Departments of Medicine and Physiology, Tulane University
School of Medicine, and Veterans Administration Medical Center, New
Orleans, Louisiana 70112 - 2699
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.
 |
INTRODUCTION |
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 |
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
, and 1.4 mM
H2PO
. 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-
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 |
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.

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 1.
The %change in mean cell volume for HET-1A cells is
plotted against bathing solution osmolality. , %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).

View larger version (13K):
[in this window]
[in a new window]
|
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. , %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+.

View larger version (22K):
[in this window]
[in a new window]
|
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. , %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.
|
|

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of a high (20 mM) K+ hyposmolal
solution (160 mosmol/kgH2O) on the regulatory volume
decrease exhibited by HET-1A cells. , %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.

View larger version (21K):
[in this window]
[in a new window]
|
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). , %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.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of 30 µM DIOA on the regulatory volume decrease
exhibited by HET-1A cells during exposure to 160 mosmol/kgH2O. , %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.
|
|

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 7.
Effect of 0.1 mM bumetanide on the regulatory volume
decrease exhibited by HET-1A cells during exposure to 160 mosmol/kgH2O. , %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.

View larger version (15K):
[in this window]
[in a new window]
|
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.

View larger version (11K):
[in this window]
[in a new window]
|
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 |
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.
This work was supported by a Veterans Affairs Merit grant and
National Institute of Diabetes and Digestive and Kidney Diseases Grant
DK-36013.
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.