Departments of 1 Medical Genetics, 2 Pathology, and 3 Surgery, Helsinki University Central Hospital, 4 Finnish Genome Center, and 5 Hospital for Children and Adolescents, Biomedicum, University of Helsinki, 00014 Helsinki, Finland; and 6 Department of Biosciences at Novum, Karolinska Institute, 14157 Huddinge, Sweden
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In inflamed colonic mucosa, the equilibrium
between absorptive and secretory functions for electrolyte and salt
transport is disturbed. We compared the expression of three major
mediators of the intestinal salt transport between healthy and inflamed colonic mucosa to understand the pathophysiology of diarrhea in inflammatory bowel disease. Expression levels of the cystic fibrosis transmembrane regulator (CFTR) (Cl channel), SLC26A3
(Cl
/HCO
secretion and by inhibiting the
epithelial NaCl absorption.
inflammation; anion transport; diarrhea; inflammatory bowel disease; cystic fibrosis transmembrane regulator
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
IN NORMAL COLONIC
MUCOSA, the absorption of salt is driven by active transporters
followed by passive movements of water. Much of the absorption of NaCl
is mediated by electroneutral pH-coupled Na+/H+ and
Cl/HCO
secretion in the colonic
epithelium (33). The intestinal phenotype of CF results
from luminal obstruction by thick mucoid secretions (15).
In contrast, overstimulation of Cl
secretion, e.g., by
bacterial enterotoxins, is associated with secretory diarrhea
(16, 28, 48).
A recent advance in the understanding of the colonic
Cl/HCO
in stools with respective defects in
intestinal HCO
/OH
and
Cl
/HCO
A distinct phenotype results from the malfunction of intestinal Na+/H+ exchangers: mice lacking SLC9A3 develop sodium diarrhea with acidosis (OMIM #600530) (40). Several compensatory mechanisms have been shown to modify the mouse phenotype (34, 40), and no human cases of sodium diarrhea have been associated with mutations in the six Na+/H+ exchangers cloned to date (36).
Little is known about the possible role of electrolyte-transport abnormalities in the pathogenesis of diarrhea associated with chronic intestinal inflammation (3, 4, 7, 44). In animal models, colitis has been associated with a reduced expression of SLC26A3 and unchanged SLC9A3 (52). Human inflammatory colon tissues may show no change in the expression of SLC26A3 (20). Although CFTR has multiple effects on cellular electrolyte and fluid homeostasis, not much is known about its expression in inflamed colonic mucosa.
The aim of this study was to examine the effect of inflammatory bowel disease (IBD) on the expression of the SLC26A3, SLC9A3, and CFTR genes. The activity of inflammation was determined by histological evaluation of the preoperative colonic samples graded in three subgroups (mild, moderate, and severe). The expression levels were measured by real-time quantitative RT-PCR, immunohistochemistry, and Western blotting.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tissue samples. Altogether, 30 preoperative tissue samples from 10 patients undergoing surgery for ulcerative colitis (UC) and 12 samples from 4 control individuals with noninflamed colon mucosa were obtained from the Department of Surgery, Helsinki University Central Hospital by permission of the respective ethical review board. Inflamed colonic tissue samples were taken from two areas: those that macroscopically appeared normal and those demonstrating the highest degree of inflammation. Control tissue samples were taken from morphologically unaltered, uninflamed colonic mucosa of benign (tubular adenoma) or malignant (two adenocarcinomas) tumors at distant nonneoplastic area, as well as of diverticulosis. Tissue samples used for mRNA and protein analysis were taken from the same area as those samples used for histopathological analysis. Tissue samples were handled immediately after surgery on ice or snap frozen in liquid nitrogen before homogenization.
Histological grading. The samples for histological analyses were fixed in 10% neutral buffered formalin immediately after surgery. Formalin-fixed paraffin-embedded samples were prepared for histology and stained by hematoxylin-eosin for histological grading. The degree of inflammation was graded as suggested by Truelove and Richards (46) on a four-point scale: normal (no significant inflammation, n = 12); mild (elevated number of mucosal leukocytes but intact epithelium, n = 11); moderate (aggregates of leucocytes with crypt abscesses and erosions but no ulceration of the epithelium, n = 7); and severe (significant ulceration of the epithelium by mononuclear cell infiltrate, n = 12). Histological grading was performed by a pathologist (M.-L. Karjalainen-Lindsberg) without knowledge of surgery or the endoscopic reports and experimental data.
Quantitative RT-PCR. Adjacent colonic tissue samples were used for histological grading and RNA extraction. Total RNA was extracted from fresh tissues using RNeasy Mini Kit (Qiagen), and 400 ng total RNA were further transcribed to cDNA with random hexamers (Taqman Reverse Transcription Reagents, Perkin-Elmer). Dilutions of the cDNA were used for real-time quantitative PCR (5' fluorogenic nuclease assay) using Perkin-Elmer's ABI Prism 7700 Sequence Detector System (30). The probes used were CCA ATC GAA TTC ATT ATG ACC GTG ATT GC for SLC26A3, ATG CAG TCT CTG GAG CAG CGA CGG for SLC9A3, and CAG AAG CGT CAT CAA AGC ATG CCA AC for CFTR. The following primers were used for amplification: SLC26A3 (100 nM), AGA CAA ACT TCC AGT GCC CAT and ACA GCC GTA GGA TAC ACC TGC; SLC9A3 (900 nM), TCC CCC AGC ACC GAC A and TCC CGG ATG CTC CGC; CFTR (300 nM), TCT TTG GTG TTT CCT ATG ATG AAT ATA GAT and GCA AAC TTG GAG ATG TCC TCT TCT. Primers were designed into exon-intron junctions to avoid amplification of genomic DNA. An S18 rRNA probe and primers were used as an endogenous control gene (S18 control kit, Perkin-Elmer). PCR conditions were: 2 min 50°C, 10 min 95°C, and 40 cycles of 15 s 95°C and 1 min 60°C. The PCR assays were performed in separate tubes, and relative quantitation of the SLC26A3, SLC9A3, and CFTR mRNAs was performed using the standard curve method according to the manufacturer's instructions (PE Applied Biosystems, User Bulletin #2: ABI PRISM 7700 Sequence Detection System).
Immunohistochemistry. Serial sections to those used for histological grading from the same blocks of tissue were used for immunohistochemistry. The peroxidase-antiperoxidase technique was performed using Vectastain Elite ABC Kit (Vector Laboratories, Burlingame, CA). For pretreatment, the deparaffinized slides were boiled in a microwave oven for 20 min in 10 mM citrate buffer (pH 6.0) or 0.01 M EDTA buffer (pH 8.0). Monoclonal CFTR antibody [Ab-2 (MM13-4), Neomarkers, Fremont, CA] was used at 3 µg/ml. Diaminobenzidine was used as the chromogenic substrate, and the slides were counterstained with hematoxylin. Normal mouse IgG (Dako, Glostrup, Denmark) at 3 µg/ml was used as a negative control on parallel sections.
Production of antisera.
The COOH-terminal antiserum was raised in rabbits against the synthetic
peptide corresponding to nucleotides 2375-2416 of the published
SLC26A3 sequence (GenBank L02785; Research Genetics, Huntsville, AL). Nucleotides 205-407, corresponding to the first 68 amino acids of the NH2-terminal region of
SLC26A3 gene, were amplified with the following primers:
5'-CGT GGA TCC ATG ATT GAA CCC TTT GGG AAT C-3'and 5'-CGA GAA TTC CGG
TAT GCT GGC AAC CAA GAT-3' by PCR, and the subsequent PCR product was
cloned into the BamH I-EcoR I sites of the
pGEX-2T expression plasmid (Amersham Pharmacia Biotech, Uppsala,
Sweden). BamH I-EcoR I recognition sequences were
added to the 5'-end of the primers. The cloned region was produced and
purified as glutathione S-transferase (GST) fusion protein
according to the manufacturer's instructions (Amersham Pharmacia
Biotech). The purified fusion protein was used as antigen in the
immunisation of two rabbits (Animal Core Facility, Viikki Biocenter,
University of Helsinki). Presera were collected before the first
immunisation. A total of five immunisations were performed at 3-wk
intervals using 400 µg of the GST fusion protein mix 1:1 with
Freund's adjuvant. Rabbits were bled after the immunization, and the
serum was divided to small aliquots for preservation at 20°C.
Western analysis. Surface epithelial cells of human colon tissues were scraped and transferred to ice-cold lysis buffer [50 mM core buffer (Roche, Basel, Switzerland), 150 mM NaCl, 0.1 mg/ml phenylmethylsulfonyl fluoride, 1 µg/ml aprotin, 1 µg/ml leupeptin, 1% Nonidet P-40, and 0.5% sodium deoxycholate]. The lysed cells were homogenized by a syringe and needle followed by centrifugation at 12,000 g for 10 min at 4°C. The supernatant was preserved for protein analyses. Protein concentration was measured using the colorimetric Bradford protein assay (Bio-Rad Laboratories, Hercules, CA). Total proteins were separated by SDS-PAGE using 9% gels and electroblotted onto Hybond C-extra membranes (Amersham Pharmacia Biotech). Nonspecific binding sites were blocked by incubating the membranes in a solution of 5% nonfat dry milk in PBS containing 0.1% Tween-20. The primary antibody sera were diluted 1:500-1:1,000, and biotin-conjugated goat anti-rabbit IgG (Roche) was used as a secondary antibody 1:2,000 in PBS. To visualize the protein bands by enhanced chemiluminescent reaction, membranes were incubated in 1:2,000 dilution of horseradish peroxidase-conjugated streptavidin (Roche). Computer-assisted scanning densitometry (Biometra, BioDocAnalyze, Göttingen, Germany) was used to analyze the intensity of the immunoreactive bands in the autographs. The optic densities were normalized to the amount of protein in lane. Statistical significance between groups was analyzed by Kruskal-Wallis nonparametric test.
Statistical analysis. Statistical analyses were performed using the PRISM Statistic Package, version 3.0 (GraphPad Software, San Diego, CA). The degree of statistical significance between two groups was calculated using the nonparametric Mann-Whitney U test. Comparisons between three or more groups were performed using the Kruskal-Wallis test. The groups were considered different at a P value <0.05. In these cases, P values for comparisons between groups were calculated using Dunn's multiple comparison as a nonparametric posttest.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To study the effect of intestinal inflammation on the expression
of the genes responsible for major NaCl and fluid movements, we
examined the relative mRNA expression of CFTR,
SLC26A3, and SLC9A3 genes in preoperative samples
from normal (n = 12) and UC patients' colons
(n = 30) by quantitative RT-PCR. Expression levels were
normalized to S18 transcripts in the same cDNA sample. In normal human
colonic mucosa, all three genes had different basal expression levels
(Fig. 1). However, a direct comparison of
mRNA levels for the three genes is not possible, because we did not
normalize the PCR assays for mRNA copy numbers. For each gene
separately, comparison of mRNA levels between the samples gives
information about their relative levels.
|
Expression of the CFTR gene was increased in inflamed compared with
normal colonic mucosa.
In inflamed colonic mucosa, the CFTR expression was increased 2.5-fold
compared with noninflamed samples (Fig. 1A;
P = 0.0002, Mann-Whitney U test). Detailed
characterization of the three subgroups of inflammation (mild,
moderate, and severe) demonstrated upregulated expression of the CFTR
mRNA in all stages of inflamed tissues: fivefold in mildly inflamed
samples (P < 0.001), 2.4-fold in moderate, and
1.5-fold in severely inflamed colon mucosa compared with normal (Fig.
2A). Most pronounced
upregulation was in mildly inflamed mucosa at 3.4-fold higher than in
severely inflamed sets of samples (Fig. 2A).
|
The expression of the SLC26A3 and SLC9A3 genes varies in inflamed colonic mucosa. In contrast to CFTR, the expression levels of the SLC26A3 and SLC9A3 mRNA were not different between normal and inflamed mucosa: the median expression of the SLC26A3 decreased 0.6-fold (P = 0.1095), and the SLC9A3 expression elevated 1.3-fold in the inflamed samples (P = 0.5130; Fig. 1, B and C). Increased expression of SLC26A3 in mild inflammation (1.4-fold) was followed by sharp reduction in moderate (1.9-fold) and severe inflammation (2.6-fold; Fig. 2B). Expression of SLC26A3 was significantly changed in the severely inflamed tissue and between the mild and severe groups (P < 0.05). In case of SLC9A3, the initial upregulation in both mild (3.3-fold) and moderate (1.5-fold) was followed by return to the same level as in noninflamed samples (1.1-fold; Fig. 2C). A statistically significant difference was seen between the mild and severe groups.
The CFTR protein is expressed in the healthy and inflamed colon
mucosa.
To assess putative alterations in the CFTR protein expression in
response to inflammation, a set of inflammatory samples was studied
using immunohistochemistry with a monoclonal CFTR antibody and compared
with the normal control. As expected, normal colon epithelium showed
CFTR expression at the bottom of the crypts and in the lower two-thirds
of the crypt epithelium, whereas the immunostaining was absent in the
upper one-third of the crypt epithelium and the lumenal surface
epithelium. The CFTR-specific immunoreactivity was detected only at the
apical edge of the cryptal epithelial cells, corresponding well
with the functional CFTR protein at the apical membrane. The
immunostaining in colon was, however, faint and sporadic compared with
pancreas that was used as a positive control (Fig.
3).
|
The SLC26A3 protein expression remains unchanged even in severely
inflamed colon mucosa.
To study whether the levels of the SLC26A3 mRNA correlate with the
protein expression level, Western blotting using similar amounts of
differently inflamed colon tissues was performed (Fig. 4A).
Antibody against the NH2-terminal part of the SLC26A3
protein detected a specific band of 85 kDa in all samples, and antibody against the COOH-terminal tail of the SLC26A3 peptide detected two
protein bands at ~85 and 75 kDa. The intensities of the bands in
mildly and severely inflamed samples appeared identical, suggesting that the expression of SLC26A3 remains unchanged even in
severely inflamed colon tissue. These results were statistically
verified by measuring altogether 30 immunoreactive bands from 10 patients by densitometric scanning of the autographs (Fig.
4B). There was no significant difference (P = 0.7841) in mean protein expression in normal vs. inflamed tissues.
The characterization of the subgroups demonstrated also that
SLC26A3 expression remains unchanged in all stages of
inflamed tissues (Fig. 4B).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Malabsorption of NaCl and water is a well-characterized feature of an inflamed intestinal mucosa and leads to diarrhea (4, 7, 10, 38). The underlying mechanisms may include activation of secretion, reduction of absorption, and a defect in epithelial barrier function (39). We examined the effect of UC on the expression of three intestinal electrolyte transporter genes suggested to account for the major colonic NaCl and fluid movements: SLC26A3, SLC9A3, and CFTR. Functional defects of these genes have been previously associated with diarrheal diseases such as CLD and sodium diarrhea; and a distinct phenotype with lack of secretory response and meconium ileus is seen in CF (19, 25, 40).
Although CFTR has a crucial role in the regulation of the intestinal
electrolyte homeostasis, not much is known about its expression or
function in inflamed colon mucosa. In general, inflammatory stimulation
results both in an increased epithelial permeability and altered
patterns of proliferation and differentiation of intestinal epithelial
cells (32, 53). Elevated CFTR expression and enhanced cAMP-dependent Cl secretion have been demonstrated in
hyperproliferated mouse intestine (47). As a
transepithelial protein on the luminal surface along the intestine, it
has a major task in mediating Cl
secretion into the
intestinal lumen. Other CFTR-regulated transport functions include
inhibition of Na+ reabsorption in intestine and activation
of Cl
/HCO
and decreased
absorption of sodium of the mucosa and ultimately contribute to diarrhea.
Previous studies with intestinal epithelial cells have produced contradictory results, because one group reported a decrease in CFTR expression, and others have observed that CFTR expression is unchanged (6, 11, 32). In our study, the expression of the CFTR protein was detected both in healthy and inflamed colon mucosa by immunohistochemistry, although the overall expression level of CFTR in colon is not abundant. Interestingly, some inflammatory samples demonstrated cytoplasmic expression of CFTR that could not be detected in normal control. It likely corresponds to a newly synthesized or retarded form of the CFTR protein in an intracellular compartment, such as endoplasmic reticulum or Golgi apparatus. Although no significant changes in the levels of CFTR expression were detected in the apical surface of the colonic epithelial cells by immunohistochemistry, the cytoplasmic expression of CFTR might suggest that the translation of the CFTR protein is enhanced in the inflamed colon mucosa.
The CFTR-induced upregulation of the SLC26A3 and SLC9A3 mRNA expression
in heterologous systems was reported recently (1, 17, 51).
Speculatively, the slight but insignificant increase in the expression
of SLC26A3 and SLC9A3 in mildly inflamed
intestine could also reflect an increased activation of CFTR.
Thus CFTR may play an important role in the pathogenesis of
inflammation-associated diarrhea by modifying the function and
properties of Cl, Na+, and K+
channels, the Cl
/HCO
The inflamed colonic mucosa contains increased levels of different
inflammatory mediators, which are capable of triggering Cl secretion. Thus the secretion of chloride and water
may contribute to the pathogenesis of diarrhea in UC (49,
50). However, there are also several human studies as well as
animal models of colitis supporting the idea that diarrhea in IBD is
likely due to antiabsorptive rather than prosecretory effects
(18). The high-lumen, negative transmucosal electrical
potential difference present in normal colon has been shown to be
decreased in UC patients, suggesting impaired electrogenic
Na+ absorption rather than enhanced Cl
secretion (37, 38). In addition, malabsorption of
Na+ and Cl
has also been associated with
inflamed colonic mucosa (5, 14, 21, 22, 45) as well as a
notably diminished activity of basolateral membrane
Na+-K+-ATPase, which is a prerequisite for
apical Cl
secretion (38). These studies
indicate that impaired water absorption secondary to impaired
Na+ and Cl
absorption rather than
Cl
secretion may be a major pathogenic factor in the
diarrhea of acute colitis. Therefore, further studies are still
required to correlate the putative functional changes in inflamed human
colon with the elevated level of CFTR and its putative intracellular redistribution found in this study.
This study supports our earlier findings demonstrating similar expression of the SLC26A3 mRNA in IBD and ischemic colitis when compared with normal colon epithelium (20). In this study, there were no significant changes in the expression of the SLC26A3 and SLC9A3 genes in UC compared with noninflamed tissues when all subgroups were analyzed together. However, there is variation in the individual levels of expression of the genes, especially with SLC9A3, which may reflect alterations in region-specific expression of the genes, because the tissue samples were collected at different locations along the colon. Furthermore, all the patients were treated with glucocorticosteroids known to alleviate intestinal inflammation and modulate the expression of some genes involved in transport processes, e.g., SLC9A3 gene (2, 26).
Characterization of the effect of disease activity on epithelial gene expression revealed slight changes for SLC26A3 and SLC9A3. An initial increase in mild disease was followed by a decrease in moderate and severe UC in case of SLC26A3. SLC9A3 expression increased more than that of SLC26A3 in mild UC but remained unchanged in severe UC. There are several reasons that may be responsible for the reduction of the SLC26A3 mRNA seen in severe UC. It may be secondary, due to a loss of epithelial cells by erosions and ulceration, or due to an expansion of undifferentiated crypt cell compartment. However, no changes in the expression of the SLC26A3 protein was noted even in severe UC colitis. In our earlier study, we have showed the expansion of the SLC26A3 expression deeper in the crypts to the cells of the proliferative cell compartment in a set of inflammatory colon samples (20). Similarly, SLC9A3 staining has been suggested to remain unchanged in a colon tissue specimen of an UC patient (52). It is likely that the protein translation machinery in inflamed intestinal cells can compensate small fluctuation in the mRNA level.
A rabbit model of chronic ileal inflammation has demonstrated that the
inhibition of coupled NaCl absorption by the villus cells occurs as a
result of diminished Cl/HCO
rather than an altered number of transporters
(12). Because there was no significant change in the
levels of SLC26A3 and SLC9A3 transporters even in severe colitis, it is
tempting to speculate that a similar model of inhibition of NaCl
absorption might exist in human colon too. However, several different
studies using different conditions or models have produced ambiguous
results regarding the regulation of the expression of different apical
and basolateral transporters (6, 11, 32, 38, 52).
Significant differences may occur in different models, and there may be
differences even between UC and other types of intestinal inflammation.
In summary, we have determined the relationship between the activity of
intestinal inflammation and the degree of the expression of the human
CFTR, SLC26A3, and SLC9A3 genes in
normal colon and in UC. Intestinal inflammation modulates the
expression of these major mediators of intestinal salt transport and
may contribute to diarrhea in UC both by increasing transepithelial
Cl secretion and by inhibiting the epithelial NaCl absorption.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank R. Eklund and A. Tallqvist for skillful assistance in laboratory work.
![]() |
FOOTNOTES |
---|
* H. Lohi and S. Mäkelä contributed equally to this work.
This study was supported by the Academy of Finland; Sigrid Juselius Foundation; Foundation for Pediatric Research, Ulla Hjelt Fund; Helsinki Univ. Central Hospital research funds; Oskar Öflund Stiftelse; Research and Science Foundation of Farmos; Paulo Foundation; and Ella and Georg Ehrnrooth's Stiftelse.
J. Kere is a member of Biocentrum Helsinki.
Address for reprint requests and other correspondence: H. Lohi, Biomedicum Helsinki, P.O. Box 63 (Haartmaninkatu 8), 00014 Univ. of Helsinki, Finland. (E-mail: hannes.lohi{at}helsinki.fi).
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.
April 10, 2002;10.1152/ajpgi.00356.2001
Received 9 August 2001; accepted in final form 3 April 2002.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Ahn, W,
Kim KH,
Lee JA,
Kim JY,
Choi JY,
Moe OW,
Milgram SL,
Muallem S,
and
Lee MG.
Regulatory interaction between the cystic fibrosis transmembrane conductance regulator and HCO
2.
Ambuhl, PM,
Yang X,
Peng Y,
Preisig PA,
Moe OW,
and
Alpern RJ.
Glucocorticoids enhance acid activation of the Na+/H+ exchanger 3 (NHE3).
J Clin Invest
103:
429-435,
1999
3.
Archampong, EQ,
Harris J,
and
Clark CG.
The absorption and secretion of water and electrolytes across the healthy and the diseased human colonic mucosa measured in vitro.
Gut
13:
880-886,
1972[ISI][Medline].
4.
Archampong, EQ,
Harris J,
and
Clark CG.
Water and electrolyte transfer by colonic mucosa studied in vitro.
Br J Surg
59:
314,
1972.
5.
Bell, CJ,
Gall DG,
and
Wallace JL.
Disruption of colonic electrolyte transport in experimental colitis.
Am J Physiol Gastrointest Liver Physiol
268:
G622-G630,
1995
6.
Besancon, F,
Przewlocki G,
Baro I,
Hongre AS,
Escande D,
and
Edelman A.
Interferon- downregulates cftr gene-expression in epithelial cells.
Am J Physiol Cell Physiol
36:
C1398-C1404,
1994[ISI].
7.
Binder, HJ,
and
Ptak T.
Jejunal absorption of water and electrolytes in inflammatory bowel disease.
J Lab Clin Med
76:
915-924,
1970[ISI][Medline].
8.
Byeon, MK,
Frankel A,
Papas TS,
Henderson KW,
and
Schweinfest CW.
Human DRA functions as a sulfate transporter in Sf9 insect cells.
Protein Expr Purif
12:
67-74,
1998[ISI][Medline].
9.
Byeon, MK,
Westerman MA,
Maroulakou IG,
Henderson KW,
Suster S,
Zhang XK,
Papas TS,
Vesely J,
Willingham MC,
Green JE,
and
Schweinfest CW.
The down-regulated in adenoma (DRA) gene encodes an intestine-specific membrane glycoprotein.
Oncogene
12:
387-396,
1996[ISI][Medline].
10.
Castro, BA,
Nepomuceno M,
Lerche NW,
Eichberg JW,
and
Levy JA.
Persistent infection of baboons and rhesus monkeys with different strains of HIV-2.
Virology
184:
219-226,
1991[ISI][Medline].
11.
Colgan, SP,
Parkos CA,
Matthews JB,
Dandrea L,
Awtrey CS,
Lichtman AH,
Delparcher C,
and
Madara JL.
Interferon- induces a cell-surface phenotype switch on T84 intestinal epithelial-cells.
Am J Physiol Cell Physiol
267:
C402-C410,
1994
12.
Coon, S,
and
Sundaram U.
Mechanism of glucocorticoid-mediated reversal of inhibition of Cl/HCO
13.
Crawford, I,
Maloney PC,
Zeitlin PL,
Guggino WB,
Hyde SC,
Turley H,
Gatter KC,
Harris A,
and
Higgins CF.
Immunocytochemical localization of the cystic fibrosis gene product CFTR.
Proc Natl Acad Sci USA
88:
9262-9266,
1991[Abstract].
14.
Edmonds, CJ,
and
Pilcher D.
Electrical potential difference and sodium and potassium fluxes across rectal mucosa in ulcerative colitis.
Gut
14:
784-789,
1973[ISI][Medline].
15.
Eggermont, E,
and
De Boeck K.
Small-intestinal abnormalities in cystic fibrosis patients.
Eur J Pediatr
150:
824-828,
1991[ISI][Medline].
16.
Gabriel, SE,
Brigman KN,
Koller BH,
Boucher RC,
and
Stutts MJ.
Cystic fibrosis heterozygote resistance to cholera toxin in the cystic fibrosis mouse model.
Science
266:
107-109,
1994[ISI][Medline].
17.
Greeley, T,
Shumaker H,
Wang ZH,
Schweinfest CW,
and
Soleimani M.
Downregulated in adenoma and putative anion transporter are regulated by CFTR in cultured pancreatic duct cells.
Am J Physiol Gastrointest Liver Physiol
281:
G1301-G1308,
2001
18.
Greig, E,
and
Sandle GI.
Diarrhea in ulcerative colitis-the role of altered colonic sodium transport.
Ann NY Acad Sci
915:
327-332,
2000
19.
Guerrant, RL,
Hughes JM,
Lima NL,
and
Crane J.
Diarrhea in developed and developing countries: magnitude, special settings, and etiologies.
Rev Infect Dis
12, Suppl1:
S41-S50,
1990[ISI][Medline].
20.
Haila, S,
Saarialho-Kere U,
Karjalainen-Lindsberg ML,
Lohi H,
Airola K,
Holmberg C,
Hästbacka J,
Kere J,
and
Höglund P.
The congenital chloride diarrhea gene is expressed in seminal vesicle, sweat gland, inflammatory colon epithelium, and in some dysplastic colon cells.
Histochem Cell Biol
113:
279-286,
2000[ISI][Medline].
21.
Harris, J,
and
Shields R.
Absorption and secretion of water and electrolytes by the intact human colon in diffuse untreated proctocolitis.
Gut
11:
27-33,
1970[ISI][Medline].
22.
Hawker, PC,
McKay JS,
and
Turnberg LA.
Electrolyte transport properties of colonic epithelium in patients with inflammatory bowel disease.
Gastroenterology
79:
508-511,
1980[ISI][Medline].
23.
Hoglund, P,
Haila S,
Socha J,
Tomaszewski L,
Saarialho-Kere U,
Karjalainen-Lindsberg ML,
Airola K,
Holmberg C,
de la Chapelle A,
and
Kere J.
Mutations of the Down-regulated in adenoma (DRA) gene cause congenital chloride diarrhoea.
Nat Genet
14:
316-319,
1996[ISI][Medline].
24.
Holmberg, C,
Perheentupa J,
and
Launiala K.
Colonic electrolyte transport in health and in congenital chloride diarrhea.
J Clin Invest
56:
302-310,
1975[ISI][Medline].
25.
Kere, J,
Lohi H,
and
Höglund P.
Genetic Disorders of Membrane Transport. III. Congenital chloride diarrhea.
Am J Physiol Gastrointest Liver Physiol
276:
G7-G13,
1999
26.
Kiela, PR,
Guner YS,
Xu H,
Collins JF,
and
Ghishan FK.
Age- and tissue-specific induction of NHE3 by glucocorticoids in the rat small intestine.
Am J Physiol Cell Physiol
278:
C629-C637,
2000
27.
Knickelbein, R,
Aronson PS,
Schron CM,
Seifter J,
and
Dobbins JW.
Sodium and chloride transport across rabbit ileal brush border. II. Evidence for Cl/HCO
28.
Kunzelmann, K,
Schreiber R,
Nitschke R,
and
Mall M.
Control of epithelial Na+ conductance by the cystic fibrosis transmembrane conductance regulator.
Pflügers Arch
440:
193-201,
2000[ISI][Medline].
29.
Lee, MG,
Choi JY,
Luo X,
Strickland E,
Thomas PJ,
and
Muallem S.
Cystic fibrosis transmembrane conductance regulator regulates luminal Cl/HCO
30.
Livak, KJ,
Flood SJ,
Marmaro J,
Giusti W,
and
Deetz K.
Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization.
PCR Methods Appl
4:
357-362,
1995[ISI][Medline].
31.
Lohi, H,
Kujala M,
Kerkelä E,
Saarialho-Kere U,
Kestilä M,
and
Kere J.
Mapping of five new putative anion transporter genes in human and characterization of SLC26A6, a candidate gene for pancreatic anion exchanger.
Genomics
70:
102-112,
2000[ISI][Medline].
32.
Madara, JL,
and
Stafford J.
Interferon- directly affects barrier function of cultured intestinal epithelial monolayers.
J Clin Invest
83:
724-727,
1989[ISI][Medline].
33.
Mall, M,
Bleich M,
Schurlein M,
Kuhr J,
Seydewitz HH,
Brandis M,
Greger R,
and
Kunzelmann K.
Cholinergic ion secretion in human colon requires coactivation by cAMP.
Am J Physiol Gastrointestinal Liver Physiology
38:
G1274-G1281,
1998.
34.
Melvin, JE,
Park K,
Richardson L,
Schultheis PJ,
and
Shull GE.
Mouse down-regulated in adenoma (DRA) is an intestinal Cl()/HCO(3)(
) exchanger and is up-regulated in colon of mice lacking the NHE3 Na(+)/H(+) exchanger.
J Biol Chem
274:
22855-22861,
1999
35.
Moseley, RH,
Höglund P,
Wu GD,
Silberg DG,
Haila S,
de la Chapelle A,
Holmberg C,
and
Kere J.
Downregulated in adenoma gene encodes a chloride transporter defective in congenital chloride diarrhea.
Am J Physiol Gastrointest Liver Physiol
276:
G185-G192,
1999
36.
Muller, T,
Wijmenga C,
Phillips AD,
Janecke A,
Houwen RHJ,
Fischer H,
Ellemunter H,
Fruhwirth M,
Offner F,
Hofer S,
Muller W,
Booth IW,
and
Heinz-Erian P.
Congenital sodium diarrhea is an autosomal recessive disorder of sodium/proton exchange but unrelated to known candidate genes.
Gastroenterology
119:
1506-1513,
2000[ISI][Medline].
37.
Sandle, GI,
Hayslett JP,
and
Binder HJ.
Effect of glucocorticoids on rectal transport in normal subjects and patients with ulcerative colitis.
Gut
27:
309-316,
1986[Abstract].
38.
Sandle, GI,
Higgs N,
Crowe P,
Marsh MN,
Venkatesan S,
and
Peters TJ.
Cellular basis for defective electrolyte transport in inflamed human colon.
Gastroenterology
99:
97-105,
1990[ISI][Medline].
39.
Schmitz, H,
Barmeyer C,
Fromm M,
Runkel N,
Foss HD,
Bentzel CJ,
Riecken EO,
and
Schulzke JD.
Altered tight junction structure contributes to the impaired epithelial barrier function in ulcerative colitis.
Gastroenterology
116:
301-309,
1999[ISI][Medline].
40.
Schultheis, PJ,
Clarke LL,
Meneton P,
Miller ML,
Soleimani M,
Gawenis LR,
Riddle TM,
Duffy JJ,
Doetschman T,
Wang T,
Giebisch G,
Aronson PS,
Lorenz JN,
and
Shull GE.
Renal and intestinal absorptive defects in mice lacking the NHE3 Na+/H+ exchanger.
Nat Genet
19:
282-285,
1998[ISI][Medline].
41.
Schweinfest, CW,
Henderson KW,
Suster S,
Kondoh N,
and
Papas TS.
Identification of a colon mucosa gene that is down-regulated in colon adenomas and adenocarcinomas.
Proc Natl Acad Sci USA
90:
4166-4170,
1993[Abstract].
42.
Silberg, DG,
Wang W,
Moseley RH,
and
Traber PG.
The downregulated in adenoma (DRA) gene encodes an intestine-specific membrane sulfate transport protein.
J Biol Chem
270:
11897-11902,
1995
43.
Stutts, MJ,
Lazarowski ER,
Paradiso AM,
and
Boucher RC.
Activation of CFTR Cl conductance in polarized T84 cells by luminal extracellular ATP.
Am J Physiol Cell Physiol
268:
C425-C433,
1995
44.
Sundaram, U,
and
West AB.
Effect of chronic inflammation on electrolyte transport in rabbit ileal villus and crypt cells.
Am J Physiol Gastrointest Liver Physiol
272:
G732-G741,
1997
45.
Sundaram, U,
Wisel S,
Rajendren VM,
and
West AB.
Mechanism of inhibition of Na+-glucose cotransport in the chronically inflamed rabbit ileum.
Am J Physiol Gastrointest Liver Physiol
273:
G913-G919,
1997
46.
Truelove, SC,
and
Richards WC.
Biopsie studies in ulcerative colitis.
BMJ
3:
1315-1318,
1956.
47.
Umar, S,
Scott J,
Sellin JH,
Dubinsky WP,
and
Morris AP.
Murine colonic mucosa hyperproliferation. I. Elevated CFTR expression and enhanced cAMP-dependent Cl secretion.
Am J Physiol Gastrointest Liver Physiol
278:
G753-G764,
2000
48.
Vaandrager, AB,
Bot AG,
and
De Jonge HR.
Guanosine 3',5'-cyclic monophosphate-dependent protein kinase II mediates heat-stable enterotoxin-provoked chloride secretion in rat intestine.
Gastroenterology
112:
437-443,
1997[ISI][Medline].
49.
Wardle, TD,
Hall L,
and
Turnberg LA.
Interrelationships between inflammatory mediators released from colonic mucosa in ulcerative-colitis and their effects on colonic secretion.
Gut
34:
503-508,
1993[Abstract].
50.
Wardle, TD,
and
Turnberg LA.
Potential role for interleukin-1 in the pathophysiology of ulcerative-colitis.
Clin Sci
86:
619-626,
1994[ISI][Medline].
51.
Wheat, VJ,
Shumaker H,
Burnham C,
Shull GE,
Yankaskas JR,
and
Soleimani M.
CFTR induces the expression of DRA along with Cl/HCO
52.
Yang, H,
Jiang W,
Furth EE,
Wen X,
Katz JP,
Sellon RK,
Silberg DG,
Antalis TM,
Schweinfest CW,
and
Wu GD.
Intestinal inflammation reduces expression of DRA, a transporter responsible for congenital chloride diarrhea.
Am J Physiol Gastrointest Liver Physiol
275:
G1445-G1453,
1998
53.
Ziambaras, T,
Rubin DC,
and
Perlmutter DH.
Regulation of sucrase-isomaltase gene expression in human intestinal epithelial cells by inflammatory cytokines.
J Biol Chem
271:
1237-1242,
1996