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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Ontogenic changes occur in intestinal brush-border membrane vesicle (BBMV) Na+/H+ exchange activity. The present studies were designed to investigate ontogenic changes in Na+/H+ exchanger (NHE) isoform 3 in rat jejunum. pH-dependent Na+ uptake was assayed in four age groups of rats in the presence of 0, 50, or 800 µM HOE-694, a specific NHE inhibitor with differential sensitivities for NHE2 [inhibition constant (Ki) = 5 µM in PS120 fibroblasts] and NHE3 (Ki = 650 µM). Results showed that NHE2 and NHE3 contribute to basal BBMV uptake at all ages. Uptake levels were highest in 6-wk-old rats, lower in adult rats, and lowest in 2-wk-old (suckling) and 3-wk-old (weanling) rats. NHE3 contribution ranged from 92% at 6 wk of age to 59% at 2 and 3 wk of age. NHE3 inhibition by 800 µM HOE-694 was 38-45%. Statistical analysis showed that HOE-694 had a significant effect at both concentrations at all ages and that differences were present between all ages except 2- and 3-wk rats (at all HOE-694 concentrations). Northern blot analyses of jejunal mucosa showed lowest NHE3 mRNA levels in 2-wk animals and higher levels in all other age groups. Polyclonal antibodies were developed against an NHE3 COOH-terminal fusion protein, and antiserum was characterized with NHE3-transfected PS120 cells and by immunohistochemistry. Western blot analyses showed lowest protein levels in 2-wk animals and higher levels in the other ages. Suckling rats were subcutaneously injected with methylprednisone (MP) for 2 days and killed 1 day later. Northern blot analyses showed a twofold increase in NHE3 mRNA expression with MP treatment. Immunoblot analyses showed a 2.5-fold increase in NHE3 immunoreactive protein levels with MP injection. Overall, these data suggest that NHE3 is regulated during ontogeny and that ontogenic changes are most apparent around the time of weaning. Furthermore, the data suggest that NHE3 is regulated at transcriptional and posttranscriptional levels during mammalian intestinal development.
rat intestinal ontogeny; sodium-hydrogen exchanger; brush-border membrane
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INTRODUCTION |
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THE SODIUM-HYDROGEN exchangers (NHEs) are integral membrane transport proteins that function to exchange cytoplasmic H+ for extracellular Na+. The first NHE identified and cloned at the molecular level was called NHE1 (21). Since that time, several other isoforms of NHEs have been identified (6, 18-20, 23-26) and designated NHE2, NHE3, and NHE4. This family of transport proteins is thought to be involved in intestinal Na+ absorption and in cell volume and intracellular pH (pHi) regulation. Additionally, the NHEs are thought to play roles in several disease states, including diarrheal disorders, hypertension, and cardiac ischemia (29). The investigation of NHEs is of clinical relevance due to this likely link between them and these disorders in humans.
NHE1 is the most ubiquitous isoform and has been cloned from several species (8, 18, 21, 25). This isoform is thought to play a role in cell volume and pHi regulation (21). NHE1 mRNA is expressed in nearly all mammalian cells, as determined by Northern blot analysis (29). Immunohistochemical analyses have localized NHE1 protein to the basolateral membrane in rabbit ileum (25), in Caco-2 and LLC-PK1 cell lines (20, 27), and in rabbit kidney epithelium (1). After the original cloning and kinetic characterization of NHE2 (6, 12), this exchanger was cloned from several species (19, 24, 26). NHE2 most likely plays a role in intestinal Na+ absorption under certain physiological conditions, and it has recently been proposed to be involved in cell volume regulation in renal medullary collecting duct cells (22). NHE2 mRNA was detected in several mammalian tissues, including kidney, liver, stomach, large intestine, jejunum, uterus, spleen, lung, brain, and testis (6). NHE2 immunoreactive protein has been localized to the brush-border membrane (BBM) in human and rabbit intestinal epithelia (22) and in renal cortical epithelium (29). NHE3 has been recently cloned from several different species (2, 18, 23). This isoform has been proposed to be involved in transepithelial Na+ absorption in the mammalian gastrointestinal tract. 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 BBM in rabbit and human ileum and in rabbit renal tubules (29). NHE4 has only been cloned from the rat (18). The NHE4 message was expressed in rat stomach, intestine, kidney, uterus, and brain. The exact function and localization of this isoform are unknown, although one recent study suggested that NHE4 protein was expressed on the basolateral membrane in renal inner medullary collecting ducts (11).
The activity of the NHEs has been investigated at the functional level for the past two decades, without a true understanding of the molecular events responsible for these observations. 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 ontogenic state in rat intestinal BBM vesicles (BBMV) (14). This previous investigation showed that pH-dependent uptake of Na+ was highest in BBMV isolated from 6-wk-old rats and significantly lower in BBMV isolated from 3- and 2-wk-old animals. However, this investigation observed the combined activity of two apical NHE isoforms, NHE2 and NHE3. Therefore, the present investigation was undertaken to expand on these previous findings. The main objective of this investigation was to determine whether variable levels of NHE activity during rat ontogeny are due to differential expression of NHE3, which is thought to be the NHE isoform involved in transepithelial Na+ absorption in the small 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. Male rats of the following ages were used for all studies: 2-wk rats (sucklings) at 14 days of age, 3-wk rats (weanlings) at 21 days of age, 6-wk rats (adolescents) at 42-45 days of age, and adult rats at 120 days of age. Additionally, groups of 18-day-old animals were subcutaneously injected with methylprednisone (MP) at a dose of 30 µg/g body wt for 2 consecutive days and killed 1 day later at 21 days of age (control animals were injected with equal volumes of phosphate-buffered saline). Animals were maintained with food and water supplied ad libitum. Animals were subjected to CO2 narcosis and killed by cervical dislocation.
Chemicals and reagents.
Poly(A)+ RNA was
isolated utilizing the FastTrack kit for tissues or the Micro FastTrack
kit for cells from Invitrogen (La Jolla, CA).
22Na+
[100-2,000 Ci/g (3.70-74.0 Tbq/g)] for uptake
studies and
[-32P]dCTP (3,000 Ci/mmol) for Northern blot analyses were purchased from DuPont 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). NHE1, NHE3,
and NHE4 cDNA-containing plasmids were kindly provided by Dr. Gary
Shull (University of Cincinnati, Cincinnati, OH).
-Actin
cDNA-containing plasmid was provided by Dr. Ray Dubois (Vanderbilt
University, Nashville, TN). HOE-694 was kindly provided by Dr. H. J. Lang (Hoechst Pharmaceuticals, Frankfurt, Germany). Plasmid DNA was
isolated from bacterial cultures with the Wizard Mini or Midi prep
systems from Promega (Madison, WI). DNA fragments were gel purified
utilizing the GeneClean kit from BIO101 (Vista, CA).
Tetramethylammonium gluconate was made by titrating solutions of
tetramethylammonium hydroxide with gluconic acid. Protein was
quantitated by a Bradford assay utilizing the Bio-Rad (Hercules, CA)
protein assay reagent and bovine serum albumin as a standard.
Lipofectamine and OPTI-MEM were purchased from GIBCO BRL (Bethesda,
MD). Dulbecco's modified Eagle's medium (DMEM) and all other cell
culture reagents were from Irvine Scientific (Irvine, CA). Solumedrol
(MP) was obtained from the Arizona Health Sciences Center pharmacy. All
other chemicals and reagents were purchased from Fisher Biotechnology
(Pittsburgh, PA) or Sigma Chemical (St. Louis, MO).
BBMV preparation. BBMV were prepared from the jejunal mucosa of groups of 10-12 animals for 2- and 3-wk rats and of groups of 4-5 for 6-wk and adult rats. BBMV were purified by the MgCl2 precipitation technique essentially as previously described (15, 16). The final BBMV pellets were resuspended in either preincubation buffer for no pH gradient condition [pHi and extracellular pH (pHo) both 7.5] or preincubation buffer with an outwardly directed pH gradient (pHi 5.2, pHo 7.5) and placed at 25°C for 1 h. Protein was quantitated by a modified Lowry protein assay. The purity and enrichment of membrane preparations were assessed by the measurement of alkaline phosphatase activity as previously described (17). Membrane preps were used on the day of preparation and were never frozen.
Uptake analysis of intestinal BBMV. Uptake of 22Na+ was measured by a rapid filtration technique as previously described (14). All incubations were done at 25°C. Briefly, transport was initiated by adding 20 µl of the final membrane suspension to 80 µl incubation solution with the addition of 31.25 µCi 22Na/10 ml. Some studies were performed in the presence of 50 or 800 µM HOE-694, a specific NHE inhibitor that allows selective inhibition of NHE2 [inhibition constant (Ki) = 5 µM in PS120 cells], without affecting NHE3 (Ki = 650 µM in PS120 cells) (9). HOE-694 was prepared for use by dissolving solid HOE-694 in dimethyl sulfoxide to make a 100 mM working stock solution and was used within 90 min. Higher concentrations of HOE-694 were not used due to our inability to consistently dissolve higher concentrations of HOE-694. The reactions were stopped after 10 s by the addition of 2 ml ice-cold stop solution. The vesicles were immediately collected on a cellulose nitrate filter and washed with 5 ml ice-cold stop solution. The amount of radioactive substrate remaining on the filter was determined in a Beckman liquid scintillation counter, with ReadySafe (Beckman, Fullerton, CA) as the liquid scintillant. Radioactivity remaining in the filters after pipetting of incubation medium into the radioactive substrate in the absence of vesicles was used as the background level and was considered in all calculations. Experiments on all groups of animals were carried out on the same day. Uptake values were determined by subtracting the uptake levels with no pH gradient condition (pHi and pHo 7.5) from those with an outwardly directed pH gradient (pHi 5.2, pHo 7.5). All values are expressed as nanomoles of Na+ uptake per milligram of vesicle protein. Multiple repetitions were performed on three membrane vesicle preps from different groups of animals at each age. For graphical representation, data are expressed as means ± SE of four uptake assays at each age group. Data were analyzed for statistical significance by two-way analysis of variance (ANOVA) with Fisher's protected least significant difference (PLSD) posttest.
Slot blot analysis of NHE isoforms.
To determine whether probes specific for one NHE isoform would
cross-hybridize with the other NHE isoforms under our conditions of
hybridization and washing, slot blots were prepared that contained NHE1, NHE2, NHE3, and NHE4 cDNAs. Briefly, 1 µg plasmid DNA in a
total volume of 20 µl water was denatured by adding 2.45 µl 1 N
NaOH and placing it at 37°C for 15 min. Samples were placed on ice,
and 1 ml ice-cold 5× saline sodium citrate (SSC) was added to the
sample. Denatured plasmid samples were then added to each of four slots
of a slot blot apparatus containing a prewetted nitrocellulose
membrane. Each slot was washed with 1 ml ice-cold 5× SSC, and the
membrane was removed, dried at room temperature, and ultraviolet
cross-linked. The membranes were prehybridized, hybridized with
radiolabeled probes (1 × 106
counts · min1 · ml
hybridization solution
1),
and washed under high-stringency conditions exactly as with the
Northern blot analyses.
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 ribonuclease and protein degraders and oligo(dT)-cellulose affinity chromatography. Northern blots were carried out as previously described, using 5 µg poly(A)+ RNA/lane (4, 5). DNA fragments were used to generate probes for hybridization that were shown to be isoform specific with the slot blot analyses, as described in the previous section (i.e., Dra I/Kpn I restriction fragment for NHE3, which spanned bp 49-4980). High-stringency washes were performed at 65°C with 0.1× SSC-0.1% sodium dodecyl sulfate (SDS), and blots were placed onto a phosphorimaging screen or film . Northern blots were stripped and subsequently reprobed with 1B15-specific probes (1B15 encodes rat cyclophilin; Ref. 10). Quantitation of hybridization signals was done by phosphorimage analysis utilizing volume integration (GS 525 molecular imager, Bio-Rad). The Northern blot experiment was performed three times with poly(A)+ RNA samples from different groups of animals, and hybridization intensities from the three experiments were averaged. NHE hybridization intensities were normalized for 1B15 levels on the same blot.
Production of polyclonal antiserum specific for NHE3. In an effort to raise antibodies against the BBM-specific NHE3 isoform, we produced a recombinant fusion protein of NHE3. NHE3 cDNA was PCR amplified between bp 2183 and 2581 (a region with no amino acid or nucleotide sequence homology to other NHE isoforms). The resulting DNA fragment spanned amino acids 699-831 from the COOH-terminal portion of the protein (known NHEs are divergent at the COOH-terminus). The PCR product was subcloned into the pCR II vector (Invitrogen) and confirmed by DNA sequencing.
NHE3 DNA fragment was further subcloned into the pBlueBacHis vector (Invitrogen), in-frame with a polyhistidine purification tag. The construct was again sequenced to confirm integrity of the manipulation and to ensure that the reading frame was correct. pBlueBacHis is a baculovirus transfer vector designed for expression and purification of recombinant proteins in insect cells. Proteins expressed from this vector are fused at the NH2-terminus to a tag of six tandem histidine residues and an enterokinase cleavage site. The histidine residues create a high-affinity metal binding site to allow purification of recombinant fusion protein on a nickel chelating resin. pBlueBacHis/NHE3 was cotransfected with Bac-N-Blue baculovirus DNA into Sf9 insect cells by cationic liposome-mediated transfection, according to the manufacturer's protocol (Invitrogen). This procedure results in recombination between homologous sequences in the viral DNA and the transfer vector (pBlueBacHis) to yield recombinant viral DNA that can infect and replicate in insect cells. Three to five days posttransfection, cell lysis was observed in confluent cell monolayers and recombinant viral DNA was detected in the culture medium by PCR analyses. This culture supernatant was utilized to infect Sf9 cells and perform plaque assays that resulted in the identification of several putative positive baculovirus clones. Six positives were screened for the presence of NHE3 cDNA sequences by PCR, and all six were shown to contain these target sequences. Furthermore, individual plaques were assayed by Western blot for the presence of immunoreactive protein, utilizing a monoclonal antiserum specific for the fusion tag derived from pBlueBacHis vector sequences. This antiserum was utilized throughout the remaining expression and purification protocol to track the recombinant proteins. From these experiments, a positive baculovirus clone was selected that showed the highest expression of NHE3 fusion protein. This clone was used for the remaining manipulations. Next, a high-titer stock had to be generated from the relatively low-titer cell culture supernatant that was isolated in the previous experiments. This high-titer stock was used for protein expression studies that involved time course analyses of recombinant protein expression levels in three cell lines that are known to vary in production of different protein types (Sf9, Sf21, and HighFive cells). For NHE3, the highest recombinant protein expression was observed with Sf21 cells at a 48-h time point. This information was then applied to a large-scale cell culture (1 liter). With the utilization of protein generated from the large-scale expression, purification by immobilized metal affinity chromatography was optimized. For NHE3, it was shown that purification was optimal under the following conditions: cell lysate preparation under nondenaturing conditions, binding with nickel chelating resin (ProBond, Invitrogen) under nondenaturing conditions, and elution with imidazole at 350 mM. Between 1.2 and 1.5 mg of recombinant protein was recovered from expression of NHE3 fusion protein per 1-liter culture volume. The recombinant protein was intradermally injected into two rabbits for production of polyclonal antibodies specific for the COOH-terminal domains of the NHE3 protein. The injection protocol was as follows: initial boost, 250 µg protein/rabbit; boost at 4 and 8 wk, 100 µg protein/rabbit. Enzyme-linked immunosorbent assay analysis of the serum collected at week 10 showed high titers against the immunogenic protein, and this serum was used for all subsequent experiments.Expression of NHE2 and NHE3 in PS120 cells. To characterize the NHE3 antiserum, we transfected PS120 cells that lacked endogenous NHEs with NHE2 and NHE3 cDNAs as follows. NHE2 cDNA was amplified by PCR with the following primer pair: left primer at bp 147, 5'-TACACTGGCGTCCTCTGCTG-3'; right primer at bp 2678, 5'-AAGTCACAACCATGCTTGCC-3'. NHE3 cDNA was similarly amplified by PCR utilizing the following primer pair: left primer at bp 33, 5'-AAGAGCGCACGAGGTACCAC-3'; right primer at bp 2717, 5'-AGTCGAAGTGCGCTCAGGTG-3'. These primer sets allowed amplification of the entire protein coding sequences for NHE2 and NHE3. PCR parameters for both NHE2 and NHE3 amplification were as follows: 94°C for 4 min (step 1), 94°C for 1 min (step 2), 58°C for 1 min (step 3), 72°C for 2 min (step 4), and cycling between step 2 and step 4 (30 times), followed by 72°C for 10 min. PCR products were subcloned into the pTarget vector (Promega), which is a eukaryotic expression vector that is designed for direct subcloning of PCR products. Ligations and bacterial cell transformation were performed according to the manufacturers' protocol. Ampicillin-resistant bacterial colonies were screened by PCR for the presence of the NHE2 or NHE3 cDNA insert. This procedure involved picking an isolated colony with a sterile pipette tip and resuspending the colony in 20 µl LB medium; 10 µl of this suspension were saved at 4°C while the remainder was centrifuged at 13,000 g for 5 min and the LB medium was removed. The bacterial pellet was next suspended in the volume of sterile water needed for a PCR reaction and boiled for 3-5 min. The tube was centrifuged briefly and placed on ice, the PCR reagents were added to the tube, and PCR was performed as usual. PCR parameters were identical to those listed above in reference to the amplification of NHE2 and NHE3 for subcloning into the pTarget vector. Primers were as follows for NHE2: left primer at bp 565, 5'-TTCGGTGTCGATGAGAAGTC-3'; right primer at bp 2343, 5'-GAACTGATCTGGCAAGAAGC-3'. NHE3 primers were as follows: left primer at bp 798, 5'-CTTCCTGCTGTCCTTGGTGA-3'; right primer at bp 2073, 5'-GCTGCTATTCCTCCGCTTCT-3'. Agarose gel electrophoresis revealed which colonies showed positive NHE2 or NHE3 PCR bands, and these colonies were selected for further analysis. Plasmid DNA was isolated from bacterial cultures by a commercially available kit.
PCR product/pTarget constructs were sequenced by the dideoxy chain-termination method, utilizing the following primers based on the pTarget vector sequence: right primer at bp 1152, 5'-GGCAAGCAAGGCGATTAAGT-3'; left primer at bp 14212, 5'-GTGTGGAATTGTGAGCGGAT-3' (note that these primers flank the multiple cloning site). PS120 cells were maintained at 37°C in a 5% CO2-95% air atmosphere in DMEM with 10% fetal calf serum (FCS) and 1% penicillin-streptomycin (PS). Cells were transfected with the NHE2/pTarget or NHE3/pTarget expression constructs when they reached 70% confluency in 35-mm dishes as follows. One microgram NHE2/pTarget or NHE3/pTarget DNA was added to 5 µl Lipofectamine, mixed with 1 ml OPTI-MEM medium, and placed at room temperature for 30 min. Cells were rinsed with OPTI-MEM twice, 1,000 µl of DNA-liposome mixture were added, and the cells were incubated for 5 h at 37°C. Then, 1 ml DMEM with 20% FCS was added, and cells were incubated overnight. Cells were then placed in DMEM-10% FCS-1% PS and incubated for 48 h. Cells containing expression plasmids (nonrecombinant and recombinant) were selected for by exposure to 800 µg/ml G418 antibiotic for 7-10 days (antibiotic resistance is carried on the pTarget vector). To further select for cells transfected with recombinant plasmids containing functional NHE2 or NHE3, cells were acid loaded as previously described (6). Briefly, cells were incubated with acid medium [in mM: 50 NH4Cl, 72 choline, 4.9 KCl, 1 CaCl2, 2.5 MgCl2, and 20 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES); pH 7.5] for 1 h at 37°C. Cells were rinsed twice with choline solution (in mM: 122 choline, 4.9 KCl, 1 CaCl2, 2.5 Mg2SO4, and 20 HEPES; pH 7.0) and incubated in NaCl solution (in mM: 122 NaCl, 4.9 KCl, 1 CaCl2, 2.5 Mg2SO4, and 20 HEPES; pH 7.5) for 1 h at 37°C. The cells were then rinsed twice with DMEM-10% FCS-1% PS and incubated at 37°C for 48 h. The acid selection protocol was repeated two to three additional times. Then, cells were scraped or trypsinized from dishes and used for poly(A)+ RNA purification for reverse transcriptase (RT)-PCR or for crude membrane protein purification for immunoblot analysis with NHE3 antiserum.RT-PCR analysis of transfected PS120 cells. To assay for NHE2 or NHE3 mRNA expression in transfected cells, the RT-PCR technique was performed as previously described (4, 6). When cells reached confluency after the final acid selection, cells were trypsinized from the dishes and poly(A)+ RNA was isolated with a commercially available kit. mRNA was converted into cDNA by Moloney murine leukemia virus RT. RT-PCR reactions were run with the NHE2 or NHE3 primer pairs that were described in the previous section in relation to the screening of bacterial colonies for recombinants. As a positive control, 20 ng NHE3-containing plasmid DNA was used in a PCR reaction, and, as a negative control, no DNA was added to the PCR reaction. PCR parameters were identical to those previously described for amplification of NHE2 and NHE3 for subcloning into pTarget vector, except that 45 cycles were run. PCR products were fractionated by 1% agarose gel electrophoresis.
Western blot analysis of PS120 cells with NHE3
antiserum.
Crude membranes were prepared from transfected and untransfected PS120
cells that were grown in 100-mm dishes. Medium was decanted, and the
cells were washed with ice-cold phosphate-buffered saline (pH 7.5).
Then, 3 ml of extraction buffer were added to cell monolayers [in
mM: 10 Tris (pH 7.5), 1 EDTA, 0.1 phenylmethylsulfonyl fluoride, 1 iodoacetamide, and 1 o-phenanthroline], and the cells were scraped from dishes and transferred to a centrifuge tube. The
cells were next homogenized with a 26-gauge needle and spun at 1,000 g for 20 min at 4°C. The
supernatant was decanted to a new tube and spun at 40,000 g for 30 min at 4°C. The resulting pellet was resuspended in Tris-EDTA buffer. The solution
was again homogenized with a 26-gauge needle, and protein assays were
performed as previously described. Proteins were solubilized in Laemmli buffer plus -mercaptoethanol, boiled for 5 min, fractionated by
SDS-polyacrylamide gel electrophoresis (PAGE), and transferred to
nitrocellulose membranes. Membranes were processed and reacted with
NHE3 antiserum at 1:4,000 dilution in an identical fashion to that
described in the next section.
Immunoblot analysis of rat jejunal BBM proteins with NHE3-specific
antiserum.
Intestinal BBM proteins were purified by a
MgCl2 precipitation method as
previously described (3, 16). Protein (20 µg) was placed in a 10-fold
excess of Laemmli solubilization buffer plus 2 mM -mercaptoethanol,
boiled for 5 min, and placed on ice. Protein samples were fractionated
by 8% SDS-PAGE and transferred onto nitrocellulose membranes. Blots
were processed as previously described (3, 6) with the Renaissance
chemiluminescent system (Dupont NEN) with 1:4,000 dilutions of
NHE3-specific antiserum. Additionally, some blots were reacted with
preimmune serum and serum that was pretreated with antigenic fusion
protein (0.4 mg/ml at 10°C for 16 h) at 1:4,000 dilution. Membranes
were stripped and subsequently reacted with
-actin antiserum at
1:2,000 dilution. NHE3 band intensities were determined by
phosphorimage analysis (utilizing chemiluminescent imaging screens,
Bio-Rad) and were normalized for
-actin band intensities on the same
blot. The experiment was performed five times with protein samples
isolated from different groups of animals.
Immunohistochemical analysis of rat intestine with NHE3 antiserum. Jejunal tissue was harvested from 2-wk-old rats, fixed in paraformaldehyde, and embedded in paraffin, and sections were cut and affixed to slides. Slides were processed utilizing an immunoperoxidase staining system (ABC Elite, Vector Labs, Burlingame, CA) exactly as previously described (7). NHE3 antiserum was reacted with sections for 16 h at 1:2,000 dilution. Some sections were reacted with preimmune serum (1:2,000) and immunogenic protein-pretreated serum (0.4 mg/ml at 10°C for 16 h) at 1:2,000. Slides were subsequently visualized and photographed by standard light microscopy.
Statistical analysis of results. Data from uptake studies were analyzed for statistical significance by two-way ANOVA with Fisher's PLSD posttest, utilizing the StatView program (version 4.53, Abacus Concepts, Berkeley, CA). Northern blot and Western blot data were analyzed for statistical significance by one-way ANOVA with Fisher's PLSD posttest, utilizing the same software package. All data are presented as means ± SE.
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RESULTS |
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Uptake analysis of intestinal BBMV.
pH-dependent uptake of
22Na+
was assayed in jejunal BBMV in four age groups of rats in the presence
of 0, 50, or 800 µM HOE-694 (Fig.
1). Alkaline phosphatase
enrichment was 10- to 12-fold in all groups, which suggested an
equivalent enrichment for BBM in all samples.
Na+ uptake (in
nmol · mg
protein1 · 10 s
1;
n = 4) with 0 µM HOE-694 was highest
in the 6-wk age group (1.80 ± 0.07), lower in adults (0.35 ± 0.5), and lowest in 2- and 3-wk rats (0.15 ± 0.03 and 0.19 ± 0.05, respectively). pH-dependent Na+ uptake in the presence of 50 µM HOE-694 was highest in 6-wk animals (1.65 ± 0.16), lower in
adults (0.27 ± 0.01), and lowest in 2- and 3-wk animals (0.10 ± 0.02 in both 2 and 3 wk). Similarly, Na+ uptake in the presence of 800 µM HOE-694 was highest in 6-wk animals (1.03 ± 0.08), lower in
adults (0.16 ± 0.01), and lowest in 2- and 3-wk animals (0.05 ± 0.01 and 0.06 ± 0.01, respectively).
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Slot blot analysis of four NHE isoforms. To determine whether probes specific for one NHE isoform cross-hybridized with other NHE isoforms under our conditions used for Northern analysis, slot blots were prepared that contained denatured plasmids with NHE cDNAs affixed to nitrocellulose membranes. When full-length NHE2 cDNA was used to generate probes, cross-hybridization was apparent with NHE4 cDNA affixed to the membrane (data not shown). However, when the NHE2 BamH I/Bgl II fragment was used to generate probes, cross-hybridization was abolished (Fig. 2, panel 1). NHE4 probes generated from a DNA fragment encompassing bp 692-2232 of the cDNA sequence showed significant cross-reactivity with NHE2 cDNA affixed to the membrane (Fig. 2, panel 2). However, when a smaller PCR fragment of NHE4 (encompassing bp 1951-2326 of the cDNA sequence) was used to generate probes, cross-hybridization with NHE2 was abolished (Fig. 2, panel 3). Greater than 95% nucleotide sequence similarity exists between NHE2 and NHE4 cDNA over a 200-bp region (between bp 1450 and 1650 of the NHE2 cDNA sequence), and presumably this is the region that leads to cross-reactivity under high-stringency hybridization and washing conditions. This area of high homology was not present in either the NHE2 or the NHE4 DNA fragments that were used to generate probes that showed no cross-reactivity. Furthermore, NHE1 probes (bp 1148-3684 of the cDNA sequence) showed no cross-reactivity with any of the other NHE isoforms [Fig. 2, panel 5; note that some background hybridization with the other NHE isoforms is seen with NHE1 probes; however, more extensive washing abolished all background hybridization (data not shown)]. Additionally, probes generated from a Dra I/Kpn I restriction enzyme fragment of NHE3 showed no cross-hybridization with any other NHE isoforms (Fig. 2, panel 4). Thus specific probes were developed for all four isoforms, and only these probes were used for Northern blot analyses.
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Northern blot analysis. Northern blots were hybridized with NHE3-specific (Dra I/Kpn I fragment) and 1B15-specific radiolabeled probes (Fig. 3A). NHE3 hybridization levels (in phosphorimage units) in the jejunal mucosa were lowest in 2-wk animals (11.38 ± 1.88) and higher in all other age groups (3 wk, 16.40 ± 0.45; 6 wk, 16.31 ± 0.31; adult, 18.75 ± 1.11; n = 3; P = 0.014 for 2 wk vs. 3 wk, P = 0.015 for 2 wk vs. 6 wk, and P = 0.002 for 2 wk vs. adult).
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Western blot analyses of intestinal BBM proteins with NHE3-specific antiserum. Polyclonal antibodies specific for an NHE3 COOH-terminal fusion protein were developed. Western blot analyses with NHE3 antiserum showed specific recognition of an 85-kDa protein, with lower intensity (in phosphorimage units) in 2-wk animals (0.17 ± 0.07) and higher levels in the other age groups (3 wk, 0.76 ± 0.18; 6 wk, 1.19 ± 0.22; adult, 0.95 ± 0.07; n = 5; P = 0.01 for 2 wk vs. 3 wk, P = 0.0002 for 2 wk vs. 6 wk, and P = 0.002 for 2 wk vs. adult). These data are depicted in Fig. 3B. Blots that were reacted with antigenic protein-blocked and preimmune sera showed no protein recognition with NHE3 serum samples (data not shown).
RT-PCR analysis of transfected PS120 cells. Transfected and untransfected cells were used to isolate mRNA, which was utilized for RT reactions. RT-PCR was then performed with NHE2- or NHE3-specific primers to assay for message expression (Fig. 4). Results showed a band of the expected size (1275 bp) in the positive control reaction and in the NHE3-transfected cells only. No bands were seen in untransfected PS120 cells, in PS120 cells transfected with vector only, or in NHE2-transfected cells. Additionally, the negative control reaction with no DNA showed no amplification (not shown). NHE2 expression was confirmed in NHE2-transfected cells by RT-PCR with NHE2-specific primers (data not shown).
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Western blot analysis of PS120 cells with NHE3 antiserum.
Crude membranes were purified from PS120 fibroblasts that had been
transfected with NHE2, NHE3, or vector only, or from untransfected cells, and Western blots were performed and reacted with NHE3 antiserum
(Fig. 5). Results showed no protein band
recognition in PS120 cells, PS120/vector cells, or PS120/NHE2 cells.
However, a strong band at ~85 kDa was detected in the PS120/NHE3
cells. All membrane preps showed a strong band at 42 kDa that
corresponds to -actin.
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Immunohistochemical analyses of rat jejunum with NHE3-specific antiserum. Jejunal tissue was excised from animals, fixed in paraformaldehyde, embedded in paraffin, sectioned, and affixed to slides. Tissue sections were reacted with NHE3-specific polyclonal antiserum. Results showed specific staining of the apical membranes of the intestinal epithelium, with no signal detected in the basolateral membranes, crypts, goblet cells, or lamina propria (Fig. 6A). Antigenic protein-blocked serum (Fig. 6B) and preimmune serum (data not shown) showed no staining of apical membranes or other cell types within the rat jejunum.
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Effect of MP on NHE3 expression in 3-wk rat jejunum. Eighteen-day-old rats were subcutaneously injected with MP for 2 days and killed 1 day later. Intestines were harvested, and mRNA or brush-border proteins were isolated from mucosal scrapings. Results of Northern blot analyses showed a twofold increase in NHE3 mRNA expression with MP treatment (saline injected, 5.28 ± 0.52 phosphorimage units; MP injected, 10.68 ± 1.25 phosphorimage units; n = 3; P = 0.03). Immunoblot analyses also showed similar results, with the MP-injected group showing ~2.5-fold increased expression of NHE3 immunoreactive protein (saline injected, 0.09 ± 0.02 densitometric units; MP injected, 0.25 ± 0.05 densitometric units; n = 3; P = 0.04). A typical experiment is depicted in Fig. 7.
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DISCUSSION |
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To investigate the relationship between apical NHE expression and ontogenic changes in intestinal BBMV Na+/H+ exchange activity, NHE activity was assayed in the rat intestine by a rapid filtration technique in the presence or absence of the NHE-inhibitory compound HOE-694. Rats at 2 wk, 3 wk, 6 wk, and 4 mo of age were studied. To determine mRNA expression levels, we isolated poly(A)+ RNA from rats at these various stages of development. RNA was fractionated by electrophoresis and transferred onto membranes. Specificity of probes for their target isoform was addressed by slot blot analyses, which allowed the development of isoform-specific probes. Northern blots were probed with NHE3- and 1B15-specific probes. Additionally, polyclonal antiserum specific for NHE3 was produced and was characterized by RT-PCR and immunoblot analyses of PS120 cells transfected with NHE2 and NHE3 and by immunohistochemistry. Subsequently, immunoblots of jejunal brush-border proteins were utilized to assess the level of protein expression at the different ages. Additional experiments were aimed at assessing the effect of glucocorticoid treatment on NHE3 expression in 3-wk-old animals.
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 markers (leucine aminopeptidase); impoverishment of membrane preparations for basolateral membrane markers (Na+-K+-ATPase), mitochondrial membrane markers (cytochrome-c oxidase), and endoplasmic reticulum membrane markers (NADPH cytochrome c reductase); level of Na+ binding to vesicles as opposed to uptake into intravesicular space; affinity of the exchanger for Na+; and rate of pH dissipation and generation of negative membrane potential. These observations suggested that the phenomenon observed in the present studies utilizing this BBMV purification and uptake method are based solely on the activity of the apical membrane NHEs.
In the present investigation, we sought to expand on these previous observations to look at initial rate of BBMV uptake in 2-, 3-, and 6-wk-old and adult rats and to attempt to assess the relative contribution of NHE3 at each age by selectively inhibiting NHE2 with HOE-694 (Ki = 5 µM in PS120 cells) (9). The NHE3 contribution may be estimated by comparing the uptake in the absence of HOE-694 (which represents the activity of both NHE2 and NHE3) with uptake rates in the presence of 50 µM HOE-694 (which represents the activity of only NHE3). Additionally, uptake was measured in the presence of 800 µM HOE-694, which should significantly inhibit NHE3 activity (Ki = 650 µM in PS120 cells) (9). At all ages, 50 µM HOE-694 had a significant effect, suggesting that NHE2 and NHE3 both contribute to basal Na+/H+ exchange. However, the relative contribution of NHE3 to total uptake was greater than that of NHE2. Additionally, the effect of age was significant between all groups with the exception of 2- and 3-wk animals.
In both the 2- and 3-wk groups, uptake was decreased by 41% with 50 µM HOE-694, suggesting that NHE2 contributed 41% to basal uptake and the remaining 59% was due to NHE3; 800 µM HOE-694 inhibited NHE3 activity by 45% in 2- and 3-wk animals. The 6-wk animals showed a 92% contribution of NHE3 to basal rates, which suggested that NHE2 contribution was very small (8%). Inhibition of NHE3 activity with 800 µM HOE-694 was 38% in 6-wk rats. Adult rats had a relative NHE3 contribution of 77% and an NHE2 contribution of 23%, whereas 800 µM HOE-694 inhibited NHE3 activity by 41%. These results show that both apical isoforms contribute to Na+/H+ exchange activity at all ages studied, with NHE3 contribution ranging from a high of 92% in 6-wk animals to a low of 59% at 2 and 3 wk of age. Conversely, NHE2 contribution was greatest in 2- and 3-wk animals (41%) and lower in 6-wk (7%) and adult (23%) animals. These data also suggest that the Ki for NHE3 is higher in rat jejunal epithelium than in PS120 fibroblasts (Ki for HOE-694 = 650 µM), since 800 µM HOE-694 only inhibited 38-45% of NHE3 activity. Furthermore, these results show that, in adolescent and adult rats, NHE3 is the isoform that chiefly contributes to basal Na+/H+ exchange activity (92 and 77% of basal uptake, respectively), whereas, in younger animals, the two apical isoforms are more equally involved (41% NHE2 and 59% NHE3).
The present uptake study results are similar to previous work in which initial rate uptake was highest in 6-wk rats and decreased in 3- and 2-wk rats (adults were not studied in the previous experiments) (14). In this previous investigation, initial rate values were proportionally similar to maximum rate (Vmax) values, and, since Vmax values had been previously determined, kinetic experiments were not repeated. The present uptake data, when considered with previous data, strongly suggest that BBM Na+/H+ exchange activity in the rat small intestine is regulated during ontogeny and that the relative contribution of NHE3 to basal activity varies during development.
To investigate differential expression of NHE3 during rat ontogeny at the molecular level, mRNA levels were determined by Northern blot analyses. Slot blots were utilized to assess the cross-reactivity of NHE isoform probes for other isoforms. These studies showed that our NHE3 probes showed no cross-hybridization with other NHEs under conditions identical to those used for Northern blot analyses. NHE3 hybridization levels on Northern blots with RNA from jejunal epithelium were lowest in 2-wk animals and increased by 1.5- to 2.0-fold in the other three age groups.
To further characterize the ontogenic expression of rat intestinal NHE3, exchanger-specific polyclonal antiserum was produced and characterized. Serum was initially tested on crude membranes from PS120 cells that had been transfected with NHE3 or NHE2 cDNA expression constructs. The presence or absence of specific NHE mRNAs in cells was determined by RT-PCR with isoform-specific primers. These experiments showed that transfected cells were expressing only the expected NHE isoform message and that untransfected cells had no NHE isoform mRNAs present. Immunoblots utilizing crude membrane proteins from the cells and NHE3 antiserum showed specific recognition of a single 85-kDa band in only the cells that had been transfected with NHE3 cDNA expression construct. This experiment exemplified the specificity of the serum for the NHE3 isoform, and it also showed that the serum did not cross-react with the other apical NHE isoform (NHE2).
On immunoblots with jejunal brush-border proteins, this antiserum also recognized a single, specific protein band that was in the 85-kDa size range. The band was blockable by pretreating the serum with antigenic protein and was not detected with preimmune serum. Furthermore, another recent investigation characterized NHE3 antiserum raised against COOH-terminal fusion protein and concluded that the NHE3 immunoreactive protein was ~85 kDa (13). 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. All of these facts make it seem likely that the antiserum is indeed recognizing the NHE3 protein.
Quantitation of bands from immunoblots with NHE3 antiserum showed the lowest band intensities in 2-wk animals, with higher intensities in the other three age groups (which were not different). These data are in agreement with NHE3 mRNA levels (which showed the lowest message expression in 2-wk rats and higher levels in the other groups) but are in contrast to NHE3 activity studies. NHE3 activity decreased dramatically between 6-wk animals and the other age groups, whereas there was either no change (adults and 3-wk animals) or a lesser decrease (2-wk animals) in mRNA and immmunoreactive protein levels. This discrepancy may be explained by the presence of NHE3 protein present at the brush border that is not functional. This inactive protein may represent a readily activatable pool that could be utilized under certain physiological conditions.
A previous investigation studied the effect of pharmacological doses of corticosteroids on intestinal NHE3 expression in adult rabbits (28). This study showed upregulation of NHE3 message in adult rabbit ileum with methylprednisolone treatment, whereas NHE2 message remained unchanged (27). In the present study, we show that treatment of 18-day-old rats with pharmacological doses of corticosteroids increases NHE3 mRNA and immunoreactive protein expression by approximately twofold. These results demonstrate that NHE3 expression is corticosteroid inducible in suckling animals and further suggest that these hormones may be in part responsible for the increase in NHE3 expression between 2 wk and 3 wk of age.
Overall, the present data show that the expression of NHE3 is most likely regulated at multiple levels during rat ontogeny, as evidenced by the variable changes in expression between age groups. The one consistent finding was that 6-wk animals clearly have the highest NHE3 expression, whereas 2- and 3-wk animals have significantly lower levels. Although it is not presently clear precisely what physiological cues may be responsible for these observed phenomenon, the MP injection studies suggest that corticosteroids play a role in this process. Furthermore, on the basis of the HOE-694 inhibition studies, it would appear that both of the known apical membrane NHE isoforms play important roles in the ontogenic development of the mammalian intestinal tract. The precise characterization of the ontogenic expression of NHE2 therefore becomes important.
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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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Address for reprint requests: F. K. Ghishan, Dept. of Pediatrics, University of Arizona Health Sciences Center, 1501 N. Campbell Ave., Tucson, AZ 85724.
Received 24 June 1997; accepted in final form 15 August 1997.
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