CURE: Digestive Diseases Research Center, West Los Angeles Veterans Affairs Medical Center, and Department of Medicine, School of Medicine, University of California, Los Angeles, California 90073
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ABSTRACT |
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Duodenal mucosal defense was assessed by
measuring blood flow and epithelial intracellular pH
(pHi) of rat proximal duodenum in vivo. Fluorescence microscopy was used to measure epithelial pHi using the trapped,
pHi-indicating dye
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM. Blood
flow was measured with laser-Doppler flowmetry. The mucosa was briefly
superfused with NH4Cl, pH 2.2 buffer, the potent
Na+/H+
exchange inhibitor
5-(N,N-dimethyl)-amiloride
(DMA), or the anion exchange and
Na+-HCO3
cotransport inhibitor DIDS. Cryostat sections localized
dye fluorescence to the villus tip. Steady-state
pHi was 7.02 ± 0.01, which
remained stable for 60 min. Interventions that load the cells with
protons without affecting superfusate pH
(NH4Cl prepulse, nigericin with
low superfusate K+ concentration,
DMA, and DIDS) all decreased pHi,
supporting our contention that the dye was faithfully measuring
pHi. An acid pulse decreased
pHi, followed by a
DIDS-inhibitable overshoot over baseline. Intracellular acidification
increased duodenal blood flow independent of superfusate pH, which was
inhibited by DMA, but not by DIDS. We conclude that we have established a novel in vivo microscopy system enabling simultaneous measurements of
pHi and blood flow of duodenal
epithelium.
Na+/H+
exchange and
Na+-HCO
3
cotransport regulate baseline duodenal epithelial
pHi. Intracellular acidification
enhances duodenal blood flow by a unique, amiloride-inhibitable,
superfusate pH-independent mechanism.
epithelial cells; in vivo microscopy; fluorescent dyes; amiloride analogs; laser-Doppler flowmetry
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INTRODUCTION |
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DUODENAL INJURY IS BELIEVED to result from a mismatch
between aggressive factors (acid) and defensive factors. Duodenal
defensive factors can be classified as they are in stomach to
preepithelial (mucus and HCO3),
epithelial (tight junctions and plasma membrane ion transport), and
postepithelial (blood flow, inflammatory cells, and neural reflexes).
Because the duodenal mucosa is alternately exposed to gastric acid and
pancreatic alkaline secretions, luminal pH in humans can rapidly vary
between pH 2 and 7 (39). Despite its importance in terms of peptic
ulceration, there are far fewer studies of duodenal mucosal defense
mechanisms than there are of gastric defense. Furthermore, perhaps due
to its complex architecture and its smaller size, duodenal mucosal defense mechanisms, with the exception of
HCO
3 secretion using perfused duodenal
loops, have never been studied in vivo in an integrated manner. In
particular, controversy surrounds the role of mucosal and submucosal
blood flow as a discrete duodenal defense mechanism (31, 44, 52).
Furthermore, impaired mucosal defense mechanisms may be a major means
by which Helicobacter pylori produces
duodenal ulceration (33, 35, 41).
Our laboratory has established a reproducible and reliable method for studying gastric mucosal defense mechanisms in vivo using fluorescence microscopy (25, 28, 36). In this paper, we describe how the technology was adapted so that duodenal epithelial intracellular pH (pHi) and duodenal blood flow can be simultaneously measured in vivo. Using this technique, we examined the regulation of epithelial pHi and duodenal blood flow by acid, hypothesizing that luminal (superfused) acid is the signal for a mucosal hyperemia, as it is in the stomach (14).
Thus the aim of this study was to establish the dynamic, simultaneous in vivo measurement of pHi of duodenal epithelial cells by in vivo microscopy and to identify both the effects of luminal acid on pHi and duodenal blood flow of the duodenal epithelium and the regulatory mechanisms underlying these effects.
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MATERIALS AND METHODS |
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Chemicals
2',7'-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) acid, BCECF-AM, and DIDS were obtained from Molecular Probes (Eugene, OR). 5-(N,N-dimethyl)-amiloride (DMA), HEPES, acridine orange, 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 superfusion, Krebs solution was titrated to pH 2.2 with 1 N HCl and adjusted to approximate isotonicity (300 mM). For the NH4Cl prepulse and nigericin studies, sodium was replaced with choline chloride and KCl, respectively. Each solution was prewarmed at 37°C using a water bath, and temperature was maintained with a heating pad during the experiment.In Vivo Microscopic Preparation
Male Sprague-Dawley rats weighing ~225-275 g (Harlan Laboratories, San Diego, CA) were fasted overnight but had free access to water. All studies were approved by the Animal Use Committee of the West Los Angeles Veterans Affairs Medical Center. An in vivo microfluorometric technique, similar to that previously used for gastric mucosa described in detail elsewhere (25, 28, 36), was adapted from a technique originally developed by Tanaka et al. (50) to measure pHi in rat duodenal epithelial cells. After Urethane (1.25 g/kg) anesthesia, the rat was placed supine on a plastic stage. Body temperature was maintained at 36-37°C by a heating pad, and rectal temperature was monitored throughout the experiment. A tracheal cannula was inserted, and warmed saline was continuously infused through the left femoral vein at a rate of 1.08 ml/h, using a Harvard infusion pump. Arterial blood pressure was monitored via a catheter placed in the left femoral artery. The abdomen was opened via a 3-cm midline incision, and the duodenum was exposed. The pylorus was tightly ligated to prevent gastric juice from entering into the proximal duodenum, and the duodenum was temporarily closed with a nylon suture proximal to the ligament of Treitz, before filling the duodenal loop with 0.5 ml saline prewarmed at 37°C. The anterior wall of the duodenum was incised distal to the pylorus to just proximal to the papilla of Vater using a miniature electrocautery to prevent bile-pancreatic juice from contaminating the observed duodenal mucosa. A concave stainless steel disk (16-mm diameter and 1-2 mm deep) with 3-mm central aperture was fixed watertight on the mucosal surface with a silicone plastic adherent (Silly Putty, Binney & Smith, Easton, PA). The serosal surface of the duodenum was supported with the laser-Doppler flow probe (described below). A thin plastic coverslip was fixed to the disk with the silicone adherent to permit closed superfusion with solutions (total volume, 50 µl; rate, 0.25 ml/min) using a Harvard infusion pump. Two PE-50 polyethylene perfusion lines were inserted into the chamber to enable rapid changes of superfusate (e.g., pH 7.0 to 2.2). The exposed mucosa was incubated with 50 µl Krebs solution (pH 7.0) containing 10 µM BCECF-AM for 15 min to load the duodenal epithelial cells before starting the experiment.Image Analysis
Fluorescence of the microscopically observed chambered segment of duodenal mucosa at 515-nm emission was recorded with a charge-coupled device color video camera (Optronics Engineering, Goleta, CA) and captured and stored using the computer's fixed disk drive. The intensity of emitted fluorescence at 495-nm stimulation is pH dependent, whereas that at 450 nm is not. Therefore, 450- and 495-nm narrow band-pass interference filters (Chroma, Brattleboro, VT) were used, and each image was captured every 5 min. Readings were taken at 10 s before and after each time point. The paired readings needed to calculate a fluorescence ratio were thus taken at a maximum of 20 s apart. Image analysis was performed on the recorded images as follows. Initially, three small areas of the duodenal epithelium were selected at random and then followed throughout the experiment. Fluorescence intensity of the selected area was measured by first capturing the image using an Intel Pentium-based IBM-compatible microcomputer with FlashPoint frame-grabbing videographic card (Integral Technologies, Silver Spring, MD) and digitized, with the area of interest defined and intensity measured using image analyzer software (Image-Pro Plus v. 1.3, Media Cybernetics, Silver Spring, MD). In vitro calibration and background compensation using an aqueous solution containing 0.2 µM BCECF free acid were performed as described previously (25). Because of compelling data produced by Boyarsky and co-workers (12), who showed that in vitro calibration of BCECF is superior to in vivo calibration, we calculated pHi according to an in vitro calibration curve (Fig. 1). The intensity at 495 nm was divided by that at 450 nm, and the resulting ratio was converted to pHi using the in vitro calibration curve as fluorescence intensity (FI) and background intensity (BGI). Figure 1 depicts a typical sigmoid curve, with linear portion between pH 5.9 and 7.3, the useful pH range for the dye. The fluorescence ratio was calculated by the equation
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Intracellular Localization of BCECF
To identify the intracellular localization of BCECF, we cut the dye-loaded mucosa into strips with a razor blade and the tissue was mounted in O.C.T. compound (Miles, Elkhart, IN) atMeasurement of Duodenal Blood Flow
For the simultaneous measurement of duodenal blood flow with pHi, a soft-tip pencil probe (model P435, Vasomedics, St. Paul, MN) was surrounded with a silicone plastic adherent and attached on the duodenal serosa just below the observed chambered mucosa. Blood flow was measured as the voltage output of the laser-Doppler instrument (LaserFlo BPM803A, Vasomedics) and expressed relative to the stable level (the basal level) 10-30 min after the superfusion started. Blood flow was recorded on the flow chart and determined at every 5 min and expressed as percentage of basal.In previous studies performed in cat small intestine (29), laser-Doppler flowmetry was shown to correlate well with mucosal-submucosal blood flow measured with labeled microspheres and a serosally placed probe measured the same blood flow as a mucosally placed probe (1). Furthermore, microsphere techniques have documented that 96% of total duodenal blood flow is distributed in mucosa and submucosa, and only 4% is in the muscularis layer (7).
To confirm whether blood flow measured from the serosal side is similar
to that measured from the mucosal side, we obtained quasisimultaneous
measurements of duodenal blood flow from the serosal and mucosal sides.
Mucosal measurements were performed with a soft-tip laser-Doppler probe
inserted through the chamber aperture and applied gently to the
duodenal mucosa. Serosal measurement was performed as described above,
with the exception that a Transonic type R right-angle probe was used
with a Transonic model BLF 21 flowmeter (Ithaca, NY). Flow measurements
were made alternately to avoid interference of the laser light emitted
between the probes. Figure 2 depicts that
there was no significant difference between serosal and mucosal
measurements of duodenal blood flow during pH 7.0 superfusion.
Furthermore, a similar hyperemic response was recorded during pH 2.2 superfusion. We thus were able to measure duodenal mucosal and
submucosal blood flow with a serosally placed laser-Doppler probe.
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Experimental Protocol of In Vivo Microscopic Study
After BCECF was loaded into duodenal epithelial cells and blood flow was stabilized with pH 7.0 Krebs buffer superfusion, the time was set as time 0. The duodenal mucosa was superfused with pH 7.0 Krebs buffer, followed by various superfusates as described below.Varying pHi with constant superfusate pH. To vary pHi with constant superfusate pH, a prepulse of NH4Cl (20 mM) or nigericin (10 µM) with three extracellular K+ concentrations ([K+]o) were used as described previously (27). The pH 7.0 Krebs buffer containing NH4Cl (20 mM) was pulsed for 10 min, followed by pH 7.0 Krebs for 10 min. The K+/H+ exchanger nigericin equilibrates pHi with pH in the superfusate [extracellular pH (pHo)] as well as intracellular K+ concentration with [K+]o. K+ solutions (2.6, 80, and 150 mM) containing 10 µM nigericin consisted of pH 7.0 HEPES in which Na+ had been replaced with K+.
Acid superfusion. To examine the effect of luminal acid on pHi and blood flow, the duodenal mucosa was superfused with pH 7.0 Krebs buffer for 10 min, followed by pH 7.0 or 2.2 solution for 10 min (acid-challenge period), followed with pH 7.0 Krebs buffer for 15 min.
Inhibition of epithelial ion transporters.
To determine the role of epithelial ion transporters such as the
Na+/H+
exchanger,
Na+-HCO3
cotransporter, and
HCO
3/Cl
exchanger in regulation of pHi and
blood flow, we added DMA (0.1 mM) or DIDS (0.5 mM) with pH 7.0 or 2.2 superfusion during the 10-min challenge period.
Statistics
Data from six rats in each group are expressed as means ± SE. Comparisons between groups were made by one-way ANOVA followed by Fisher's least significant differences test. Bartlett's test was used to calculate the significance of the correlation between two measures. P < 0.05 was taken as significant. ![]() |
RESULTS |
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Initial experiments were directed toward confirming the intracellular localization and fidelity of reporting pHi of the BCECF-loaded cells.
Imaging of Loaded Mucosa In Vivo and in Dye-Loaded Frozen Sections
An examination of mucosa loaded with BCECF in vivo produced images such as those depicted in Fig. 3A. Note that the fluorescence appears to be limited to the villus epithelial cells. Dye localization was confirmed with frozen sections of tissue loaded in vivo with BCECF. In the sections, fluorescence was limited to the cytoplasm of the villus tip epithelial cells, and to a few, yet unidentified cells in the lamina propria mucosa. No fluorescence was observed in the mucus gel or other parts of the mucosa (Fig. 3B). In contrast, the preparation, when loaded with 10 µM acridine orange in vivo, was diffusely fluorescent (Fig. 3C). Examination of the frozen sections of the mucosa loaded with acridine orange in vivo demonstrated that the dye was present in the nucleus and cytoplasm of the epithelial cells, in addition to cells in the lamina propria and mucus (Fig. 3D).
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Measurement of pHi and Duodenal Blood Flow: Stability of In Vivo Preparation
Figure 4 depicts an experiment in which pHi and duodenal blood flow were measured in six rats. Blood flow gradually decreased by 12% of baseline over the 1-h observation period, whereas pHi and mean arterial blood pressure were stable, with mean values of 7.01 ± 0.01 and 98.5 ± 0.8 mmHg, respectively. Rectal temperature was stable at 36.4 ± 0.03°C throughout the observation period (data not shown).
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Effect of an NH4Cl Prepulse
After baseline stabilization, mucosal superfusion with 20 mM NH4Cl, buffered to pH 7.0 promptly increased pHi (Fig. 5A). After 10 min of incubation, the NH4Cl superfusion was stopped, promptly and transiently decreasing pHi below baseline (undershoot). Intracellular acidification was associated with a 33.3% increase in duodenal blood flow, although intracellular alkalinization had no effect on blood flow (Fig. 5B).
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Effect of Varying [K+]o in the Presence of Nigericin
The K+-selective ionophore was used to equilibrate K+ and proton gradients across the plasma membranes of BCECF-loaded duodenal epithelial cells (Fig. 6A). In the presence of a low superfusate K+ concentration (2.6 mM), pHi promptly fell, consistent with a rapid proton influx that balanced the K+ efflux. Exposure to a superfusate containing 80 mM KCl, the estimated intracellular K+ concentration, minimally affected pHi, whereas a high (150 mM) K+ concentration raised pHi, consistent with adequate functioning of the dye-ionophore system and the "null point" concept (27). Interestingly, duodenal blood flow was increased concurrently with the fall in pHi in the presence of nigericin and the low superfusate K+ concentration but was decreased with the increased pHi observed in the presence of nigericin and high superfusate K+ concentration (Fig. 6B).
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Effect of DIDS and DMA
Superfusion with DIDS (0.5 mM; pH 7.0)-containing Krebs solution significantly lowered pHi; recovery to baseline was observed after DIDS removal (Fig. 7A). Duodenal blood flow increased during DIDS-induced intracellular acidification (Fig. 7B). Superfusion with DMA (0.1 mM; pH 7.0)-containing Krebs solution also significantly lowered pHi to a new level followed by recovery to baseline (Fig. 7A). An increase in duodenal blood flow was not observed during DMA-induced intracellular acidification (Fig. 7B).
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Effect of an Acid Pulse
pH 2.2 superfusion for 10 min reduced pHi to 6.26 ± 0.05. After acid removal, pHi increased to above the baseline (overshoot) 10 min after superfusate pH was returned to 7.0 (Fig. 8A). pH 2.2 superfusion in the presence of DMA and/or DIDS significantly reduced pHi during acid challenge, whereas DIDS and DIDS plus DMA, but not DMA alone, inhibited the recovery of pHi to baseline after superfusate was returned to 7.0 at the 25-min point only and completely blocked the overshoot phenomenon (Fig. 8A). Superfused acid transiently increased duodenal blood flow, an effect abolished by DMA, but not affected by DIDS (Fig. 8B).
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Correlation of pHi and Duodenal Blood Flow
Figure 9 depicts a graph in which pHi and duodenal blood flow are plotted for all of the experiments shown in Figs. 4-8. Blood flow and pHi values were measured 5 min (2 min in the case of NH4Cl) after superfusate change (e.g., acid, inhibitor, NH4Cl, and nigericin). Note that there is an inverse correlation between duodenal blood flow and pHi in the absence of DMA (slope =
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DISCUSSION |
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Using a technique modeled on our previous studies of gastric surface cell pHi (25, 28, 36), we loaded the dye BCECF into the duodenal epithelium in vivo, with its intracellular localization confirmed by a variety of complementary, independent techniques. The presence of intracellular dye enabled pHi to be reliably and reproducibly measured in vivo with a fluorometric technique. Furthermore, we found that duodenal epithelial cells, unlike gastric surface cells, are readily acidified during superfusion with moderately (e.g., pH 2.2) acidic solutions. Moreover, the simultaneous measurement of duodenal blood flow by laser-Doppler flowmetry and pHi demonstrated for the first time that the hyperemic response to luminal acid is related to pHi, which is correlated with duodenal blood flow independent of luminal pH. This is the first study in which duodenal epithelial pHi was measured in vivo and the first in which intestinal blood flow and pHi were measured simultaneously, which in turn enabled us to make a novel observation regarding the regulation of duodenal blood flow.
Verification of the intracellular localization of trapped,
intracellular fluorescent dyes is accomplished by demonstrating that
the dye behaves in a manner that would be predicted if the dye was
recording cytoplasmic rather than
pHo. The usual means is to use an
ammonium prepulse, a well-described phenomenon (10, 27) in which
pHi is elevated during exposure to
ammonium-containing solutions and then drops rapidly on the withdrawal
of ammonium. A second means is to vary
pHi solely by changing
[K+]o
in the presence of nigericin. A third means is to expose the cells to
the
Na+/H+
exchange inhibitor DMA, one of the amiloride derivatives, or DIDS, an
inhibitor of
Na+-HCO3
cotransport and
HCO
3/Cl
exchange. In all of these studies,
pHi behaved in the predicted manner, consistent with the intracellular localization of the dye.
Furthermore, in all of these instances,
pHi was changed without changing
superfusate pH, which again would only be expected if the dye was
intracellular. The second method we used was to either visualize the
dye in vivo or to observe frozen sections of tissue that had been
loaded in vivo. Imaging of the loaded tissue demonstrated fluorescence
only in the area corresponding to the villus tip epithelial cells.
Notably, the area between the cells lacked fluorescence. This was
confirmed with frozen sections, which demonstrated dye fluorescence
only in the cytoplasm of the villus tip cells, and in a few
unidentified cells in the lamina propria mucosa. We also stained the
mucosa in vivo with the fluorescent dye acridine orange, which has the
advantages of being excited at a similar wavelength as BCECF but which
also nonspecifically stains cellular and noncellular structures.
Visualization of mucosa stained in vivo with acridine orange revealed
diffuse staining of the villi and all of the surrounding area. Frozen
sections of this area revealed the genesis of this image in that the
mucus, epithelial cells, and subepithelial structures were all stained
by acridine orange. Acridine orange stained the mucus gel and lamina
propria mucosa, in clear contrast to the selective staining of the
villus tip epithelial cells by BCECF, which further and convincingly
supports the intracellular localization of BCECF. It is not clear why
BCECF localized to the villus tip cells only; the acridine orange
staining pattern suggests that dye access was not rate limiting
although dye access may have been different with BCECF. Furthermore,
both villus and crypt cells could be loaded with BCECF in vitro (2).
Although some cells at the villus tip are apoptotic, dye hydrolysis and
retention are highly consistent with the presence of the intact plasma
membrane of a living cell (13, 48). The terminally differentiated
functions unique to villus cells may be prerequisite for successful dye loading and retention. The rounded, nonfluorescent inclusions at the
villus tip seen in Fig. 3B may
represent areas of cytoplasm and apical membrane that will be
"pinched off" as part of the cell turnover process, as described
by Iwanaga et al. (24). This conjecture is supported by the finding
that the mucus gel covering the villus tips contained an increasing
number of fluorescent vesicles as the experiment progressed, which are
likely to be the luminal spherical, microvillus-covered cell fragments
described by Iwanaga et al. (24).
We chose pH 2.2 for the acid challenge because prolonged mucosal superfusion with solutions at this pH failed to produce epithelial cell damage (34, 37) and also because it is a minimal duodenal bulb pH level typically measured clinically (39). Interestingly, pH 2.2 produces a more profound acidification in duodenum than in stomach. The reason for this difference between the two organs is unknown. Nevertheless, it is well known that the small intestine, including the duodenum, is considerably "leakier" than the stomach due to the markedly decreased tight junctional permeability in stomach (37, 38). This argument presupposes that intercellular tight junctions are rate limiting for proton influx into the epithelial cells, as has been advanced by Hirst (17) and Powell (42). The other major possibility is that the apical membrane proton permeability is markedly different between the two organs, a possibility that has been confirmed in isolated gastric glands (10) but not studied extensively in the duodenal epithelium.
Only four previous published studies address the measurement of
duodenal epithelial pHi. Paimela
and co-workers (40), working with isolated duodenal mucosa obtained
from the amphibian Necturus maculosus,
accomplished the first published study of duodenal epithelial
pHi in a polarized system. A mean
baseline pHi of 7.05 ± 0.07 was found (40), which is close to our mean baseline
pHi of 7.02 ± 0.01. Paimela et
al. (40) found that superfusion of pH 2.7 over the mucosal surface
solution decreased pHi to 6.69 ± 0.08, which compares well with the pH of 6.26 reached in our studies during superfusion with pH 2.2 solution. In the study by
Paimela et al. (40) and in our study,
pHi promptly recovered on
returning the mucosal superfusate to 7.0. Superfusion with amiloride
and SITS, a
Cl/OH
exchange inhibitor similar to DIDS, produced an exaggerated fall in
pHi to mucosal acidification as we
observed, but, unlike our study, failed to alter baseline
pHi in the absence of mucosal acid. BCECF was used to measure duodenal
pHi in two other studies (2, 22),
in which microfluorometry was used to obtain fluorescence ratios in
freshly isolated collagen-plated duodenal epithelial cells. In
HCO
3 buffer, which resembles the
cellular milieu in vivo, baseline
pHi of villus cells was ~7.0,
and the cells acidified normally in response to an ammonium prepulse. A
recent study (51) describes a technique in which a laser scanning confocal microscope was used to measure
pHi in dispersed epithelial cells
and in intact rat duodenal epithelium in vitro using BCECF fluorescence. In this study (51) baseline
pHi was 7.37 ± 0.03, which
fell to 6.84 ± 0.08 with exposure to 0.01 M HCl, similar to our
results. Of interest was that exposure of the intact mucosa to 60 mM
NH4Cl in the luminal solution was
needed to alkalinize pHi, which
did not undershoot after NH4Cl
withdrawal (51). Furthermore, luminal loading of the epithelial cells
with 40 µM BCECF in situ was unsuccessful, requiring loading by
subepithelial dye injection (51). This contrasts with our experience,
in which a 20 mM NH4Cl prepulse
produced the expected alkalinization-acidification sequence, and our
success with luminal dye loading with 10 µM BCECF. In preliminary
experiments, we used higher BCECF concentrations (e.g., 100 µM) for
loading but found that pHi
variations in response to
nigericin/K+ and
NH4Cl to be blunted in comparison
with the data shown, perhaps due to dye-induced buffering. Another
explanation is that the increased intensity of whole cell fluorescence
compared with confocal-generated sections enables imaging of fainter
signals and thus the use of lower dye concentrations. In any case, the
epithelial cells loaded well with luminally placed 10 µM BCECF. Basal
pHi and
pHi in response to superfused
acid, NH4Cl, DMA, DIDS, and
nigericin with varying [K+]o
all produced predicted and consistent
pHi changes, providing further
evidence of the authenticity of the measurements.
Duodenal epithelial ion transporters consist of apical and basolateral
Na+/H+
exchange, basolateral
Na+-HCO3
cotransport, and apical
HCO
3/Cl
exchange (2, 22, 40). Our findings are consistent with the existence of
an amiloride-sensitive basolateral
Na+/H+
exchanger and a DIDS-sensitive basolateral
Na+-HCO
3
cotransport in this system, with both ion transporters participating in
the regulation of pHi during pH
7.0 superfusion, confirming an earlier observation made in Necturus duodenum (40). Moreover, acid
challenge in the presence of DMA and DIDS shows that both ion
transporters are necessary for maintenance of
pHi during acid challenge.
Furthermore, an overshoot was observed after removing the acid pulse.
This overshoot is reminiscent of the overshoot observed in other cell
types after a brief pulse of exposure to a
HCO
3/CO2
buffer system, which in turn is due to excess base loading via
Na+-HCO
3
cotransport (9). The observation that DIDS applied in the superfusate
abolished the overshoot whereas DMA did not suggests that the mechanism
underlying the overshoot likely involves a DIDS-sensitive, basolateral
Na+-HCO
3
cotransporter. The lack of apparent inhibition of an apical
Cl
/OH
exchanger may be due to this transporter's relative lack of activity at acidic pHi and its apparently
minor role in overall pHi
homeostasis and transcellular HCO
3
secretion (15, 46). It is possible that cellular
HCO
3 loading induced by rapid
variations in luminal pH may constitute an acute cellular defensive
mechanism, as well as a prelude to
HCO
3 secretion, which occurs
10-15 min after an acid challenge (16, 18, 23). Inhibition of
duodenal HCO
3 secretion by apical DIDS
has been observed in vitro, which was initially assumed to be due to
DIDS-induced inhibition of
Cl
/OH
exchange (53), but which also may be due to inhibition of the basolateral
Na+-HCO
3
cotransporter as well if DIDS permeated across the epithelium.
Mucosal blood flow has only infrequently been measured in the duodenum. Depending on species and technique used, there is agreement that luminal acid produces hyperemia in the superfused duodenal segment, as demonstrated early on by Starlinger et al. (49) and later by Leung and co-workers (26, 30, 45). The protective nature of this duodenal hyperemic response, as mentioned previously, is still a matter of contention among investigators (3, 4, 31, 45).
One of our most surprising and novel findings was that pHi and duodenal blood flow were correlated independent of luminal pH. Because pHi and duodenal blood flow had not heretofore been measured simultaneously, it had been assumed that luminal acid was a major stimulus for mucosal hyperemia (19, 30, 37). In our studies to confirm the intracellular localization of BCECF, we altered pHi without altering luminal pH and discovered that intracellular acidification increased duodenal blood flow. Furthermore, DMA inhibited the hyperemic response. One likely explanation for this phenomenon is that extracellular acidification stimulates capsaicin-sensitive afferent nerves, which in turn increase blood flow through a well-described mechanism (20, 21, 30, 32). Supporting this hypothesis is the observation that extracellular acidification stimulates capsaicin-sensitive nerves in vitro (8). Activation of Na+/H+ exchange expels cellular protons at the basolateral membrane, acidifying the extracellular milieu, which in turn sensitizes capsaicin-sensitive afferent nerves, increasing duodenal blood flow. We thus propose that the final common pathway of protons for afferent nerve activation is expulsion via NHE-1, the Na+/H+ exchanger implicated in cellular pH homeostasis, across the epithelial cell basolateral membrane, regardless of the cause of intracellular acidification. This proposed mechanism resembles that of other intestinal chemosensitive neurons, such as the glucose sensor, which appear to require prior transepithelial carrier-mediated transport of the ligand for optimal activation of submucosal afferent chemosensitive nerves (H.-R. Berthoud and H. Raybould, personal communication). Because NHE-1 is present in all cell types, it is possible that DMA may also exert its inhibitory effect directly on the microvasculature or on nerves, although evidence supporting the involvement of NHE-1 on smooth muscle tone is insubstantial (43, 47). Furthermore, extracellular acidification decreases vascular smooth muscle tone in isolated preparations, suggesting that a decrease of pHo might produce hyperemia by directly relaxing smooth muscle (5, 6). This explanation, however, is not supported by the observation that duodenal acid-related hyperemia is medicated by capsaicin-sensitive afferent nerves (30). The preponderance of the data thus supports that acid-related hyperemia in the duodenum is mediated by afferent nerves, as has been well established in the stomach (for review, see Ref. 20).
In summary, duodenal mucosal pHi
could be reliably and reproducibly measured in intact rat duodenum in
vivo. Intracellular acidification produced an amiloride-inhibitable
hyperemic response, suggesting that a previously unsuspected mechanism
regulates duodenal blood flow. Basolateral
Na+-HCO3
cotransporter and
Na+/H+
exchanger regulate pHi of duodenal
epithelial cells, in the presence of acidic and neutral
superfusates. The ability to simultaneously measure
another potential defensive mechanism such as blood flow provides a set
of powerful new tools that should be of value in elucidating important
clinical questions such as those having to do with the role of host
defenses in duodenal ulcer pathogenesis.
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ACKNOWLEDGEMENTS |
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We thank Drs. Paul Guth and Eli Engel for helpful and thoughtful comments and Jonathan Lee, Igor Nastaskin, and Dipty Shah for technical assistance.
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FOOTNOTES |
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This study was supported by Veterans Affairs Merit Review Funding.
The methodology underlying this study has been previously published in abstract form (Ref. 50).
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: J. D. Kaunitz, Bldg. 114, Rm. 217, West Los Angeles Veterans Affairs Medical Center, 11301 Wilshire Blvd., Los Angeles, CA 90073.
Received 11 June 1998; accepted in final form 8 October 1998.
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