Human duodenal mucosal brush border Na+/H+ exchangers NHE2 and NHE3 alter net bicarbonate movement

Maltin Repishti, Daniel L. Hogan, Vijaya Pratha, Laura Davydova, Mark Donowitz, C. M. Tse, and Jon I. Isenberg

Department of Medicine, University of California at San Diego, San Diego, California 92103-8413; and Department of Medicine, John Hopkins University School of Medicine, Baltimore, Maryland 21205-2195


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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The proximal duodenal mucosa secretes HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> that serves to protect the epithelium from injury. In isolated human duodenal enterocytes in vitro, multiple luminal membrane proteins are involved in acid/base transport. We postulated that one or more isoforms of the Na+/H+ exchanger (NHE) family is located on the apical surface of human duodenal mucosal epithelial cells and thereby contributes to duodenal mucosal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport. Duodenal biopsies were obtained from human volunteers, and the presence of NHE2 and NHE3 was determined by using previously characterized polyclonal antibodies (Ab 597 for NHE2 and Ab 1381 for NHE3). In addition, proximal duodenal mucosal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport was measured in humans in vivo in response to luminal perfusion of graded doses of amiloride; 10-5-10-4 M amiloride was used to inhibit NHE2 and 10-3 M amiloride to inhibit NHE3. Both NHE2 and NHE3 were localized principally to the brush border of duodenal villus cells. Sequential doses of amiloride resulted in significant, step-wise increases in net duodenal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> output. Inhibition of NHE2 with 10-5 M and 10-4 M amiloride significantly increased net HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> output. Moreover, there was an additional, equivalent increase (P < 0.05) in duodenal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> output with 10-3 M amiloride, which inhibited NHE3. We conclude that 1) NHE2 and NHE3 are localized principally to the brush border of human duodenal villus epithelial cells; 2) sequential inhibition of NHE2 and NHE3 isoforms resulted in step-wise increases in net HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> output; 3) NHE2 and NHE3 participate in human duodenal villus cell HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport; and 4) the contribution of NHE-related transport events should be considered when studying duodenal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport processes.

sodium/hydrogen exchange; sodium/hydrogen exchangers; duodenum; intestine; transport


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE DUODENAL MUCOSA of all mammalian species secretes HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> with a steep proximal-to-distal gradient (3, 10). Surface epithelial HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion is involved in preventing mucosal acid peptic damage (8). Moreover, duodenal ulcer patients infected with Helicobacter pylori have diminished basal and stimulated HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion that normalizes after H. pylori eradication (11). Duodenal mucosa in vitro obtained from patients with cystic fibrosis has decreased resting and cAMP-stimulated HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion (18). Thus duodenal mucosa bicarbonate secretion (DMBS) is important in both health and disease.

Similar to other cells that are involved in ion transport, duodenal enterocytes contain apical and basolateral membrane transporters that serve to maintain the intracellular pH near 7.1 (1). To date, the acid/base transporters that have been identified in animal and human duodenal enterocytes include 1) an amiloride-sensitive Na+/H+ exchanger (NHE) that functions largely as an acid extruder; 2) a stilbene-sensitive NaHCO3 cotransporter that functions as a base loader; and 3) a stilbene-sensitive Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> antiporter that is a base extruder (1, 2). Additionally, the cystic fibrosis transmembrane regulator (CFTR) has also been implicated in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion, in addition to Cl- conductance (17, 20). There are, however, limited in vivo human studies that integrate mucosal structure with the function of acid/base transporters. Nyberg et al. (16) demonstrated in humans that PGE2-stimulated DMBS was decreased significantly by putative blockade of the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger with luminal perfusion of the stilbene derivative DIDS, whereas theophylline-stimulated DMBS (presumably acting by inhibition of cyclic nucleotide phosphodiesterases and thereby increasing cAMP content) was unaltered. These findings suggest that separate luminal transport mechanisms are involved in human PGE2- and cAMP-stimulated duodenal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport.

The present study was based on the hypothesis that one or more of the epithelial, amiloride-sensitive NHE isoforms is located and functional on the human duodenal enterocyte apical surface. Thus immunofluorescence studies for NHE2 and NHE3 were performed on proximal duodenal mucosal biopsies. Furthermore, the effect of graded doses of amiloride that selectively inhibit NHE2 and NHE3 were determined. We observed that 1) both NHE2 and NHE3 are located principally on the apical surface of human duodenal villus cells and 2) suppression of NHE2 activity (10-5-10-4 M amiloride) significantly increased net duodenal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> output; moreover, inhibition of NHE3 (by 10-3 M amiloride) resulted in an additional significant increase in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> output.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Subjects. Five male subjects aged 32-55 yr participated in these studies. Each was in excellent health, taking no medications, and free of any acute or chronic disease. The experimental protocol was approved by the University of California at San Diego Human Subjects Committee, and each subject gave signed informed consent. Duodenal biopsies and measurement of DMBS were performed on days separated by at least 2 wk.

Duodenal mucosal biopsies and immunofluorescence of NHE2 and 3. Two to three endoscopic biopsies (Radial Jaw, Microvasive; Boston Scientific, Watertown, MA) were obtained in the midportion of the duodenal bulb. Tissues were fixed in 3% neutral buffered formaldehyde, processed on a Technicon, and embedded in paraffin. Immunohistochemistry was performed as described previously (12). After being embedded in paraffin, sections were cut at 4 nm and mounted on gelatin-coated slides. The slides were dewaxed in xylene, rehydrated in ethanol, and rinsed in PBS buffer; endogenous peroxide was blocked by incubation in 0.3% H2O2 in methanol. The slides were blocked in PBS buffer (1% NFDM) with goat serum and incubated overnight at 4°C with primary rabbit polyclonal antibodies Ab 597 (rabbit polyclonal anti-GST-C terminal 87 AA of NHE2) or Ab 1381 (rabbit polyclonal anti-GST-C terminal 85 AA of NHE3) or, as control, secondary antibody alone (goat anti-rabbit IgG; Vector Laboratories, Burlingame, CA). Each of these antibodies has been characterized previously (12, 21). Labeling was visualized by light microscopy and horseradish peroxidase by experts in this technique using the Vectostain ABC kit (Vector Laboratories, Burlingame, CA).

Duodenal mucosal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion. Proximal duodenal mucosal net HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> output (i.e., by the duodenal bulb; DMBS) was measured with well-documented and validated methods described previously (11, 13, 14). In brief, 3 cm of proximal duodenum was isolated by two balloons that straddled the pylorus and a third that was 3 cm beyond. The isolated segment was perfused with 154 mM NaCl at 2 ml/min containing 14C-labeled polyethylene glycol as a nonabsorbable marker, and the effluent was collected by gravity. HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration ([HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>]) was determined in duplicate on samples obtained anaerobically. Measurements of pH and PCO2 were obtained (IL 1420, BG3 PCO2 electrode, blood gas analyzer; Instrument Laboratories, Lexington, MA), and [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>] was calculated by the Henderson-Hasselbalch equation (11, 13, 14). HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> outputs were calculated as the product of [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>] times the [14C]polyethylene glycol-corrected volume (>= 85% of the infusates were recovered). Gastric and distal duodenal markers (phenol red and trypsin, respectively) were also infused continuously to assess for potential contamination of the isolated segment (11, 13, 14).

After measurement of basal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion for three 15-min periods, graded doses of amiloride (10-5, 10-4, and 10-3 M; Sigma, St. Louis, MO) were infused into the test segment in increasing concentrations, each for 30 min, and the effects on net HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> output were determined. Moreover, additional control experiments were performed on a separate day in three subjects to determine whether there were time-related alterations in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion; thus isosmolar NaCl was infused alone for the 135-min test period.

Statistics. Data are presented as means ± SE and 95% confidence intervals (CI). Results were analyzed by repeated-measures analysis of variance and the Tukey-Kramer multiple-comparisons test. P values <0.05 were considered significant.


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Immunofluorescence of NHE2 and NHE3. Light microscopic sections of the duodenal biopsies were examined by using previously characterized polyclonal antibodies raised in rabbits. These antibodies had been shown to demonstrate the presence of NHE2 and NHE3 in multiple species, for example, in intestine [human (jejunum, ileum, and colon), rat (ileum and colon), chicken (small intestine and colon), and mouse (ileum and colon)] (22-24). As shown in Fig. 1, NHE2 and NHE3 were present principally in the brush border of villus cells from human proximal duodenum; a modest amount of staining extended into the upper crypt region. Neither NHE2 nor NHE3 was present on the basolateral surface of any epithelial cells. In each of the five subjects studied the immunofluorescence patterns were identical. In addition, no staining for either NHE2 or NHE3 was observed in the absence of primary antibody.


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Fig. 1.   Na+/H+ exchanger (NHE)2 and NHE3 are human duodenal cell brush border proteins: immunolocalization of NHE2 and NHE3 in human duodenum. Human duodenal biopsies were immunostained with anti-NHE2 antibody 597 (1:100 dilution) and anti-NHE3 antibody 1380 (1:100 dilution) and detected with horseradish peroxidase. Secondary antibody alone was used as a negative control and gave no signal. A: NHE2. B: NHE3. C: NHE2 with no primary antibody. D: NHE3 with no primary antibody. NHE2 and NHE3 staining were localized principally to the duodenal villus cells, with limited staining in the upper crypt cells. Magnification ×200. Results were similar in biopsies from 4 other normal healthy subjects.

Duodenal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion. Basal DMBS was 355 ± 19 µmol · cm-1 · h-1 (95% CI: 300-410 µmol · cm-1 · h-1). Amiloride resulted in significant (P < 0.003) concentration-dependent increases in duodenal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> output compared with basal output (Fig. 2). The net mean (95% CI) incremental increases above baseline in response to 10-5, 10-4, and 10-3 M amiloride were 47 (11-82), 67 (32-103), and 111 (75) µmol · cm-1 · h-1, respectively. In addition, although DMBS in response to each dose of amiloride was significantly greater than basal secretion, the DMBS response to 10-3 M amiloride was significantly greater than the response to either the 10-5 M or the 10-4 M infusion, which were not significantly different from one another. Furthermore, DMBS in the subjects in whom an additional NaCl infusion control test was performed revealed that basal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion was 336 (CI 289-383) µmol · cm-1 · h-1 and decreased only modestly to 306 (242) µmol · cm-1 · h-1 (P = not significant) during the final 30 min.


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Fig. 2.   Effect of amiloride on mean ± SE proximal duodenal mucosal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion in normal healthy subjects. A: after basal secretion was measured for 45 min, amiloride (n = 5 subjects) or saline (n = 3 subjects) was perfused into the isolated duodenal segment in increasing sequential doses (10-5-10-3 M; each dose perfused for 30 min). Perfusion of amiloride significantly increased duodenal mucosa HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion; *P < 0.05 vs. basal. B: net (basal subtracted) HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> output. *P < 0.05 vs. 10-5 M and 10-4 M amiloride.

In addition, HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration in the duodenal effluent (the infusate was nominally HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> free) increased significantly from basal concentration in response to 10-4 M and 10-3 M amiloride, and the effluent volume also increased modestly (Table 1). The increase in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration was caused primarily by an increase in PCO2 (basal: 13.70 ± 0.59 mmHg, 10-3 M amiloride: 15.75 ± 0.67 mmHg; P < 0.02). Thus, during amiloride perfusion, PCO2, [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>], and net volume increased. An increase in effluent pH also occurred but did not attain statistical significance (basal: 7.22 ± 0.01, 10-3 M amiloride: 7.30 ± 0.02; P = 0.08).

                              
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Table 1.   Effect of amiloride on duodenal effluent [HCO<UP><SUB>3</SUB><SUP><UP>−</UP></SUP></UP>] and secretion


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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The results of this study indicate that NHE2 and NHE3 are localized principally to the apical membrane of human proximal duodenal villus cells and that the sequential inhibition of each NHE by amiloride results in stepwise and significant increases in net duodenal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> output. These findings indicate that NHE2 and NHE3 affect luminal duodenal pH by altering HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion and that human duodenal villus cell function contributes to net duodenal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport. Thus, similar to the mediation of renal proximal tubule HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption by apical membrane Na+/H+ exchange, duodenal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport is also influenced by apical NHEs (19). This process is likely caused by proton transport into the lumen via Na+/H+ exchange, neutralizing HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and resulting in H2O and CO2 production, the latter being partially permeable across the cell membrane. The intracellular CO2 is then catalyzed by carbonic anhydrases to produce intracellular HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and H+, which in turn are available for transport by their respective membrane acid/base antiporters. A potential alternative process would be inhibition of apically located NHEs resulting in intracellular acidification (due to decreased Na+/H+ exchange), thereby activating basolateral NaHCO3 cotransport resulting in increased HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> entry and transport into the lumen. However, it is also possible that the basolateral "housekeeping" NHE1 would transport H+ out of the cell before activation of NaHCO3 cotransport.

In health there is a sharp pH gradient from the non-acid-secreting antral portion of the stomach, where the luminal pH may be as low as 0.85, and the most proximal duodenum, where the luminal pH is near neutral, except for brief (~30 s) transient periods when the pH decreases to ~2-3 (5, 9). The mechanisms that contribute to duodenal neutrality are HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion by the proximal duodenal mucosa and pancreaticobiliary HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (25, 26). This study indicates that intraduodenal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration is affected by Na+/H+ exchange that can result in neutralization of luminal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. It is possible that under physiological conditions the exchangers may change direction. That is, when the lumen is very acidic, as occurs in the bulb, NHEs may take up protons and transport HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and Na+ into the lumen. However, it has been demonstrated that a pH gradient is present in the in vivo duodenum and that the juxtamucosal pH is ~7 (3).

The cellular events that result in net HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion by the intestine, as well as other organs, have been the subject of intense study (15). In the intestine, the key transporters involved in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport across the apical membrane are 1) the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger, 2) CFTR, and, as this study demonstrates, 3) NHE2 and NHE3. CFTR conducts Cl- that can be exchanged after secretion into the lumen across the apical membrane for intracellular HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> by the brush border Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger. In addition, there is accumulating evidence that CFTR also has a HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> conductance. There are fewer HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transporters across the basolateral membrane; HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> enters the cell principally via the basolateral NaHCO3 cotransporter (12, 22). However, the location of an anion exchanger in the duodenum is in question. A recent report by Alper et al. (4) describes in mice that AE2 immunostaining of enterocytes was restricted to a basolateral distribution. Further work is needed to delineate membrane transporters in human duodenum.

These findings implicate NHE2 and NHE3 in the overall regulation of human proximal DMBS. The inhibitory constant values of NHE2 and NHE3 to amiloride are ~1 and 90 µM, respectively (23, 24). Thus 10-5 M and 10-4 M amiloride inhibited NHE2 but had minimal if any effect on NHE3 in the presence of 154 mM Na+, whereas 10-3 M amiloride inhibited NHE3 (27). The amiloride dose-dependent increases in net HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion (due to inhibition of H+ transport caused by NHE2 and NHE3) and apical location of both NHE isoforms indicate that both NHE2 and NHE3 can contribute to overall duodenal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> output. Although it would be prudent to use more specific NHE3 and NHE2 inhibitors, such as S3226 and HOE694, their use in humans has not been approved. Amiloride-sensitive Na+ uptake is a widely used definition of Na+/H+ exchange in settings in which no other recognized amiloride-sensitive Na+ uptake processes are present. Whereas the epithelial Na+ channel is in the colon, this transporter has not been localized in duodenum. Therefore, the amiloride-sensitive Na+ uptake processes appear to be limited to NHEs.

The relative amounts of NHE-induced CO2 absorption and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion can be estimated from these studies in which basal net HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion (which is made up of both basal CO2 absorption and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion) was 355 µmol · cm-1 · h-1. Assuming that 10-3 M amiloride blocks both apical NHEs and CO2 absorption, it can be estimated that basal CO2 absorption accounts for ~111 µmol · cm-1 · h-1 of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, because this represents the net increase in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion above basal levels caused by the 10-3 M amiloride. Furthermore, it can be estimated that basal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion is ~466 µmol · cm-1 · h-1, calculated from the net basal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport rate of 355 µmol · cm-1 · h-1 plus that estimated to be due to CO2 absorption, 111 µmol · cm-1 · h-1. Thus the contribution of CO2 absorption by NHEs is estimated to be ~24% of the basal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretory rate. Moreover, these studies suggest that NHE2 and NHE3 contribute approximately equally to basal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion. Additional studies are required to determine whether the increase in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport was caused by increased HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> conductance and/or by Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange.

Thus the results suggest that both NHE2 and NHE3 contribute to human duodenal intestinal Na+ absorption. Although this was observed in rabbit ileum, in which both apical NHE2 and NHE3 contributed approximately equally to basal Na+ absorption (27), in other species one or another NHE may predominate in a given intestinal segment. For instance, in chicken small intestine and colon, NHE2 and not NHE3 accounts for the majority of basal Na+ absorption (6). Although the contribution of apical NHE2 versus NHE3 in human intestinal Na+ or proton-induced CO2 absorption has not been compared in other intestinal segments, both are present in the brush border of villus cells in human jejunum, ileum, and colon, although in the colon NHE2 message is considerably larger (7).

Given the importance of understanding the regulation of duodenal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion in health and disease, it now becomes important to recognize that regulation of both HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretory and absorptive processes must be considered and examined separately. Moreover, in carrying out studies of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion, experiments must be done in a way that the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorptive and secretory processes can be measured separately.


    ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health Grants RO1-DK-33491 (J. I. Isenberg), RO1-DK-26523 and RO1-DK-44484 (M. Donowitz), and RO1-DK-51116 (M. Tse), in part by NIH Grant M01-RR-00827 (UCSD Clinical Research Center), and by an educational grant from The Miles and Shirley Fiterman Foundation (L. Davydova and M. Repishti).


    FOOTNOTES

Address for reprint requests and other correspondence: J. I. Isenberg, Gastroenterology Div., UCSD Medical Center, 200 W. Arbor Dr., San Diego, CA 92103-8413 (E-mail: jisenberg{at}ucsd.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 13 October 2000; accepted in final form 27 February 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastrointest Liver Physiol 281(1):G159-G163
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