Regulation of intracellular pH and blood flow in rat duodenal epithelium in vivo

Yasutada Akiba and Jonathan D. Kaunitz

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


    ABSTRACT
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Abstract
Introduction
Materials and methods
Results
Discussion
References

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+-HCO-3 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


    INTRODUCTION
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Abstract
Introduction
Materials and methods
Results
Discussion
References

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 HCO-3), 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.


    MATERIALS AND METHODS
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Abstract
Introduction
Materials and methods
Results
Discussion
References

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
Fluorescence ratio = (FI<SUB>495</SUB> − BGI<SUB>495</SUB>)/(FI<SUB>450</SUB> − BGI<SUB>450</SUB>)
BGI was defined as the intensity of nonfluorescent area between each villus near the selected area. BCECF fluorescence remained above background at a level sufficient to obtain reproducible fluorescence ratios for 60 min. Mean of the ratio from the three selected areas was defined as the ratio at the period, with standard error for the mean of the three points <2.5%.


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Fig. 1.   In vitro calibration of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Solutions containing BCECF acid were prepared in pH range 5.0-8.0. Solutions were imaged using the microscopic image analysis system as described in MATERIALS AND METHODS. The curve is fitted to the standard titration equation as described by Boyarsky and co-workers (11), with a and b denoting the calculated minimum and maximum asymptotes, respectively. The usable pH range for intracellular measurements is taken from the quasilinear portion of the curve and is set as 5.9-7.3 (n = 4-6). FI, fluorescence intensity. pK, dissociation constant.

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) at -20°C. Frozen cryostat sections (10 µm) were made and mounted on nonfluorescent glass slides (Fisher Scientific, Willard, OH) and observed by a Zeiss MPS fluorescent microscope with a BH filter set (Carl Zeiss). In separate experiments, acridine orange (10 µM), which nonselectively stains nuclei, cytoplasm, and mucus, was loaded into the exposed duodenal mucosa, with frozen sections made as described above.

Measurement 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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Fig. 2.   Duodenal blood flow measurements from serosal and mucosal sides by laser-Doppler flowmetry. Simultaneous measurements of duodenal blood flow from serosal and mucosal sides were performed by laser-Doppler flowmetry. Similar hyperemic responses to pH 2.2 superfusion were observed regardless of probe placement, suggesting that a serosally placed probe was faithfully recording mucosal and submucosal blood flow. * P < 0.05 vs. pH 7.0 group.

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+-HCO-3 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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Abstract
Introduction
Materials and methods
Results
Discussion
References

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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Fig. 3.   Fluorescence loading to duodenal mucosa in vivo. A: in vivo image of BCECF-loaded mucosa. Note that dye localizes only in epithelial cells of villus tips. B: frozen section of mucosa in which BCECF was loaded in vivo. Dye clearly localizes in cytoplasm of villus tip cells. A few unidentified cells are also stained in the lamina propria mucosa. C: in vivo image of acridine orange-loaded mucosa. Fluorescence is observed diffusely over mucosa, appearing as a green haze. D: frozen section of mucosa in which acridine orange was loaded in vivo. Acridine orange nonspecifically stains in nuclei, cytoplasm, subepithelial space, and mucus layer. Bars, 100 µm.

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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Fig. 4.   Intracellular pH (pHi), duodenal blood flow, and mean arterial blood pressure during a 60-min pH 7.0 superfusion. pHi (A) and duodenal blood flow (B) were monitored as described in MATERIALS AND METHODS while mean arterial blood pressure (C) was measured with an intra-arterial catheter. Note the stability of the measurements, with minimal decrease in duodenal blood flow, confirming usefulness of this system for prolonged simultaneous measurements of defensive mechanisms. Absence of visible error bars indicates that SE is less than the height of the symbol representing the mean (n = 6 rats/group).

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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Fig. 5.   Effects of an NH4Cl pulse on pHi and duodenal blood flow. A: a 10-min pulse of NH4Cl (20 mM, pH 7.0) superfusion () raised pHi; removal of NH4Cl subsequently lowered pHi below baseline (undershoot). , Krebs buffer only. B: increased duodenal blood flow occurs only during intracellular acidification. Absence of visible error bars indicates that SE is less than the height of the symbol representing the mean. * P < 0.05 vs. Krebs only group (n = 6 rats/group).

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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Fig. 6.   Effects of nigericin with varying extracellular K+ concentrations ([K+]o) on pHi and duodenal blood flow. A: in the presence of nigericin, a typical [K+]o (2.6 mM; bullet ) decreases pHi, whereas high [K+]o (150 mM; open circle ) increases pHi. The estimated [K+]o of 80 mM () has little effect on pHi (null effect). B: increased duodenal blood flow occurs only during intracellular acidification, whereas duodenal blood flow decreases with cytoplasmic alkalinization produced by high [K+]o. Absence of visible error bars indicates that SE is less than the height of the symbol representing the mean. * P < 0.05 vs. 80 mM [K+]o group (n = 6 rats/group).

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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Fig. 7.   Effect of DIDS or 5-(N,N-dimethyl)-amiloride (DMA) with constant superfusate pH on pHi and duodenal blood flow. A: inhibition of basolateral Na+-HCO-3 cotransport by DIDS (0.5 mM) decreased pHi; note that pHi recovers during DIDS superfusion. DMA (0.1 mM) superfusion decreased pHi with no recovery evident. , Krebs buffer only. B: only DIDS increases duodenal blood flow during intracellular acidification. Absence of visible error bars indicates that SE is less than the height of the symbol representing the mean. * P < 0.05 vs. Krebs only group (n = 6 rats/group).

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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Fig. 8.   Effect of superfused acid on pHi and duodenal blood flow. A: duodenal cells were exposed to pH 2.2 superfusate either alone or in the presence of DMA, DIDS, DMA + DIDS, or pH 7.0 Krebs only (). Note that pHi decreases rapidly, reaching a new plateau, and then increases over baseline (overshoot). DMA + DIDS reduced pHi during acid challenge (t = 20) and abolished overshoot (t = 30-35). At 25 min, recovery is significantly impaired in DIDS and DMA + DIDS groups. B: duodenal blood flow increases only during luminal acidification in pH 2.2 and DIDS groups. No increase in duodenal blood flow is seen in the DMA or DMA + DIDS groups even though intracellular acidification occurs. Absence of visible error bars indicates that SE is less than the height of the symbol representing the mean. * P < 0.05 vs. pH 7.0 group; # P < 0.05 vs. pH 2.2 group (n = 6 rats/group).

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 = -31.9, r = 0.706, P < 0.0001), but there is no correlation (slope = -0.49, r = 0.022, P = 0.996) between pHi and duodenal blood flow in the experiments in which DMA was used.


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Fig. 9.   Relationship between pHi and duodenal blood flow. For all of the experiments depicted in Figs. 4-8, pHi and duodenal blood flow measured 5 min (2 min in the case of NH4Cl) after the change of superfusate (e.g., acid, inhibitor, NH4Cl, and nigericin) are plotted; n = 18 from 3 groups for DMA experiments; n = 54 from 8 groups for experiments without DMA. Note the inverse correlation between duodenal blood flow and pHi that exists only in experiments in which DMA is not used.


    DISCUSSION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

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+-HCO-3 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+-HCO-3 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+-HCO-3 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.


    ACKNOWLEDGEMENTS

We thank Drs. Paul Guth and Eli Engel for helpful and thoughtful comments and Jonathan Lee, Igor Nastaskin, and Dipty Shah for technical assistance.


    FOOTNOTES

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.


    REFERENCES
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

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