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Mechanisms of acid injury to rabbit esophageal epithelium
GASTROENTEROLOGY
1991;101:1220-1228
Mechanisms of Acid Injury to Rabbit Esophageal Epithelium Role of Basolateral Cell Membrane Acidification NELIA A. TOBEY
and ROY C. ORLANDO
Department of Medicine, University of North Carolina School of Medicine, Chapel Hill, North Carolina
Because mucosal HCl traverses the paracellular pathway before significant damage develops within esophageal epithelium, the effects of mucosal and serosal acidification were compared on Ussingchambered rabbit esophageal epithelia. Notably serosa], but not mucosal, acidification was associated with cell necrosis, and the latter was accompanied by abolition of potential difference and short-circuit current. The reason for this difference was explored by exposing tissues serosally to ouabain, chloridefree solution, 4-acetamido-a’-isothiocyanatostilbene2-2’-disulfonic acid (SITS), or amiloride. The results show that serosal acidification, but not ouabainization, is associated with cell necrosis and that cell necrosis induced by serosal acidification can be blocked by SITS and chloride-free solution, but not by amiloride. These findings are compatible with the hypothesis that serosal acidification is more damaging than mucosal acidification because of the greater rate with which hydrogen ions can traverse the basolateral membrane; also, the route for more rapid entry appears to involve a SITS-sensitive, chloride-dependent mechanism (e.g., WHCO, ana tiport). he major cause of esophagitis in humans is the prolonged contact of esophageal epithelium with refluxed gastric (hydrochloric) acid. However, the mechanisms by which mucosal (luminal) HCl damages esophageal epithelium remain incompletely understood. Experimentally, one of the earliest signs of epithelial damage in acid-perfused esophagus is a decline in the transmural electrical potential difference (PD) (l-3), a decline that has been shown in rabbit esophagus to reflect increased permeability across the intercellular junctions (l-3). Further, after mucosal HCl increases paracellular permeability, epithelial necrosis can be prevented by the presence of buffers within the extracellular space (4). This latter
T
observation is particularly noteworthy, because it adds support to the hypothesis that the paracellular pathway is the major route by which mucosal HCl enters and then damages the esophageal epithelium. Based on the concepts of acid injury discussed above, it is evident that a consequence of HCl entering the paracellular pathway, before the development of epithelial necrosis, is the lowering of extracellular pH below the level of the (intercellular) junctional complex, i.e., the acidification of the region adjacent to the basolateral membrane (BLM). To understand the significance of BLM acidification as it relates to the development of epithelial necrosis, we studied the effects of BLM acidification on rabbit esophageal epithelial structure and function in the Ussing chamber and compared the effects with the effects of apical membrane (AM) acidification. Apical membrane acidification was produced in the traditional way by the addition of acid to the mucosal bathing solution, whereas BLM acidification was achieved by the addition of acid to the serosal bathing solution. Materials
and Methods
New Zealand white rabbits weighing between 8 and 9 lb were killed by administering an IV overdose of pentobarbital (60 mg/mL). The esophagus was excised, opened, and pinned mucosal surface down in a paraffin tray containing ice-cold oxygenated normal Ringer’s solution. The submucosa was grasped with hemostats, lifted up, and dissected free of the underlying mucosa with a scalpel. This process yielded a sheet of tissue consisting of stratified squamous epithelium and a small amount of underlying connective tissue. From this tissue, four sections were cut and mounted
Abbreviations used in this paper: AM, apical membrane; BLM, basolateral membrane; kc, short-circuit current; R, resistance; SITS, 4-acetamido-4’-isothiocyanatostilbene-2-2’-disulfonic acid. o 1991 by the American Gastroenterological Association 0016-5085/91/$3.00
November 1991
as flat sheets between Lucite half-chambers with an aperture of 1.13 cm2 for measurements of PD, short-circuit current (IX), and resistance (R). Tissues were bathed with normal Ringer’s solution composed of the following (in mmol/L): Na’, 140; Cl-, 119.8; K’, 5.2; HCO,-, 25; Ca’+, 1.2; M$‘. 1.2; HPOi-, 2.4; H,PO,-, 0.4; and 268 mOsm/kg H,O, with pH 7.5 when gassed with 95% 0,/5% CO, at 37°C. Mucosal and serosal solutions were connected to calomel and Ag-AgCl electrodes with Ringer’sagar bridges for measurements of PD and automatic short-circuiting of the tissue with a voltage clamp (World Precision Instruments Inc., Sarasota, FL). Tissues were continuously short-circuited except for 5-lo-second periods when the open-circuit PD was read. Electrical resistance was calculated using Ohm’s law from the open-circuit PD and the Isc or from the current deflection to imposed voltage. Forty-five minutes after mounting (equilibration period], tissues paired by R (R within 25%) had HCl added to either the mucosal or serosal solution to reduce pH stepwise at 15-minute increments from 7.5 to 1.5 or to reduce and maintain solution pH at 2.9 for 2 hours. At the end of the periods of acid exposure, the mucosal and serosal solutions were drained and replaced with nonacidified solutions of similar pH and ion composition to those initially present (washout). Potential difference, Isc, and R were monitored before, during, and after acidification. For experiments designed to study the effect of chloridefree solution on epithelial protection against serosal acid, tissues were washed, stripped, and mounted in Ussing chambers in Cl-free Ringer’s solution composed of the following (in mmol/L): Na’, 140; isethionate, 115; K’, 5.2: HCO,jm, 25; Ca’+, 1.2; Mg”, 1.2; HPO,*-, 2.4; H,PO,-, 0.4; SO,‘., 2.4; with pH 7.5 when gassed with 95% 0,/5% CO, at 37°C. Forty-five minutes later, tissues paired by R were acidified (titrated) serosally to pH 2.0 with either H,SO, (to maintain the Cl-free environment) or HCl (to provide the Cl-containing environment). This required the addition of approximately 22.5 mmol/L H,SO, or 45 mmol/L HCl to the serosal bathing solutions. The resulting bathing solutions had similar osmolalities at 264 and 280 mOsm/kg H,O, respectively. Acidification was maintained for 1 hour. After acidification, tissues were fixed in the chamber and processed for light microscopy. Junction potentials were determined for all solutions by a modification of the method of Read and Fordtran (5). Solutions reflecting junction potentials present under experimental conditions were placed in separate beakers, and the beakers were used in pairs to reflect mucosal or serosal bathing solutions for tissues mounted in the Ussing chamber. The two solutions were connected by having one end of a 3 mol/L KC1 agar bridge placed in each beaker. Each beaker also contained one end of a Ringers-agar bridge. The other end of each Ringers-agar bridge was placed in a separate beaker containing 3 mol/L KC1 and one of a pair of matched calomel electrodes. The calomel electrodes were connected to a voltmeter for measurements of PD. Junction potentials between bathing solutions and the Ringer’s_agar bridges were 2 1 mV for all experimental conditions except those experiments involving solutions with pH 1.5. At pH 1.5 the junction potential was 5.6 mV, with the potential being positive for experiments involving mucosal acidification
MECHANISMS OF ACID INJURY TO RABBIT ESOPHAGUS 1221
and negative for those involving serosal acidification. Therefore, corrections for junction potentials were only made under conditions in which pH 1.5 was present (Figure 1).
Morphology Morphology was assessed by light microscopy on tissues fixed with 2% paraformaldehyde and 4% glutaraldehyde in 0.1 mol/L phosphate buffer, pH 7.4, and stained
B
I
c
I WASHOUT
1
4200
1.300 t
:-I-1,,_ 0
30
60
90
120
ill0
TIME (mm)
Figure 1. The effect of mucosal
[apical membrane) or serosal (basolateral membrane) acidification with HCI on the PD (A), Isc (B), and R (C) of rabbit esophageal epithelium. Stepwise mucosal and serosal acidification are observed to have dissimilar effects on PD and Isc but similar effects on R. Unlike mucosal acidification, semsal acidification abolished PD and Isc and did so irreversibly because replacement (washout) of the acidified bathing solution with normal Ringer’s did not result in recovery. The dotted line indicates the PD and Isc after a 5.6-mV correction for junction potentials at pH 1.5. (n = 5).
GASTROENTEROLOGYVol. 101.No. 5
1222 TOBEY AND ORLANDO
with H&E. Injury was assessed by an observer who had no knowledge of treatment groups. The scoring system was as follows: 0, normal; 1, tissue edema; 2, patchy necrosis; 3, diffuse necrosis; and 4, transmucosal necrosis [ulceration).
Statistics
Statistical significance was determined using either Student’s t test or the Wilcoxon signed ranks test. Data are reported as the mean + SE. The animal protocol has been reviewed and approved by our Animal Welfare Committee.
morphology. Tissues exposed to progressive mucosal acidification were undamaged, whereas tissues exposed to serosal acid had marked edema and cell necrosis located predominantly within those cell layers closest to the acidified bath, i.e., the stratum germinativum and lower layers of stratum spinosum [injury scores, 0.6 & 0.3 vs. 3.2 ? 0.3, respectively; n = 5; P < 0.05). An illustration of the type and extent of damage from these exposures is presented in Figure 2. Tissues exposed continuously to mucosal or serosal acid, pH 2, for 2 hours, were also evaluated. Similar to the results above with stepwise acidification, mucosal
Results When tissues were mounted in the Ussing chamber and bathed by normal Ringer’s solution at pH 7.5, there was a gradual decline in PD and Isc to approximately 50% of baseline over 3.25 hours. Over this same period, R increased slightly or remained stable (Figure 1). A change of bathing solution [from normal Ringer’s to normal Ringer’s) at 2.15 hours is noted to have no effect on PD, Isc, or R. Similar to tissues bathed by normal Ringer’s, progressive mucosal acidification with HCl from pH 7.5 to 2.5 produced no significant change in PD or Isc. However, initially increased R from pH mucosal acidification
nonacidified
controls
(Figure 1). Progressive
serosal
serosal pH reached 2.5, PD and Isc were abolished and remained so throughout the subsequent periods of acidification. Also, the changes in PD, Isc, and R with serosal acidification were, unlike mucosal acidification, shown to be irreversible because they failed to increase significantly after washout. Differences in electrical responses between mucosally or serosally acidified tissues were mirrored by differences in
Figure 2. A representative example of the effect of stepwise mucosal (apical membrane) or serosal (basolateral membrane) acidification on the light microscopic morphology of rabbit esophageal epithelium. There is no discernable difference between tissues exposed to (A) normal Ringer’s (controls) and (B) stepwise mucosal acidification from pH 7.5 to pH 1.5. In contrast, there is evidence of diffuse edema and cell necrosis within the lower layers for tissues exposed to (C) stepwise serosal acidification from pH 7.5 to 1.5 (H&E; original magnification x250).
MECHANISMS
1991
November
and serosal acidification lowered R to a similar degree (maximum decline after 2 hours of 44% k 2% with mucosal acidification vs. decline of 53% + 7% with serosal acidification; n = 5; P > 0.05) (Figure 3). Also, mucosal acidification to pH 2 had no effect on PD and Isc, whereas serosal acidification resulted in the
OF ACID INTURY TO RABBIT
I
A
HCI pH2
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ESOPHAGUS
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a -4
+WASHOUT
+HCI , pH2
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L-41 ’
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+ HCI. pH2
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+HCI ,pH2
i
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L-4,-
c
MUCOSAL ACIDIFICATION SEROSAL ACIDIFICATION
SEROSAL OUABAIN D------o SEROSAL OUABAIN
I HCI. pH2
+HCl,pH2
-
1200
-
400
-
H
c WASH OUT
O
SEROSAL ‘p
I 0
I 30
ACIDIFICATION
WASHOUl
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1600
MUCOSAL 1104 MI
Figure 4. The effects of abolishing PD (A) and Isc (II) by serosal exposure to either HCl at pH 2 or ouabain on the R (C) of rabbit esophageal epithelium. Serosal acidification unlike ouabainization results in a progressive decline in R with time at pH 2, although this decline does not reach statistical significance by 2 hours. Further, although the addition of mucosal acid (pH 2 for 2 hours) to ouabinized tissues also reduced R, the decline was similar to that seen with mucosal (or serosal) acidification alone (n = 5).
ACIDIFICATION
MUCOSAL
VS. SEROSAL
I 60
I 90
TIME
(mlnl
I 120
ACIDIFICATION I 150
J I80
(min)
Figure 2. The effect of mucosal (apical membrane) or serosal (basolateral membrane) acidification with HCI, pH 2 for 2 hours, on PD (A), Isc (II) and R (C) of rabbit esophageal epithelium. Mucosal and serosal acidification are observed to have dissimilar effects on PD and Isc but similar effects on R. Unlike mucosal acidification, serosal acidification abolished PD and Isc and did so irreversibly because replacement (washout) of the acidified bathing solution with normal Ringer’s did not result in recovery. The small gradual increase in PD and Isc noted after their abolition may reflect diffusion of chloride from serosa to mucosa in tissues damaged by serosal acid (n = 5).
immediate abolition of both PD and IX (Figure 3). Further, the abolition of PD and Isc and the reduction in R with serosal acidification were irreversible, as shown by little or no recovery after replacement of the acidic bath with normal Ringer’s (washout) (Figure 3). After a similar washout, the reduction in R after mucosal acidification increased 23% & 4% within 30 minutes. Morphology, which was similar to changes shown in Figure 2 for the two treatment groups, also confirmed the greater damaging effects of serosal acidification than mucosal acidification (injury scores, 3.2 k 0.2 vs. 0.7 k 0.3, respectively: n = 5; P < 0.05).
1224
TOBEY AND ORLANDO
Because the above experiments clearly establish the greater potential for cell necrosis to occur with serosal (BLM) as opposed to mucosal (AM) acidification, additional studies were performed to clarify whether specific membrane proteins presumed present on the BLM but not AM of esophageal epithelium were responsible for these disparate effects. Those studied were the ouabain-inhibitable sodium-potassiumstimulated adenosinetriphosphatase (Na’,K’-ATPase), the amiloride-inhibitable Na/H antiport, and the 4,4’diisothiocyanatostilbene-2,2’-disulfonic acid (DIDS)/4acetamido-4’-isothiocyanatostilbene-2-2’-disulfonic acid (SITS)-inhibitable Cl/HCO, antiport. A role for inhibition of the enzyme, Na’,K+-ATPase, in the production of cell necrosis was assessed by comparing abolition of PD and Isc by serosal acid with abolition produced by the addition of ouabain, lo-* mol/L, to the serosal solution. In addition, mucosal acidification, pH 2, for 2 hours was superimposed on some tissues in which PD and Isc were abolished by ouabain. As shown in Figure 4, abolition of PD and Isc by both serosal acid or ouabain was irreversible in that neither recovered after washout. However, R in ouabainized tissues was unchanged in contrast to serosal acidification, which reduced R progressively but not significantly with time. Morphological comparison also showed marked differences between prolonged abolition of PD and Isc by ouabain compared with abolition produced by serosal acid; ouabainized tissues had no morphological injury, whereas serosally acidified tissues had marked edema and cell necrosis (injury scores, 0.8 + 0.3 vs. 2.9 ? 0.2, respectively; n = 5; P < 0.05) (Figure 5). Further, tissues in which PD and Isc were abolished by ouabain and which were then mucosally acidified for 2 hours showed no significant morphological damage (injury scores, 0.5 + 0 mucosal acid plus ouabain vs. 0.8 f 0.3
GASTROENTEROLOGY
Vol. 101. No. 5
ouabain alone vs. 0.3 & 0.1 mucosal acid alone; n = 5; P > 0.05 for all comparisons). Further, there was a similar recovery in the acid-induced decline in R whether or not mucosally acidified tissues had PD and Isc previously abolished by ouabain (an increase of 20% + 8% mucosal acid alone vs. 28% 2 3% mucosal acid plus ouabain; n = 5; P > 0.05) (Figure 4). A role for an amiloride-inhibitable Na/H exchanger and/or a SITS-inhibitable Cl/HCO, exchanger in the development of epithelial necrosis was assessed by pretreating tissues bathed in normal Ringer’s for 1 hour with either 1 mmol/L amiloride, 4 mmol/L SITS, or both amiloride and SITS. Control tissues contained neither agent. After pretreatment all tissues were serosally acidified with HCl pH 2 for 2 hours. The effect of various treatments on the electrical parameters and injury scores in these tissues is presented in Table 1. As shown, PD, Isc, and R were essentially similar for all tissues before pretreatment with drugs. During the pretreatment period, tissues exposed to amiloride (with or without SITS), but not SITS alone, showed a significant decline in PD and Isc, presumably because of amiloride’s reported ability to inhibit Na’,K’-ATPase activity (6). After serosal acidification to pH 2, PD and Isc were immediately abolished in tissues from all groups, whereas R declined in all but the SITS plus amiloride group. Tissues pretreated with SITS alone had less of a reduction in R than controls, but this did not reach statistical significance (Table 1). However, the injury scores of tissues exposed to SITS in the presence or absence of amiloride were notably significantly lower than scores of either acid-exposed (untreated) controls or tissues exposed to amiloride alone. An example of the morphological protection observed in the SITS-treated groups is shown in Figure 6. Note that whereas SITS protection was evident at 4 mmol/L, this beneficial effect was not
Figure 5. A representative example of the light microscopic morphology of rabbit esophageal tissues with PD and Isc abolished for 2 hours by serosal exposures to either (A) HCl, pH 2, or (II) ouabain, 1Oe4mol/L. Serosal acid, but not ouabainization, is shown to result in diffise edema and cell necrosis. Mucosally acidified tissues pretreated with ouabain (C) show no more damage than those mucosally acidified with no pretreatment (D) (H&E; original magnification x 250).
November
MECHANISMS
1991
OF ACID INJURY TO RABBIT
ESOPHAGUS
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Table 1. The Effect of Serosal Pretreatment With Amiloride, SITS or Amiloride Plus SITS on the Potential Difference,
Short-Circuit Current, and Electrical Resistance of Rabbit Esophageal Epithelium Serosally Acidified With HCl, pH 2 PD @V) Agent
Baseline
(n = 6) -15.7 Amiloride Control (n = 6) -13.2 SITS (n = 8) -13.8 Control (n = 8) -14.4 Amiloride -C SITS (n = 6) -16.1 Control (n = 6) -19.0
Postdrug
t 2.6 -4.7 2 2.1 -12.7 +- 1.6 -13.5
Isc (pA/cm”) Postacid
f 2.3
-14.4
f. 2 k ?
0.6” 2.1 1.3 2.5
k 0.7 2 2.4
-4.6 -17.9
k 0.2” 2 2.6
-0.7 -0.4 -0.1 -0.3
2 2 2 2
Baseline 0.3 0.2 0.3 0.2
11 k 2 g-t1 10 +- 1 11 t 1
0.4 2 0.3 0.2 k 0.2
13 2 2 13 -c 2
Postdrug 3 7 8 9
2 -+ ” +
1” 1 1 1
R(Wcm
Postacid 1.0 0.3 0.3 0.3
rf: 1.0 2 0.4 + 0.2 + 0.2
Baseline 1572 1539 1587 1478
255 275 123 92
4 + 1” 0.2 + 0.2 1441 + 213 10 + 2 0.6 2 0.2 1390 ” 259
NOTE. Values are expressed as means ? SEM. Amount of amiloride, 1 mmol/L; SITS, 4 mmol/L. 2, patchy necrosis; 3, diffuse necrosis; and 4, transepithelial necrosis or ulceration. “P < 0.05 compared with paired controls.
observed at lower doses. We speculate that this loss of effect reflects SITS’ dilution as it diffuses through the intercellular spaces of this multilayered epithelium (Table 2). Further, because SITS, but not amiloride, was protective, making inhibition of Cl/HCO, exchange a more likely possibility, we tested whether inhibition of Cl/HCO, exchange by exposure to serosal acid in a Cl-free environment was also protective. This was performed by comparing the morphology of tissues exposed to serosal acid, pH 2, for 1 hour in the presence or absence of Cl (Table 3). To achieve this comparison, all tissues were initially mounted in Cl-free Ringer’s solution. After pairing by R, tissues to remain in a Cl-free environment were acidified to pH 2 serosally with H,SO,, whereas tissues to be exposed to Cl were acidified serosally to pH 2 with HCl. After acidification, morphological comparisons of tissues showed significant protection in tissues exposed to a Cl-free environment compared with tissues in a Clcontaining environment (injury scores, 0.7 + 0.3 vs. 2.1 ? 0.2, respectively; n = 10, P < 0.05). These morphological results are shown in Figure 7.
2 ? 2 f
Injury
’)
Postdrug 1743 1800 1813 1787
k 332 + 283 IT 111 -t 155
1545 k 303 1662 2 360
Postacid 1224 1426 1613 1405
f 2 2 k
235 191 95 123
Injury score 3.0 2.7 1.4 2.7
+ + + t
0.2 0.2 0.3” 0.2
1666 -c 237” 2.0 t 0.3” 1282 2 306 2.7 2 0.4
scores are 0, normal:
1, tissue edema:
Discussion The results of the present investigation show that, in contrast to acidification of the AM surface of esophageal epithelium, acidification of the BLM surface is poorly tolerated. The relative acid-resistance of AMs was documented by the inability of mucosal HCl, pH 2.5 for 15 minutes and pH 2.0 for 30 minutes, to alter tissue structure (normal histology by light microscopy) or function (normal PD, Isc, and R, reflecting cellular transport and epithelial barrier properties, respectively). Only when mucosal pH was lowered to 2.0 for 30 minutes with continuous exposure or to 1.5 in the stepwise experiments did permeability increase (decline in R); this was readily reversed when mucosal pH was restored to neutrality. This resistance to damage when the AM surface of esophageal epithelium is exposed to mucosal HCl has been documented previously by us and others (1,2,711). Furthermore, this resistance to damage with exposure of the AM surface of esophageal epithelium to mucosal HCl is not the result of a mucusbicarbonate barrier creating a significant pH gradient
Figure 6. A representative example of the effect of serosal pretreatment with amiloride, SITS, or amiloride plus SITS, on the degree of injury to rabbit esophageal epitbelium exposed serosally to HCI, pH 2.0 for 2 hours. Tissues exposed to (A) SITS alone or (B) SITS plus amiloride are shown to be significantly less damaged than (C) those receiving no pretreatment or (0) pretreatment with amiloride alone (H&E; original magnification x 250).
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TOBEY AND ORLANDO
GASTROENTEROLOGY
Table 2. The Effect of Varying Concentrations of Serosal SITS on Electrical Resistance and Tissue Injury of Rabbit Esophageal Epithelium Exposed Serosally to HCI, pH 2 Postacid.
1h
Resistance SITS concentration
(mmoZ/L)
6 6 6 6 7 7 8 6
0.01 Control
0.10 Control
1.0 Control 4.0 Control
(Wcm’)
n
14212 1640? 1428 k 1781k 1458 2 1689 k 1613 k 1405 +
255 198 232 175 169 174 95" 123"
Injury
score
2.2 k 0.2 2.6 k 0.3 2.5 Ii0.2 2.7 + 0.2 2.3 + 0.2 2.6 f 0.2 1.4 + 0.3".' 2.7 f 0.2"
NOTE. Values are expressed as means + SEM. Amount mmol/L. For injury scores, see Table 1. “Values from Table 1. *P < 0.05 compared with paired controls.
of SITS, 4
from lumen-to-cell surface. This is because, unlike stomach and duodenum, the rabbit esophagus has neither a mucus layer nor the ability of the epithelium to secrete bicarbonate (10,12-14). Indeed, Quigley and Turnberg have shown in humans that, unlike gastric fundic and duodenal epithelia, perfusion of the esophagus with HCl resulted in little or no lumento-surface gradient across the pH range of 7-2 (15). In contrast to the acid-resistance of the AM surface, the BLM surface was shown to be very sensitive to the damaging effects of acid. This was documented by the ability of even a modest decline in serosal pH (6.5) to inhibit esophageal transport (PD and Isc) and lower serosal pHs ( I 2.0) to irreversibly abolish esophageal transport. Also, although in the present experiments, a similar degree of acidity with both apical and BLM acidification (pH 2 for 30 minutes or pH 1.5 for 15 minutes) impaired esophageal barrier function (reduction in R), only BLM acidification with its coincident abolition of transport was associated with epithelial cell necrosis. Similar to our results, Sanders et al., in canine gastric chief cell monolayers, have documented that acidification of the BLM is poorly tolerated compared with AM acidification (16). However,
in their experiments the pH-impairing barrier function, like that for inhibition of cell transport, was lower with apical (pH 2-2.5) than with basolateral (pH 4.0) acidification. This difference may reflect the equal access of acid from the mucosal and serosal bathing solutions to the junctions of chief cell monolayers as opposed to the unequal access of acid to the junctional complex of the approximately 30 celllayered esophageal epithelium due to the greater distances through which hydrogen ions (H’) must diffuse with serosal acidification. [The intercellular permeability barrier in rabbit esophageal epithelium has been localized using electron microscopy in horseradish peroxidase-exposed tissues to the most luminal g-lo-cell layers (14).] In this investigation we also explored some of the possible mechanisms responsible for the ready occurrence of cell death with serosal, but not mucosal, acidification. One possible mechanism considered for cell death was the inhibition of the BLM enzyme Na’,K’-ATPase and with it inhibition of cell Na transport. In this scenario the loss of Na transport would cause cell death through loss of cell osmoregulation or pH regulation. Support for this is provided by this and prior investigations showing that abolition of PD (the PD generated principally by active Na transport in esophageal epithelium) accompanies cell necrosis (1,3). However, against this concept is the present demonstration that abolition of PD by ouabainization, even when accompanied by mucosal acidification, was not associated with cell necrosis (Figures 4 and 5). This indicates that the abolition of Na transport observed with serosal acidification accompanies but does not cause cell death in this model. Another possible mechanism for cell death from serosal acidification may be more effective entry of H’ into the cell across the BLM compared with the AM. This concept is supported by studies in Necturus antrum by Ashley et al. (11). Using pH microelectrodes in Ussing-chambered tissues, they observed significant declines in pH, with serosal acidification, whereas similar degrees of mucosal acidification had no effect on intracellular pH (pH,). Furthermore, if there
Table 3. Comparison of Cl-Free Versus Cl-Containing Serosal Solution on the Electrical Parameters Rabbit Esophageal Epithelium Exposed Serosally to HCI, pH 2
Cl-free (n = 10) Cl-containing (n = 10)
PD (mV) -22.3 k 1.8 -20.6 k 2.3
Isc (fi/cm*) +5.a r 0.3 +5.1 + 0.4
and Tissue Injury of
Postacid
Initial Solution
Vol. 101. No. 5
R(0/cm*) 38815 373 4045 + 363
PD (mV) +2.5 k 0.3" +0.8 2 0.3
Isc (+4/cm’) -0.8 k 0.1 -0.2 -c 0.1
R(Ucm2) 4372 ?444 4312 2 417
Injury
score
0.7 + 0.3" 2.1 + 0.2
NOTE. Values are expressed as means ? SEM. Initial values are similar and R high for all tissues because of mounting in Cl-free Ringer’s solution. Serosal acidification was performed with HCl for Cl-containing solutions and with H,SO, for Cl-free solutions. After acid exposure for 1 hour, the small differences in PDs reflect small differences in diffusion and/or junction potentials between Cl-containing and SO,-containing serosal solutions and their respective mucosal solutions containing isethionate as Cl replacement. “P < 0.05 compared with Cl-containing solutions.
November
1991
MECHANISMS
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ESOPHAGUS
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Figure 7. A representative example of the effect of serosal acidification in (A) Cl-free solution (acidification to pH 2 with H,SO,) or (B) Cl-containing solution (acidification to pH 2 with HCl) on the morphology of esophageal epithelium. Tissues exposed to serosal acid in Cl-free solution show mild edema but were protected against the cell necrosis evident in the lower layers of esophagus exposed to acid in Cl-containing solution (H&E; original magnkcation x 200).
are greater rates of H’ entry into esophageal cells across the BLM, then mechanisms for H’ transport on the BLM must be present that are not present on the AM of esophageal cells. Two such candidate mechanisms present in stratified squamous epithelia are the amiloride-sensitive Na/H antiport and SITS(DIDS)Both antiports sensitive Cl/HCO, antiport (17,18). have now been identified in esophageal cells of the rabbit (1%21), and although neither has been localized to BLM or AM, both are capable of moving H’ across the membrane and into the cell. [The Cl/HCO, antiport normally operates as a cell acidifier by transporting HCO, out of the cell in exchange for Cl, whereas the Na/H antiport, which normally operates as a cell alkalinizer, can be reversed by changing ion gradients to favor H’ entry in exchange for Na movement out of the cell (22,23).] Documentation for participation of these mechanisms in cell acidification during serosal (BLM) acidification has been provided by the studies of Starlinger et al. in isolated rabbit gastric glands (24). They observed that blockade of the Cl/HCO, antiport either alone (by SITS or Cl-free solution) or in combination with blockade of the Na/H antiport (NaCl-free solution) was associated with limited cell acidification as pH of the external bathing solution was lowered from 7.8 to 6.4. In the present investigation, esophageal epithelia treated with SITS, alone or in combination with amiloride, but not amiloride alone, were protected against damage upon exposure to serosal HCl. Because these results were consistent with the concept that cell necrosis was mediated by H+ entry through a Cl/HCO, antiport located on the BLM, we also tested the ability of a Cl-free environment (a maneuver that, like SITS, inhibits Cl/HCO, exchange) to block the damage associated with serosal acidification. As shown in Figure 7, serosal acidification (with H,SO, to maintain the Cl-free environment) produced less damage to esophageal epithelia than Cl-containing solutions acidified to the same degree and for similar lengths of time. These results add further support to
the concept that serosal acidification damages esophageal tissues by cell acidification and that the pathway for cell acidification across the BLM appears to involve a SITS-sensitive, Cl-dependent pathway such as the Cl/HCO, antiport. * References 1. Orlando
acid
RC, Powell DW, Carney CN. Pathophysiology of acute injury in rabbit esophageal epithelium. J Clin Invest
1981;68:286-293. 2. Orlando
3.
4.
5.
6.
7. 8. 9.
10.
RC, Bryson JC. Powell DW. Mechanism of H’ injury in rabbit esophageal epithelium. Am J Physiol 1984;246:G718G724. Carney CN, Orlando RC. Powell DW. Dotson MM. Morphologic alteration in early acid-induced epithelial injury of rabbit esophagus. Lab Invest 1981:45:198-208. Tobey NA. Powell DW, Schreiner VJ, Orlando RC. Serosal bicarbonate protects against acid injury to rabbit esophagus. Gastroenterology 1989;96:1466-1477. Read NW, Fordtran JS. The role of intraluminal junction potentials in the generation of the gastric potential difference in man. Gastroenterology 1979;76:932-938. Soltoff SP, Mandel LJ. Amiloride directly inhibits the Na,KATPase activity of rabbit kidney proximal tubules. Science 1983;220:957-959. Salo J, Kivilaakso E. Role of luminal H’ in the pathogenesis of experimental esophagitis. Surgery 1982;92:61-68. Chung RSK, Magri JK. DenBesten L. Hydrogen ion transport in the rabbit esophagus. Am J Physiol 1975:229:496-500. Harmon JW, Johnson LF, Maydonovitch CL. Effects of acid and bile salts on the rabbit esophageal mucosa. Dig Dis Sci 1981:26: 65-72. Silen W. Gastric mucosal defense and repair. In: Johnson LR, Christensen J, Jacobson ED, Jackson MJ, Walsh JH, eds. Physiology of the gastrointestinal tract. Volume 2. 2nd ed. New York: Raven, 1987:1055-1069.
*Whereas the concept that cell acidification can destroy cells is not in doubt, the levels at which lowering pH, become noxious are likely to vary among tissues. Arvidsson et al. (25) lowered pH, in secreting frog oxynticopeptic cells to pH 6.4 through acidification of the BLM but failed to show structural (normal histology) or functional [normal PD and R) evidence of damage. However, when pH, was similarly reduced in nonsecreting cells (cell secretion inhibited by omeprazole or cimetidinel. there was extensive and irreversible tissue damage.
1228
TOBEY AND ORLANDO
11. Ashley SW, Soybel DI, Moore D, Cheung LY. Intracellular pH (pH,) in gastric surface epithelium is more susceptible to serosal than mucosal acidification. Surgery 1987;102:371-379. 12. Flemstrom G. Gastric and duodenal mucosal bicarbonate secretion. In: Johnson LR, Christensen J, Jacobson ED, Jackson MJ, Walsh JH, eds. Physiology of the gastrointestinal tract. Volume 2.2nd ed. New York: Raven, 1987:1011-1029. 13. Hamilton BH. Orlando RC. In vivo alkaline secretion by mammalian esophagus. Gastroenterology 1989;97:640-648. 14. Lacy ER, Tobey NA, Cowart K, Orlando RC. The esophageal mucosal barrier: structural correlates (abstr). Gastroenterology 1989;96:A281. 15. Quigley EMM, Turnberg LA. pH of the microclimate lining the human gastric and duodenal mucosa in vivo-studies in control subjects and in duodenal ulcer patients. Gastroenterology 1987;92:1876-1884. 16. Sanders MJ, Ayalon A, Roll M, Sol1 AH. The apical surface of canine chief cell monolayers resists H’ back-diffusion. Nature 1985;313:52-54. 17. Civan MM, Cragoe EJ, Peterson-Yantorno K. Intracellular pH in frog skin: effects of Nat, volume and CAMP. Am J Physiol 1988;255:F126-F134. 18. Drewnowska K, Cragoe EJ, Biber T. pH in principal cells of frog skin (Rana pipiens): dependence on extracellular Na+. Am J Physiol 1988;255:F930-F935. 19. Tobey NA, Khalbuss WE, Keku TO, Orlando RC. Cll/HCO; antiport activity in rabbit squamous and basal esophageal cells in primary culture (abstr). Gastroenterology 1991;100:706.
GASTROENTEROLOGY
Vol. 101. No. ii
20. Tobey NA, Khalbuss WE, Keku TO, Reddy SP, Hamilton BH. Orlando RC. Nat/H+ antiport activity and intrinsic buffering capacity in rabbit squamous and basal esophageal cells in primary culture (abstr). Gastroenterology 1991;100:707. 21. Agnone LM, Schmidt LN, Goldstein JL, Layden TJ. Regulation of rabbit esophageal cell pH by a Nat/H+ antiport (abstr). Gastroenterology 1989;96:A3. 22. Roos A, Boron WF. Intracellular pH. Physiol Rev 1981:61:296434. 23. Boron WF. Intracellular pH regulation in epithelial cells. Ann Rev Physiol 1986;48:377-388. 24. Starlinger M, Paradiso AM, Machen TE. Steady state regulation of intracellular pH in isolated rabbit gastric glands. Gastroenterology 1987;92:957-965. 25. Arvidsson S, Carter K. Yanaka A, Ito S, Silen W. Effect of basolateral acidification on the frog oxynticopeptic cell. Am J Physiol 1990;259:G564-G570.
Received June 27,199O. Accepted April 16,1991. Address requests for reprints to: Roy C. Orlando, M.D., Division of Digestive Diseases, CB no. 7080, 324 Burnett Womack Building, Chapel Hill, North Carolina 27599-7080. The project was supported in part by National Institutes of Health grant no. 2 ROl-DK-36013-04. The authors thank Virginia J. Schreiner, Angela D. Semones, and Lara E. Glenn for their expert technical assistance.
1991;101:1220-1228
Mechanisms of Acid Injury to Rabbit Esophageal Epithelium Role of Basolateral Cell Membrane Acidification NELIA A. TOBEY
and ROY C. ORLANDO
Department of Medicine, University of North Carolina School of Medicine, Chapel Hill, North Carolina
Because mucosal HCl traverses the paracellular pathway before significant damage develops within esophageal epithelium, the effects of mucosal and serosal acidification were compared on Ussingchambered rabbit esophageal epithelia. Notably serosa], but not mucosal, acidification was associated with cell necrosis, and the latter was accompanied by abolition of potential difference and short-circuit current. The reason for this difference was explored by exposing tissues serosally to ouabain, chloridefree solution, 4-acetamido-a’-isothiocyanatostilbene2-2’-disulfonic acid (SITS), or amiloride. The results show that serosal acidification, but not ouabainization, is associated with cell necrosis and that cell necrosis induced by serosal acidification can be blocked by SITS and chloride-free solution, but not by amiloride. These findings are compatible with the hypothesis that serosal acidification is more damaging than mucosal acidification because of the greater rate with which hydrogen ions can traverse the basolateral membrane; also, the route for more rapid entry appears to involve a SITS-sensitive, chloride-dependent mechanism (e.g., WHCO, ana tiport). he major cause of esophagitis in humans is the prolonged contact of esophageal epithelium with refluxed gastric (hydrochloric) acid. However, the mechanisms by which mucosal (luminal) HCl damages esophageal epithelium remain incompletely understood. Experimentally, one of the earliest signs of epithelial damage in acid-perfused esophagus is a decline in the transmural electrical potential difference (PD) (l-3), a decline that has been shown in rabbit esophagus to reflect increased permeability across the intercellular junctions (l-3). Further, after mucosal HCl increases paracellular permeability, epithelial necrosis can be prevented by the presence of buffers within the extracellular space (4). This latter
T
observation is particularly noteworthy, because it adds support to the hypothesis that the paracellular pathway is the major route by which mucosal HCl enters and then damages the esophageal epithelium. Based on the concepts of acid injury discussed above, it is evident that a consequence of HCl entering the paracellular pathway, before the development of epithelial necrosis, is the lowering of extracellular pH below the level of the (intercellular) junctional complex, i.e., the acidification of the region adjacent to the basolateral membrane (BLM). To understand the significance of BLM acidification as it relates to the development of epithelial necrosis, we studied the effects of BLM acidification on rabbit esophageal epithelial structure and function in the Ussing chamber and compared the effects with the effects of apical membrane (AM) acidification. Apical membrane acidification was produced in the traditional way by the addition of acid to the mucosal bathing solution, whereas BLM acidification was achieved by the addition of acid to the serosal bathing solution. Materials
and Methods
New Zealand white rabbits weighing between 8 and 9 lb were killed by administering an IV overdose of pentobarbital (60 mg/mL). The esophagus was excised, opened, and pinned mucosal surface down in a paraffin tray containing ice-cold oxygenated normal Ringer’s solution. The submucosa was grasped with hemostats, lifted up, and dissected free of the underlying mucosa with a scalpel. This process yielded a sheet of tissue consisting of stratified squamous epithelium and a small amount of underlying connective tissue. From this tissue, four sections were cut and mounted
Abbreviations used in this paper: AM, apical membrane; BLM, basolateral membrane; kc, short-circuit current; R, resistance; SITS, 4-acetamido-4’-isothiocyanatostilbene-2-2’-disulfonic acid. o 1991 by the American Gastroenterological Association 0016-5085/91/$3.00
November 1991
as flat sheets between Lucite half-chambers with an aperture of 1.13 cm2 for measurements of PD, short-circuit current (IX), and resistance (R). Tissues were bathed with normal Ringer’s solution composed of the following (in mmol/L): Na’, 140; Cl-, 119.8; K’, 5.2; HCO,-, 25; Ca’+, 1.2; M$‘. 1.2; HPOi-, 2.4; H,PO,-, 0.4; and 268 mOsm/kg H,O, with pH 7.5 when gassed with 95% 0,/5% CO, at 37°C. Mucosal and serosal solutions were connected to calomel and Ag-AgCl electrodes with Ringer’sagar bridges for measurements of PD and automatic short-circuiting of the tissue with a voltage clamp (World Precision Instruments Inc., Sarasota, FL). Tissues were continuously short-circuited except for 5-lo-second periods when the open-circuit PD was read. Electrical resistance was calculated using Ohm’s law from the open-circuit PD and the Isc or from the current deflection to imposed voltage. Forty-five minutes after mounting (equilibration period], tissues paired by R (R within 25%) had HCl added to either the mucosal or serosal solution to reduce pH stepwise at 15-minute increments from 7.5 to 1.5 or to reduce and maintain solution pH at 2.9 for 2 hours. At the end of the periods of acid exposure, the mucosal and serosal solutions were drained and replaced with nonacidified solutions of similar pH and ion composition to those initially present (washout). Potential difference, Isc, and R were monitored before, during, and after acidification. For experiments designed to study the effect of chloridefree solution on epithelial protection against serosal acid, tissues were washed, stripped, and mounted in Ussing chambers in Cl-free Ringer’s solution composed of the following (in mmol/L): Na’, 140; isethionate, 115; K’, 5.2: HCO,jm, 25; Ca’+, 1.2; Mg”, 1.2; HPO,*-, 2.4; H,PO,-, 0.4; SO,‘., 2.4; with pH 7.5 when gassed with 95% 0,/5% CO, at 37°C. Forty-five minutes later, tissues paired by R were acidified (titrated) serosally to pH 2.0 with either H,SO, (to maintain the Cl-free environment) or HCl (to provide the Cl-containing environment). This required the addition of approximately 22.5 mmol/L H,SO, or 45 mmol/L HCl to the serosal bathing solutions. The resulting bathing solutions had similar osmolalities at 264 and 280 mOsm/kg H,O, respectively. Acidification was maintained for 1 hour. After acidification, tissues were fixed in the chamber and processed for light microscopy. Junction potentials were determined for all solutions by a modification of the method of Read and Fordtran (5). Solutions reflecting junction potentials present under experimental conditions were placed in separate beakers, and the beakers were used in pairs to reflect mucosal or serosal bathing solutions for tissues mounted in the Ussing chamber. The two solutions were connected by having one end of a 3 mol/L KC1 agar bridge placed in each beaker. Each beaker also contained one end of a Ringers-agar bridge. The other end of each Ringers-agar bridge was placed in a separate beaker containing 3 mol/L KC1 and one of a pair of matched calomel electrodes. The calomel electrodes were connected to a voltmeter for measurements of PD. Junction potentials between bathing solutions and the Ringer’s_agar bridges were 2 1 mV for all experimental conditions except those experiments involving solutions with pH 1.5. At pH 1.5 the junction potential was 5.6 mV, with the potential being positive for experiments involving mucosal acidification
MECHANISMS OF ACID INJURY TO RABBIT ESOPHAGUS 1221
and negative for those involving serosal acidification. Therefore, corrections for junction potentials were only made under conditions in which pH 1.5 was present (Figure 1).
Morphology Morphology was assessed by light microscopy on tissues fixed with 2% paraformaldehyde and 4% glutaraldehyde in 0.1 mol/L phosphate buffer, pH 7.4, and stained
B
I
c
I WASHOUT
1
4200
1.300 t
:-I-1,,_ 0
30
60
90
120
ill0
TIME (mm)
Figure 1. The effect of mucosal
[apical membrane) or serosal (basolateral membrane) acidification with HCI on the PD (A), Isc (B), and R (C) of rabbit esophageal epithelium. Stepwise mucosal and serosal acidification are observed to have dissimilar effects on PD and Isc but similar effects on R. Unlike mucosal acidification, semsal acidification abolished PD and Isc and did so irreversibly because replacement (washout) of the acidified bathing solution with normal Ringer’s did not result in recovery. The dotted line indicates the PD and Isc after a 5.6-mV correction for junction potentials at pH 1.5. (n = 5).
GASTROENTEROLOGYVol. 101.No. 5
1222 TOBEY AND ORLANDO
with H&E. Injury was assessed by an observer who had no knowledge of treatment groups. The scoring system was as follows: 0, normal; 1, tissue edema; 2, patchy necrosis; 3, diffuse necrosis; and 4, transmucosal necrosis [ulceration).
Statistics
Statistical significance was determined using either Student’s t test or the Wilcoxon signed ranks test. Data are reported as the mean + SE. The animal protocol has been reviewed and approved by our Animal Welfare Committee.
morphology. Tissues exposed to progressive mucosal acidification were undamaged, whereas tissues exposed to serosal acid had marked edema and cell necrosis located predominantly within those cell layers closest to the acidified bath, i.e., the stratum germinativum and lower layers of stratum spinosum [injury scores, 0.6 & 0.3 vs. 3.2 ? 0.3, respectively; n = 5; P < 0.05). An illustration of the type and extent of damage from these exposures is presented in Figure 2. Tissues exposed continuously to mucosal or serosal acid, pH 2, for 2 hours, were also evaluated. Similar to the results above with stepwise acidification, mucosal
Results When tissues were mounted in the Ussing chamber and bathed by normal Ringer’s solution at pH 7.5, there was a gradual decline in PD and Isc to approximately 50% of baseline over 3.25 hours. Over this same period, R increased slightly or remained stable (Figure 1). A change of bathing solution [from normal Ringer’s to normal Ringer’s) at 2.15 hours is noted to have no effect on PD, Isc, or R. Similar to tissues bathed by normal Ringer’s, progressive mucosal acidification with HCl from pH 7.5 to 2.5 produced no significant change in PD or Isc. However, initially increased R from pH mucosal acidification
nonacidified
controls
(Figure 1). Progressive
serosal
serosal pH reached 2.5, PD and Isc were abolished and remained so throughout the subsequent periods of acidification. Also, the changes in PD, Isc, and R with serosal acidification were, unlike mucosal acidification, shown to be irreversible because they failed to increase significantly after washout. Differences in electrical responses between mucosally or serosally acidified tissues were mirrored by differences in
Figure 2. A representative example of the effect of stepwise mucosal (apical membrane) or serosal (basolateral membrane) acidification on the light microscopic morphology of rabbit esophageal epithelium. There is no discernable difference between tissues exposed to (A) normal Ringer’s (controls) and (B) stepwise mucosal acidification from pH 7.5 to pH 1.5. In contrast, there is evidence of diffuse edema and cell necrosis within the lower layers for tissues exposed to (C) stepwise serosal acidification from pH 7.5 to 1.5 (H&E; original magnification x250).
MECHANISMS
1991
November
and serosal acidification lowered R to a similar degree (maximum decline after 2 hours of 44% k 2% with mucosal acidification vs. decline of 53% + 7% with serosal acidification; n = 5; P > 0.05) (Figure 3). Also, mucosal acidification to pH 2 had no effect on PD and Isc, whereas serosal acidification resulted in the
OF ACID INTURY TO RABBIT
I
A
HCI pH2
1223
ESOPHAGUS
WASHOUT I
-20 1 -16 c -12 -
P
r
-8 -
0
a -4
+WASHOUT
+HCI , pH2
-
o-
L-41 ’
I
I
I
I
I
I
I
B WASHOUl
+ HCI. pH2
I
I
I
I
I
+HCI ,pH2
i
I
I
I
WASHOUT
L-4,-
c
MUCOSAL ACIDIFICATION SEROSAL ACIDIFICATION
SEROSAL OUABAIN D------o SEROSAL OUABAIN
I HCI. pH2
+HCl,pH2
-
1200
-
400
-
H
c WASH OUT
O
SEROSAL ‘p
I 0
I 30
ACIDIFICATION
WASHOUl
TIME
1600
MUCOSAL 1104 MI
Figure 4. The effects of abolishing PD (A) and Isc (II) by serosal exposure to either HCl at pH 2 or ouabain on the R (C) of rabbit esophageal epithelium. Serosal acidification unlike ouabainization results in a progressive decline in R with time at pH 2, although this decline does not reach statistical significance by 2 hours. Further, although the addition of mucosal acid (pH 2 for 2 hours) to ouabinized tissues also reduced R, the decline was similar to that seen with mucosal (or serosal) acidification alone (n = 5).
ACIDIFICATION
MUCOSAL
VS. SEROSAL
I 60
I 90
TIME
(mlnl
I 120
ACIDIFICATION I 150
J I80
(min)
Figure 2. The effect of mucosal (apical membrane) or serosal (basolateral membrane) acidification with HCI, pH 2 for 2 hours, on PD (A), Isc (II) and R (C) of rabbit esophageal epithelium. Mucosal and serosal acidification are observed to have dissimilar effects on PD and Isc but similar effects on R. Unlike mucosal acidification, serosal acidification abolished PD and Isc and did so irreversibly because replacement (washout) of the acidified bathing solution with normal Ringer’s did not result in recovery. The small gradual increase in PD and Isc noted after their abolition may reflect diffusion of chloride from serosa to mucosa in tissues damaged by serosal acid (n = 5).
immediate abolition of both PD and IX (Figure 3). Further, the abolition of PD and Isc and the reduction in R with serosal acidification were irreversible, as shown by little or no recovery after replacement of the acidic bath with normal Ringer’s (washout) (Figure 3). After a similar washout, the reduction in R after mucosal acidification increased 23% & 4% within 30 minutes. Morphology, which was similar to changes shown in Figure 2 for the two treatment groups, also confirmed the greater damaging effects of serosal acidification than mucosal acidification (injury scores, 3.2 k 0.2 vs. 0.7 k 0.3, respectively: n = 5; P < 0.05).
1224
TOBEY AND ORLANDO
Because the above experiments clearly establish the greater potential for cell necrosis to occur with serosal (BLM) as opposed to mucosal (AM) acidification, additional studies were performed to clarify whether specific membrane proteins presumed present on the BLM but not AM of esophageal epithelium were responsible for these disparate effects. Those studied were the ouabain-inhibitable sodium-potassiumstimulated adenosinetriphosphatase (Na’,K’-ATPase), the amiloride-inhibitable Na/H antiport, and the 4,4’diisothiocyanatostilbene-2,2’-disulfonic acid (DIDS)/4acetamido-4’-isothiocyanatostilbene-2-2’-disulfonic acid (SITS)-inhibitable Cl/HCO, antiport. A role for inhibition of the enzyme, Na’,K+-ATPase, in the production of cell necrosis was assessed by comparing abolition of PD and Isc by serosal acid with abolition produced by the addition of ouabain, lo-* mol/L, to the serosal solution. In addition, mucosal acidification, pH 2, for 2 hours was superimposed on some tissues in which PD and Isc were abolished by ouabain. As shown in Figure 4, abolition of PD and Isc by both serosal acid or ouabain was irreversible in that neither recovered after washout. However, R in ouabainized tissues was unchanged in contrast to serosal acidification, which reduced R progressively but not significantly with time. Morphological comparison also showed marked differences between prolonged abolition of PD and Isc by ouabain compared with abolition produced by serosal acid; ouabainized tissues had no morphological injury, whereas serosally acidified tissues had marked edema and cell necrosis (injury scores, 0.8 + 0.3 vs. 2.9 ? 0.2, respectively; n = 5; P < 0.05) (Figure 5). Further, tissues in which PD and Isc were abolished by ouabain and which were then mucosally acidified for 2 hours showed no significant morphological damage (injury scores, 0.5 + 0 mucosal acid plus ouabain vs. 0.8 f 0.3
GASTROENTEROLOGY
Vol. 101. No. 5
ouabain alone vs. 0.3 & 0.1 mucosal acid alone; n = 5; P > 0.05 for all comparisons). Further, there was a similar recovery in the acid-induced decline in R whether or not mucosally acidified tissues had PD and Isc previously abolished by ouabain (an increase of 20% + 8% mucosal acid alone vs. 28% 2 3% mucosal acid plus ouabain; n = 5; P > 0.05) (Figure 4). A role for an amiloride-inhibitable Na/H exchanger and/or a SITS-inhibitable Cl/HCO, exchanger in the development of epithelial necrosis was assessed by pretreating tissues bathed in normal Ringer’s for 1 hour with either 1 mmol/L amiloride, 4 mmol/L SITS, or both amiloride and SITS. Control tissues contained neither agent. After pretreatment all tissues were serosally acidified with HCl pH 2 for 2 hours. The effect of various treatments on the electrical parameters and injury scores in these tissues is presented in Table 1. As shown, PD, Isc, and R were essentially similar for all tissues before pretreatment with drugs. During the pretreatment period, tissues exposed to amiloride (with or without SITS), but not SITS alone, showed a significant decline in PD and Isc, presumably because of amiloride’s reported ability to inhibit Na’,K’-ATPase activity (6). After serosal acidification to pH 2, PD and Isc were immediately abolished in tissues from all groups, whereas R declined in all but the SITS plus amiloride group. Tissues pretreated with SITS alone had less of a reduction in R than controls, but this did not reach statistical significance (Table 1). However, the injury scores of tissues exposed to SITS in the presence or absence of amiloride were notably significantly lower than scores of either acid-exposed (untreated) controls or tissues exposed to amiloride alone. An example of the morphological protection observed in the SITS-treated groups is shown in Figure 6. Note that whereas SITS protection was evident at 4 mmol/L, this beneficial effect was not
Figure 5. A representative example of the light microscopic morphology of rabbit esophageal tissues with PD and Isc abolished for 2 hours by serosal exposures to either (A) HCl, pH 2, or (II) ouabain, 1Oe4mol/L. Serosal acid, but not ouabainization, is shown to result in diffise edema and cell necrosis. Mucosally acidified tissues pretreated with ouabain (C) show no more damage than those mucosally acidified with no pretreatment (D) (H&E; original magnification x 250).
November
MECHANISMS
1991
OF ACID INJURY TO RABBIT
ESOPHAGUS
1225
Table 1. The Effect of Serosal Pretreatment With Amiloride, SITS or Amiloride Plus SITS on the Potential Difference,
Short-Circuit Current, and Electrical Resistance of Rabbit Esophageal Epithelium Serosally Acidified With HCl, pH 2 PD @V) Agent
Baseline
(n = 6) -15.7 Amiloride Control (n = 6) -13.2 SITS (n = 8) -13.8 Control (n = 8) -14.4 Amiloride -C SITS (n = 6) -16.1 Control (n = 6) -19.0
Postdrug
t 2.6 -4.7 2 2.1 -12.7 +- 1.6 -13.5
Isc (pA/cm”) Postacid
f 2.3
-14.4
f. 2 k ?
0.6” 2.1 1.3 2.5
k 0.7 2 2.4
-4.6 -17.9
k 0.2” 2 2.6
-0.7 -0.4 -0.1 -0.3
2 2 2 2
Baseline 0.3 0.2 0.3 0.2
11 k 2 g-t1 10 +- 1 11 t 1
0.4 2 0.3 0.2 k 0.2
13 2 2 13 -c 2
Postdrug 3 7 8 9
2 -+ ” +
1” 1 1 1
R(Wcm
Postacid 1.0 0.3 0.3 0.3
rf: 1.0 2 0.4 + 0.2 + 0.2
Baseline 1572 1539 1587 1478
255 275 123 92
4 + 1” 0.2 + 0.2 1441 + 213 10 + 2 0.6 2 0.2 1390 ” 259
NOTE. Values are expressed as means ? SEM. Amount of amiloride, 1 mmol/L; SITS, 4 mmol/L. 2, patchy necrosis; 3, diffuse necrosis; and 4, transepithelial necrosis or ulceration. “P < 0.05 compared with paired controls.
observed at lower doses. We speculate that this loss of effect reflects SITS’ dilution as it diffuses through the intercellular spaces of this multilayered epithelium (Table 2). Further, because SITS, but not amiloride, was protective, making inhibition of Cl/HCO, exchange a more likely possibility, we tested whether inhibition of Cl/HCO, exchange by exposure to serosal acid in a Cl-free environment was also protective. This was performed by comparing the morphology of tissues exposed to serosal acid, pH 2, for 1 hour in the presence or absence of Cl (Table 3). To achieve this comparison, all tissues were initially mounted in Cl-free Ringer’s solution. After pairing by R, tissues to remain in a Cl-free environment were acidified to pH 2 serosally with H,SO,, whereas tissues to be exposed to Cl were acidified serosally to pH 2 with HCl. After acidification, morphological comparisons of tissues showed significant protection in tissues exposed to a Cl-free environment compared with tissues in a Clcontaining environment (injury scores, 0.7 + 0.3 vs. 2.1 ? 0.2, respectively; n = 10, P < 0.05). These morphological results are shown in Figure 7.
2 ? 2 f
Injury
’)
Postdrug 1743 1800 1813 1787
k 332 + 283 IT 111 -t 155
1545 k 303 1662 2 360
Postacid 1224 1426 1613 1405
f 2 2 k
235 191 95 123
Injury score 3.0 2.7 1.4 2.7
+ + + t
0.2 0.2 0.3” 0.2
1666 -c 237” 2.0 t 0.3” 1282 2 306 2.7 2 0.4
scores are 0, normal:
1, tissue edema:
Discussion The results of the present investigation show that, in contrast to acidification of the AM surface of esophageal epithelium, acidification of the BLM surface is poorly tolerated. The relative acid-resistance of AMs was documented by the inability of mucosal HCl, pH 2.5 for 15 minutes and pH 2.0 for 30 minutes, to alter tissue structure (normal histology by light microscopy) or function (normal PD, Isc, and R, reflecting cellular transport and epithelial barrier properties, respectively). Only when mucosal pH was lowered to 2.0 for 30 minutes with continuous exposure or to 1.5 in the stepwise experiments did permeability increase (decline in R); this was readily reversed when mucosal pH was restored to neutrality. This resistance to damage when the AM surface of esophageal epithelium is exposed to mucosal HCl has been documented previously by us and others (1,2,711). Furthermore, this resistance to damage with exposure of the AM surface of esophageal epithelium to mucosal HCl is not the result of a mucusbicarbonate barrier creating a significant pH gradient
Figure 6. A representative example of the effect of serosal pretreatment with amiloride, SITS, or amiloride plus SITS, on the degree of injury to rabbit esophageal epitbelium exposed serosally to HCI, pH 2.0 for 2 hours. Tissues exposed to (A) SITS alone or (B) SITS plus amiloride are shown to be significantly less damaged than (C) those receiving no pretreatment or (0) pretreatment with amiloride alone (H&E; original magnification x 250).
1226
TOBEY AND ORLANDO
GASTROENTEROLOGY
Table 2. The Effect of Varying Concentrations of Serosal SITS on Electrical Resistance and Tissue Injury of Rabbit Esophageal Epithelium Exposed Serosally to HCI, pH 2 Postacid.
1h
Resistance SITS concentration
(mmoZ/L)
6 6 6 6 7 7 8 6
0.01 Control
0.10 Control
1.0 Control 4.0 Control
(Wcm’)
n
14212 1640? 1428 k 1781k 1458 2 1689 k 1613 k 1405 +
255 198 232 175 169 174 95" 123"
Injury
score
2.2 k 0.2 2.6 k 0.3 2.5 Ii0.2 2.7 + 0.2 2.3 + 0.2 2.6 f 0.2 1.4 + 0.3".' 2.7 f 0.2"
NOTE. Values are expressed as means + SEM. Amount mmol/L. For injury scores, see Table 1. “Values from Table 1. *P < 0.05 compared with paired controls.
of SITS, 4
from lumen-to-cell surface. This is because, unlike stomach and duodenum, the rabbit esophagus has neither a mucus layer nor the ability of the epithelium to secrete bicarbonate (10,12-14). Indeed, Quigley and Turnberg have shown in humans that, unlike gastric fundic and duodenal epithelia, perfusion of the esophagus with HCl resulted in little or no lumento-surface gradient across the pH range of 7-2 (15). In contrast to the acid-resistance of the AM surface, the BLM surface was shown to be very sensitive to the damaging effects of acid. This was documented by the ability of even a modest decline in serosal pH (6.5) to inhibit esophageal transport (PD and Isc) and lower serosal pHs ( I 2.0) to irreversibly abolish esophageal transport. Also, although in the present experiments, a similar degree of acidity with both apical and BLM acidification (pH 2 for 30 minutes or pH 1.5 for 15 minutes) impaired esophageal barrier function (reduction in R), only BLM acidification with its coincident abolition of transport was associated with epithelial cell necrosis. Similar to our results, Sanders et al., in canine gastric chief cell monolayers, have documented that acidification of the BLM is poorly tolerated compared with AM acidification (16). However,
in their experiments the pH-impairing barrier function, like that for inhibition of cell transport, was lower with apical (pH 2-2.5) than with basolateral (pH 4.0) acidification. This difference may reflect the equal access of acid from the mucosal and serosal bathing solutions to the junctions of chief cell monolayers as opposed to the unequal access of acid to the junctional complex of the approximately 30 celllayered esophageal epithelium due to the greater distances through which hydrogen ions (H’) must diffuse with serosal acidification. [The intercellular permeability barrier in rabbit esophageal epithelium has been localized using electron microscopy in horseradish peroxidase-exposed tissues to the most luminal g-lo-cell layers (14).] In this investigation we also explored some of the possible mechanisms responsible for the ready occurrence of cell death with serosal, but not mucosal, acidification. One possible mechanism considered for cell death was the inhibition of the BLM enzyme Na’,K’-ATPase and with it inhibition of cell Na transport. In this scenario the loss of Na transport would cause cell death through loss of cell osmoregulation or pH regulation. Support for this is provided by this and prior investigations showing that abolition of PD (the PD generated principally by active Na transport in esophageal epithelium) accompanies cell necrosis (1,3). However, against this concept is the present demonstration that abolition of PD by ouabainization, even when accompanied by mucosal acidification, was not associated with cell necrosis (Figures 4 and 5). This indicates that the abolition of Na transport observed with serosal acidification accompanies but does not cause cell death in this model. Another possible mechanism for cell death from serosal acidification may be more effective entry of H’ into the cell across the BLM compared with the AM. This concept is supported by studies in Necturus antrum by Ashley et al. (11). Using pH microelectrodes in Ussing-chambered tissues, they observed significant declines in pH, with serosal acidification, whereas similar degrees of mucosal acidification had no effect on intracellular pH (pH,). Furthermore, if there
Table 3. Comparison of Cl-Free Versus Cl-Containing Serosal Solution on the Electrical Parameters Rabbit Esophageal Epithelium Exposed Serosally to HCI, pH 2
Cl-free (n = 10) Cl-containing (n = 10)
PD (mV) -22.3 k 1.8 -20.6 k 2.3
Isc (fi/cm*) +5.a r 0.3 +5.1 + 0.4
and Tissue Injury of
Postacid
Initial Solution
Vol. 101. No. 5
R(0/cm*) 38815 373 4045 + 363
PD (mV) +2.5 k 0.3" +0.8 2 0.3
Isc (+4/cm’) -0.8 k 0.1 -0.2 -c 0.1
R(Ucm2) 4372 ?444 4312 2 417
Injury
score
0.7 + 0.3" 2.1 + 0.2
NOTE. Values are expressed as means ? SEM. Initial values are similar and R high for all tissues because of mounting in Cl-free Ringer’s solution. Serosal acidification was performed with HCl for Cl-containing solutions and with H,SO, for Cl-free solutions. After acid exposure for 1 hour, the small differences in PDs reflect small differences in diffusion and/or junction potentials between Cl-containing and SO,-containing serosal solutions and their respective mucosal solutions containing isethionate as Cl replacement. “P < 0.05 compared with Cl-containing solutions.
November
1991
MECHANISMS
OF ACID INJURY TO RABBIT
ESOPHAGUS
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Figure 7. A representative example of the effect of serosal acidification in (A) Cl-free solution (acidification to pH 2 with H,SO,) or (B) Cl-containing solution (acidification to pH 2 with HCl) on the morphology of esophageal epithelium. Tissues exposed to serosal acid in Cl-free solution show mild edema but were protected against the cell necrosis evident in the lower layers of esophagus exposed to acid in Cl-containing solution (H&E; original magnkcation x 200).
are greater rates of H’ entry into esophageal cells across the BLM, then mechanisms for H’ transport on the BLM must be present that are not present on the AM of esophageal cells. Two such candidate mechanisms present in stratified squamous epithelia are the amiloride-sensitive Na/H antiport and SITS(DIDS)Both antiports sensitive Cl/HCO, antiport (17,18). have now been identified in esophageal cells of the rabbit (1%21), and although neither has been localized to BLM or AM, both are capable of moving H’ across the membrane and into the cell. [The Cl/HCO, antiport normally operates as a cell acidifier by transporting HCO, out of the cell in exchange for Cl, whereas the Na/H antiport, which normally operates as a cell alkalinizer, can be reversed by changing ion gradients to favor H’ entry in exchange for Na movement out of the cell (22,23).] Documentation for participation of these mechanisms in cell acidification during serosal (BLM) acidification has been provided by the studies of Starlinger et al. in isolated rabbit gastric glands (24). They observed that blockade of the Cl/HCO, antiport either alone (by SITS or Cl-free solution) or in combination with blockade of the Na/H antiport (NaCl-free solution) was associated with limited cell acidification as pH of the external bathing solution was lowered from 7.8 to 6.4. In the present investigation, esophageal epithelia treated with SITS, alone or in combination with amiloride, but not amiloride alone, were protected against damage upon exposure to serosal HCl. Because these results were consistent with the concept that cell necrosis was mediated by H+ entry through a Cl/HCO, antiport located on the BLM, we also tested the ability of a Cl-free environment (a maneuver that, like SITS, inhibits Cl/HCO, exchange) to block the damage associated with serosal acidification. As shown in Figure 7, serosal acidification (with H,SO, to maintain the Cl-free environment) produced less damage to esophageal epithelia than Cl-containing solutions acidified to the same degree and for similar lengths of time. These results add further support to
the concept that serosal acidification damages esophageal tissues by cell acidification and that the pathway for cell acidification across the BLM appears to involve a SITS-sensitive, Cl-dependent pathway such as the Cl/HCO, antiport. * References 1. Orlando
acid
RC, Powell DW, Carney CN. Pathophysiology of acute injury in rabbit esophageal epithelium. J Clin Invest
1981;68:286-293. 2. Orlando
3.
4.
5.
6.
7. 8. 9.
10.
RC, Bryson JC. Powell DW. Mechanism of H’ injury in rabbit esophageal epithelium. Am J Physiol 1984;246:G718G724. Carney CN, Orlando RC. Powell DW. Dotson MM. Morphologic alteration in early acid-induced epithelial injury of rabbit esophagus. Lab Invest 1981:45:198-208. Tobey NA. Powell DW, Schreiner VJ, Orlando RC. Serosal bicarbonate protects against acid injury to rabbit esophagus. Gastroenterology 1989;96:1466-1477. Read NW, Fordtran JS. The role of intraluminal junction potentials in the generation of the gastric potential difference in man. Gastroenterology 1979;76:932-938. Soltoff SP, Mandel LJ. Amiloride directly inhibits the Na,KATPase activity of rabbit kidney proximal tubules. Science 1983;220:957-959. Salo J, Kivilaakso E. Role of luminal H’ in the pathogenesis of experimental esophagitis. Surgery 1982;92:61-68. Chung RSK, Magri JK. DenBesten L. Hydrogen ion transport in the rabbit esophagus. Am J Physiol 1975:229:496-500. Harmon JW, Johnson LF, Maydonovitch CL. Effects of acid and bile salts on the rabbit esophageal mucosa. Dig Dis Sci 1981:26: 65-72. Silen W. Gastric mucosal defense and repair. In: Johnson LR, Christensen J, Jacobson ED, Jackson MJ, Walsh JH, eds. Physiology of the gastrointestinal tract. Volume 2. 2nd ed. New York: Raven, 1987:1055-1069.
*Whereas the concept that cell acidification can destroy cells is not in doubt, the levels at which lowering pH, become noxious are likely to vary among tissues. Arvidsson et al. (25) lowered pH, in secreting frog oxynticopeptic cells to pH 6.4 through acidification of the BLM but failed to show structural (normal histology) or functional [normal PD and R) evidence of damage. However, when pH, was similarly reduced in nonsecreting cells (cell secretion inhibited by omeprazole or cimetidinel. there was extensive and irreversible tissue damage.
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11. Ashley SW, Soybel DI, Moore D, Cheung LY. Intracellular pH (pH,) in gastric surface epithelium is more susceptible to serosal than mucosal acidification. Surgery 1987;102:371-379. 12. Flemstrom G. Gastric and duodenal mucosal bicarbonate secretion. In: Johnson LR, Christensen J, Jacobson ED, Jackson MJ, Walsh JH, eds. Physiology of the gastrointestinal tract. Volume 2.2nd ed. New York: Raven, 1987:1011-1029. 13. Hamilton BH. Orlando RC. In vivo alkaline secretion by mammalian esophagus. Gastroenterology 1989;97:640-648. 14. Lacy ER, Tobey NA, Cowart K, Orlando RC. The esophageal mucosal barrier: structural correlates (abstr). Gastroenterology 1989;96:A281. 15. Quigley EMM, Turnberg LA. pH of the microclimate lining the human gastric and duodenal mucosa in vivo-studies in control subjects and in duodenal ulcer patients. Gastroenterology 1987;92:1876-1884. 16. Sanders MJ, Ayalon A, Roll M, Sol1 AH. The apical surface of canine chief cell monolayers resists H’ back-diffusion. Nature 1985;313:52-54. 17. Civan MM, Cragoe EJ, Peterson-Yantorno K. Intracellular pH in frog skin: effects of Nat, volume and CAMP. Am J Physiol 1988;255:F126-F134. 18. Drewnowska K, Cragoe EJ, Biber T. pH in principal cells of frog skin (Rana pipiens): dependence on extracellular Na+. Am J Physiol 1988;255:F930-F935. 19. Tobey NA, Khalbuss WE, Keku TO, Orlando RC. Cll/HCO; antiport activity in rabbit squamous and basal esophageal cells in primary culture (abstr). Gastroenterology 1991;100:706.
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20. Tobey NA, Khalbuss WE, Keku TO, Reddy SP, Hamilton BH. Orlando RC. Nat/H+ antiport activity and intrinsic buffering capacity in rabbit squamous and basal esophageal cells in primary culture (abstr). Gastroenterology 1991;100:707. 21. Agnone LM, Schmidt LN, Goldstein JL, Layden TJ. Regulation of rabbit esophageal cell pH by a Nat/H+ antiport (abstr). Gastroenterology 1989;96:A3. 22. Roos A, Boron WF. Intracellular pH. Physiol Rev 1981:61:296434. 23. Boron WF. Intracellular pH regulation in epithelial cells. Ann Rev Physiol 1986;48:377-388. 24. Starlinger M, Paradiso AM, Machen TE. Steady state regulation of intracellular pH in isolated rabbit gastric glands. Gastroenterology 1987;92:957-965. 25. Arvidsson S, Carter K. Yanaka A, Ito S, Silen W. Effect of basolateral acidification on the frog oxynticopeptic cell. Am J Physiol 1990;259:G564-G570.
Received June 27,199O. Accepted April 16,1991. Address requests for reprints to: Roy C. Orlando, M.D., Division of Digestive Diseases, CB no. 7080, 324 Burnett Womack Building, Chapel Hill, North Carolina 27599-7080. The project was supported in part by National Institutes of Health grant no. 2 ROl-DK-36013-04. The authors thank Virginia J. Schreiner, Angela D. Semones, and Lara E. Glenn for their expert technical assistance.