Department of Medicine, Tulane University School of Medicine,
and the Veterans Affairs Medical Center, New Orleans, Louisiana 70112
Hot beverages
expose the esophageal epithelium to temperatures as high as 58°C.
To study the impact of such temperatures, rabbit esophageal epithelium
was exposed to luminal heat or both luminal and serosal heat while
mounted in Ussing chambers. Luminal heat, mimicking exposure to hot
beverages, reduced potential difference (PD) and resistance
(R) when applied at
49°C and
reduced short-circuit current
(Isc) at
60°C. At
60°C, subepithelial blisters developed. Higher
temperatures reduced R only moderately
and reversibly. In contrast, the
Isc declined
sharply and irreversibly once threshold was reached. Luminal and
serosal heat also reduced PD,
Isc, and R, although the threshold for
reduction in Isc
was now similar to that for R.
Additionally, luminal and serosal heat reduced Isc more than
R for any given temperature and
resulted in blisters at lower temperatures (50°C) than luminal heat
alone. The heat-induced decline in
Isc was
attributed in part to inactivation of Na-K-ATPase activity, although
other transport systems could have been equally affected, and the
decline in R to an increase in
paracellular permeability. The latter effect on
R also contributed to an increase in
tissue sensitivity to luminal acid damage. Consumption of hot beverages
exposes the esophagus to temperatures that can negatively impact
epithelial structure and function. Impaired barrier function by heat
increases the risk of esophageal damage by subsequent contact with
(refluxed) gastric acid. These findings help explain in part the
association between esophageal disease and consumption of hot beverages.
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INTRODUCTION |
HUMANS ARE UNIQUE in their preference for eating food
and drinking beverages at high temperatures. Indeed in restaurants
within the United States, hot beverages are served at temperatures up to 80°C (176°F; investigator survey), and yerba maté is
eaten in Argentina at similar temperatures (5). In England, three separate studies indicated that healthy subjects prefer their hot
coffee or tea at temperatures as high as 65°C, 68°C, and
76°C, with two reports noting that temperature preferences did not
differ among males and females (2, 6, 14). Other studies indicate that
22% of English, 43% of Dutch, and 14% of Swedish subjects have
temperature preferences for food and beverages that exceed 60°C.
Notably, however, when hot beverages are consumed, the oral cavity
modulates these temperatures so that the esophagus in actuality experiences temperatures well below those of the ingested liquid. For
example, De Jong and colleagues (3) showed that sipping a hot beverage
at temperatures ranging from 55 to 65°C resulted in increases in
distal esophageal (luminal) temperatures averaging from 5 to 12°C
above body temperature depending on bolus size (minimum 5 ml to a
maximum of 20 ml), with the maximum intraluminal temperature observed
for any individual being 53°C. Because this study demonstrated a
linear relationship between temperature of the ingested bolus and
temperature in the lumen in the distal esophagus, ingestion of hot
beverages up to 80°C could be extrapolated from these data to
elevate distal esophageal luminal temperature and so expose the
esophageal epithelium to temperatures as high as 58°C (range
48-58°C).
Because it is known that the tertiary structure of proteins is
destroyed at 43°C and cultured cells are destroyed at temperatures of 47°C (8, 14), it has long been recognized that ingestion of such
hot substances has the potential to damage the tissues in its path.
Indeed, consumption of hot beverages has been previously linked with a
number of diseases of the upper gastrointestinal tract, including
reflux esophagitis, gastritis, gastric ulcer, duodenal ulcer, and
esophageal carcinoma (4, 14, 18, 19). Particularly notable was a study
by Pearson and McCloy (14) in which they determined the temperature
preference for consumption of hot beverages among patients with various
disorders of the upper gastrointestinal tract and compared
them to healthy controls. The results indicated that subjects
with acid-peptic disease of esophagus, stomach, and duodenum preferred
their beverages at significantly hotter temperatures than did the
healthy subjects, with median temperature preference of those with
esophageal disease (63.5°C) being the hottest for any group.
Moreover this difference could not be accounted for by any difference
in age, sex, smoking history, or choice of beverage (i.e.,
coffee or tea) among the groups.
Despite these intriguing observations, little is known about the
effects of heat on the esophageal epithelium. For this reason, we
exposed the esophageal epithelium of the rabbit to heat in the Ussing
chamber and did so in two different ways. One method was to expose it
to heat from the luminal side only to mimick the exposure as it occurs
in vivo in humans consuming hot beverages. The second method was to
expose it to heat from both luminal and serosal sides, a means of heat
clamping tissues to study the impact of specific temperatures.
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MATERIALS AND METHODS |
New Zealand White male rabbits weighing between 8 and 9 pounds were
killed by administering intravenously an overdose of pentobarbital (60 mg/ml). The esophagus was excised, opened, and stripped of its muscle
layers in a paraffin tray containing ice-cold oxygenated Ringer
solution so that a sheet of tissue was obtained consisting of
stratified squamous epithelium and a small amount of underlying connective tissue. From this tissue, four sections were cut and mounted
as flat sheets between Lucite half-chambers with an aperture of 1.13 cm2 for measurements of potential
difference (PD), short-circuit current
(Isc), and
calculation of electrical resistance
(R). All tissues were bathed with
Ringer solution (composition in mM): 140 Na+, 119.8 Cl
, 5.2 K+, 25 HCO
3, 1.2 Ca2+, 1.2 Mg2+, 2.4 HPO2
4, and 0.4 H2PO
4, and gassed with 95%
O2-5%
CO2. Mucosal and serosal solutions
were connected to calomel and Ag/AgCl electrodes with Ringer agar
bridges for measurements of PD and automatic short-circuiting except
for 5-10 s when the open circuit PD was read.
R was calculated using Ohm's law by
dividing the open circuit PD by the
Isc (or from the
current deflection to imposed voltage). (The current deflection to
imposed voltage was used for resistance calculations in cases in which
Isc was
immeasurably low and so could result in an inaccurate calculation of
transepithelial resistance).
After mounting and time for equilibration, moist heat stress was
applied by heating either the luminal bath alone (to mimick human
consumption of a hot beverage) or both luminal and serosal baths (to
heat clamp the tissue to a given temperature). Bathing solutions were
heated indirectly by heating the water circulating in the outer water
jacket surrounding the bathing solutions to a fixed temperature and
then allowing the bathing solution (or solutions) temperature to
equilibrate with it. Bathing solution temperatures were directly and
independently monitored by the presence of separate thermometers
(Bestell-NR-ET34,MGW Lauda, Brinkmann Instruments, Westbury, NY) in the
luminal and serosal solutions. In the case in which both luminal and
serosal bathing solutions were heated to the same temperature (heat
clamped), bathing solution temperature was assumed to be equivalent to
tissue temperature. In heat-clamped experiments in which the
reversibility of the heat stress on the tissue was being monitored,
bathing solution temperatures were efficiently lowered (back to
37°C) by the addition of cold packs to the bath containing the
water circulating in the water jacket. In the case in which luminal bathing solutions only were heated, luminal heat was applied rapidly by
switching the tubing from the original luminal bath (37°C) to one
of an adjacent Ussing chamber whose bathing solution was preheated to
the desired higher temperature. Therefore, by disconnecting and
reconnecting the bathing solution tubing, the luminal bath temperature
could be rapidly raised and then lowered back to 37°C.
Luminal acid exposure.
In some experiments the effect of heat stress on the ability of the
tissue to resist luminal acid injury was assessed in the Ussing
chamber. This was done by initially pairing three tissues by
R (within 25%; note 4-5 tissues
can be obtained from the same rabbit esophagus) and then heat clamping
two of the three to a fixed temperature. After a fixed time period, the
unheated tissue (unheated, acid-treated control) and one of the pair of
heat-clamped tissues were acidified by the luminal addition of 60 mM
HCl (pH 1.7) for 1 h. The other heated tissue was used as a
nonacidified, heat-clamped control. Equimolar choline chloride was
added to the serosal bath during HCl exposure to reduce osmolar effects and to balance the chloride concentration. Tissue damage was then assessed by monitoring subsequent changes in
R and by morphology. Tissues evaluated
morphologically were fixed for light microscopy in the chamber using
3% gluteraldehyde and later stained using hematoxylin and eosin.
Morphological injury was assessed by an observer who had no knowledge
of treatment groups using the scoring system: 0, normal; 1, intracellular or intercellular edema; 2, patchy necrosis; 3, diffuse
necrosis; and 4, transmucosal necrosis (ulceration).
Mannitol flux measurements.
Mannitol fluxes were measured in some experiments by the addition of
both "cold" mannitol to Ringer solution to bring its concentration to 10 mM and "hot"
[14C]mannitol (ICN,
Irvine, CA; 10 µCi) to the luminal bath. After taking the "hot"
side sample, 45 min were allowed for equilibration before sampling the
serosal bathing solution at two consecutive 45-min intervals. Mannitol
fluxes were determined using counts obtained from a liquid
scintillation counter. The mean value for the two 45-min fluxes was
reported as the value for the tissue.
Na-K-ATPase measurements.
The effects of varying times and temperature on Na-K-ATPase activity
was performed on a commercially available rabbit kidney Na-K-ATPase
(Sigma, St. Louis, MO) using the phosphate-determination method of Yoda
and Hokin (20). The enzyme was initially dissolved in 10 mM
Tris · Cl and 1 mM EDTA, pH 7.4, buffer and diluted
1:10 in 50 mM Tris · Cl, 2.5 mM
MgCl2, and 12.5 mM KCl, pH 7.4. The solution containing enzyme was then heated for a fixed time and at
a fixed temperature, and the solution then was allowed to return to
37°C before assessment of enzyme activity. Enzyme activity was
assessed by adding 2 mM ATP at 37°C for 1 h, and the reaction was
stopped with a solution containing 4% molybdate and 12% perchloric acid. Butyl acetate was added, and the absorbance of the top layer of
butyl acetate was measured at 320 nm in a Beckman DU-640
spectrophotometer and compared with that of a blank containing
Tris · Cl buffer and ATP, thereby accounting for
nonspecific phosphate. The absorbances were converted to phosphate
liberated by Na-K-ATPase using a standard curve of
KH2PO4
concentration vs. absorbance at 320 nm.
Statistics.
Statistical significance was determined using Student's
t-test for parametric data and the
Mann-Whitney test for nonparametric data (injury assessment). All data
were reported as the means ± SE. The protocol has been reviewed and
approved by the institutional Animal Welfare Committee.
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RESULTS |
To mimick esophageal exposure to hot beverage ingestion, rabbit
esophageal epithelium mounted in Ussing chambers was initially exposed
to heat by warming the luminal, but not the serosal, bathing solution;
rapid changes in temperature were achieved by switching the tubing of
the original bath at 37°C to one from an adjacent Ussing chamber
preheated to the desired higher temperature. This was done in three
sets of experiments. In one set of experiments luminal heat was applied
once for 5 min up to a temperature of 70°C to establish a
temperature-dependent response, and this exposure was followed by
restoration of bathing solution temperature to body temperature
(37°C) to assess reversibility of the observed changes (Fig.
1). Figure 1 depicts the effects of
different temperatures on a single tissue. The findings, however, were
reproducible in that for each temperature two to four similar
experiments yielded the same results. In a second set of experiments,
luminal heat was applied for 5 min at 49°C in an additional four
tissues to document the reproducibility of the effects at this modest
level of heat exposure (Fig. 2). (In these
experiments tissues exposed luminally to 49°C for 5 min were found
to have a mean serosal surface tissue temperature of 41°C; this is
a reflection of heat transfer from the luminal bath, determined by
applying a miniature temperature probe to the serosal surface,
n = 4.) In a third set of experiments,
luminal heat up to 70°C was applied for 1 min, and after
restoration of 37°C for 5 min, a 1-min period of heat was repeated.
This 6-min cycle was repeated six times so that the esophageal
epithelium was exposed to heat for 6 min over a 36-min period (Fig.
3). As shown in Figs. 1 and 3, exposure to luminal heat reduces PD,
Isc, and
R in a temperature-dependent manner,
although the temperature thresholds at which reductions occur for
R, reflecting epithelial permeability
or barrier function, and
Isc, reflecting
epithelial active ion transport, differ considerably. [PD, being
the product of
Isc and
R (Ohm's law), simply reflects the
net change in Isc
and R.] Thus it is observed that
luminal heat reduced R more readily
than Isc,
R with a single 5-min exposure rapidly
declining below baseline at a temperature of 49°C and Isc remaining
uninhibited until the luminal solution temperature reached 60°C
(Figs. 1 and 2). (The small, transient, but significant, rise in
Isc at 1 min was
not reproduced in subsequent experiments and so may in part be
artifact). Moreover, once the threshold temperature was reached,
luminal heat reduced R rapidly but
modestly to its new value and there it remained despite continued
exposure to the same level of heat. Additionally, luminal heat-induced reduction in R was a reversible
phenomenon, R rising to or toward preheat levels after temperature was restored to 37°C. In contrast, luminal heat, once threshold temperature was reached, inhibited Isc more
dramatically and, based on failure of
Isc to return
toward preheat levels on restoration of 37°C, for the most part
irreversibly (Figs. 1-3). Interestingly, tissue morphology was
unremarkable at levels in which luminal heat did not inhibit the
Isc; however, at
levels of luminal heat that
Isc was
inhibited, i.e.,
60°C whether by single exposure for 5 min or
repeated 1-min exposures for 6 min over a 36-min period, blistering of
the epithelium was noted (Fig. 4). The
plane of cleavage of the blister was localized to the region of the
basement membrane, with the basal cells being the roof of the blister
and the lamina propria being the floor. Also, in some areas there was
evidence of cell edema.



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Fig. 1.
Effect of single 5-min exposure to luminal heat on potential difference
(PD, A), short-circuit current
(Isc,
B) and electrical resistance
(R,
C) of rabbit esophageal epithelium
in Ussing chambers. Effects are plotted as percent change from baseline
values (37°C), and each line represents that of 1 tissue response
out of 2-4 experiments at each temperature. After 5 min of luminal
heat, all luminal solution temperatures were restored to 37°C
(second arrow) to monitor reversibility of heat-induced changes.
Serosal temperatures were maintained at 37°C throughout the
experiment. Initial values for exposures from 37 to 70°C for PD
ranged from 13.5 to 19 mV, for
Isc from 4 to 8 µA/cm2, and for
R from 2,376 to 3,251 · cm2.
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Fig. 2.
Effect of single 5-min exposure to luminal heat on PD
(A),
Isc
(B), and
R
(C) of rabbit esophageal epithelium
in Ussing chambers. Effects are plotted as percent change from baseline
values for each tissue at 37°C. After 5 min of luminal heat,
luminal solution temperatures were restored to 37°C. Serosal
temperatures were maintained at 37°C throughout experiment.
* P < 0.05 compared with
non-heat-stressed controls at 37°C;
n = 4. Absolute baseline PD was
15.7 ± 0.2 and 15.9 ± 0.6 mV,
Isc was 7 ± 0.3 and 8 ± 0.6 µA/cm2, and
R was 2,641 ± 115 and 2,275 ± 130 · cm2
for controls and heat-stressed tissues, respectively,
P > 0.05 for each comparison.
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Fig. 3.
Effect of series of 1-min exposures to luminal heat on the PD
(A),
Isc
(B), and
R
(C) of rabbit esophageal epithelium
in Ussing chambers. Effects are plotted as percent change from baseline
values for each tissue at 37°C. Each temperature plot represents
single tissue. After each 1-min exposure to luminal heat (arrows), all
luminal solution temperatures were restored to 37°C to monitor
reversibility of heat-induced changes. Serosal temperatures were
maintained at 37°C throughout experiment. Initial values for
exposures from 37°C to 70°C for PD ranged from 15 to
19 mV, for
Isc from 4 to 8 µA/cm2, and for
R from 2,352 to 4,290 · cm2.
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Fig. 4.
Light micrograph of rabbit esophageal epithelium exposed for 5 min to
luminal temperature of 70°C. Note subepithelial blister formation
with epithelium lifted off lamina propria. There is also some evidence
for cellular edema in some sections of tissue. Hematoxylin and eosin
×200 magnification.
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Given the variability in rate and direction of heat transfer and
dissipation, the technique of luminal heating left unknown the impact
of specific temperatures on the esophageal epithelium. For this reason
in some experiments luminal and serosal solutions were simultaneously
heated to the same temperature, thereby heat clamping the tissue to a
particular level. As shown in Fig. 5, heat
clamping tissues over the range of 38-52°C reduced PD,
Isc, and
R, with
Isc being as heat
sensitive as R in terms of threshold for effect. Additionally, the impact of heat clamping on
Isc was significantly greater than on R at any
given temperature, and so much so that, while
R was reversibly reduced by
10-15% at 52°C, Isc was
irreversibly abolished. Heat clamping to
50°C was also associated
with epithelial blisters (data not shown).



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Fig. 5.
Effect of both luminal and serosal heat (heat clamping) on PD
(A),
Isc
(B), and
R
(C) of rabbit esophageal epithelium
in Ussing chambers. Effects are illustrated for control group of
tissues (only exposed to 37°C) and for those exposed to varying
temperatures, and results are plotted as percent change from baseline
value of each tissue at 37°C. Each temperature plot represents mean
value for 3 tissues. Bathing solution and tissue temperatures at
various times are indicated by arrows. After exposure for 5 min at
52°C, bathing solution temperatures of all tissues were restored to
37°C to monitor reversibility of heat-induced changes.
* P < 0.05 compared with
controls at 37°C. Note as a frame of reference initial PD values
were 11.9 ± 3.6 and 15.0 ± 3 mV,
Isc 6 ± 1 and
10 ± 1 µA/cm2 and
R 1,881 ± 493 and 1,463 ± 294 · cm2 for
control and heat-stressed tissues, respectively.
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Because the effect of luminal heat alone or heat clamping the tissues
was to inhibit
Isc and reduce
R, studies were done to clarify the
mechanism for the impairment in active transport
(Isc) and
reduction in barrier function (R).
Specifically, because the Isc of esophageal
epithelium is predominantly due to active Na transport (15), and
enzymes such as Na-K-ATPase are known to be temperature sensitive, we
initially sought to directly measure the effect of heat on esophageal
Na-K-ATPase activity in homogenized sections of Ussing-chambered
heat-exposed epithelium. However, when this proved unreliable
technically, we chose instead to use a commercially available rabbit
kidney Na-K-ATPase as a model to investigate the effects of heat on
Na-K-ATPase activity. As shown in Fig. 6,
the enzyme Na-K-ATPase was heat sensitive in a temperature-dependent
and time-dependent manner. For example, Na-K-ATPase activity gradually
declined as temperature was raised from 37 to 52°C and then
declined sharply thereafter until complete inhibition was observed at
60°C. In Fig. 6B, using a
temperature of 49°C, it was observed that Na-K-ATPase activity
declined from 5 to 30% below baseline over 10 min, whereupon it
plateaued and declined no further. Also, to explore the locus for the
decline in R on exposure to heat,
tissues were heat clamped at 49°C and [14C]mannitol flux
measurements were performed and compared with tissues exposed to
37°C. As shown in Table 1, tissues
heated to 49°C had a 29 ± 5% decline in
R compared with an increase for those
at 37°C and more than double the mannitol flux of those at
37°C. Additionally, under these conditions there was
no morphological change by light microscopy to the cells of the barrier
layer, although there was evidence of subepithelial blistering.


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Fig. 6.
Effect of varying degrees of heat
(A) and time of heating
(B) on rabbit kidney Na-K-ATPase
activity in vitro. Na-K-ATPase activity is shown as percent change in
absorbance compared with baseline values for activity at 37°C.
* P < 0.05 compared with
baseline at 37°C; n = 3.
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Table 1.
Effect of heat stress on electrical resistance and transepithelial
mannitol flux in Ussing-chambered rabbit esophageal epithelium
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Consumption of hot food and beverages coincidently exposes the
esophageal epithelium to other noxious environments, most notably to
gastric acid due to the well-known postprandial increase in gastroesophageal reflux. For this reason, we investigated the impact of
prior heat exposure on the ability of the esophageal epithelium to
resist injury on exposure to luminal acid. This was done by monitoring
R of heat-clamped tissues in the
Ussing chamber in the presence or absence of luminal HCl, pH 1.7. Tissues clamped at 37°C and similarly exposed to HCl served as
unheated controls. [R was
monitored because prior study has shown it to be the most sensitive
marker of acid injury to esophageal epithelium (13)]. As shown in
Fig. 7, heat clamping tissues to 49°C
reduced R by ~20%, whereas tissues
clamped at 37°C exhibited a small increase in
R. After luminal acidification,
however, tissues at 37°C exhibited an acid-induced decline of 28 ± 7% vs. an acid-induced decline of 58 ± 4% for tissues
exposed to 49°C (n = 6, P < 0.05). Notably, the acid-induced
decline in R for heat-stressed tissue
was not only lower than the acidified, non-heat-stressed tissue, but
lower than that decline combined with the decline from unacidified, heat-stressed tissue alone (58 ± 4%,
n = 6 vs. 44 ± 6%,
n = 12, P < 0.05). Interestingly, although
differing in R, these tissues were not
different morphologically (injury scores for acid alone, 0.8 ± 0.2 vs. heat plus acid, 1.0 ± 0.2, respectively,
n = 6, P > 0.05). Furthermore, if heat-
clamped tissues at 49°C were returned to 37°C,
R not only returned to baseline but
subsequent luminal acidification was only able to lower
R to the same degree as acidified,
non-heat-stressed (37°C) controls (Fig.
8).

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Fig. 7.
Effect of heat stress on ability of rabbit esophageal epithelium in
Ussing chambers to resist damage on exposure to luminal acid. Damage
was monitored by effects on R, marker
of epithelial barrier function. Tissues were either heat clamped to
49°C or heat clamped at body temperature of 37°C. In tissues
paired initially by R, heat stress was
administered sufficiently to lower R
to a new stable value. After this, one of heat-stressed pair and
non-heat-stressed control at 37°C were luminally acidified with HCl
to pH 1.7 for 1 h. R was monitored and
plotted as percent change from initial (preheat stress) values.
* P < 0.05 compared with
tissues at 37°C and
t P < 0.05 compared with heat-stressed, unacidified controls;
n = 6. Note initial
R values for each group were similar;
2,079 ± 280 · cm2 for
non-heat-stressed tissues, 2,591 ± 205 · cm2 for
heat-stressed, acidified tissues, and 2,018 ± 219 · cm2 for
heat-stressed, unacidified tissues.
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Fig. 8.
Effect of remote heat stress on ability of rabbit esophageal epithelium
in Ussing chambers to resist damage on exposure to luminal acid. Damage
was monitored by effects on R, a
marker of epithelial barrier function. Tissues were initially heat
clamped to 49°C (heat-stressed) or heat clamped at body temperature
of 37°C (non-heat-stressed control). In tissues paired initially by
R, heat stress was administered
sufficiently to lower R to new stable
value. After this, heat stress at 49°C was removed and
R was allowed to recover over ensuing
15 min before both were luminally acidified with HCl, pH 1.7, for 1 h.
R was monitored and plotted as percent
change from initial (preheat stress) values.
* P < 0.05 compared with
tissues at 37°C; n = 4. Initial
R values were similar at 1,370 ± 379 · cm2
for heat-stressed and 1,188 ± 182 · cm2 for
non-heat-stressed controls.
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DISCUSSION |
Hot beverages are consumed at temperatures as high as 80°C, and,
according to (and extrapolated from) the work of De Jong et al. (3),
this could expose the esophageal epithelium within the distal esophagus
to temperatures as high as 58°C. (De Jong only studied ingestion of
hot liquids up to 65°C and found it to elevate distal esophageal
temperature as high as 53°C.) In this study, we examined the impact
of temperatures covering this range and above on the esophageal
epithelium. When the esophageal epithelium was mounted in Ussing
chambers and exposed to heat from the luminal side only, mimicking the
method of heat delivery on oral ingestion of hot beverages,
temperatures of
49°C reduced PD and
R and temperatures of
60°C
reduced Isc.
Because R is a reflection of
permeability and
Isc a reflection
of active (ion) transport, these data indicate that under appropriate
circumstances luminal heat can impair both epithelial barrier and
transport functions. Moreover, once the threshold temperature was
reached, impairment in barrier and transport functions could occur
rapidly and with short exposure times (
1 min). (Although esophageal
luminal bolus clearance is on the order 5-10 s per bolus, this
time frame may be within reach given ingestion of repeated boluses of
hot liquid and the absorption and perhaps accumulation of heat energy transmitted from each bolus to the epithelium). Interestingly, however,
these two functions responded differently to luminal heat. First, as
already noted, they exhibited different temperature thresholds for
impairment by heat, barrier function being the more temperature
sensitive than transport. Second, although having a lower temperature
threshold, barrier function even at the highest temperatures studied
(70°C) stabilized rapidly and was at worst only moderately impaired
by luminal heat, whereas transport fell more precipitously once
threshold was reached and was abolished at 70°C. Third, impairment
of barrier function by luminal heat was generally reversible, whereas
this was not the case for the heat-induced inhibition of active transport.
One plausible reason for the differences in response to luminal heat
for barrier and transport functions is the location of the cells
primarily responsible for these activities. Thus, for example, it would
be expected that functions carried out by the cells of the most
lumenward layers, i.e., stratum corneum, which come into direct contact
with the luminal solution, would be the first to experience the impact
of raising luminal temperature. This in fact is the case because
barrier function was affected at lower temperatures than transport, and
it is the cells of the stratum corneum, through their combination of
apical membranes and junctional complex comprised of tight junctions
and intercellular glycoconjugates, that are principally involved with
barrier function (12). Also, it would be expected that active transport
would be less temperature sensitive than barrier function because the cells responsible for this activity are primarily localized within the
stratum spinosum and stratum germinativum, two layers physically removed from contact with the heated luminal solution and further protected from luminal heat by heat dissipation-absorption as it
traverses the more lumenward cells of the stratum corneum (3, 11).
Experimental evidence that supports this concept includes both the
lower temperature threshold for inhibition of transport with heat
clamping and its greater impairment compared with barrier function when
tissues were heat clamped, a process that delivered heat to the tissue
from the serosal (as well as luminal) side. Under these circumstances,
i.e., serosal heating, the transporting cells (and their enzymes, e.g.,
Na-K-ATPase; see below) in the stratum spinosum and stratum
germinativum can come into direct contact with the heated solution or,
in the absence of an interposing stratum, have less heat dissipated
before contact is made with the cells. Consistent with this
interpretation is the observation that barrier function, as reflected
in R, was equally impaired at a given
temperature whether heat was delivered only luminally or both luminally
and serosally. This suggests that the addition of serosal heat to the
tissue had little effect on barrier function because this function is
carried out by the more distant cells of the luminal stratum (stratum corneum).
How an increase in tissue temperature produces changes in transport and
barrier functions was also investigated. First, because the esophageal epithelium is primarily a sodium transporting
(absorbing) tissue, we investigated the impact of heat on Na-K-ATPase
activity (15). When the rabbit kidney Na-K-ATPase was used as a model, it was shown that this enzyme is highly susceptible to inhibition at
temperatures used in our experiments. For example, active transport by
the cells of the stratum spinosum and stratum germinativum was
significantly inhibited with even brief exposure to luminal temperatures of 60°C or to serosal (and luminal) temperatures of
52°C, and at temperatures in this range (50-60°C), it was
observed that Na-K-ATPase activity was either markedly inhibited or
completely abolished. For this reason, the temperature-induced
inhibition in transport in esophageal epithelium was likely a
reflection of inhibition of Na-K-ATPase activity and other unmeasured
heat-sensitive transport proteins.
Second, because the barrier function of the multilayered esophageal
epithelium, as reflected in R, is most
readily changed by alterations in its junctional complex (11, 13), we
performed transepithelial mannitol fluxes in heat-exposed tissues. As
noted in Table 1, the heat-induced reduction in
R was accompanied by increases in
transepithelial mannitol flux, and under a condition that produced no
morphological change (by light microscopy) in the cells of the barrier
layer. These results indicate that heat alters esophageal epithelial
barrier function by increasing the permeability across the paracellular
pathway. Mosely and colleagues (10) also reported similar results with
heat stress to cultured monolayers of MDCK cells; that is, heat
reversibly increased paracellular permeability without altering tissue
transport or viability. Although the mechanism for this occurrence was
unknown, among the possibilities considered by them and others were
activation of phospholipase C, increased intracellular calcium, altered
cytoskeletal elements, altered membrane fluidity, and/or increased
production of oxidants (7, 10).
The permeability across the paracellular pathway in rabbit esophageal
epithelium is controlled by a series of tight junctions and
intercellular glycoconjugates (12), and the present study suggests that
the proteins of this barrier are highly sensitive to luminal heat. This
sensitivity to luminal heat, however, may be viewed as having both good
and bad effects. On the positive side, the ability of luminal heat to
increase permeability across the paracellular pathway may serve a
protective function, by permitting greater shunting of heat around
rather than through the cells of the barrier layers. Alternatively, and
on the negative side, our experiments showed that when heat increases
the permeability across the paracellular pathway
(R reduced), the risk of damage from
luminal acidity is also increased. This observation is not surprising
because the major path for acid entry into the esophageal epithelium is
via this route, and other factors, e.g., hypertonicity and ethanol,
that increase paracellular permeability in esophagus similarly increase
the damaging effects of luminal acidity (1, 9, 17). That these
observations with heat stress and acidity may have clinical relevance
is supported by the fact that the risk of esophageal exposure to
gastric acid through gastroesophageal reflux is markedly increased
under the very same conditions that the esophagus is exposed to luminal
heat, i.e., the process of eating and drinking of hot substances (or
substances at any temperature).
Finally, luminal heat had little effect on tissue morphology by light
microscopy until temperatures reached 60°C. At temperatures of
60°C or higher, subepithelial blisters were noted, as is reported to occur with temperatures above 50°C producing second-degree burns
to skin, another stratified squamous epithelium (16). Because the
experiments with serosal heat (heat clamping) produced similar blisters
at lower temperatures (52°C), blister formation was likely due to a
direct effect of heat on the protein fibrils anchoring the epithelium
to the underlying lamina propria.
In summary, the present study suggests that human consumption of hot
beverages can impair esophageal epithelial structure and function and
that the impairment in barrier function increases the risk of damage
from contact with refluxed gastric acid. Thus esophageal exposure to
high luminal temperature can damage the epithelium both directly and
indirectly. These observations in part may help explain the reported
association between esophageal disease and human consumption of hot beverages.
This project was funded in part 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 and other correspondence: N. A. Tobey,
Tulane Univ. Medical Center, Gastroenterology-SL 35, 1430 Tulane
Ave., New Orleans, LA 70112.