Introduction

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NHE transports extracellular Na⁺ for intracellular H⁺ electroneutrally and is present in every mammalian cell (Bianchini and Pouyssegur, 1995; Noel and Pouyssegur, 1995). Recent studies have demonstrated four different genes encoding the isozymes NHE-1, NHE-2, NHE-3 and NHE-4 (Bookstein et al., 1994; Collins et al., 1993; Orlowski et al., 1992; Wang et al., 1993). These isoforms exhibit differential distribution in the GI tract. The NHE-1 isoform is located on the basolateral side of the epithelial cell and is considered a main regulator of pH_i and cell volume, whereas the apical isoforms NHE-2 and NHE-3 are known to regulate NaCl absorption (Levine et al., 1993; Maher et al., 1997; Tse et al., 1992). These isoforms differ in their sensitivity to amiloride and in their regulation by hormones and growth factors (Bianchini and Pouyssegur, 1995; Noel and Pouyssegur, 1995; Tse et al., 1992; Tse et al., 1993).

Intestinal epithelial cells act as an important site of absorption of the luminal contents. They are also the primary target of insults that cause IBDs. These cells actively participate in inflammatory reactions by producing cytokines, growth factors (Justinichi et al., 1992; Volker et al., 1995) or derivatives of arachidonic acid (Dias et al., 1992). These mediators alter the membrane permeability and activity of ion-transporting proteins such as Na/K-ATPase during IBDs in human and in animal models (Allgayer et al., 1988; Khan and Collins, 1993; Wild and Murray, 1989). In Crohn's disease, the pH_i of colonocytes is decreased (Rowe and Bobar, 1995). Because NHE-1 is the main regulator of pH_i, cellular growth and differentiation, in the present study we examined expression of the NHE-1 isoform. Animal models have proved useful in understanding the underlying mechanisms of altered physiological functions. However, the changes seen during colitis may be influenced by the nature of the colitides as well. Therefore, in this study we investigated the mechanism of NHE-1 expression in HAC- and TNBS-induced colitis. Further, to identify the factors responsible for the expression of NHE-1 during colitis, we also examined the effect of dexamethasone on the NHE-1, NHE-2 and NHE-3 levels of mRNA.

The specific aim of the present study was to investigate the underlying mechanism of NHE-1 expression during colitis induced by HAC or TNBS. We employed the RT-PCR and slot blot analysis for quantitation of the mRNA levels and the ECL Western blot analysis for protein estimation.

	Materials and Methods

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Induction of colitis. Sprague-Dawley male rats weighing 230 to 280 g and maintained by the Faculty of Medicine, Kuwait University, were used in this study. Colitis was induced by intrarectal administration HAC (Grossi et al., 1993; Khan and Al-Awadi, 1997; McPherson and Pfeiffer, 1977) or TNBS (Fluka Bucks, Switzerland) as described (Morris et al., 1989). Rats that received PBS and rats that received 50% ethanol vehicle served as controls for HAC- and TNBS-induced colitis, respectively. Animals were sacrificed by cervical dislocation after 1, 2, 5 and 7 days of HAC and after 7 days of TNBS administration. A colonic segment 6 cm long was taken from the site of inflammation and cleaned with sterile PBS. Mucosa was scraped off and stored frozen at 70°C until use. The mucosal scrapping contained mucosa and submucosa from both the control and the inflamed colon. However, the tissue was not homogeneously pure because of the presence of other cell types. The mucosal scrappings so obtained were used in the experiments reported in this study. The rats were treated i.p. with dexamethasone (1 mg/kg body weight) 2 h before HAC or TNBS administration, and the dose was repeated every 24 h. On the third day after HAC administration or on the seventh day after TNBS administration, animals were sacrificed and tissues used.

MPO activity. MPO activity was estimated in the whole colonic tissue obtained from the rats (Bradley et al., 1982; Khan and Al-Awadi, 1997). MPO activity was estimated in the whole colonic segment containing mucosa and muscle layers. Tissue was taken from the site of inflammation, minced finely with scissors in (1 gm/10 ml) 50 mM potassium phosphate buffer, pH 6.0, containing 14 mM hexadecyltrimethylammonium bromide (Sigma, UK). Tissues were homogenized with a polytron (Janke and Kunkle Staufen, FRG) for 1 min. Lysates were frozen in liquid nitrogen and thawed once. The lysates were centrifuged for 2 min in cold room at 15,000 × g (Eppendorf). Supernatants were used to estimate the MPO activity in the presence of o-dianisidine-HCl (Sigma, UK) and 0.0005% H₂O₂ (Khan and Al-Awadi, 1997). Optical density was recorded at 415 nm every 15 s over a period of 1 min using a spectrophotometer, DU700 (Beckman, CA). MPO activity was expressed as units per minute per milligram of tissue. The enzyme unit is defined as the conversion of 1 µmol H₂O₂/minute/mg of tissue at room temperature.

Extraction of total cellular RNA. RNA was extracted from the frozen colonic mucosa by the guanidinium isothiocyanate-cesium chloride centrifugation method (Chirgwin et al., 1979; Khan and Al-Awadi, 1997). Frozen colonic mucosa (1 g/10 ml) was thawed in 4 M guanidinium isothiocyanate (Sigma), minced finely with scissors and homogenized with the polytron. The homogenates were centrifuged at 10,000 × g (J2-MI, Beckman) at 4°C for 10 min. The supernatants were layered on 3.5 ml of 5.7 M cesium chloride solution (Sigma) and centrifuged at 110,000 × g for 24 h at 16°C using an SW41 rotor (LX-80 Beckman, CA). The pellet of RNA was dissolved in diethylpyrocarbonate (DEPC, Sigma)-treated and autoclaved distilled water and was extracted once with phenol-chloroform. RNA was precipitated from the aqueous layer with 2.5 vol of absolute ethanol for 2 h at 70°C (Khan and Al-Awadi, 1997). RNA was recovered by centrifugation of the samples in cold room at 14,000 rpm (5415C Eppendorf) for 20 min and dissolved in the RNase-free water. The concentration of RNA was determined spectrophotometrically at 260/280 nm. The quality of RNA was routinely electrophoretically analyzed on a formaldehyde-agarose (1.4%) gel (Khan and Al-Awadi, 1997; Khan and Collins, 1993; Sambrook et al., 1989). All solutions and glassware were made RNase-free by DEPC treatment followed by autoclaving. Heat-sensitive solutions were made in RNase-free water and filtered subsequently through a 0.45-µm millipore filter.

RT. Aliquots (5 µg) of total RNA were mixed with 250 ng oligodT primer (Promega, WI) and heated at 75°C for 10 min. Annealing was performed by slow cooling of the heated sample to 37°C (Khan and Al-Awadi, 1997; Khan et al., 1992). RT was carried out in the presence of 15 to 20 units of AMV reverse transcriptase (Pharmacia, Uppsala, Sweden) and 15 units of RNA guard (Pharmacia, Uppsala, Sweden) using a buffer containing (mmol/l): 10 Tris-HCl, pH 8.4, at 42°C, 10 MgCl₂, 70 KCl, and 10 dithiothreitol for 30 min at 37°C followed by a 30-min incubation at 42°C (Khan et al., 1992; Khan and Al-Awadi, 1997). The reverse reaction was terminated by heating the samples at 90°C for 10 min. The cDNA so obtained was routinely employed for amplification of the NHE-1 mRNA isoform using the specific primers (table 1). The primers were selected on the basis of rat kidney, brain or stomach cDNA information (Orlowski et al., 1992). Whether there is an intron present in the region between the primers is not known.

PCR. Aliquots (5 µl) of cDNA were routinely amplified by PCR using 1 to 2 units of Amplitaq (Perkin-Elmer, Norwalk, CT) and 50 pmol of each primer (table 1). The conditions were optimized in the linear range of amplification, in accordance with the basic PCR protocol described elsewhere (Innis et al., 1990; Khan and Al-Awadi, 1997; Khan et al., 1992). The following PCR conditions were used: denaturation 94°C × 30 s; annealing 55°C × 30 s; extension 74°C × 60 s. Aliquots (5 µl) of PCR products were analyzed electrophoretically using a 7% polyacrylamide gel (Khan and Collins, 1993; Khan et al., 1992). The gels were stained with ethidium bromide and photographed on a gel documentation system (Stratagene, CA). To ensure that the PCR bands were not due to genomic contamination, two controls were used, one containing RNA without RT and the other without cDNA or RNA.

Quantitation of mRNA. The area of the PCR band was obtained by scanning the negatives on a densitometer (Pharmacia LKB, UltroScan-XL), and the ratio NHE-1 mRNA:internal control, alfa-1 isoform of sodium pump (Khan and Collins, 1993) was calculated. OligodT primer was used for RT, and from the same cDNA sample, NHE-1 and internal control were amplified separately. This technique was chosen to minimize tube-tube variations during the reverse reaction.

Slot blot analysis. The RT-PCR data were validated using slot blot analysis. Aliquots of 20 µg total RNA were spotted on to the nitrocellulose filter (Bio-rad, CA) using a slot blot apparatus (Bio-rad). RNA was immobilized by heating the filters for 2 h at 80°C. The filters were prehybridized for 2 h and hybridized with biotinylated downstream primer (Genosys, UK) listed in table 1. Hybridization was carried out at 42°C in 5XSSC. Subsequently, the filters were washed with 2XSSC buffer at 40°C and incubated with streptavidin-horseradish peroxidase (1:2000) for 1 h at ambient temperature. The filters were washed with 2XSSC three times at ambient temperature. Signals were developed using the ECL kit components 1 and 2 for 1 min. The autoradiogram was scanned on a densitometer to obtain the area of the bands.

Western blot analysis. NHE-1 protein was measured using NHE-1 antibodies (a kind gift from Dr. E. B. Chang). Crude plasma membranes were prepared from mucosal scrapings, following the standard method with modifications (Shallat et al., 1996). Briefly, mucosal scrapings were homogenized with a polytron for 60 s in 250 mM sucrose and 20 mM MOPS buffer, pH 7.5, and centrifuged for 10 min at 450 × g at 4°C; the resulting supernatant and pellet were designated supernatant 1 and pellet 1. Pellet 1 was rehomogenized and centrifuged. The resulting supernatant 1 and supernatant 2 were pooled and centrifuged at 20,000 × g in the JA20 rotor of a Beckman high-speed centrifuge for 45 min at 4°C. Supernatant was discarded and pellet 3 was collected. Pellet 2 was used to check the loss of the NHE-1 protein. Pellet 3 was suspended in the sucrose-MOPS buffer and homogenized. Total protein was measured using a dye-binding assay kit (Bio-rad). Pellet 3 samples were used to measure the NHE-1 protein level. Aliquots containing 50 µg total protein from each sample were separated on a 7.5% polyacrylamide gel electrophoretically under denaturing conditions (Laemmli, 1970) and transferred onto the nitrocellulose filter (Bio-rad) electrophoretically. Filters were blocked with 5% nonfat dry milk in PBS for 2 h at ambient temperature. Antiserum of NHE-1 was added to the blocked filters, incubated at ambient temperature for 2 h and washed thoroughly with PBS. Anti-rabbit secondary antibodies conjugated with horseradish peroxidase (Sigma) were added (1:5000) to the filters and incubated for 1 h at ambient temperature. Subsequently, the filters were washed with PBS and incubated with components 1 and 2 of the ECL kit (Amersham) for 1 min. The filters were exposed to X-ray film (Kodak) for 5 to 20 s, and the signals were developed. The bands were scanned on the densitometer to obtain the band area.

Analysis of data. The area of the bands was obtained by scanning the negatives on a densitometer (Pharmacia LKB, Uppsala, Sweden; UltroScan-XL). Statistical analysis was performed using the SPSS program. A value of P < .05 was considered statistically significant.

	Results

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MPO activity. MPO activity is considered a reliable marker of inflammation; therefore, we estimated MPO activity to monitor colitis. MPO activity increased in a time-dependent manner during 1, 2 and 5 days (fig. 1) and returned to the basal level after 7 days of HAC administration. MPO activity was also significantly higher after 7 days of TNBS administration than in the control untreated rats (fig. 2). MPO activity was suppressed significantly after the dexamethasone treatment of the HAC- or TNBS-induced colitis (fig. 2).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Bar diagram showing MPO activity in the colon of rats that received HAC on the days indicated before assessment. Data are mean ± S.E.M. (n = 10). * P < .002.

View larger version (33K): [in this window] [in a new window]   Fig. 2.   The MPO activity (hatched bars), NHE-1 mRNA measured by RT-PCR (open bars) and slot blot analysis (closed bars) in PBS (control), HAC (3 days after the administration of HAC), HD (3 days after the administration of HAC + dexamethasone), ET (ethanol control), TNBS (7 days after the administration of TNBS) and TD (7 days after the administration of TNBS + dexamethasone). Data are mean ± S.E.M. (n = 8). * P < .02 vs. controls.

Restriction analysis and sequencing. It was necessary to characterize the NHE-1 PCR fragment before estimating the mRNA level. Restriction endonucleases recognize specific sequences in a DNA fragment, so the NHE-1 PCR fragment was identified using AvaII restriction enzyme. AvaII restriction sites were identified using DNASIS software (Pharmacia) in the PCR fragment, which on digestion was expected to yield 7-, 49-, 51-, 56- and 105-bp fragments. But we obtained only two bands; one probably contained 49-, 51- and 56-bp fragments migrating together (fig. 3, lane 2, lower band) and the other corresponded to 105 bp (fig. 3, lane 2, upper band). Because it was difficult to separate and match the size of 49, 51 and 56 bp of AvaII fragments accurately, we further identified the NHE-1 PCR fragment by nucleotide sequencing using a nonradioactive method. The PCR fragment was purified using a DNA purification kit (U.S. Biochemical) and sequenced. Briefly, 200 ng of the purified PCR-fragment was mixed with 3.2 pmol of sense or antisense primer (table 1) and amplified (9600 Thermocycler, Perkin-Elmer) using the fluorescent dye terminators and Taq DNA polymerase mix (Perkin-Elmer) for 40 cycles. The PCR conditions were denaturation 94°C × 30 s, annealing 50°C × 30 s and extension 60°C × 90 sec. After amplification, the samples were extracted once with phenol-chloroform and dissolved in the loading buffer. The samples were run on an automated DNA sequencer (Applied Biosystems) and analyzed. The derived nucleotide sequence showed 100% identity with the published sequence (data not shown).

View larger version (56K): [in this window] [in a new window]   Fig. 3.   Amplification of the NHE-1 PCR fragment (208 bp) in the colonic mucosa of the control rats (lane 1). The fragments resulting from the complete digestion of the 208-bp fragment with AvaII are shown in lane 2, upper bright band (105 bp) and lower band (49-56 bp). The unlabeled lane contains a 100-bp DNA ladder.

Yield of total cellular RNA. To rule out the possibility that differences in the yield of total RNA from the control and the inflamed colon contribute to the changes in the mRNA levels, we estimated total RNA. The amount of total RNA (2.0 ± 0.25 mg/g) in the control rats did not differ significantly from the amounts of RNA [(mg/g): 1.7 ± 0.20, 1.9 ± 0.17, 1.6 ± 0.20 and 1.5 ± 0.32] obtained from the colonic mucosa of rats after 1, 2, 5 and 7 days of HAC administration, respectively. The quality of total RNA prepared each time was routinely analyzed (fig. 4A) electrophoretically using a 1.2% formaldehyde-agarose gel (Sambrook et al., 1989). The quality of total RNA was consistent in all the samples, as shown by the intensity of the 28s and 18s ribosomal RNA bands (fig. 4A). The yields of total RNA [(mg/gm tissue): 1.7 ± 0.20, 1.8 ± 0.15 and 1.5 ± 0.14] from the rats that received PBS, ethanol and TNBS, respectively, were also not significantly different.

View larger version (50K): [in this window] [in a new window]   Fig. 4.   Representative gel picture shows the quality of total RNA. RNA extracted from the control rats is shown in lane 1, and RNA from the rats that received HAC 1, 2, 5 and 7 days earlier is shown in lanes 2, 3, 4 and 5, respectively. Each lane contains 5 µg total RNA. The positions of 28s (upper arrow) and 18s (lower arrow) ribosomal RNAs are indicated (fig. 4A). Ethidium bromide-stained gel picture showing the PCR amplification of the NHE-1 (Figure 4B) and alfa-1 isoform (fig. 4C). NHE-1 mRNA levels estimated by slot blot analysis are shown in figure 4D. In each figure, the level of mRNA in the control rats is shown in lane 1, and the level of mRNA in the rats that received HAC 1, 2, 5 and 7 days earlier is shown in lanes 2, 3, 4 and 5, respectively. Lane 6 (fig. 4D) is a negative control, and the unlabeled lane (fig. 4B) contains the PBR322xHaeIII size marker. Each lane shows the level of PCR product in 250 ng of total RNA from colonic mucosa after amplifying for 21 cycles. Figure 4E shows that the percent increases calculated from the ratios NHE-1:SD by RT-PCR (closed bars) on the days indicated as post-administration of HAC were not significantly different from those calculated from slot blot analysis (hatched bars). Cycle-dependent amplification of the NHE-1 () and sodium pump mRNA (). Data are mean ± S.E.M. of duplicate determinations (fig. 4F). Representative gel picture (fig. 4G) showing PCR amplification of the indicated isoforms of the NHE and internal control sodium pump (SD). Lanes 1 (control rats), 2 (rats that received HAC 3 days earlier) and 3 (dexamethasone treatment of the rats that received HAC 3 days earlier) contain equal amounts of total RNA as the starting material (fig. 4G).

Profile of NHE-1 mRNA. The basal level of NHE-1 (fig. 4B, lane 1) measured by RT-PCR increased in a time-dependent manner during 1, 2, 5 and 7 days after HAC administration (fig. 4B, lanes 2-5, respectively). The level of alfa-1 RNA internal control decreased in the same samples (fig. 4C, lanes 2-5) as compared with the untreated control rats (fig. 4C, lane 1). Slot blot analysis (fig. 4D) also showed increased levels at 1, 2, 5 and 7 days (fig. 4D, lanes 2-5) after HAC administration as compared with the control rats (fig. 4D, lane 1). Both methods produced comparable percent increases in the level of NHE-1 mRNA (fig. 4E). The PCR conditions were standardized in the linear range of amplification of both NHE and sodium pump internal control using different cycle numbers in the presence of 2 mM MgCl₂ (fig. 4F).

Western blot analysis. To investigate whether changes seen at the mRNA level are also translated into protein, we estimated levels of the NHE-1 protein by ECL Western blot analysis using NHE-1 antibodies raised in rabbit (a kind gift from Dr. E. B. Chang). Because dexamethasone did not suppress the level of NHE-1 mRNA, we did not measure the levels of the protein. The NHE-1 antibodies reacted with the 90- to 92-kD band corresponding to the size of the NHE-1 protein (fig. 5A). The antibodies also showed some cross-reaction in colonic preparations, but the reaction was more specific and intense with the kidney positive control (fig. 5A). In the same amount of crude membranes from colonic mucosa, the amount of NHE-1 protein increased as assessed at 1, 2, 5 and 7 days after HAC administration (fig. 5, A and B) as compared with the control rats. There was no loss of NHE-1 protein in pellet 2 (data not shown). Similarly, in an equal amount of crude membranes, the level of NHE-1 protein was increased in the colonic mucosa from rats that had colitis induced by TNBS (fig. 6, A and B) as compared with the PBS or ethanol control group.

View larger version (38K): [in this window] [in a new window]   Fig. 5.   ECL Western blot analysis showing the NHE-1 protein in the crude membranes from control rats (lane 1) and from rats that received HAC 1 (lane 2), 2 (lane 3), 5 (lane 4) and 7 (lane 5) days earlier. Lane 6 shows NHE-1 from the kidney microsomes positive control. Each lane contains 50 µg of crude membrane proteins. Position of NHE-1 is shown. Bar diagram showing (on the y-axis) a time-dependent increase in the NHE-1 protein levels (band area) in rats that received HAC 1 (bar 1), 2 (bar 2), 5 (bar 5) and 7 (bar 7) days earlier as compared with control rats (bar 0) shown on the x-axis. Data are mean ± S.E.M. (n = 6, P < .04).

View larger version (40K): [in this window] [in a new window]   Fig. 6.   ECL Western blot analysis showing the NHE-1 protein levels (band area) in the mucosal crude membranes (50 µg) from rats that received PBS (lane 1), ethanol (lane 2) or TNBS (lane 3) 7 days earlier. Position of NHE-1 is shown. Bar diagram showing (on the y-axis) increases in the NHE-1 protein levels (band area) in the crude membranes from rats that received TNBS (bar 2) 7 days earlier as compared with the PBS control (bar 0) or the ethanol control (bar 1) on the x-axis. Data are mean ± S.E.M. (n = 6, P < .03).

	Discussion

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Four NHE isozymes are known at present, among which NHE-1 is a ubiquitous isozyme. It is located on the basolateral side of the epithelial cells in the GI tract (Bianchini and Pouyssegur, 1995; Noel and Pouyssegur, 1995). We demonstrate that the isoforms NHE-1 (present study), NHE-2, NHE-3 and NHE-4 mRNA (data not shown) are expressed in the colonic mucosa at varying levels. Our findings show increased levels of NHE-1 mRNA and protein in HAC-induced colitis. Physiologically, NHE induction may be important in maintaining the pH_i of colonocytes acidified by HAC. It is relevant to mention that the colonocytes from patients with Crohn's disease show decreased pH_i (Rowe and Bobar, 1995). However, at this point we do not know whether the pH_i of the colonocytes is decreased in the models of colitis used in the present study. Acidosis induces the NHE-1 activity (L'Allemain et al., 1984). Whether pH_i is a primary or a secondary factor in the induction of NHE-1 remains to be known. Because other isoforms are also increased by colitis, we suggest that acidosis may be a secondary phenomenon and that therefore NHE-1 should contribute significantly to the pH_i, cell growth and tissue repair during IBD. This notion is further supported by a similar increase in mRNA during TNBS-induced colitis. These findings suggest that the induction of NHE-1 is independent of the way colitis is induced. NHE plays an important role in cellular injury, hypertrophy and proliferation of smooth muscle cells and other cell types (Takewaki et al., 1995). It is relevant to mention that in intestinal inflammation, hyperplasia of smooth muscle cells and epithelial cells has been reported (Blennerhassett et al., 1992, MacDonald, 1992). We believe that NHE-1 might be beneficial in the repair of tissue injury caused by the colitides. Although our mucosal scrapings contained predominantly epithelial cells, we also noticed a large infiltration of inflammatory cells in the inflamed colon (data not shown). Therefore, changes in the NHE-1 do not reflect epithelial cells exclusively. The underlying mechanism of the induction of mRNA may involve transcription activation and or stabilization of mRNA; this hypothesis requires further confirmation. Both models were characterized by measuring the MPO activity, which was suppressed significantly by dexamethasone. We also examined histological changes, which included epithelial erosion, loss of crypts and infiltration of inflammatory cells in lamina propria and muscularis mucosa (data not shown). These findings, together with the induction of MPO, confirm the induction of colitis by the colitides.

We employed RT-PCR to estimate the level of NHE-1 mRNA, using as internal control the alfa-1 isoform of the sodium pump (Khan and Collins, 1993), which was amplified under similar conditions from each sample. Because its expression is decreased during colitis, it was considered suitable to prove the specificity of changes. For quantitation, the conditions were optimized in the linear range of amplification, using different cycle numbers. The ratio NHE:internal control was used to indicate the level of NHE-1 mRNA. Elevation of NHE-1 mRNA does not reflect a general increase in the poly A⁺ RNA content because 1) the level of internal control decreased, 2) the difference in yield of total RNA between control and treated rats was not significant and 3) the quality and quantity of the RNA samples used in RT-PCR were comparable as seen on the agarose gel. In addition, slot blot analysis yielded increases in the NHE-1 mRNA levels that were not significantly different from the RT-PCR data. In several studies, Northern blot analysis used for NHE-1 mRNA analysis (Bookstein et al., 1994; Cho et al., 1994) has shown one specific NHE-1 band. However, inconsistencies of the transfer step or poly A⁺ bias are difficult to address by Northern blot analysis. Therefore, we used slot blot analysis in the present study. Taken together, these findings suggest that the changes in the NHE-1 expression are specific. PCR being supersensitive poses a problem of carry-over, or genomic contamination. This was addressed by amplifying RNA without RT, which failed to amplify the specific PCR fragment. In addition, the PCR fragment was characterized by restriction analysis and nucleotide sequencing. These findings rule out contamination of the genomic DNA in the RNA samples, or carry-over. It was difficult to control the number of epithelial cells in the mucosal preparation, so changes shown here are normalized to the total cellular RNA but not the number of cells.

Because it has been suggested that proinflammatory cytokines stimulate NHE in other systems (Benos et al., 1994), in the present study we were interested in identifying the role of inflammation in the induction of NHE-mRNA. Therefore, we examined the effect of dexamethasone 3 days after HAC and 7 days after TNBS administration. In both models, the MPO activity was significantly suppressed by dexamethasone treatment, whereas the NHE-1 mRNA remained unchanged. We selected the condition of HAC for the following reasons. First, this time-point was in the linear range of MPO activity profile. Second, on days 1 and 2 after administration, the colon was badly damaged. Whereas in the HAC- or TNBS-inflamed colon MPO activity was suppressed significantly, the level of NHE isoforms remained unaltered by dexamethsone treatment. Because dexamethsone inhibits the synthesis of cytokines, these findings suggest that the cytokines may be a secondary factor in the induction of NHE-1 during colitis. Dexamethasone acts through its interactions with the glucocorticoid responsive element (GRE) module in a gene promoter and activates the NHE-3 isoform (Cho et al., 1994; Yun et al., 1993).

In conclusion, we demonstrate that the NHE-1 mRNA and protein are induced in both models of colitis, which suggests that NHE-1 expression is independent of the nature of the colitide. Because colitis-induced NHE-2 and NHE-3 mRNA levels were not suppressed by dexamethasone treatment of colitis, pH_i and cytokines may be a secondary factor in the induction of NHE-1 mRNA. Increases in the expression of NHE-1 should contribute to pH_i and to cell growth and repair during colitis and, therefore, may be beneficial. The regulation of the expression of NHE-1 might involve pretranslational steps that do not seem to be regulated primarily by cytokines or pH_i.