Lumen-to-surface pH gradients in opossum and rabbit
esophagi: role of submucosal glands
Solange
Abdulnour-Nakhoul,
Nazih L.
Nakhoul, and
Roy C.
Orlando
Departments of Medicine and Physiology, Tulane University School of
Medicine, and Veterans Administration Medical Center, New Orleans,
Louisiana 70112-2699
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ABSTRACT |
The opossum esophagus, like that of humans, contains a
network of submucosal glands with the capacity to secrete bicarbonate ions into the esophageal lumen. To evaluate the role of these glands in
protecting the epithelial surface from acid insult, we measured the
lumen-to-surface pH gradient in opossum esophagus at different luminal
pH and compared it to that of rabbit esophagus, an organ devoid of
submucosal glands. Sections of opossum and rabbit esophageal epithelium
were mounted luminal side up in a modified Ussing chamber. pH-sensitive
microelectrodes, positioned within 5 µm of the epithelial cell
surface, were used to monitor surface pH during perfusion with
solutions of different pH. At luminal pH 7.5, the pHs of
both opossum and rabbit were similar (pHs = 7.5). Lowering
luminal pH from 7.5 to 3.5 in opossum decreased pHs to 4.2 ± 0.16, a value significantly higher than pH of perfusate, whereas in
rabbit this maneuver decreased pHs to 3.69 ± 0.08, a
value not significantly different from pH of perfusate. In opossum but
not in rabbit, addition of carbachol to the serosal solution increased
basal pHs to 7.8 ± 0.1 and significantly blunted the decline in pHs on perfusion with acidic Ringer solution (pH
3.5), with pHs falling to 5.6 ± 0.45. The effect of
carbachol on surface buffering was inhibited by prior treatment with
atropine. Luminal acidification to pH 2.0 in opossum (as in rabbit)
abolished the lumen-to-surface pH gradient even after addition of
serosal carbachol. We conclude that the presence of submucosal glands
in esophagus contributes through bicarbonate secretion to creation of a
lumen-to-surface pH gradient. Although this gradient can be modulated
by carbachol, its capacity to buffer (and therefore to protect) the
epithelial surface against back-diffusing H+ is limited and
dissipated at pH 2.0.
carbachol; alkaline secretion; microelectrodes; acid
reflux
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INTRODUCTION |
ONE OF THE DEFENSES that upper gastrointestinal
epithelia use for protection against injury by high concentrations of
luminal acid is the establishment and maintenance of a lumen-to-surface pH gradient. This gradient protects by limiting the degree of acidity
that comes in contact with surface epithelial cells. Multiple factors
determine the magnitude and resilience of the gradient among species
and among organs within a species, but the most important factors
appear to be the capacity of the tissue to secrete mucus and
bicarbonate. It is therefore not surprising that the stomach and
duodenum, with both mucus and bicarbonate-secreting surface epithelial
cells, have been shown in both animals and humans to maintain a sizable
lumen-to-surface pH gradient in the presence of high luminal acidity
(1, 6, 7, 12, 17, 18, 20). In contrast to stomach and duodenum, the
moist stratified squamous epithelium lining the esophagus has neither
mucus nor bicarbonate-secreting cells and as such appears to be
disadvantaged in its capacity to produce or maintain a sizable
lumen-to-surface pH gradient. However, humans and some animal species
like opossum have submucosal glands (SMGs) within the esophageal wall
(2, 8), which are reported to secrete both mucin and bicarbonate into
the esophageal lumen (4, 9, 11, 15, 16). However, the role of the
secretions from these glandular structures to create and support a
lumen-to-surface pH gradient in esophagus has not been evaluated yet.
We therefore characterized the magnitude of the lumen-to-surface pH
gradient in the opossum, a species with a rich network of submucosal
glands, and compared its capacity for protection against luminal
acidity to that of the rabbit, a species whose esophagus is devoid of
such glands. The results of this comparison demonstrate that the
presence of SMGs, through bicarbonate secretion, contributes to the
ability of the esophagus to limit back-diffusing luminal acid from
reaching the epithelial surface; however, this capacity is limited and
can be overwhelmed at low pH.
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METHODS |
Animal and tissue preparation.
American opossums (Northeastern Wildlife, South Plymouth, NY) and New
Zealand White rabbits were killed by administration of an intravenous
overdose of pentobarbital sodium (60 mg/ml). The esophagus was excised,
opened longitudinally, and pinned mucosal side down in a paraffin tray
containing ice-cold oxygenated Ringer. The muscle layers were lifted up
with forceps, and the underlying mucosa was dissected free with a
scalpel. The sheet of mucosa obtained was cut, and a section was
mounted apical side up, horizontally in a modified Ussing chamber with
an aperture of 1.13 cm2. The chamber allowed continuous and
independent perfusion of the apical and the serosal sides of the
tissue. The fluid for the perfusion of the tissue was delivered by
gravity, and all solutions were placed at the same height from the
chamber. The perfusion solutions could be switched quickly and with
minimal dead space by means of a combination of six-way rotary valves and four-way pneumatically activated slider valves (Rainin, Emeryville, CA). In this arrangement, one of six experimental solutions could flow
to each side of the chamber. The volume of fluid in the upper and in
the lower sides of the chamber was ~0.25 ml. The rate of flow of the
solution to the upper chamber was 2 ml/min, which yielded a turnover
rate (i.e., time for a solution to be washed out completely from the
chamber) of ~8 s. Perfusion fluid to the lower part of the chamber
was delivered at 2 ml/min; however, only part of this fluid (~1
ml/min) entered the chamber, the rest leaving through a drain that was
kept at a fixed level. The turnover rate in the lower side of the
chamber was ~15 s. The solutions were prewarmed and delivered to the
chamber at 37°C.
Electrodes.
Transepithelial potential difference (VTE) was
measured as the voltage difference between a free-flowing KCl electrode
(tip <10 µm) placed in the bath fluid of the serosal side and a
similar electrode placed in the bath fluid of the apical side. Both
electrodes were fitted with an Ag-AgCl wire, and the leads were
connected to the amplifier of a voltage clamp (Physiologic Instruments, San Diego, CA). The voltage clamp was also used to deliver a
direct-current pulse of 5-15 µA via platinum wires located in
each side of the chamber. This allowed us to determine the
transepithelial resistance (RTE) from the voltage
deflection (
VTE) as follows
where
I is current. Given the use of free-flowing KCl electrodes to
measure VTE, junction potentials produced during
exposure to solutions of varying composition, including pH 2.0 Ringer
solution, were negligible and therefore were not corrected for.
The pH-sensitive microelectrodes were of the liquid ion exchanger type.
Alumino-silicate glass tubes (1.2 mm OD × 0.86 mm ID; Frederick
Haer, Brunswick, MD) were pulled on a vertical microelectrode puller
(David Kopf, Tujunga ,CA) to a tip of ~1-2 µm and dried in an
oven at 200°C for 2 h. The electrodes were exposed in a closed
vessel to tri-n-butyl-chlorosilane fumes for 2 min, after which
the silane fumes were vented, and the electrodes were left in the oven
for an additional 30 min. The exchanger (Fluka, Hydrogen Ionophore II,
cocktail A) was then introduced into the tip of the electrodes
by means of a very fine glass capillary. The electrodes were then
backfilled with a buffer solution containing 0.04 M KH2PO4, 0.023 M NaOH, and 0.015 M NaCl, pH 7.0 (3) and calibrated in standard buffer solutions of pH 2, 4, and 7. The
average slope was 57.8 ± 0.4 mV/pH unit (n = 15).
For measurements of pH at the epithelial surface (pHs), the
pH-sensitive electrode was advanced using a motorized micromanipulator (Merhauzer, Fine Science Tools) and under microscopic control at
×140 (Stereozoom7, Leica) to a position immediately above, but
without touching, the tissue. The pH electrode was then further advanced using the micromanipulator until the electrode was observed microscopically to touch the apical membrane of a surface cell, an
event that was accompanied by an increase in the magnitude of the
direct- current pulses. The tip size of the microelectrode was big
enough not to allow intracellular impalements of the epithelial cells.
The pH electrode was then withdrawn in steps of 1-2 µm until the
direct-current pulse returned to that observed in free solution (12).
This permitted localization of the pH electrode within 5 µm of the
epithelial cell surface, a position that was maintained throughout the
course of the experiments. All electrical and pH readings were recorded
on a three-channel strip chart recorder (Kipp & Zonen, Bohemia, NY).
Solutions.
The composition of the solutions is shown in Table
1. The chemicals were obtained from Sigma.
Statistical analysis.
The results are presented as means ± SE. Data were analyzed using the
two-tailed paired Student's t-test unless otherwise indicated;
n is the number of observations. The initial rates of
pHs change were determined from the slope of a linear
regression fit of pHs vs. time. Only the initial portion of
pHs change was taken, which amounted to 30-40 s,
during which the data could be tightly fit to a straight line.
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RESULTS |
Both the opossum and rabbit esophagi are lined by a moist stratified
squamous epithelium; however, the opossum esophagus contains an
extensive network of tubuloacinar SMGs (8, 13, 14), whereas that of the
rabbit is completely devoid of SMGs (Fig. 1). Using pH-selective microelectrodes
positioned within 5 µm of the esophageal epithelium, we measured
pHs in both rabbit and opossum esophagi and monitored the
changes during luminal perfusion with acidic Ringer solutions of pH 3.5 or 2.0 (Table 1). RTE and VTE
were monitored as well.

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Fig. 1.
Light micrographs (×100) of opossum (A) and rabbit
(B) esophageal mucosa. Although both esophagi possess
stratified squamous epithelia (uppermost layer), only the
opossum esophagus shows an extensive network of submucosal glands
(arrows).
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Resting condition.
Esophageal tissues obtained from six opossums and five rabbits were
perfused with control Ringer (pH 7.5) in the lumen and in the serosal
bath. Under basal conditions in opossum, pHs was 7.5 ± 0.03, VTE was
7.6 ± 0.94 mV, and
RTE was 1,616 ± 214
· cm2 (n = 13, 1-3
tissues were used from each animal). In the rabbit, pHs was
7.5 ± 0.04, VTE was
12.0 ± 0.94 mV, and
RTE was 2,001 ± 157
· cm2 (n = 7). Both
RTE and VTE were significantly
higher in the rabbit than in the opossum (P < 0.004, unpaired
t-test).
Effect of luminal acidification to pH 3.5.
After baseline recordings, luminal pH was lowered by switching the
luminal solution to acidic Ringer, pH 3.5. In the opossum, this
maneuver resulted in a small depolarization of VTE
(1.9 ± 0.4 mV, P < 0.001) but no significant change in
RTE. However, acidification of the lumen resulted
in a progressive decline in pHs, over 7.4 ± 1.14 min,
to a new pHs of 4.2 ± 0.16 (Fig.
2, segment ab). The initial rate of
decline in pHs during perfusion with pH 3.5 was 1.78 ± 0.48 pH/min, and
pHs was 3.3 ± 0.15 pH units. Thus
pHs at the new steady state was 0.64 ± 0.16 pH units higher than that of the luminal perfusate pH 3.5 (n = 13, P < 0.002). When the luminal bathing solution was switched
back to bicarbonate-Ringer solution, pH 7.5, the changes were
reversible and pHs returned to its previous baseline value
(Fig. 2, segment bc).

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Fig. 2.
Tracing of an experiment showing the changes in surface pH
(pHs) (top trace) and transepithelial potential
difference (VTE) (bottom trace) of the
opossum esophagus on exposure of the lumen to a Ringer solution of pH
3.5 in the absence (segment ab) and in the presence of
carbachol (segment ef) and in the presence of carbachol and
atropine (segment hi). The addition of carbachol to the serosal
bath caused an increase in pHs (segment de). Also,
in the presence of carbachol, the decrease in pHs and the
rate of acidification were smaller than in control. In the presence of
atropine, the effect of carbachol is partially inhibited. The break in
the tracing is ~20 min.
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In the rabbit esophagus, perfusion of the lumen with acidic Ringer, pH
3.5 (solution 2), produced no change in VTE
(
12.8 ± 1.25 mV) or RTE (1,928 ± 180
· cm2; n = 7, P
> 0.05). However, pHs declined at an initial rate of 2.58 ± 0.78 pH/min, by 3.81 ± 0.07 units (n = 6) to reach a
plateau value of 3.69 ± 0.08 (Fig. 3,
segment ab). This latter pHs value was not
significantly different from pH 3.5 of perfusate (plateau reached
within 5.1 ± 1.01 min). These changes were reversible on switching
back the luminal solution to a control Ringer solution (Fig. 3,
segment bc).

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Fig. 3.
Tracing of an experiment showing the changes in pHs
(top trace) and VTE (bottom trace)
in the rabbit esophagus on exposure of the lumen to a solution of pH
3.5 in the absence (segment ab) and the presence of carbachol
(segment ef). In the presence of carbachol, both the decrease
in pHs and the rate of acidification are not significantly
different from control. This is in contrast to the findings in the
opossum (see Fig. 2).
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Effect of carbachol on pHs.
Carbachol, a cholinergic agonist, has been reported to stimulate
bicarbonate secretion in the opossum esophagus (10). Therefore, the
effect of serosal carbachol on pHs was studied in the
opossum and, for comparison, in the rabbit. Perfusing the serosal side of opossum esophagus with a Ringer solution containing carbachol (10
7 M) increased pHs by 0.3 ± 0.11 pH units (Fig. 2, segment de), decreased
RTE by 236 ± 49
· cm2, and had no effect on
VTE (n = 9, P < 0.01). These data
are summarized in Fig. 4. In contrast to
the opossum, the addition of carbachol to the serosal bath of rabbit
esophagus had no effect on pHs (pHs remaining
7.5 ± 0.1; Fig. 3, segment de), RTE
(2,043 ± 125
· cm2), or
VTE (
12 ± 0.95 mV; n = 9, P > 0.05). These data are summarized in Fig. 4.


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Fig. 4.
Effect of carbachol on steady-state pHs (A),
VTE (B), and transepithelial resistance
(RTE; C) in the opossum and rabbit
esophagi. In the opossum but not in the rabbit, addition of carbachol
(10 7 M) to the serosal bath increased
pHs and decreased RTE.
VTE remained unchanged. * Significantly
different from control (P < 0.05, paired data);
§ significantly different from opossum (P < 0.05, unpaired t-test).
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Effect of luminal acidification to pH 3.5 in the presence of
carbachol.
When tissues perfused with carbachol in opossum were subsequently
exposed to acidic Ringer, pH 3.5, pHs dropped only by 2.2 ± 0.36 pH units, a value significantly smaller than the decline in
pHs of 3.27 ± 0.23 (P < 0.01, n = 8)
observed in the absence of carbachol (Fig. 2, segment ef, and
Fig. 5). Hence, at luminal pH 3.5, pHs in opossum was 5.6 ± 0.45 vs. 4.2 ± 0.24 in the
absence of carbachol (n = 8, P < 0.01). Also, in the presence of carbachol, the initial rate of
acidification was slower, at 0.58 ± 0.2 pH/min, than in its absence
(1.78 ± 0.48 pH/min), and the time it took to reach the new
steady-state value for pHs was longer (11.5 ± 0.5 min vs.
7.4 ± 1.14 min) than in the absence of carbachol (n = 8, P < 0.03 for each comparison). pHs values at
defined time points of 1, 3, and 5 min after acidification are shown in
Table 2. Luminal perfusion with pH 3.5 in
the presence or absence of carbachol resulted similarly in a
depolarization of VTE of 2.32 ± 0.77 mV and no
change in RTE.

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Fig. 5.
Changes in pHs ( pHs, A) and initial
rate of acidification (B) of the opossum and rabbit esophagi on
exposure to acidic Ringer of pH 3.5 in the absence or the presence of
carbachol. In the opossum, in the presence of carbachol, the decline in
pHs was significantly smaller than the decline in
pHs observed in the absence of carbachol. Also, in the
presence of carbachol, initial rate of acidification was slower than in
its absence. In the rabbit, decrease in pHs was not
significantly different from that observed in the absence of carbachol.
Also, in the rabbit, there was no difference in the initial rate of
decline of pHs at luminal pH 3.5 in the presence of
carbachol compared with its absence. * Significantly different from
control (P < 0.05, paired data); § significantly
different from opossum (P < 0.05, unpaired t-test).
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Table 2.
Surface pH in the opossum at defined time points after luminal
acidification to pH 3.5 in the absence and presence of carbachol
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In contrast to the opossum, luminal exposure to pH 3.5 in the rabbit,
in the presence of serosal carbachol, resulted in a decrease of
pHs to 3.9 ± 0.18 (Fig. 3, segment ef), a value
not significantly different from that observed in the absence of
carbachol (pHs = 3.7 ± 0.1) or from that of the luminal
perfusate (n = 5, P > 0.05 for both comparisons; Fig.
5). Also, in the rabbit, there was no difference in the initial rate of
decline of pHs at luminal pH 3.5 (2.58 ± 0.78 pH/min) or
time to plateau (4.9 ± 1.12 min) in the presence of carbachol
compared with the values recorded in its absence (P > 0.05 for each comparison; Fig. 5).
Because serosal carbachol had a marked effect on the lumen-to-surface
pH gradient in opossum, we assessed the effect of atropine, a
cholinergic antagonist, on this parameter. This was done by simultaneously exposing the tissue to both serosal carbachol and atropine (10
6 M) and then acidifying the
luminal bath with Ringer at pH 3.5. As shown in Fig. 2, the ability of
carbachol alone to reduce the rate and degree of decline in
pHs with luminal acidification (segment ef) was
markedly reduced by the addition of atropine to the serosal solution
(segment hi). Indeed, in the presence of atropine, the change
of pHs and the rate of acidification in response to pH 3.5 in the lumen were similar to the changes obtained in control conditions. Thus, in tissues from four opossums when the lumen was
acidified to pH 3.5, pHs decreased to 4.0 ± 0.09 in control, 6.1 ± 0.54 in the presence of carbachol (n = 4, P < 0.05), and 5.1 ± 0.6 for atropine plus
carbachol. This is reflected in different
pHs shown in
Fig. 6. Also observed is that the
initial rate of acidification in the presence of atropine (1.1 ± 0.61 pH/min) was not significantly different from the rate of 1.3 ± 0.4 pH/min in the control period (Fig. 6).

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Fig. 6.
Changes in pHs (A) and the rate of acidification
(B) of the opossum esophagus on exposure of the lumen to a
solution of pH 3.5 in the absence or presence of carbachol and in the
presence of carbachol and atropine. Although in the presence of
carbachol pHs and the rate of acidification were
significantly smaller than control, the addition of atropine (1 µM)
reversed, at least partially, the effect of carbachol.
* Significantly different from control (P < 0.05, paired
data).
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Effect of luminal acidification to pH 2.0 in the absence and
presence of carbachol.
Because the opossum esophagus maintained a significant lumen-to-surface
pH gradient during luminal perfusion at pH 3.5, we investigated its
ability to handle an increased acid load by perfusing the lumen with an
acidic Ringer at pH 2.0, in both the presence and absence of serosal
carbachol. In the opossum, when the luminal perfusate was switched to
an acidic Ringer at pH 2.0, there was a rapid decline in
pHs, which reached a value similar to that of the
perfusate (
pHs of 5.43 ± 0.15 units) over a period of 2.5 ± 0.67 min (Fig. 7, segment
ab). Although the presence of carbachol was able to reduce the
initial rate of surface acidification (1.70 ± 0.5 pH/min);
compared with its absence (3.45 ± 0.9 pH/min; n = 4, P < 0.05), acidifying the luminal solution to pH 2.0 in the
presence or absence of carbachol resulted in similarly substantial declines in pHs (5.45 ± 0.2 units vs. 5.6 ± 0.28 pH
units, respectively; n = 4, P > 0.05) such that both
reached new steady-state values that were not significantly different
from the luminal perfusate of pH 2.0 (Fig. 7, segment ef).
These data are summarized in Fig. 8. In
these experiments, it was also noted that luminal perfusion at pH 2.0 decreased RTE significantly from 1,347 ± 244 to
1,239 ± 240
· cm2 (P < 0.03, n = 9) in the absence of carbachol and from 1,449 ± 302 to 1,166 ± 262
· cm2 (P < 0.03, n = 7) in the presence of carbachol.

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Fig. 7.
Tracing of an experiment showing the changes in pHs
(top trace) and VTE (bottom trace)
in the opossum esophagus on exposure of the lumen to a solution of pH
2.0 in the absence and in the presence of carbachol. When the luminal
perfusate was switched to an acidic Ringer at pH 2.0, there was a rapid
decline in pHs, which reached a value similar to that of
the perfusate within ~3 min (segment ab). Although the
presence of carbachol was able to reduce the initial rate of surface
acidification compared with its absence, acidifying the luminal
solution to pH 2.0 in the presence (segment ef) or absence of
carbachol (segment ab) resulted in similarly substantial
declines in pHs. Inset: superimposed tracings
showing the initial decrease in pHs in response to luminal
pH 2.0 in the absence and presence of carbachol.
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Fig. 8.
Changes in pHs (A) and the rate of acidification
(B) of the opossum and rabbit esophagi on exposure to a
solution of pH 2.0 in the absence and presence of carbachol. In the
opossum, although the presence of carbachol was able to reduce the
initial rate of surface acidification compared with its absence,
acidifying the luminal solution to pH 2.0 in the presence or absence of
carbachol resulted in similarly substantial declines in pHs
within ~3 min from acidification. * Significantly different from
control (P < 0.05, paired data).
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In the rabbit, when the lumen was similarly perfused with acidic Ringer
(pH 2.0), pHs declined to 1.94 ± 0.17 in 1.7 ± 0.5 min
and at an initial rate of acidification of 3.43 ± 0.32 pH/min. Furthermore, these values in the rabbit were not altered by the presence of serosal carbachol: pHs declined to 2.3 ± 0.3 in 2.6 ± 1.3 min and at an initial rate of 4.13 ± 1.05 pH/min
(n = 5, P > 0.05 for each comparison).
These data are summarized in Fig. 8. In the rabbit, acidification of
the lumen to pH 2.0 hyperpolarized VTE by
2.7 ± 0.32 mV, whereas RTE remained unchanged.
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DISCUSSION |
In the present investigation, pH-sensitive microelectrodes were used to
measure pHs in two species, the opossum (submucosal glands
bearing) and the rabbit (submucosal glands free), under two conditions:
1) in the presence of two different levels of luminal acidity
(pH 3.5 and 2.0), and 2) in the presence of a known stimulant
of bicarbonate secretion by the submucosal glands.
As shown during luminal acidification at pH 3.5 of the rabbit
esophagus, the absence of SMGs results in little to no capacity to
protect the epithelium from being accessed by luminal acid even at this
relatively modest level of acidity. Thus luminal acidity at pH 3.5 resulted in the rapid decline in pHs until it reached a
value similar to that of the luminal perfusate.
In contrast to rabbit esophagus, the opossum esophagus responded to
luminal acid perfusion of pH 3.5 with a slower rate of acidification
and a longer time to reach a new steady-state pHs value
that was higher than that of the rabbit esophagus and significantly above that of the luminal perfusate by a little more than one-half a pH
unit. Because neither the rabbit nor the opossum stratified squamous
esophageal epithelium secretes mucus or bicarbonate, this indicates
that the superior lumen-to-surface pH gradient maintained by the
opossum esophagus is due either to secretion of bicarbonate (and/or
mucus, see below) by the submucosal glands or to greater passive
paracellular diffusion of bicarbonate from blood to lumen. This latter
possibility cannot be ignored, given that the transepithelial
electrical resistance of opossum esophageal epithelium is significantly
lower than that of rabbit and that this parameter is dominated by the
ion permeability across the paracellular route (19).
Documentation that secretion from the esophageal submucosal glands is a
major contributor to the lumen-to-surface pH gradient (and so
preepithelial defense) in opossum was established by serosal exposure to carbachol, a known stimulant of submucosal gland secretion. Notably, the addition of carbachol both raised the resting
pHs at luminal pH 7.5 and markedly enhanced the magnitude
of the lumen-to-surface pH gradient at luminal pH 3.5 in opossum
esophagus. In contrast, carbachol neither raised the resting
pHs at luminal pH 7.5 nor did it improve the ability of the
rabbit esophagus to establish a lumen-to-surface pH gradient in the
presence of luminal acid of pH 3.5. Carbachol has no effect on
esophageal stratified squamous epithelium (5), and so these
observations indicate that the source of the enhanced lumen-to-surface
pH gradient in opossum but not rabbit is due to its ability to
stimulate secretion (bicarbonate) by the SMGs. Furthermore,
bicarbonate, not mucin, is the likely factor in the secretion to be
responsible for these observations, since mucin secretion, by creating
a larger diffusion barrier, might slow the rate of H+
diffusion but would not probably change the ultimate pHs
reached on luminal acidification. In effect, the ability of carbachol to produce a new higher steady-state value for pHs over
that of the perfusate indicates that the secretion of bicarbonate from the submucosal glands was the likely component accounting for protection. Indeed, from the change in pHs, the increase in
buffer capacity produced by carbachol can be estimated at 1.75 times over basal levels. This increase in buffering power can be calculated using the formula
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where
carb and
control indicate the buffering
power of the surface layer in the presence of carbachol and in control
respectively. Moreover, Hamilton and Orlando (9) have shown that
carbachol stimulation increases the basal rate of bicarbonate secretion in the opossum esophagus from 0.39 ± 0.03 to 0.74 ± 0.07 µeq · h
1 · cm
2.
Assuming that this increase results in a proportionate increase in
bicarbonate concentration in the surface unstirred layer, on the basis
of the Henderson-Hasselbach equation, this increase in secretory rate
could account for an increase in pHs of ~0.27 pH units.
Thus our experiments document that the presence of submucosal glands
can contribute to and modulate the protective capacity of the
preepithelial defense against luminal acid and that such modulation by
cholinergic agonists is clearly absent in esophagi devoid of such
glands. Moreover, because atropine could block the enhancement in the
gradient produced by carbachol, it is evident that cholinergic
muscarinic receptors in the tissue mediate the response to carbachol in
vitro just as they have been documented to do in vivo (15).
The effect of exposing the rabbit and opossum esophagi to luminal pH
2.0 was also of interest. In the absence of carbachol, luminal pH 2.0 resulted in a similar pattern of acidification in the opossum as in the
rabbit esophagus, i.e., there was rapid equilibration of the
pHs to that of the luminal solution. When stimulated by
carbachol, however, the opossum esophagus was able to reduce the
initial rate of acidification but not stop the ultimate decline in
pHs to a value similar to that of the luminal perfusate. It
is conceivable that below pH 3.5 most of the secreted or diffused bicarbonate would be consumed by an overwhelming H+ influx,
causing an abrupt decrease in pHs. Nonetheless, together, these data suggest that, under both basal and stimulated conditions, the SMG-bearing esophagus (opossum) has a limited capacity to resist
acidification of its surface cells and that at luminal acidity in the
range of pH 2.0, which occurs commonly during episodes of
gastroesophageal reflux in humans, the protective capacity of the
lumen-to-surface pH gradient is overwhelmed.
In summary, this study establishes that the presence of submucosal
glands in the esophageal wall is an important contributor to the
preepithelial (buffering) defense against luminal acid. This defense is
generated in large measure through the ability of these glands to
secrete bicarbonate, and so the capacity for protection against luminal
acidity varies and is dependent in part on the degree of glandular
secretion to protect the epithelial surface against back-diffusing
H+. However, the capacity of the glandular secretion to
maintain a lumen-to-surface pH gradient is limited. In humans, whose
SMG-bearing esophagus is often exposed to luminal pH
2.0, additional intrinsic (epithelium proper) and extrinsic (acid clearance
mechanisms) esophageal defenses are necessary for protection against
gastroesophageal reflux disease.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grant 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: S. Abdulnour-Nakhoul, Dept. of Medicine, Section of Gastroenterology, SL
35, 1430 Tulane Ave., New Orleans, LA 70112-2699 (E-mail:
solange{at}mailhost.tcs.tulane.edu).
Received 24 June 1999; accepted in final form 6 October
1999.
 |
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