Departments of Pediatrics and Physiology, Steele Memorial Children's Research Center, University of Arizona Health Sciences Center, Tucson, Arizona 85724
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ABSTRACT |
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We have previously described changes in intestinal brush-border membrane vesicle (BBMV) Na+/H+ exchange activity and characterized Na+/H+ exchanger (NHE3) expression during rat ontogeny. The current studies were designed to investigate developmental changes in NHE2 expression in rat intestine. In previous studies, pH-dependent uptake of Na+ in jejunal BBMV utilizing HOE-694 inhibition demonstrated that NHE2 functional protein levels were lowest in 2-wk-old rats, higher in 3-wk-old and adult rats, and highest in 6-wk-old rats [Collins et al. Am. J. Physiol. 273 (Cell Physiol. 42): C1937-C1946, 1997]. In the current investigation, Northern blot analyses showed that NHE2 mRNA levels in the jejunum were similar in 6-wk-old, adult, and 3-wk-old rats and three- to fivefold lower in 2-wk-old rats. In situ hybridization of 2- and 6-wk-old rat intestine with NHE2-specific probes confirmed Northern blot observations. Polyclonal antibodies were developed against an NHE2-specific peptide from amino acids 652-661. Western blots with NHE2 antiserum showed that the intensity of a specific 90-kDa band was lowest in 2-wk-old animals and four- to sixfold higher in 3- and 6-wk-old and adult animals. Immunohistochemical analysis showed specific staining of NHE2 antiserum to only the apical intestinal membrane. Furthermore, nuclear run-on analyses showed a 1.7-fold higher NHE2 transcription rate in 6-wk-old rats than in 2-wk-old rats. Overall, the current data suggest that increases in NHE2 expression upon weaning are mediated by increased gene transcription.
rat intestinal development; sodium/hydrogen exchanger; brush-border membrane; transcription rate analysis; jejunum
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INTRODUCTION |
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THE SODIUM/HYDROGEN exchangers (NHE) are plasma-membrane-bound transport proteins that function to exchange cytoplasmic H+ for extracellular Na+. Of several identified isoforms, NHE3 and NHE2 are known to be expressed on the apical membrane in the intestinal epithelium (12). NHE3 has been cloned from several species (1, 18, 21), and this isoform is thought to be involved in transepithelial Na+ absorption. Northern blot analyses detected NHE3 message in rabbit kidney cortex and medulla, jejunum, ileum, ascending colon, and stomach (18, 23). The immunoreactive NHE3 protein has been localized to the brush-border membrane (BBM) in rabbit, rat, and human ileum (7, 8, 12).
After the original cloning and kinetic characterization of NHE2 (5, 11), this exchanger was cloned from several species (19, 22, 23). Previous studies have shown that NHE2 mRNA is expressed in several mammalian tissues including kidney, liver, stomach, large intestine, jejunum, uterus, spleen, lung, brain, and testis (5). NHE2 immunoreactive protein has been localized to the BBM in human and rabbit intestinal epithelium (21). Despite precise localization in the intestinal epithelium, the physiological role of intestinal NHE2 remains unknown. It was shown, however, that, when transfected into mutagenized Chinese hamster ovary cells devoid of endogenous NHE activity, NHE2 was capable of regulating intracellular pH, cell volume, and cell proliferation in a manner similar to NHE1 (13). However, the physiological significance of these observations remains unclear.
The activity of the NHEs has been investigated at the functional level for the past two decades, without a true understanding of the underlying molecular events. With the recent cloning of several isoforms of these transport proteins, these investigations may now proceed at a molecular level. It has been previously documented that Na+/H+ exchange activity varies with postnatal development in rat intestinal BBM vesicles (BBMV) (14). It was unclear, however, whether these observations were the result of the activity of NHE2 and/or NHE3. We have previously characterized the contribution of NHE3 to basal Na+ uptake and NHE3 mRNA and immunoreactive protein expression during rat intestinal development. These data demonstrated that NHE3 is likely regulated at transcriptional and posttranscriptional levels during ontogeny (7). We also found that both NHE2 and NHE3 contribute almost equally to basal Na+/H+ exchange in 2- and 3-wk-old animals, whereas in older rats the NHE2 contribution is minimal (7). However, the molecular expression and regulation of NHE2 during intestinal development have not been previously investigated. The current studies were undertaken to define the molecular expression of intestinal NHE2 during postnatal development in rats in an effort to elucidate the physiological role of NHE2 in the mammalian intestine.
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METHODS |
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Animals. Sprague-Dawley rats were used in groups of at least four animals for all experiments except in situ hybridization and immunohistochemical analyses. Male rats of the following ages were used for all studies: 2 wk (sucklings) at 14 days of age, 3 wk (weanlings) at 21 days of age, 6 wk (adolescents) at 42-45 days of age, and adult at 4-6 mo of age. Animals were maintained in overhanging cages, with food and water supplied ad libitum. Animals were subjected to CO2 narcosis and were killed by cervical dislocation.
Chemicals and reagents.
Poly(A)+ RNA was isolated
utilizing the FastTrack kit from Invitrogen (La Jolla, CA).
22Na [100-2,000 Ci
(3.70-74.0 TBq)/g] for uptake studies and
[-32P]dCTP
(3,000Ci/mmol) for Northern blot analyses were purchased from NEN
(Boston, MA). Radioactive probes for Northern blot analyses were
generated by random prime labeling using the RediPrime system from
Amersham Life Science (Piscataway, NJ). Nitrocellulose membranes (Nitroplus) were from Micron Separations (Westboro, MA). DNA
fragments were gel purified utilizing the GeneClean kit from BIO101
(Vista, CA). Protein was quantitated by a Bradford assay utilizing the Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA). All other chemicals and reagents were purchased from Fisher Biotechnology (Pittsburgh, PA) or Sigma Chemical (St. Louis, MO).
Northern blot analysis. Poly(A)+ RNA was isolated from the jejunum of at least four rats per group, utilizing a commercially available kit. This method uses a tissue lysis buffer containing RNase and protein degraders and oligo(dT)-cellulose affinity chromatography. Northern blots were carried out as previously described using 5 µg poly(A)+ RNA per lane (3, 4). DNA fragments were used to generate probes for hybridization that were shown to be isoform specific by slot-blot analyses as described previously (a BamH I/Bgl II NHE2 restriction fragment that spanned bp 2105-3573 and was shown to not cross-react with other NHE isoforms) (7). Additionally, some blots were reacted with NHE4-specific probes (from a region of the cDNA that has no nucleotide sequence homology with NHE2) (7). High-stringency washes were performed at 65°C with 0.1× standard sodium citrate (SSC), 0.1% SDS, and blots were placed on a phosphorimaging screen or film. Northern blots were stripped and subsequently reprobed with 1B15 (encoding rat cyclophilin)-specific probes (9). Quantitation of hybridization signals was done by phosphorimage analysis utilizing volume integration (GS 525 molecular imager; Bio-Rad). The Northern blot experiment was performed in triplicate with poly(A)+ RNA samples from different groups of animals, with hybridization intensities being averaged from the three experiments. Hybridization intensities were normalized for 1B15 levels on the same blot.
In situ hybridization. RNA probes were
produced from the 5' and 3' ends of NHE2, regions that show
no nucleotide sequence similarity with the other NHE isoforms. Probes
were generated in the presence of
[35S]UTP, and 5 × 105
counts · min1(cpm) · µl
1
of reaction solution were used for hybridizations to
paraformaldehyde-fixed, paraffin-embedded tissue sections. The entire
experimental protocol has been extensively described previously (4),
and it was followed exactly in the present investigation.
Production of polyclonal antiserum specific for NHE2. A multiple antigen peptide (MAP) corresponding to amino acids 652-661 of the putative NHE2 protein (encoded by bp 2140-2169 of the nucleotide sequence) (23) was synthesized by Research Genetics (Huntsville, AL). This region of the molecule showed no amino acid sequence homology to other NHE isoforms and, furthermore, using a GenBank search, we did not identify other proteins with significant sequence identity to this peptide. The peptide was sent to Lampire Biological Laboratories (Pippersville, PA) for polyclonal antibody production in rabbits. Crude serum-coded Ab6450 was used for Western blot and immunohistochemical analyses.
Western blot analysis of rat jejunal BBM proteins with
NHE2-specific antiserum. Intestinal BBM proteins were
purified by a MgCl2 precipitation
method as previously described (6, 15-17). Additionally,
basolateral membrane vesicles were prepared by a previously described
method (8). Twenty micrograms of protein were placed in a twofold
excess of Laemmli solubilization buffer plus 2 mM -mercaptoethanol,
boiled for 4 min, and placed on ice. Protein samples were fractionated
by 4-12% gradient SDS-PAGE and transferred onto nitrocellulose
membranes. Blots were processed as previously described (2, 6) with the
Renaissance chemiluminescent system (DuPont NEN) with 1:500 dilutions
of NHE2-specific antiserum. Additionally, some blots were reacted with
NHE3 serum (7, 8), preimmune NHE2 serum, and serum that was pretreated
with antigenic peptide (400 µg/ml at 25°C overnight) at 1:500
dilution. Membranes were stripped and subsequently reacted with
-actin antiserum (Sigma) at 1:5,000 dilution. NHE2-specific
hybridization band intensities were determined by densitometric
analysis [utilizing GS-700 imaging densitometer and molecular
analyst software (Bio-Rad)] and were normalized for
-actin
hybridization intensities on the same blot. The experiment was repeated
six times with protein samples isolated from different groups of
animals.
Immunohistochemical analysis of rat intestine with NHE2-specific antiserum. Jejunal tissue was harvested from 3-wk-old rats, fixed in paraformaldehyde, and embedded in paraffin, and sections were cut and affixed to slides. Slides were blocked by overnight incubation with 5% normal goat serum (Vector Labs, Burlingame, CA) at room temperature in a humidified chamber. NHE2 antiserum was reacted with sections for 1 h at 1:100 dilution in 0.1% normal goat serum. Some sections were reacted with preimmune serum (1:100) and immunogenic peptide-pretreated serum (400 µg/ml at 25°C overnight) at 1:100 dilution. Slides were subsequently reacted with indocarbocyanine-labeled anti-rabbit antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) at 1:1,000 dilution and visualized by fluorescent light microscopy. Pictures were captured with the Metamorph imaging system and software (version 2.0; Universal Imaging, West Chester, PA) using identical settings for both samples and are presented in pseudocolor. Slides were analyzed by two "blinded" observers to gain independent opinions about staining intensity and localization.
Nuclei isolation and nuclear run-on analysis of the NHE2 gene in rat jejunum. Jejunums were excised from animals, mucosal scrapings were taken, and 1 g of tissue was used for nuclei isolation. Transcriptionally active nuclei were isolated from at least four rats per group exactly as previously described (6); 1-5 × 107 nuclei were routinely obtained per gram of tissue. Radiolabeled transcription reactions (nuclear run-ons) were performed with at least 1 × 107 nuclei as previously described (6). Equal numbers of counts per minute were used for hybridizations.
pBluescript plasmids containing the NHE2 cDNA (5), -actin, or
pBluescript plasmids with no cDNA insert were denatured and added to
each of two slots on a slot-blot apparatus. Prehybridization was
carried out for 16 h, and hybridization with radiolabeled transcription
reaction products (at least 1 × 106 cpm/ml hybridization solution)
was carried out as previously described for 3-5 days at 42°C
(6). Posthybridization washes were done several times for 45-60
min under high-stringency conditions (0.1× SSC, 0.1% SDS, 1 mM
EDTA at 42°C). Membranes were then exposed to a phosphorimaging
screen for 2-4 days. Hybridization band intensities were
determined by volume integration using phosphorimage analysis (GS 525 molecular imager; Bio-Rad Life Science). Three independent experiments
were performed, with nuclei isolated from different groups of animals.
Statistical analysis of results. Data from Northern and Western blot analyses were analyzed for statistical significance by one-way ANOVA, utilizing the StatView program (Abacus Concepts, Berkeley, CA) and are presented as means ± SE, with n and P values reported. Data from nuclear run-on analyses were analyzed by the unpaired two-tailed t-test utilizing the same program.
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RESULTS |
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Northern blot analysis. Northern blots were hybridized with NHE2 (BamH I/Bgl II fragment) and 1B15 specific radiolabeled probes. NHE2 hybridization levels (as normalized for 1B15 levels) in the jejunum were similar in 6-wk-old (51.46 ± 8.87 phosphorimage units), adult (47.86 ± 7.73), and 3-wk-old (33.04 ± 5.02) rats and were significantly lower in 2-wk-old rats (10.54 ± 1.62; n = 3; P = 0.0389 for 2 wk-old vs. 3-wk-old, P = 0.0020 for 2-wk-old vs. 6-wk-old, P = 0.0035 for 2-wk-old vs. adult; other groups not different; Fig. 1). Furthermore, NHE4-specific probes showed no hybridization in intestinal mRNA samples even under reduced stringency conditions (data not shown).
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In situ hybridization. Jejunal tissue was harvested from 2- and 6-wk-old rats, fixed in paraformaldehyde, embedded in paraffin, sectioned, and placed on slides. NHE2 sense and antisense RNA probes were hybridized with the tissue sections for 18 h. NHE2 antisense RNA probes showed specific hybridization to the epithelium of the jejunum in 6-wk-old animals, and the signal was visible along the villus-crypt axis (Fig. 2, panel 2). However, the signal was not visible in the crypt or the lamina propria. Antisense RNA probes also hybridized to the jejunal epithelium in 2-wk samples (Fig. 2, panel 1); however, signal intensity was extremely low compared with 6-wk tissue sections. Sense RNA probes showed no hybridization signal in 2- or 6-wk-old rat jejunal epithelia (6-wk sense hybridization is depicted in Fig. 2, panel 3).
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Western blot analyses of intestinal BBM proteins with
NHE2-specific antiserum. Polyclonal antibodies were
developed against an NHE2-specific peptide. Preimmune serum and
antigen-pretreated Ab6450 serum recognized no protein band in either
intestinal brush-border or basolateral membranes (Fig.
3). However, Ab6450 serum recognized a
single band of ~90 kDa in intestinal brush-border but not in basolateral membrane preparation. This band was distinct from the
85-kDa band recognized by NHE3-specific antibody (Fig. 3) (7). Western
blot analyses of jejunal brush-border protein from different age groups
with NHE2-specific Ab6450 serum showed specific recognition of a 90-kDa
band (Fig. 4), with lowest signal intensity
in 2-wk-old animals (0.035 ± 0.009 densitometric units) and
significantly higher intensities in 3-wk-old (0.197 ± 0.01), 6-wk-old (0.164 ± 0.019), and adult animals (0.157 ± 0.026; n = 6;
P < 0.0001 between all groups and
2-wk-old group; other groups not different; Fig. 4). Protein samples
that were reacted with antigenic protein blocked and preimmune serum
showed no protein recognition on immunoblots (data not shown). All
samples showed positive reaction with -actin serum.
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Immunohistochemical analyses of rat jejunum with NHE2-specific antiserum. To further exemplify the specificity of Ab6450 serum, jejunal tissue was excised from animals, fixed in paraformaldehyde, embedded in paraffin, sectioned, and affixed to slides. Slides were reacted with NHE2-specific Ab6450 serum. Results showed specific staining of the apical membranes of the intestinal epithelium, with highest intensities at the villi tips (Fig. 5A). No signal was detected at the basolateral membranes or in any other cell type within the intestinal epithelium. Antigenic peptide blocked serum (400 µg/ml, 25°C for 16 h; not shown), and preimmune serum (Fig. 5B) showed no staining of apical membranes or other cell types within the jejunal epithelium.
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Nuclear run-on analyses. Radiolabeled nuclear run-on reactions (transcription reactions) were performed with nuclei isolated from different age group rats and were hybridized to cDNAs immobilized on nitrocellulose membranes. The groups studied were those with the highest and lowest NHE2 expression as determined by Northern blot analyses and BBMV uptake analyses (i.e., 6- and 2-wk jejunum). Results showed a higher relative NHE2 transcription rate in 6-wk rat intestine (0.78 ± 0.08 phosphorimage units) compared with 2 wk rat intestine (0.46 ± 0.03; n = 3, P = 0.02; Fig. 6).
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DISCUSSION |
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To investigate the relationship between NHE2 expression and ontogenic changes in intestinal BBMV Na+/H+ exchange activity, a series of experiments in rats at 2-, 3-, and 6-wk and 4-6 mo of age were performed. To determine mRNA expression levels, we isolated poly(A)+ RNA from rats at these various stages of development. Northern blots were probed with NHE2- and 1B15-specific probes. On the basis of the differences observed in previous uptake studies and on Northern blots, 2- and 6-wk-old age groups were selected to perform in situ hybridizations. Additionally, polyclonal peptide antiserum specific for NHE2 was produced and characterized. Western blots were utilized to assess the level of immunoreactive protein expression at the different ages. Furthermore, nuclear run-on analyses were performed to determine whether the observed molecular changes between 2-wk-old rats and older animals are due to changes in NHE2 gene transcription rates.
An earlier study by our group investigated intestinal BBMV Na+ uptake in 2-, 3-, and 6-wk-old rats (14). These previous investigations determined that 10-s uptake represented the initial rate in all groups and that there were no differences between age groups in the following experimental parameters: enrichment of membrane preparations for BBM marker (alkaline phosphatase), impoverishment of membrane preparations for a basolateral membrane marker (Na+-K+-ATPase), a mitochondrial membrane marker (cytochrome c-oxidase) and an endoplasmic reticulum membrane marker (NADPH cytochrome c reductase), level of Na+ binding to vesicles as opposed to uptake into intravesicular space, affinity of the exchanger for Na+ (Km), and rate of pH dissipation and generation of negative membrane potential. These observations suggested that the phenomenon observed utilizing this method is based solely on the activity of the apical membrane NHEs. Data showed highest NHE activity in 6-wk-old animals and lower activity in the 3- and 2-wk-old groups, with apparent changes due to maximum velocity (Vmax) differences between groups.
In another recent investigation, we sought to expand on these previous observations to look at initial rate BBMV uptake in 2-, 3-, and 6-wk-old and adult rats and to attempt to assess the relative contribution of NHE3 and NHE2 at each age by comparing uptake without HOE-694 and with 50 and 800 µM HOE-694 (7). The NHE2 contribution was estimated by subtracting the uptake rates in the absence of HOE-694 (which represents the activity of both NHE2 and NHE3) from uptake rates in the presence of 50 µM HOE-694 (which represents the activity of only NHE3). At all ages, 50 µM HOE-694 had a significant effect, suggesting that NHE2 and NHE3 both contribute to basal Na+/H+ exchange activity. However, in the 2- and 3-wk-old age groups, NHE2 contribution was ~41% of basal uptake. Six-week animals showed a very small contribution of NHE2 to basal rates (8%), whereas adult rats had a slightly higher relative NHE2 contribution of 23%. These results demonstrate that, in suckling and recently weaned animals, NHE2 and NHE3 contribute more or less equally to basal Na+/H+ exchange activity, whereas, in older animals, NHE2 contributes to a much lesser extent. Additionally, we found that there was an approximately fourfold increase in functional NHE2 activity between 2 and 6 wk of age (7).
When considered together, these previous observations strongly suggested that BBM Na+/H+ exchange activity in the rat small intestine is regulated during ontogeny and that the relative contribution of both apical isoforms to basal activity varies during development. However, the most significant previous finding was that NHE2 represents a significant portion of basal NHE activity in young animals. This observation suggested that early in life NHE2 plays an important role in transepithelial Na+ absorption. Another recent study, which produced a mouse strain with a null mutation in the NHE2 gene, showed that mutated animals had no intestinal absorptive defect (20). However, since this study did not focus on characterizing intestinal NHE expression during development, it is difficult to make strong conclusions concerning the physiological role of NHE2 expression in the developing mammalian intestine.
To understand how NHE2 expression is regulated during development, further studies were performed to look at NHE2 mRNA levels by Northern blot analyses. NHE2 hybridization levels in the jejunum were highest in 3- and 6-wk-old and adult rats, and approximately three- to fivefold lower in 2-wk-old animals compared with the other ages. To confirm differential expression of the NHE2 message with age, in situ hybridization was performed with NHE2-specific sense and antisense RNA probes on paraformaldehyde-fixed jejunal sections from the groups that showed highest (6 wk) and lowest (2 wk) NHE2 mRNA expression. Very strong signal intensity was noted with antisense probes only in 6-wk jejunum, and very low (almost undetectable) levels were noted in 2-wk jejunum. The hybridization signal was concentrated along the villus-crypt axis, with no hybridization apparent in the crypts or lamina propria.
To further characterize the molecular expression of rat intestinal NHE2 during development, an exchanger-specific polyclonal antiserum was produced and characterized. Initially, immunoblots were performed on BBM proteins utilizing this peptide antiserum (Ab6450). The antiserum recognized a single, specific protein band on Western blots of BBM proteins that was ~90 kDa. This single band was not detected in basolateral membrane protein preparations, was blockable by pretreating the serum with antigenic peptide, and was not detected with preimmune serum. Additionally, immunohistochemical analyses showed specific, blockable staining of apical membranes in the rat intestinal epithelium, with no staining apparent in basolateral membranes, crypts, goblet cells, or lamina propria. The intensity of staining was the strongest at the tips of villi, which is in agreement with previously published data (10). In this previous study, mRNA was purified from enterocytes isolated from along the crypt-villus axis, and Northern blots were performed and probed with NHE2-specific probes. Results demonstrated highest NHE2 message abundance in the fraction corresponding to the villus tip (10). All of these observations confirm that the antiserum is indeed recognizing the NHE2 protein.
Western blot analyses of jejunal brush-border proteins from different age groups showed specific recognition of a 90-kDa band with the signal intensity in 2-wk-old animals being decreased four- to fivefold compared with other age groups. These values parallel the data obtained in the functional studies and by Northern blot and reflect significant increases in NHE2 levels around weaning. Together, these data led us to hypothesize that increases in NHE2 gene transcription rates were likely responsible for increased NHE2 expression during ontogeny.
In an attempt to further test this hypothesis, we performed nuclear run-on analyses to assess the relative transcription rates of the NHE2 gene in 6- and 2-wk-old rat intestine. This experiment was designed based on the fact that NHE2 mRNA expression was highest in 6-wk-old rats and lowest in 2-wk-old animals. For this assay, plasmids containing NHE2 cDNAs were denatured and affixed to nitrocellulose membranes. Membranes were hybridized with radiolabeled transcription reactions (run-ons) from nuclei isolated from 2- and 6-wk rat intestinal mucosa. Full-length NHE2 cDNA was utilized due to the fact that we detected no NHE4 mRNA expression in rat jejunum and this is the only isoform with nucleotide sequence similarity to NHE2 that may cause cross-reactivity (7). Results showed a 1.7-fold higher relative NHE2 transcription rate in 6-wk-old animals compared with 2-wk-old animals. Because of the relative insensitivity of the nuclear run-on technique, one could expect that the obtained difference is underestimated (due to the fact that only differences in the 3- to 5-fold range or greater are detectable utilizing this experimental methodology). These data more strongly suggested that observed differences in ontogenic NHE2 expression are related to differential gene transcription rates.
Overall, the current data show that NHE2 is likely regulated at a transcriptional level in the developing rat jejunum, as evidenced by functional studies, Northern blots, immunoblots, and nuclear run-on analyses. Therefore, the current data provide a mechanistic explanation for observed increases in NHE2 expression between suckling and older animals. These findings also exemplify the importance of designing subsequent studies to identify the genetic elements responsible for transcriptional regulation of NHE2 during ontogeny. In this sense, NHE2 may serve as a "model gene" with which to understand the precise molecular mechanisms that regulate gene expression in the developing mammalian small intestine. Overall, these current and proposed future studies will help further elucidate the physiological role of intestinal NHE2 and should also provide important information concerning intestinal gene regulation during development.
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ACKNOWLEDGEMENTS |
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This investigation was funded by National Institute of Diabetes and Digestive and Kidney Diseases Grant 2R01-DK-41274.
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FOOTNOTES |
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Some of the material in this paper has appeared previously in abstract form [J. F. Collins, H. Xu, and F. K. Ghishan. Ontogeny of the apical sodium-hydrogen exchanger (NHE) isoforms 2 and 3 in rat jejunum and kidney. Gastroenterology 110: A796, 1996].
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: F. K. Ghishan, Dept. of Pediatrics, Univ. of Arizona Health Sciences Center, 1501 N. Campbell Ave., Tucson, AZ 85724.
Received 21 May 1998; accepted in final form 1 July 1998.
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