Acute adaptive cellular base uptake in rat duodenal
epithelium
Yasutada
Akiba2,5,
Osamu
Furukawa2,5,
Paul H.
Guth1,
Eli
Engel2,3,
Igor
Nastaskin4, and
Jonathan D.
Kaunitz1,2,5
1 Greater Los Angeles Veterans Affairs Healthcare System,
2 School of Medicine, and 3 Department of Biomathematics
and 4 College of Letters and Science, University of California
Los Angeles 90024; and 5 CURE: Digestive Diseases Research
Center, Los Angeles, California 90073
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ABSTRACT |
We studied the role of duodenal cellular ion transport
in epithelial defense mechanisms in response to rapid shifts of luminal pH. We used in vivo microscopy to measure duodenal epithelial cell
intracellular pH (pHi), mucus gel thickness, blood flow, and HCO
secretion in anesthetized rats with
or without the Na+/H+ exchange inhibitor
5-(N,N-dimethyl)-amiloride (DMA) or the anion transport inhibitor DIDS. During acid perfusion pHi
decreased, whereas mucus gel thickness and blood flow increased, with
pHi increasing to over baseline (overshoot) and blood flow
and gel thickness returning to basal levels during subsequent neutral solution perfusion. During a second brief acid challenge,
pHi decrease was lessened (adaptation). These are best
explained by augmented cellular HCO
uptake in
response to perfused acid. DIDS, but not DMA, abolished the overshoot
and pHi adaptation and decreased acid-enhanced
HCO
secretion. In perfused duodenum, effluent total
CO2 output was not increased by acid perfusion, despite a
massive increase of titratable alkalinity, consistent with substantial
acid back diffusion and modest CO2 back diffusion during
acid perfusions. Rapid shifts of luminal pH increased duodenal
epithelial buffering power, which protected the cells from perfused
acid, presumably by activation of
Na+-HCO
cotransport. This adaptation may
be a novel, important, and early duodenal protective mechanism against
rapid physiological shifts of luminal acidity.
intracellular pH; bicarbonate secretion; mucosal defense; mucus
secretion; mucosal blood flow
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INTRODUCTION |
THE DUODENAL MUCOSA IS
REGULARLY exposed to intermittent pulses of gastric acid, with
luminal pH varying rapidly between 2 and 7 (23). Without
protective mechanisms in place, the duodenal cells, like other cells in
the upper gastrointestinal tract, are believed to irreversibly acidify
in the presence of acidic luminal contents, injuring the epithelium
(4, 21). With the measurement of robust epithelial
HCO
secretion and a neutral pH in the
juxtamucosal mucus gel despite the presence of luminal acid, the
"bicarbonate hypothesis" was developed, wherein HCO
, secreted by the epithelial cells, completely
neutralized luminal acid diffusing through the mucus gel toward the
epithelium (9). Correlation of HCO
secretion with mucosal protection from acid-related injury further bolstered this hypothesis (10, 12, 30).
One means of defending the mucosa against rapid shifts of pH is the
phenomenon of acute adaptive protection, wherein exposure to a low
concentration of acid or other substance decreases injury from a
subsequent challenge with a higher concentration of the same or other
substance. This phenomenon, although extensively studied in
experimental gastroprotection models (13), has been investigated only twice in the duodenum (16, 17).
Furthermore, both prior studies addressing this phenomenon used injury,
and not alterations of defensive factors, as an endpoint. It is not known, for example, whether duodenal adaptive protective mechanisms primarily result from an enhancement of preepithelial
mechanisms such as increased HCO
secretion, from thickening of the overlying mucus gel, from postepithelial
mechanisms such as hyperemia, or from an augmentation of intrinsic
cellular mechanisms such as cellular buffering power. Furthermore, it
was previously assumed that preexposure to a low concentration of injurious substance was required for inducing adaptive changes. An
alternative explanation is that rapid shifts of luminal pH per se, and
not the "mild-strong" exposure sequence, is of primary importance
in inducing acid-related adaptive changes.
HCO
secretion is the most studied duodenal
defense mechanism. Despite its obvious appeal as a means of
neutralizing gastric acid, most studies have addressed its measurement
in the absence of luminal acid, when its acid neutralizing effect is
unnecessary. One purpose of this study was therefore to measure
HCO
secretion during acid exposure and to determine
its relative importance in mucosal defense in general and adaptive
protection in particular.
Our laboratory has recently developed a technique in which the
intracellular pH (pHi) and duodenal blood flow are measured simultaneously in the rat duodenum (3). We demonstrated
that a 10-min pulse of strongly acidic perfusate (pH 2.2) increased cellular buffering by a DIDS-sensitive mechanism, suggesting that rapid
shifts of perfusate pH enhanced resistance to a subsequent acid
challenge and, by extrapolation, injury. We hypothesized that rapid
shifts of perfusate pH enhance intrinsic cellular defense mechanisms
and further speculated that these pH shifts may underlie the phenomenon
of acute adaptive cytoprotection. We have also established a technique
for measurement of duodenal mucus gel thickness (MGT) in our
system, which we showed was affected by the balance between mucus
secretion and exudation (2).
We studied the effects of varying perfusate pH on duodenal mucosal
defense mechanisms to test the hypothesis that adaptive changes are
produced by rapid shifts of perfusate pH and that a major duodenal
protective mechanism is an increase of cellular buffering power induced
by the activation of epithelial ion transport.
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MATERIALS AND METHODS |
Chemicals
2',7'-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein
(BCECF) acid, BCECF-AM, and DIDS were obtained from Molecular Probes
(Eugene, OR). Two-micrometer pink fluorescent microspheres (excitation 575 nm, emission 600 nm) were obtained from Bangs Laboratories (Fishers, IN). 5-(N,N-dimethyl)-amiloride (DMA),
HEPES, and other chemicals were obtained from Sigma Chemical (St.
Louis, MO). Krebs solution contained (in mM) 136 NaCl, 2.6 KCl, 1.8 CaCl2, and 10 HEPES at pH 7.0. For acid perfusion, Krebs
solution was titrated to pH 6.4, 4.5, 3.5, or 2.2 with 0.2 or 1 N HCl
and adjusted to isotonicity (300 mM). Each solution was prewarmed at
37°C using a water bath, and temperature was maintained with a
heating pad during the experiment.
Experimental Protocol for In Vivo Microscopic Study
Animal preparation and measurement of pHi, blood
flow, and MGT were performed according to previously published methods
(1-3). After loading BCECF, applying fluorescent
microspheres on the gel surface, and blood flow stabilization with pH
7.0 Krebs buffer perfusion, time was set as t = 0. Perfusate pH was then varied in 10-, 15-, or 30-min time intervals as
described below.
Acid perfusion.
To examine the effect of sustained luminal acid on pHi,
blood flow, and MGT, the duodenal mucosa was perfused with pH 7.0 Krebs
buffer for 15 min, followed with either pH 7.0, 4.5, 3.5, or 2.2 solution for an additional 30 min (single acid challenge period),
followed by a 15-min recovery period with pH 7.0 Krebs buffer.
Sequential perfusion of acids of different concentrations.
Mild (pH 4.5) and strong (pH 2.2) acid concentrations were perfused
over the mucosa in 15-min increments. After a 15-min perfusion with pH
7.0 Krebs buffer, we exposed the mucosa to mild
strong or strong
mild acid concentrations, with exposure at each pH lasting
15 min, followed by a 15-min recovery period at pH 7.0. Thus exposure
to mild
strong acid would be as follows: pH 7.0 from
t = 0-15 min (baseline); pH 4.5 from
t = 15-30 min (the 1st acid challenge period); pH
2.2 from t = 30-45 min (the 2nd acid challenge
period); and pH 7.0 from t = 45-60 min (recovery
period). Adaptive changes are defined as differences in
pHi, MGT, and blood flow in a group in which perfusate pH
is changed at 15-min intervals with respect to groups in which the same
perfusate pH was held constant for 30 min.
Repeated acid exposure and effects of ion transport inhibitors.
In another experimental series, two pulses of strong acid were used to
provoke adaptive responses. Perfusate pH was changed from pH 7.0 for 10 min (t = 0-10 min) to pH 2.2 for 15 min (t = 10-25 min; the 1st acid challenge period), followed by pH
7.0 for 10 min (t = 25-35 min; the 1st recovery
period), followed by pH 2.2 for 15 min again (t = 35-50 min; the 2nd acid challenge period), and returned to pH 7.0 for 15 min (t = 50-65 min; the 2nd recovery
period). To determine the role of epithelial ion transport in
regulation of pHi, blood flow, and MGT, DMA (0.1 mM), which
inhibits Na+/H+ exchange, or DIDS (0.5 mM),
which inhibits Na+-HCO
cotransport and
HCO
/Cl
exchange, was added with the pH
2.2 perfusion during the first acid challenge period. Both inhibitors
exert their effects on the epithelial cells primarily on the serosal
membrane transporters (3). Adaptive changes are defined as
differences in pHi, MGT, and blood flow during the second
acid challenge compared with those during the first acid challenge.
Measurement of Duodenal Loop
HCO
Secretion
Preparation of the duodenal loop.
In a separate experiment, a duodenal loop was prepared and perfused to
measure duodenal HCO
secretion, as modified from
previously described methods (26, 27). In urethane-anesthetized rats, the stomach and duodenum were exposed and
the forestomach wall was incised 0.5 cm using a miniature electrocautery. A polyethylene tube (diameter 5 mm) was inserted through the incision until it was 0.5 cm caudal from the pyloric ring,
where it was secured with a nylon ligature. The distal duodenum was
ligated proximal to the ligament of Treitz before the duodenal loop was
filled with 1 ml saline prewarmed at 37°C. The distal duodenum was
then incised, and another polyethylene tube was inserted through the
incision and sutured into place. To prevent contamination of the
perfusate from bile or pancreatic juice, the pancreaticobiliary duct
was ligated just proximal to its insertion into the duodenal wall. The
resultant closed proximal duodenal loop (perfused length 2 cm) was
perfused with prewarmed saline by using a peristaltic pump (Cole-Parmer
Instrument, Vernon Hills, IL) at 1 ml/min. Input (perfusate) and
effluent of the duodenal loop were circulated through a reservoir in
which the perfusate was bubbled with 100% O2. The pH of
the perfusate was kept constant at pH 7.0 with a pH stat (models PHM290
and ABU901; Radiometer Analytical, Lyon, France).
Back titration.
HCO
secretion was measured by two complementary
methods: back titration and direct CO2 measurement. For
back titration measurements, the amount of 0.01 N HCl added to maintain
constant pH was considered equivalent to duodenal HCO
secretion. For pH 7.0 perfusion, a pH stat
technique was used with perfusate recirculation. Manual back titration
was used for pH 2.2 perfusates. Preliminary in vitro studies indicated
an excellent correlation between perfusate HCO
concentration and titratable base measured in the effluent (Fig.
1A). For duodenal
HCO
measurement, a 30-min stabilization with pH 7.0 saline (t =
35 to
5) was followed by baseline
measurements with pH 7.0 saline (t =
5 to 10). Acid
solution was perfused with a Harvard infusion pump at 1 ml/min. For
experiments involving repeated acid exposure, solutions were perfused
identically to the protocol described in Repeated acid exposure
and effects of ion transport inhibitors as follows: pH
7.0 saline was perfused for 10 min (t = 0-10;
baseline), followed by pH 2.2 saline for 15 min (t = 10-25; 1st acid challenge period), followed by pH 7.0 saline for
10 min (t = 25-35; 1st recovery period), followed
by pH 2.2 saline for 15 min (t = 35-50; 2nd acid
challenge period), and then pH 7.0 saline for 15 min (t = 50-65; 2nd recovery period). O2 gas-bubbled pH 7.0 saline was recirculated with a peristaltic pump, whereas pH 2.2 saline was perfused via syringe pump. The duodenal loop solution was gently
flushed with 5 ml of perfusate to rapidly change the perfusate composition at t = 10, 25, 35, and 50 min. Samples from
acid exposure periods were collected in tubes every 5 min and analyzed
for HCO
secretion by back titration to pH 2.2 with
0.1 N HCl.

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Fig. 1.
Calibration of HCO measurement
systems. A: pH stat. Standard HCO
solutions were circulated through the perfusion system tubes.
Alkalinity was inferred from the amount of HCl added to maintain
starting pH. B: CO2 electrode. A CO2
electrode as described in MATERIALS AND METHODS measured
total CO2 content of HCO solutions.
Lines denote the range of measured HCO
concentrations. Note that at the lowest concentration, a 33%
overestimate occurred, which disappeared when HCO
concentration exceeded 0.5 mM, compared with the line of identity
(dotted). All data are expressed as means of 2-3 experiments.
C: stability of CO2-containing solutions in an
open system. 10 mM NaHCO3 solutions were prepared, and
total dissolved CO2 concentration
([CO2]t) was measured with a CO2
electrode. At t = 0, HCl (0.1 M) was added to titrate
to pH 2.0, 4.5, or 7.0. [CO2]t was measured
at t = 5, 30, and 60 min.
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CO2 measurement.
To determine the relative contributions of HCO
secretion and H+ back diffusion during acid exposure, total
dissolved CO2 was measured with a CO2 electrode
(FK1501CO2; Radiometer America, Westlake, OH) connected to a pH meter
(PHM 62; Radiometer, Copenhagen, Denmark). The duodenal loops were
prepared and perfused as described in Preparation of the duodenal
loop. Every 5 min, effluent pH was measured immediately
after collection. One-half milliliter of 1 M citrate buffer (pH 4.5)
was then added to the sample (5 ml) to convert free
HCO
to CO2, followed by measurement of
electrode potential (mV) with the CO2 electrode. Total
dissolved CO2 concentration
([CO2]t) was calculated according to a
calibration curve using freshly prepared 0.1, 1, and 10 mM NaHCO3 solutions as standards, which generate 0.1, 1, and
10 mM [CO2]t, respectively, at pH
4.8-5.0. Since the calibration curve had a slope ~56 mV/log
[CO2]t within the range of 0.1-10 mM
at 20-25°C, all samples were analyzed at 25°C.
[CO2]t and pH in the perfusate provide
CO2 concentration ([CO2]) and
HCO
concentration ([HCO
]) from
the Henderson-Hasselbach formula
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(1)
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(2)
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Thus
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(3)
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(4)
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According to Henry's law
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(5)
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Substituting Eq. 3 into Eq. 5,
PCO2 can be calculated from
[CO2]t and pH
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(6)
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where pKa, the first dissociation constant for
carbonic acid in saline, is 6.2 at 25°C (25), and
Kh, the Henry solubility constant for
subsaturating CO2 concentrations in weak electrolyte solutions, is 0.047 mM/mmHg at 25°C (15). To examine the
accuracy of CO2 measurements through the perfusion system,
[CO2]t of the perfusate was measured in
O2-bubbled saline containing 0.1, 1, or 10 mM
NaHCO3 that was perfused through the system using the pump
and tubing used for duodenal perfusions. Neither addition nor loss of
CO2 was observed when the afferent
[HCO
] was above ~0.5 mM, but there was a 33%
overestimation at the lowest measured CO2 concentration
(0.21 mM), with no difference between measured and predicted
CO2 at the highest measured concentration (1.06 mM; Fig.
1B). Further experiments were done to test the stability of
dissolved CO2 over time in an open system. Ten-millimolar HCO
solutions were prepared, their [CO2]t was measured, they were titrated to pH
2.0, 4.5, or 7.0 at t = 0, and they were kept at 25°C
in the same containers used to collect the effluent. Aliquots were
removed at t = 5 min, 30 min, and 60 min. Figure
1C reveals that there was an initial decrease of
[CO2]t in the first 5 min, probably
reflecting liberation of CO2 during acid titration, but
[CO2]t was stable for 55 min thereafter.
Prostaglandin injection was used to augment HCO
secretion in the absence of acid as a positive control for duodenal
HCO
secretion. PGE2 (Oxford Biochemical,
Oxford, MI) was administered with a single intravenous injection (0.3 mg/kg) as previously described (26). Baseline
HCO
output, measured by pH stat, and total
CO2 output, measured by the CO2 electrode during pH 7.0 saline perfusion, were 0.10 ± 0.02 and 0.20 ± 0.01 µmol · min
1 · cm
1,
respectively. PGE2 increased HCO
and
CO2 output in parallel (0.39 ± 0.11 and 0.72 ± 0.17, respectively; P < 0.05), confirming that
titratable alkalinity and measured CO2 output increased
concordantly. Furthermore, to measure CO2 and
H+ back diffusion into the mucosa, solutions of nominal pH
2.2, 6.4, and 7.0 and varying [CO2]t were
perfused through the duodenal loop (Table
1). Net loss of H+ and
CO2 was calculated from changes of H+
concentration and [CO2]t between perfusate
and effluent, with PCO2 (mmHg) calculated from
pH and [CO2]t of the solutions according to
Eq. 6.
Statistics
All data from six rats in each group are expressed as means ± SE. Comparisons between groups were made by one-way ANOVA followed by Fischer's least significant difference test. Comparisons of two
time points were assessed by paired, one-tailed t-test.
P values of <0.05 were taken as significant.
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RESULTS |
Effect of Sustained Acid Perfusion
Blood flow and pHi were stabilized with a 1-h
perfusion of pH 7.0 Krebs buffer solution as previously described
(3). MGT was also stable during this period. Figure
2 depicts pHi, blood flow,
and MGT at baseline (t = 0) and 5 min after acid
exposure (t = 20). Acid exposure rapidly and
significantly decreased pHi to a new steady state during
acid perfusion (Fig. 2A), with return to baseline after acid
removal (Fig. 3). Steady-state
pHi was perfusate pH dependent (Fig. 2A). Blood
flow and MGT rapidly increased during perfusion with pH 3.5 or pH 2.2 but not during pH 4.5 perfusion (Fig. 2, B and
C).

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Fig. 2.
Effect of sustained acid perfusion on intracellular pH
(pHi), blood flow, and mucosal gel thickness (MGT).
pHi was measured at baseline (t = 0) and 5 min after acid perfusion (t = 20). pHi
decreased rapidly and significantly at t = 20, the
magnitude relative to perfusate pH (A). Blood flow (BF;
B) and MGT (C) increased during pH 3.5 or 2.2 perfusion but not perfusion at pH 4.5. *P < 0.05 vs.
pH 7.0 Krebs group. Values are means ± SE from 6 rats.
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Fig. 3.
Effect of mild strong or strong mild acid
exposure on pHi. In the mild strong group
( ), pHi during the 2nd acid challenge
period was higher than in the constant pH 2.2 group, followed by
pHi recovery to baseline and subsequent overshoot at 60 min
(A). In the strong mild group ( ),
pHi during the 2nd acid challenge period gradually
increased; an overshoot is observed at 45 min (B). Constant
pH 4.5 (pH 4.5-4.5; ) and constant pH 2.2 (pH
2.2-2.2; ) perfusion groups are shown for
comparison. *P < 0.05 vs. the corresponding constant
pH group. Values are means ± SE from 6 rats.
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Effect of Rapid Shifts of Luminal Acid
In the mild
strong group, pHi during the second
acid challenge period was higher than in the constant pH 2.2 group
(Fig. 3A). pHi recovery to baseline levels and
subsequent alkalinization were also observed after acid removal. No
adaptive change was seen in blood flow and MGT during the second acid
challenge period compared with the corresponding constant pH group
(data not shown).
In the strong
mild acid group, pHi gradually increased
after the perfusate was changed to pH 4.5, and alkalinization to above
the predicted levels occurred during the second acid challenge period
(Fig. 3B). No adaptive changes occurred in blood flow and MGT in the strong
mild acid group (data not shown).
Effect of Repeated Acid Exposure With or Without DMA
or DIDS
Since both the mild
strong and strong
mild perfusion
sequences produced adaptive pHi changes, we hypothesized
that rapid shifts of perfusate pH, but not a mild
strong sequence
per se, produced adaptation. To test this hypothesis, we exposed the
mucosa to two pulses of strong acid. Two 15-min acid pulses separated by a 10-min recovery period produced an overshoot during the recovery periods and an adaptive response during the second acid challenge, in
which the fall of pHi during the second acid challenge was attenuated (Fig. 4). DIDS but not DMA
exposure during the first acid challenge inhibited pHi
recovery during the first recovery period and abolished the
pHi adaptive response during the second acid challenge.
Blood flow increased during the first and second acid challenges (Fig.
5); DIDS had no effect on the blood flow response during the first or second acid challenges. DMA abolished the
hyperemic response during both acid challenge periods, similar to our
previous description of a single acid challenge (3). MGT
increased during the first acid challenge and further increased during
the second acid challenge (Fig. 6). DIDS
reduced the MGT response during the second acid challenge, whereas DMA
had no effect.

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Fig. 4.
Effect of repeated acid exposure with or without
5-(N,N-dimethyl)-amiloride (DMA) or DIDS on
pHi. Two acid pulses separated by a 10-min recovery period
( ) produced an overshoot after the 1st and 2nd acid
challenges at 35 and 55 min compared with the pH 7.0 Krebs group
( ). An adaptive response of pHi during the
2nd acid challenge at 40-50 min was observed. Although DMA
( ) and DIDS ( ) with pH 2.2 further
deceased pHi during the 1st acid challenge at 20 min, DIDS
but not DMA abolished the overshoot after the 1st acid challenge and
pHi adaptation during the 2nd acid challenge.
*P < 0.05 vs. pH 7.0 Krebs group; P < 0.05 vs. repeated pH 2.2 alone group by ANOVA; P < 0.05 vs. the corresponding time during the 1st acid challenge period
by paired t-test.
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Fig. 5.
Effect of repeated acid exposure with or without DMA or
DIDS on blood flow. Blood flow increased during the 1st and 2nd acid
challenge periods ( ). DMA ( ), but not
DIDS ( ), abolished the hyperemic response during both
acid challenge periods. *P < 0.05 vs. pH 7.0 Krebs
group; P < 0.05 vs. repeated pH 2.2 alone group by
ANOVA; P < 0.05 vs. the corresponding time during
the 1st acid challenge period by paired t-test. Values are
means ± SE from 6 rats.
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Fig. 6.
Effect of repeated acid exposure with or without DMA or
DIDS on MGT. Acid exposure increased MGT during the 1st acid challenge
and further increased during the 2nd acid challenge ( ).
Although DMA ( ) has no effect on MGT, DIDS
( ) reduced MGT response during the 2nd acid challenge.
*P < 0.05 vs. pH 7.0 Krebs group; P < 0.05 vs. repeated pH 2.2 alone group by ANOVA; P < 0.05 vs. the corresponding time during the 1st acid challenge period
by paired t-test. Values are means ± SE from 6 rats.
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Spontaneous HCO
secretion was measured during pH 7.0 saline perfusion (Fig. 7A).
Acid exposure increased titratable alkalinity to extreme levels during
the first and second acid challenges. During the recovery periods, acid loss in the pH 2.2 saline group was higher than in the pH 7.0 saline
group, consistent with post-acid augmented HCO
secretion. This stimulated secretion was reduced by DIDS but not by
DMA. Although DIDS had no effect on acid loss during the acid challenges, DMA reduced acid loss during the second acid challenge. Figure 7B depicts total CO2 output in the
perfusate of pH 7.0 saline and repeated acid exposure groups. Total
CO2 output stabilized during pH 7.0 saline perfusion but
decreased during both acid challenges and progressively increased
during both recovery periods. Decreased total CO2 output
compared with the extreme increase of titratable alkalinity during acid
exposures suggests that loss of luminal acidity during acid perfusion
may be due to H+ back diffusion rather than
HCO
secretion or CO2 loss. In contrast,
increased total CO2 output during the recovery periods was
consistent with post-acid augmented HCO
secretion.

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Fig. 7.
Effect of repeated acid exposure with or without DMA or
DIDS on effluent alkalinization (acid disappearance) and
[CO2]t. A: Back titration. Acid
increased titratable alkalinity during both challenges and during both
recovery periods ( ). Although DMA ( )
and DIDS ( ) had no effect on acid loss during the 1st
challenge period, DMA reduced acid loss during the 2nd acid challenge
and DIDS, but not DMA, reduced HCO secretion during
the recovery periods. B: CO2 measurements.
Separate experiments indicated that total CO2 output was
somewhat higher during neutral perfusion than was estimated by back
diffusion. During the 1st and the last point of the 2nd acid challenge
periods, CO2 output was suppressed, indicating that the
90-95% acid loss during this period resulted mostly from
H+ back diffusion. Inset:
PCO2 was calculated from
[CO2]t and pH (see eq. 6).
*P < 0.05 vs. pH 7.0 Krebs group; P < 0.05 vs. repeated pH 2.2 alone group by ANOVA. Values are means ± SE from 6 rats.
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H+ and
CO2 Back Diffusion in Duodenal Loops
Figure 8 depicts pH and
[CO2]t and calculated
PCO2 in the perfusates and effluents.
[CO2]t loss was 20.6-27.5% and
H+ loss was 12.3-27.5% as measured in the perfusate
and effluent, respectively. These data are similar to those of
Feitelberg et al. (6), in which
PCO2 in fixed pH perfusates (pH 5) collected from human proximal duodenal segments decreased 23 and 27% in solutions bubbled with 10 and 20% CO2, respectively.

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Fig. 8.
Changes of H+ concentration ([H+]) and
[CO2]t in perfusates and effluents of
different pH and [CO2]t solutions. Measured
pH, measured [CO2]t, and calculated
PCO2 of perfusates and effluents for solutions
of nominal pH 7.0, 2.2, and 6.4 are shown. The numbers over the closed
columns indicate %net loss of [CO2]t.
*P < 0.05 vs. perfusate in pH and
[CO2]t by ANOVA. Values are means ± SE
from 3 rats.
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DISCUSSION |
Simultaneous and parallel measurements of rat duodenal defenses in
vivo provided a useful means of examining the response of the mucosa to
luminal acid. Alteration of perfusate pH or a brief acid challenge
induced cellular base uptake, protecting the epithelial cells from
acidification during subsequent acid exposure. Responses of MGT and
blood flow to the acid challenges showed little such adaptation to the
second acid challenge or to a change of perfusate pH. Adaptive
pHi changes were abolished by DIDS, which had no effect on
blood flow but did prevent the increase of MGT during the second acid
challenge. Furthermore, DIDS inhibited the post-acid challenge
increases of HCO
secretion. Conversely, DMA
inhibited the blood flow increase during the first and second acid
challenges but had no effect on MGT or on HCO
secretion following acid challenge. Measurement of
HCO
secretion by back titration and total
CO2 measurement revealed that with pH 2.2 perfusate, most
acid disappearance was due to H+ back diffusion and not
alkaline secretion. Furthermore, DMA decreased the rate of acid back
diffusion, presumably by inhibiting the hyperemic response to acid
(3).
Augmented HCO
secretion following acid perfusion is
currently accepted as the most important duodenal defense mechanism
(7, 30). The mechanism by which HCO
secretion protects the mucosa has generally been assumed to be complete
neutralization of luminal acid diffusing toward the mucosa within the
mucus gel. Since duodenal epithelial cells readily acidify in the
presence of even moderate concentrations of perfused acid, it is
doubtful that complete neutralization of back-diffusing acid occurs in
the preepithelial mucus gel. If acid is able to enter the cells,
perhaps an alternate mucosal protective mechanism exists: intracellular
buffering or pHi regulation. If pHi regulation is important, then HCO
secretion may not need to
increase in the presence of luminal acid. This prospect prompted our
measurement of HCO
secretion during the same
repeated acid exposure that produced pHi adaptation. We
hence endeavored to ascertain the relative contributions of acid back
diffusion, of CO2 back diffusion, and of
HCO
secretion toward the disappearance of luminal
acid in duodenal perfusions. With the use of back titration, the amount
of titratable alkalinity increased 10-fold during acid perfusion, in
agreement with Flemström and Kivilaakso (8),
although the relative increase was greater than that found by Nylander
and colleagues (18, 19). To further study this increase of
titratable alkalinity during acid perfusion, we measured
[CO2]t. Measurement of
[CO2]t is a more direct means of measuring
HCO
secretion than is back titration since it has
the advantage of not being affected by secretion of other alkali or
loss of H+ due to back diffusion. The concordant increase
of effluent [CO2]t and titratable alkalinity
in PGE2-treated rats confirms the validity of these
measurements in the absence of luminal acid. As long as total
PCO2 is <10 mmHg at 25°C (the highest
[CO2]t was <2 mM in this study), the aqueous
solubility of CO2 is such that little loss of
CO2 to the atmosphere occurs over the measurement period, as we demonstrated in preliminary studies. The rapid perfusion technique has been applied successfully by Olbe and co-workers (5), who found that the accuracy of clinical gastric
HCO
secretion measurements with a CO2
electrode was improved by increasing the perfusion rate, which ensured
that CO2 concentrations would remain well below the Henry
solubility limit. Furthermore, the 20-30% loss of
[CO2]t and ~12-28% loss of
H+ between perfusate and effluent of duodenal perfusions is
in close agreement with published measurements of CO2 loss
from duodenally perfused HCO
solutions
(6), confirming that the duodenum has a measurable, finite
permeability to CO2 and H+. The rapid increase
of titratable alkalinity measured during acid perfusion was not
accompanied by a commensurate increase of effluent
[CO2]t, strongly consistent with our
hypothesis that HCO
secretion is not augmented
during acid perfusion but rather that luminal acid disappears via
H+ back diffusion. To explain these data, we hypothesized
that, during luminal acid perfusion, either: 1)
HCO
secretion increases ~1,000% within 5 min,
HCO
is converted by acid to CO2 gas,
>90% of the CO2 is lost due to back diffusion, and
HCO
secretion decreases to near baseline within 5 min, or 2) HCO
secretion is unchanged,
~25% of H+ is lost, and a further ~25%
CO2 is lost due to conversion to CO2 gas with
back diffusion. Since the data depicted in Fig. 8 and the work of
others indicate that CO2 loss, even in solutions with high
PCO2, in a rapidly perfused system is ~25%
and that increases and decreases of the rate of HCO
secretion generally develop over a 30- to 60-min time period, the
latter H+ back diffusion model fits most closely with the
data. Furthermore, we demonstrated that, even in an open system,
CO2 loss cannot account for the discrepancy between
[CO2]t measurements and acid disappearance.
These measurements add confidence to our contention that
HCO
secretion was not augmented, although
H+ back diffusion was substantially increased during
luminal acid perfusion.
Importantly, since the only time that HCO
secretion
is necessary for mucosal protection is during acid exposure, its lack
of increase by acid diminishes its importance as a mucosal defense
mechanism. During acid stress, it would seem more logical for the cell
to retain its protective HCO
buffer rather than
release it into the lumen, as would be predicted by models implicating
HCO
secretion during acid exposure as a major
defensive factor. From these studies, we could conclude that most of
the luminal acid loss during acid challenge is from H+ back
diffusion, suggesting that HCO
secretion of
itself cannot defend pHi against acid challenge and may
merely serve as an easily measurable sequelum to prior cellular base uptake.
The pHi response to repeated acid challenge differed
between the first and second challenges. During the first challenge, cells acidified more in the presence of DIDS and DMA. Following acid
challenge, DIDS attenuated pHi recovery and overshoot,
coincident with its inhibition of cellular base uptake. During the
second acid challenge, DIDS also abolished the adaptive pHi
response, although an early overshoot was observed. These observations
are in full agreement with those of Paimela et al. (20),
who noted that addition of the DIDS-related stilbene derivative SITS or removal of HCO
from the serosal bathing solution of
isolated Necturus duodenal mucosa increased cellular acidification during luminal acid challenge. Our interpretation differs
from theirs, however, in that we believe that the increased susceptibility to acidification more likely represents suppression of
cellular base uptake than it does inhibition of HCO
secretion. In the absence of perfused acid, augmented
HCO
secretion during the first and second recovery
periods was attenuated by DIDS but not by DMA, indicating that
DIDS-inhibitable HCO
secretion is correlated with
DIDS-inhibitable cellular HCO
uptake.
As shown previously (1, 3), the duodenum has a predictable
and robust hyperemic response to perfused acid, even though there is no
evidence of augmentation of this response to repeated acid challenges.
Although the hyperemic response has been unquestionably implicated in
the reduction of gastric injury susceptibility (11), the
protective role of blood flow in the duodenum is controversial (14, 24, 30). One explanation for the lower amount of acid back diffusion in DMA-treated rats during the second acid challenge is
that suppression of acid-related hyperemia by DMA reduced acid back
diffusion during the second acid challenge. The most comprehensive study of the relationship between duodenal blood flow and injury susceptibility was published by Lugea et al. (16, 17), who found that a 1-ml intraduodenal bolus of 100 mM HCl (pH 1)
significantly reduced damage due to a bolus of 400 mM HCl (pH 0.4)
given 30 min later. Although they measured a significant increase of
duodenal blood flow in response to a bolus of 100 mM HCl, they did not measure blood flow during the mild
strong acid sequence. It is thus
difficult to ascertain the contribution of blood flow to the observed
adaptive protection from injury observed in that study. The suppression
of the large increase of titratable alkalinity by DMA, which also
suppressed acid-related hyperemia (3), underscores the
putative role of blood flow in removing mucosal (luminal) acid via back
diffusion. In other words, if the function of acid-related hyperemia is
to carry away back-diffusing acid, one would predict that suppression
of the hyperemic response would decrease the amount of acid back
diffusion, as was observed in this study.
MGT also increased during acid perfusion, as we have
previously demonstrated. Augmentation of MGT during acid perfusion
indirectly supports its defensive role against acid. It is likely that
blood flow, MGT increase, and cellular base uptake, all of which are increased by luminal acid, act in concert to increase duodenal resistance to acid.
One of the most interesting aspects of this study was the demonstration
that rapid shifts of perfusate pH per se, and not necessarily the mild
strong exposure sequence, induced the adaptive responses observed.
To our knowledge, this possibility has heretofore never been explored,
although our data, in which adaptive responses of pHi were
observed after reversing the mild
strong sequence or exposing the
mucosa to repeated pulses of strong acid, convincingly suggests that,
at least in terms of HCO
uptake, the rapid
shifts of perfusate pH are all that are necessary. These shifts
simulate the physiological state of the duodenum, wherein the mucosa is
exposed alternately to peristaltically conveyed waves of gastric acid
and bursts of pancreatic HCO
after meal ingestion.
Measurements of duodenal pH indicate shifts of 4-5 pH units over
<1 min (23). Although we have not proven that the
observed resistance to acidification truly reflects protection from
injury, numerous studies of gastric epithelium have confirmed that
resistance to mucosal acidification during acid challenge consistently
correlates with decreased injury susceptibility in standard models
(22, 28, 29).
In summary, we found that acute adaptive responses occurred when
perfusate pH was shifted every 10-15 min, regardless of the sequence of acid concentrations used. Furthermore, the only consistent acute adaptive alteration of a defense mechanism was presumably increased cellular HCO
uptake, which produced
intracellular alkalinization and resistance to acidification from
perfused acid. Significant back diffusion of perfused acid correlated
with increased mucosal blood flow. We propose that intracellular
HCO
uptake via a DIDS-sensitive basolateral
Na+-HCO
cotransporter, which temporally precedes HCO
secretion in response to rapid shifts
of luminal pH, is an early acute duodenal response to luminal acid.
Furthermore, we postulate that HCO
secretion may not
be the primary mucosal defense mechanism. The reason why
HCO
secretion correlates well with duodenal acid
resistance may signify its role in restoring epithelial pHi
after acid-induced base uptake. We conclude that cellular
HCO
uptake is likely to be an important duodenal
defense mechanism that is induced by physiological postprandial rapid
shifts of luminal pH and precedes active HCO
secretion.
 |
ACKNOWLEDGEMENTS |
We thank Dipty Shah for her assistance with the experimental procedures.
 |
FOOTNOTES |
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grant RO1-DK-54221 and Veterans Affairs Merit Award funds.
Current address for Y. Akiba: Department of Internal Medicine, Division
of Gastroenterology and Hepatology, Keio University School of Medicine,
35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
Address for reprint requests and other correspondence: J. D. Kaunitz, West Los Angeles VA Medical Center, Bldg. 114, Rm. 217, 11301 Wilshire Blvd., Los Angeles, CA 90073 (e-mail:
jake{at}ucla.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 7 July 2000; accepted in final form 9 January 2001.
 |
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