Effect of heat stress on rabbit esophageal epithelium

Nelia A. Tobey, Dipali Sikka, Esteban Marten, Canan Caymaz-Bor, S. Seraj Hosseini, and Roy C. Orlando

Department of Medicine, Tulane University School of Medicine, and the Veterans Affairs Medical Center, New Orleans, Louisiana 70112


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

hydrochloric acid; potential difference; resistance; short-circuit current


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


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

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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 Omega  · 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 Omega  · 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 Omega  · 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.

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 Omega  · cm2 for control and heat-stressed tissues, respectively.

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

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 Omega  · cm2 for non-heat-stressed tissues, 2,591 ± 205 Omega  · cm2 for heat-stressed, acidified tissues, and 2,018 ± 219 Omega  · 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 Omega  · cm2 for heat-stressed and 1,188 ± 182 Omega  · cm2 for non-heat-stressed controls.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGEMENTS

This project was funded in part by the National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-36013.


    FOOTNOTES

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.

Received 10 November 1998; accepted in final form 18 February 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastroint Liver Physiol 276(6):G1322-G1330
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