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
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ABSTRACT |
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The
proximal duodenal mucosa secretes HCO5-10
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
5 M and 10
4 M amiloride significantly
increased net HCO
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
sodium/hydrogen exchange; sodium/hydrogen exchangers; duodenum; intestine; transport
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INTRODUCTION |
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THE DUODENAL
MUCOSA of all mammalian species secretes
HCO
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
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
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 (105-10
4
M amiloride) significantly increased net duodenal
HCO
3 M amiloride) resulted in an additional significant
increase in HCO
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METHODS |
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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 HCO85% 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).
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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RESULTS |
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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.
|
Duodenal HCO1 · h
1
(95% CI: 300-410
µmol · cm
1 · h
1).
Amiloride resulted in significant (P < 0.003)
concentration-dependent increases in duodenal HCO
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
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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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
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
The cellular events that result in net HCO/HCO
that can be
exchanged after secretion into the lumen across the apical membrane for
intracellular HCO
/HCO
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 105 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
The relative amounts of NHE-induced CO2 absorption and
HCO1 · 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
3 M amiloride. Furthermore, it can be estimated that
basal HCO
1 · h
1,
calculated from the net basal HCO
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
/HCO
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
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ACKNOWLEDGEMENTS |
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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).
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FOOTNOTES |
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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.
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