N-methyl-D-glucosamine (NMDG). This suspension or stock sample had a concentration of 1 × 10 6 cells/ml. Cell viability was >95% as determined by trypan blue exclusion. Before use, all cell suspensions were allowed to stabilize for at least 60 min. All reagents, unless specified, were purchased from Sigma (St. Louis, MO).

Cell volume determination. Cell volume was determined at room temperature using a Coulter Counter Multisizer II (Coulter Electronics, Limited Beds, UK) with a 100-µm orifice. This counter sizes particles based on the ability of particles to displace an electrolyte volume as they pass through the orifice that is proportional to particle size. After calibration of the instrument using 20-µm latex beads, baseline cell volume was obtained for HET-1A cell populations by passage of 5,000 cells through the aperture and recording the individual readings on a computer using Coulter Acucomp software. From a graph of this data can be read the mean cell volume for the specific HET-1A cell population under study. The changes in mean cell volume over time for any given perturbation are determined after baseline measurement by passage through the aperture of a series of samples containing 5,000 suspended cells from the sample population.

Hyposmolal stress. To study the response to hyposmolal stress, a 300-lambda aliquot of stock HET-1A cells in isotonic HEPES buffer was diluted in 10 ml of isotonic HEPES buffer. After baseline determination of mean cell volume from the sample in the Coulter Counter, the population was exposed to a hyposmolal stress by adding to the isotonic HEPES buffer, varying amounts of a Na-free HEPES buffer whose osmolality was 40 mosmol/kgH2O and whose composition was (in mM/l) 5 K+, 1 Ca2+, 1 Mg2+, 13.5 Cl-, 10 HEPES, 5 D-glucose, and 4.5 NMDG, pH 7.4 after being adjusted with NMDG. The hyposmolal stress created by this means ranged from 118 to 280 mosmol/kgH2O. After hyposmolal stress, mean cell volume was serially recorded over a 30-min period. At each time point, mean cell volume was determined in triplicate (differing from each by only a few seconds in time) and the mean cell volume plotted for any given time point is the average of the triplicate runs.

For some studies on RVD, cell populations were preincubated in the following pharmacologic agents for 15 min before measurement of mean cell volume: barium chloride (BaCl2), H2DIDS, R+-butylindazone (DIOA), indanyloxyacetic acid-94 (IAA), N-phenylanthranilic acid (DPC), and bumetanide (BUM). All chemicals were from Sigma except DPC, which was purchased from Fluka (Milwaukee, WI), BaCl2, which was purchased from Specialties Chemicals Division (Morristown, NJ), and DIOA, which was purchased from Research Biochemicals (Natick, MA).

Statistical significance was determined using Student's t-test for paired samples unless otherwise indicated.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The mean cell volume for HET-1A cells in isotonic HEPES buffer at room temperature averaged 1,702 ± 28 µm3 (n = 160). After exposure to osmolalities ranging from 280 to 118 mosmol/kgH2O, the cells swelled in direct proportion to the magnitude of the hyposmolal stress, behaving effectively like osmometers (Fig. 1). Notably, at the lowest osmolality, 118 mosmol/kgH2O, there was greater variability in mean cell volume, a reflection of cell lysis as evidenced by the appearance of cell fragments whose recording resulted in the averaging of smaller particles with the much larger swollen cells. Because cell fragility was clearly in evidence at 118 mosmol/kgH2O, but not at 160 mosmol/kgH2O, additional experiments were carried out using exposures at the latter osmolality.


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Fig. 1.   The %change in mean cell volume for HET-1A cells is plotted against bathing solution osmolality. Delta , %change in mean cell volume in µm3 from cells in isosmolal solution.

Notably, HET-1A cells exhibited RVD in response to hyposmolal stress. As illustrated in Fig. 2, hyposmolal stress (160 mosmol/kgH2O) initially resulted in a period of rapid cell swelling that peaked 15-20% above baseline ~3-5 min postexposure. After the rapid increase in cell volume and despite continued exposure to hyposmolal stress, HET-1A cells exhibited a gradual and progressive decline until cell volume returned to baseline, which could require <= 25-30 min postexposure. After reaching baseline, cell volume continued to decline modestly (~5%) over the ensuing 5-10 min before increasing and stabilizing at the initial baseline value (data not shown).


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Fig. 2.   Effect of a hyposmolal stress of 160 mosmol/kgH2O on mean cell volume for HET-1A cells. After the rapid period of swelling, HET-1A cells exhibited regulatory volume decrease, returning to baseline values by 30 min. Delta , %change in mean cell volume from initial volume in isosmolal solution; * P < 0.05 compared with isosmolal controls; n = 10/group. Mean cell volume for HET-1A cells in isosmolal solution declined modestly during the experiment.

After establishing the presence of RVD after hyposmolal stress in HET-1A cells, the transmembrane pathways responsible for the process were investigated by monitoring RVD in the presence of pharmacologic agents affecting known mechanisms for RVD in other cell types (5, 13). Specifically, the presence of a K+ conductance pathway was assessed by monitoring RVD in HET-1A cells exposed to hyposmolal stress in the presence of the K+ channel inhibitor BaCl2 (5 mM). As depicted in Fig. 3, BaCl2 in isosmotic solution had no significant effect on resting cell volume, whereas exposure to BaCl2 during hyposmolal stress resulted in both greater swelling and reduced capacity for RVD than untreated controls. Furthermore, and supporting a role for the outward movement of K+ in RVD in HET-1A cells, RVD under hyposmolal stress was monitored during exposure to high (20 mM) K+ buffer. As shown in Fig. 4, high K+ in isosmolal solution had no effect on resting cell volume, whereas high K+ during hyposmolal stress resulted in both greater swelling and inhibition of RVD compared with (control) cells in the standard buffer with 5 mM K+.


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Fig. 3.   Effect of 5 mM barium chloride (BaCl2) on the regulatory volume decrease exhibited by HET-1A cells during exposure to 160 mosmol/kgH2O. Delta , %change in mean cell volume from initial volume in isosmolal solution; * P < 0.05 compared with untreated hyposmolal controls; n = 9 cells/group. Mean cell volume for HET-1A cells in isosmolal solution modestly declined during the experiment, and BaCl2 in isosmolal solution had no significant effect on resting mean cell volume.



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Fig. 4.   Effect of a high (20 mM) K+ hyposmolal solution (160 mosmol/kgH2O) on the regulatory volume decrease exhibited by HET-1A cells. Delta , %change in mean cell volume from initial volume in isosmolal solution; * P < 0.05 compared with cells exposed to a normal (5 mM) K+ hyposmolal solution; n = 3 cells/group. Mean cell volume for HET-1A cells in isosmolal solution modestly declined over the time period of the experiment, and a high K+ isosmolal solution had no significant effect on resting mean cell volume.

If the loss of K+ was through K+ channels during RVD, these channels would require for maintenance of electrochemical equilibrium a parallel conductive loss of anions, and specifically, conductive Cl- loss. Therefore, RVD in HET-1A cells during hyposmolal stress (160 mosmol/kgH2O) was monitored in the presence of three known inhibitors of Cl- conductance pathways: H2DIDS, IAA, and DPC. H2DIDS (0.2 mM), IAA (50 µM), and DPC (50 µM) had no effect on resting cell volume in HET-1A cells in isosmolal solution (data not shown). However, H2DIDS and IAA, but not DPC, significantly inhibited RVD, and the lack of inhibition of RVD by DPC was not a result of the absence of membrane charge, because repeat experiments in a low (1 mM) K+ buffer produced the same outcome (Fig. 5, A and B). Also, whereas both H2DIDS and IAA produced significantly greater increases in cell volume, compared with controls within the first 2-5 min of hyposmolal stress (Fig. 5A), only the inhibitory effect of H2DIDS on RVD persisted at significant levels by 30 min (Fig. 5B). In an attempt to further establish dependence of RVD on Cl- loss in HET-1A cells, cells were Cl-depleted by incubation in an isosmotic Cl-free HEPES solution (gluconate substitution for Cl- and Ca2+ increased to compensate for reduction by gluconate), pH 7.4, for 30 min. These maneuvers, however, resulted in marked cell instability, such that exposure to hyposmolal stress resulted in cell shrinkage and/or fragmentation. Moreover, similar effects occurred when Cl-depletion was attempted using low Cl- (75% reduction) as opposed to zero-Cl- solution, thereby precluding us from using ion manipulation for direct confirmation of the dependence of RVD on Cl efflux.


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Fig. 5.   Effects of chloride channel blockers, H2DIDS (0.2 mM), indanyloxyacetic acid-94 (IAA; 50 µM), and N-phenylanthranilic acid (DPC; 50 µM), the latter in both normal (5 mM) and low K DPC (1 mM DPC-LK) solutions, on mean cell volume in HET-1A cells after hyposmolal stress (160 mosmol/kgH2O) is shown for the early maximal change in cell volume above baseline at 2-5 min (A) and for the change in cell volume at 30 min (B). Delta , %change in mean cell volume from initial volume in isosmolal solution; * P < 0.05 compared with untreated hyposmolal stressed controls; n = 4-7 cells/group. Mean cell volume for HET-1A cells in isosmolal solution modestly declined during the experiment, and neither H2DIDS, IAA, nor DPC in isosmolal solution had any significant effect on resting mean cell volume (data not shown).

Inhibition of RVD by high K+ solution (Fig. 4) supports the importance of outward movement of K+, but this is not specific for K+ loss via K+ channels, i.e., this same maneuver would also inhibit RVD mediated by KCl cotransport. A role for KCl cotransport in RVD in HET-1A cells was assessed by exposing cells to a dose of 30 µM DIOA, reported to be relatively selective for inhibition of this transporter (Fig. 6) (3). As shown in the figure, DIOA had no effect on cell volume in isosmolal solution nor did it result in an early (2-5 min) increase in cell swelling on exposure to hyposmolal stress (160 mosmol/kgH2O). However, DIOA, like BaCl2, H2DIDS, and IAA, produced significant inhibition of RVD at the 30-min time period. This suggests that in addition to conductive K+ and Cl- loss, RVD is dependent on the activity of a KCl cotransporter. To further establish that the response to DIOA was through inhibition of a KCl cotransporter rather than through cell swelling-induced reversal of a Na-K-2Cl cotransporter, cells were exposed to 0.1 mM BUM, during hyposmolal stress (160 mosmol/kgH2O) (Fig. 7). BUM, which at this dose is known to inhibit Na-K-2Cl cotransport in esophageal cells (16), had no effect on cell volume in isosmolal solution. BUM also had no effect on cell swelling or RVD after hyposmolal stress.


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Fig. 6.   Effect of 30 µM DIOA on the regulatory volume decrease exhibited by HET-1A cells during exposure to 160 mosmol/kgH2O. Delta , %change in mean cell volume from initial volume in isosmolal solution; * P < 0.05 compared with untreated hyposmolal controls; n = 8 cells/group. Mean cell volume for HET-1A cells in isosmolal solution modestly declined during the experiment, and R+-butylindazone (DIOA) in isosmolal solution had no significant effect on resting mean cell volume.



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Fig. 7.   Effect of 0.1 mM bumetanide on the regulatory volume decrease exhibited by HET-1A cells during exposure to 160 mosmol/kgH2O. Delta , %change in mean cell volume from initial volume in isosmolal solution; * P < 0.05 compared with untreated hyposmolal controls; n = 3 cells/group. Mean cell volume for HET-1A cells in isosmolal solution modestly declined during the experiment, and bumetanide produced a small but statistically insignificant increase in resting cell volume after 20 min in isosmolal solution.

The above experiments clearly suggest that two different mechanisms are operative for RVD under hyposmolal stress in HET-1A cells, parallel K+ and Cl- channels, and a KCl cotransporter. Support for this concept can also be generated by a comparison of the kinetics of RVD in the presence and absence of the various blockers. Notably, as shown in Fig. 8, K+ channel blockade (with Ba++ data from experiments depicted in Fig. 3) and Cl- channel blockade (with H2DIDS and IAA data from experiments depicted in Fig. 5, A-B) resulted in substantial early (2-5 min) inhibition of RVD; this is illustrated by the significant difference in cell volume between blocker and controls (0 baseline). In contrast, the KCl cotransport blockade (with DIOA data from experiments depicted in Fig. 6) had essentially no effect on RVD at the 2-5 min time frame, whereas at 30 min, DIOA exerted a dramatic inhibitory effect on RVD (Fig. 8). On the other hand, the effects of channel blockade by Ba++, H2DIDS, and IAA, although still significant, were below those at 2-5 min and so on the decline. Furthermore, high K+ solution, which inhibits RVD mediated by both K+ channels and KCl cotransporter had early (2-5 min) effects similar to K+ channel blockade with Ba++ and late effects (30 min) similar to KCl cotransport blockade with DIOA (Fig. 8). These data support the presence of two different mechanisms for RVD in esophageal cells---a rapidly activated K+ and Cl- channel-mediated response and a slower KCl cotransport-mediated response.


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Fig. 8.   Comparison of the early maximal increase in cell volume (2-5 min) and late increase in cell volume (30 min) after hyposmolal stress for K+ channel blockade (Ba++) and Cl- channel blockade (H2DIDS and IAA) vs. KCl cotransport blockade (DIOA). DIOA had no inhibitory effect on regulatory volume decrease (RVD) early (at 2-5 min) as opposed to Ba++, H2DIDS, and IAA; but DIOA significantly inhibited RVD late (at 30 min). In addition, the inhibitory effects of Ba++, H2DIDS, and IAA declined with time and were significantly less than DIOA at 30 min. Moreover, high K+ solution, which inhibits both K+ channels and KCl cotransport is observed to inhibit RVD early like Ba++ and late like DIOA. * P < 0.05 inhibitor vs. DIOA. Data were extrapolated from experiments reported in Figs. 3, 5 and 6.

In the presence of a broken epithelial barrier, such as in reflux disease, esophageal epithelial cells may be called on to exhibit RVD under conditions in which the extracellular environment is more acidic than normal. To determine the effect of lowering extracellular pH on RVD, HET-1A cells were exposed to hyposmolal stress (160 mosmol/kgH2O) at a neutral pH of 7.4 or an acidic pH of 6.6. As shown in Fig. 9, this degree of acidity significantly delayed, but did not abolish, RVD.


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Fig. 9.   The effect of extracellular acidity (pH 6.6) on the time (min) for RVD in HET-1A cells exposed to hyposmolal stress (160 mosmol/kgH2O). * P < 0.05 compared with HET-1A cells at pH 7.4; n = 7/group.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The major findings in the present investigation are that human esophageal epithelial cells, as represented by the noncancer-derived basal cell type HET-1A cell line have the capacity for RVD when challenged by hyposmolal stress and that this response appears to be mediated by loss of cytosolic K+ and Cl- through two mechanisms: 1) a rapid loss via parallel K+ and Cl- channels and 2) slow onset loss via a KCl cotransporter. The presence of RVD in esophageal epithelial cells was initially documented by Snow et al. (12) using a freshly isolated basal cell-enriched population from the rabbit esophagus. In these experiments, the investigators showed that RVD was inhibited by barium and high extracellular K+, supporting loss of K+ through a K+ channel, and by depletion of cell Cl-, H2DIDS, and DPC, supporting loss of Cl- through a DPC-sensitive Cl- channel. In our experiments, RVD in HET-1A cells was blocked by barium, high K+ solution, H2DIDS, and IAA, all supporting the loss of KCl through parallel K+ and Cl- channels. In HET-1A cells, unlike rabbit basal cells, the chloride channel blocker IAA but not DPC was effective in blocking RVD. DPC was ineffective even in low K+ solution, which maximizes membrane charge. Nonetheless, although the nature of the Cl- channels involved with RVD appear to differ, these results indicate that both human and rabbit esophageal epithelial cells exhibit RVD and have at least one mechanism, parallel operation of K+ and Cl- channels, in common.

RVD was also inhibited in HET-1A cells by DIOA, but not by BUM, suggesting that in addition to parallel K+ and Cl- channels for RVD, there is a second mechanism compatible with a KCl cotransporter. The presence of a KCl cotransporter was also supported by an analysis of the kinetics of RVD in the presence of various inhibitors. Specifically, channel blockade with Ba++ or H2DIDS, but not with the KCl cotransport inhibitor DIOA, resulted in an early (2-5 min) inhibition in RVD, whereas inhibition of KCl cotransport by DIOA exerted a significant inhibitory effect on RVD later (at 30 min). These data indicate there are two mechanisms for RVD in hyposmolal-stressed HET-1A cells---an early response due to parallel operations of K+ and Cl- channels---and joining them, a late response due to the activation of a KCl cotransporter. Dual mechanisms for RVD have been reported previously (5, 14, 6) in such diverse cell types as Necturus gallbladder cells, Ehrlich ascites tumor cells, and human retinal pigmented epithelial cells. It is presently unknown whether rabbit esophageal epithelial cells also possess a KCl cotransporter. It is of interest, however, that the basal cell population of rabbit esophageal epithelial cells responded rapidly to the hyposmolal challenge with swelling, and RVD was completed within 5 min (12). A fast response such as this, according to Spring and Hoffman (13) suggests the dominance of parallel K+ and Cl- channels for KCl loss during RVD. In contrast, the HET-1A cell population swelled rapidly but then could require <= 30 min before completion of RVD. Although this difference in RVD between rabbit basal cells and HET-1A cells may represent differences in species, phenotype, or methodology, this difference could also reflect a fundamental difference in the mechanism(s) for RVD. For example the slow response of RVD in HET-1A cells reflects the predominance of the KCl cotransporter, as is the case in duck red blood cells in which the KCl cotransporter is the exclusive means of RVD in response to hyposmolal stress and which requires ~90 min for completion (8, 7).

The importance of RVD as a mechanism for defense in human esophageal epithelial cells is worth emphasizing. For instance, in the pathologic setting of GERD when there is a breakdown in epithelial barrier function, cells of the lower layers may be exposed to luminal contents (e.g., tap water) whose osmolality is low enough for cell destruction by volume-induced lysis. RVD, therefore, represents in this setting, a potential protective defense against cell death by enabling cells to tolerate the hyposmolal environments for considerable periods long enough to permit clearance of the noxious environment from the lumen by swallow-induced peristalsis. Yet, protection against hyposmolal stress by RVD under the complex pathologic conditions produced by GERD should not be assumed, because RVD is also dependent on environmental pH. For instance, Snow et al. (12) have shown that the rapid RVD in rabbit basal cells is inhibited under mildly acidic conditions (pH 6.8). This inhibitory effect, however, may be limited or transient in cells with dual mechanisms, such as the HET-1A cells, because it has been shown in Erhlich ascites tumor cells that acidic pH may shift the operation of RVD from parallel K+ and Cl- channels to the KCl cotransporter (5, 7). In this respect, it was notable in our studies that RVD was inhibited but not abolished in HET-1A cells exposed to extracellular acidity (pH 6.6). This suggests that the apparent redundancy in mechanisms for RVD, and in particular, the presence of a KCl cotransporter, may serve as an additional protective role in human esophageal epithelial cells under hyposmolal stress in an acidic environment.


    ACKNOWLEDGEMENTS

This work was supported by a Veterans Affairs Merit grant and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-36013.


    FOOTNOTES

Address for reprint requests and other correspondence: G. Orlando, Tulane University, Dept. of Medicine, Section of Gastroenterology SL 35, 1430 Tulane Ave., New Orleans, LA 70112-2699 (E-mail: gorland{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.

10.1152/ajpgi.00455.2001

Received 13 November 2001; accepted in final form 25 May 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastrointest Liver Physiol 283(4):G932-G937