1Department of Gastroenterology, Hepatology, and Endocrinology and 3Department of Anatomy I, Hannover Medical School, 30623 Hannover; and 2Department of Internal Medicine I, University of Tübingen, Tübingen, Germany; and 4Department of Molecular Genetics, University of Cincinnati, Cincinnati, Ohio
Submitted 4 August 2003 ; accepted in final form 9 February 2004
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ABSTRACT |
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NHE2 knockout mice; pH regulation; colon; ion transport
The physiological role of NHE2 has remained obscure. Early studies have described a high expression and apical localization of NHE2 in the ileum and colon, and some studies have reported a similar role for NHE2 as for NHE3, namely mediating the electroneutral absorption of NaCl (35). Consequently, a major intestinal phenotype was expected in NHE2 deficient mice. However, the small intestinal mucosa of NHE2 knockout mice displays neither gross morphological changes (29) nor an obvious absorptive defect (13, 29). On the other hand, NHE2 (/) mice develop gastric mucosal atrophy. We recently studied the expression and function of NHE1, NHE2, NHE3, and NHE4 in the different epithelial cell types in the gastric mucosa and, surprisingly, found that the expression pattern and regulatory characteristics of NHE2 resembled that of NHE1 (25), suggesting a potential role in pHi- and volume-regulatory mechanisms. We therefore speculated that this isoform may have a role in enterocyte pHi and volume regulation as well. We studied NHE13 expression and transport function in isolated murine colonic crypts and surface cells, as well as the regulation of the different isoforms by low pHi, hyperosmolarity, and cAMP. When we found that NHE2 has a major role in cryptal pHi regulation, we wondered about the compensatory changes that may occur in NHE2 (/) colon and studied NHE1 and NHE3 expression levels and NHE1- and NHE3-mediated acid-activated proton efflux rates in crypts isolated from NHE2 (/) mice and their normal littermates.
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MATERIALS AND METHODS |
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HOE-642 was prepared by Aventis (Frankfurt, Germany), bovine serum albumin (BSA; cell culture grade) was from Paesel und Lorei (Frankfurt, Germany), and 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) from Molecular Probes (Leiden, the Netherlands). All other chemicals were either obtained from Sigma (Deisenhofen, Germany) or from Merck (Darmstadt, Germany) at tissue culture grade or the highest grade available.
Animal breeding and genotyping.
The NHE2 knockout mouse strain had been generated and characterized in the laboratories of Shull and colleagues (29). The animals were raised in the animal facilities of the University of Tübingen and the Medical School of Hannover and PCR-genotyped as previously described (31) using the primers outlined in Table 1 for detection of the wild-type and mutant sequence.
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After the mice were killed by CO2-narcosis and cervical dislocation, crypts and surface cells were isolated from an everted segment of proximal colon exactly as previously described (2, 3), using a combination of Ca2+ chelation [solution composition (in mM): 127 NaCl, 5 KCl, 1 MgCl2, 5 Na-pyruvate, 10 HEPES, 5 EDTA, 1% BSA, pH 7.4] and mechanical vibration. Surface epithelial sheet fragments and intact crypts were harvested by low-speed centrifugation, and their viability was tested by trypan blue exclusion. This preparation results in the isolation of intact crypts that possess all the cell types along the crypt length, as well as isolated surface cells or surface cell sheets. The protocols for raising and killing the mice were approved by the local committee for the welfare of animals.
Quantitative RT-PCR in relation to 18S rRNA.
Homologous primers for murine NHE13 were deduced from published sequence information (see Table 1). The identity of the PCR fragments was confirmed by restriction analysis or cloning and sequencing. RT-PCR was performed as described previously (2). As an internal control, an 18S rRNA fragment was amplified with the use of primers supplied by Ambion (Austin, TX). Briefly, the relationship of the amplification efficiency of the gene of interest vs. 18S rRNA during the exponential phase of the reaction was calculated after semilogarithmic plotting of the integrated optical density (ODI) values of the digitized bands against the cycle number (Fig. 1A). To adjust the measured values to the expression levels of the NHE isoforms, the OD values of the different PCR products were corrected according to their length.
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pHi was measured fluometrically after loading crypts or surface cells for 30 min with 5 µM BCECF in buffer A (in mM: 120 NaCl, 14 HEPES, 7 Tris, 3 KH2PO4, 2 K2HPO4, 1.2 MgSO4, 1.2 Ca2+-gluconate, 20 glucose, pH 7.4), fixing them between a glass coverslip and a 0.3 µM polycarbonate membrane (Osmonics, Minnetonka, MN) in a custom-made perfusion chamber on the stage of an inverted microscope (Nikon Diaphot TMD, Nikon, Düsseldorf, Germany), and perfusion with different buffers following the appropriate protocol {buffer A, buffer B (40 mM NaCl were replaced by NH4Cl), buffer C [containing 120 mM tetramethylammonium chloride (TMA-Cl) instead of NaCl], buffer D (containing 120 mM sodium gluconate instead of NaCl), buffer E [containing 40 mM (NH4)2SO4 and 80 mM sodium gluconate instead of NaCl], and buffer F (containing 120 mM TMA-hydroxide and 120 mM gluconic acid lactone instead of NaCl)}. Hyperosmolar solution contained 100 mM mannitol in addition. Calibration and determination of the intracellular buffering power (i) was performed as described previously (2);
i curves (see Fig. 2B) were neither different between crypts and surface cells nor between cells from NHE2 (+/+) and NHE2 (/) mice.
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Immediately after removal, samples of the proximal colon from NHE2 (/) mice and their normal littermates for histological and morphometric analysis were fixed in 3% glutaraldehyde buffered in 0.1 M Na-cacodylate/HCl, cut into smaller pieces and stored in the fixative solution at 4°C. After washing in 0.1 M Na-cacodylate/HCl buffer, the specimens were postfixed for 90 min at room temperature in 2% OsO4 in the same buffer. Subsequently, they were dehydrated in graded ethanols, immersed in the intermedium toluol, and finally embedded in epoxy resin (Serva, Heidelberg, Germany). Semithin (0.75 µm) and thin (
70 nm) sections were cut with an ultramicrotome Reichert Ultracut E (Reichert, Vienna, Austria). Semithin sections were stained with 1% alkalinized toluidine blue. Thin sections were collected on formvar-coated copper slot membranes or grids, stained with uranyl acetate and lead citrate, and examined in an electron microscope Zeiss EM 10 CR (Zeiss, Jena, Germany) at an acceleration voltage of 80 kV.
Morphometric analysis.
The Videoplan image analysis system (Kontron-Elektronik, Munich, Germany) equipped with a light microscope (Zeiss Axioskop, Zeiss) and a color video camera (Sony DXC-101P) was used to determine the crypt depth. In well-oriented semithin sections orthogonal to the mucosa, the depth of crypts cut through their length axis was measured from the luminal border to their base.
Statistics and calculation of proton fluxes.
Results are given as means ± SE. Proton fluxes (see Fig. 2C) were calculated by multiplying the initial steep pHi slope after readdition of sodium (see Fig. 2A), which was determined by regression analysis, with the buffering capacity (i, see Fig. 2B) at the initial pHi. Statistical significance was determined using Student's t-test. For morphometric analysis, statistics were performed using the statistical package of the Videoplan image analysis system. Due to the non-Gaussian distribution of the values as assessed by the application of the
2-approximation test, the differences among the datasets were determined by the nonparametric (distribution free) test of Kolmogoroff and Smirnoff. The level of significance was taken as P < 0.05. The results were plotted as histograms of relative frequency using Excel 5.0 software (Microsoft, Redmond, WA).
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RESULTS |
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NHE13 expression levels were measured in isolated crypts and surface cells using a quantitative PCR protocol. These experiments revealed an even distribution of NHE1 mRNA expression in crypt vs. surface cells (Fig. 1B), significantly higher NHE2 mRNA expression levels in colonic crypts than in surface cells (Fig. 1C), and much higher surface cell than crypt expression for NHE3 mRNA (Fig. 1D).
Inhibition curve for HOE-642 on acid-activated Na+/H+ exchange.
Because existing inhibition curves for HOE-642 consisted of 22Na+ flux experiments with low extracellular sodium (22, 28), we performed a dose-response study for the inhibition of HOE-642 on acid-activated Na+/H+ exchange under 120 mM extracellular Na+.(Fig. 2D). The rationale of the experiments was the following. If the chosen concentration of 1 µM HOE-642, found to be selective for NHE1 in 22Na+ flux experiments (28), was indeed fully sufficient for NHE1 inhibition, but not high enough to inhibit NHE2, then a shift of the concentration to 0.7 or 3 µM should not cause a change in the percent Na+/H+ exchange inhibition. This was indeed the case. The same applied for 50 µM HOE-642 for NHE2 inhibition. These results demonstrate that the chosen concentrations of HOE-642 and 5-(N,N-dimethyl) amiloride (DMA) are good discriminators of NHE13 activity. Most importantly, they also demonstrate that NHE2 is either located basolaterally in colonic crypts or, if apically, is reached by the inhibitors in the crypt lumen. These conclusions are strengthened by the experiments performed in the NHE2 (/) crypts.
Differential activation of NHE13 by intracellular acidification in crypt and surface cells.
Total Na+/H+ exchange rate in crypts was 18.7 ± 1.7 mM/min, and NHE1-selective inhibition with 1 µM HOE-642 reduced this rate by 40% (Fig. 3, A and B), additional NHE2 inhibition with 50 µM HOE-642 by another 48%, and 700 µM DMA resulted in complete inhibition (Fig. 3B). In surface cells, the total acid extrusion rate (Fig. 3C) was 9.31 ± 0.29. The NHE1 and the NHE1 plus NHE2 selective concentrations of HOE-642 each blocked 20% of the acid extrusion rate; the remaining 55% could only be blocked by 700 µM DMA (Fig. 3D). Thus the major part of pHi recovery from an acid load is mediated by NHE2 in colonic crypts and by NHE3 in surface cells.
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A Cl-dependent Na+/H+ exchanger has been functionally (24) and molecularly (26) identified in rat colonic epithelium. To assess its potential presence in murine colon, we performed pHi recovery experiments in the absence of Cl (Fig. 3E). Cells were washed several times with buffer D and perfused with this buffer for 15 min to achieve intracellular Cl depletion. Using the same experimental protocol as above (replacing buffer A for buffer D, buffer B for buffer E, and buffer C for buffer F), acid-activated proton efflux rates were not significantly different from the Cl-containing control. This argues against the presence of Cl-dependent Na+/H+ exchange in murine crypts.
An increase in intracellular cAMP activates NHE1 and NHE2 in colonic crypts.
Next, we measured the effect of a rise in intracellular cAMP on the acid-activated Na+/H+ exchange rate (Fig. 3F). Forskolin (105 M) stimulation increased acid-activated Na+/H+ exchange, and this activation was partially preserved in the presence of NHE1 inhibition and blocked by DMA, suggesting that a rise in cAMP stimulated both NHE1 and NHE2, either directly or via cryptal shrinkage after stimulation of anion secretion. This demonstrates that NHE2 is an unlikely candidate for the residual EIPA-sensitive Na+-resorption mechanism found in the small intestine of NHE3 (/) mice, because this mechanism was inhibited by cAMP (5).
Hyperosmolarity activates only NHE1 and NHE2 in both crypt and surface cells.
Hyperosmolarity caused an activation of colonic Na+/H+ exchange (Fig. 4, A and B). In crypts, inhibition of NHE1 by 1 µM HOE-642 reduced this Na+/H+ exchange rate by 40% and NHE1 and NHE2 inhibition by >90% (Fig. 4B). In surface cells (Fig. 4, C and D), NHE1 inhibition reduced the rate by 60% and additional NHE2 inhibition by >95%. Blocking NHE3 by DMA had no further effect (Fig. 4D), consistent with the inhibition of NHE3 by shrinkage, as found by others (18, 23). After Na+/H+ exchange inhibition by DMA, concomitant Cl/OH exchange activation is unmasked and causes a slight intracellular acidification.
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To investigate potential compensatory mechanisms, we studied pHi recovery in colonic crypts of NHE2 (/) mice (Fig. 5A).
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What could be the compensatory mechanisms that allow normal pHi recovery in NHE2 (/) crypts? One such mechanism is a higher percentage of NHE1-mediated Na+/H+ exchange. However, we also found a higher HOE-642-insensitive, DMA-sensitive Na+/H+ exchange in NHE2 (/) compared with (+/+) crypts, likely mediated by NHE3. NHE1 and NHE3 mRNA expression levels were determined in NHE2 (/) crypts and (+/+) littermates. NHE1 mRNA expression levels were not different, and NHE3 mRNA levels were significantly higher in the former (Fig. 5, B and C). Thus it appears that cryptal NHE3 expression seen in normal mice is upregulated in NHE2 deficient colonic crypts.
Light and transmission electron microscopy.
Light microscopy of semithin sections from colon specimens of NHE2 (+/+) (Fig. 6, A and B) and NHE2 (/) mice (Fig. 6, C and D) showed a regular colon mucosa, and no differences could be detected between the two groups of animals. In both, the mucosa surface bordering the lumen and the upper parts of the crypts were predominantly covered by simple columnar epithelial cells, the principal cells or absorptive cells. Large numbers of mucus-producing goblet cells could be especially detected in deeper crypt regions. In addition, occasional enteroendocrine cells could be seen within the epithelial lining.
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Morphometric analysis.
In control animals morphometric analysis revealed a minimal crypt depth of 67.4 µm and a maximum depth of 147.5 µm. In NHE2 knockout mice the values ranged between 83.6 and 127.8 µm. However, relative frequency distribution of the values (Fig. 6E) showed that in both groups of animals the bulk of the crypts were approximately 100125 µm in depth, and statistical analysis revealed no significant differences between the datasets.
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DISCUSSION |
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NHE localization.
A number of studies have localized NHEs in the colonic epithelium (12, 16). Whereas all studies agreed on the predominantly surface cell localization of NHE3 (4, 16), NHE2 localization studies yielded controversial results (16, 33). In situ hybridization experiments in rat intestinal epithelium have shown a predominant signal in the villus or surface cell region and a relatively weak signal in the crypts (6), whereas an even distribution was found in human colon (12). Our method is very sensitive for measuring relative differences in expression levels of the same mRNA species in different samples (surface cells vs. crypts in this case) and therefore support the recent report by Chu et al. (9) of the presence of NHE2 in the colonic crypt region. They further demonstrate that NHE2 is the NHE isoform with the highest expression level in that area of the colonic epithelium.
Contribution of NHE13 to acid-, hyperosmolarity-, and forskolin-induced Na+/H+ exchange.
Given the fact that different half-lives of NHE13 in the plasma membrane have been described (7), expression levels may not correlate with functional relevance. We therefore monitored Na+/H+ exchange activity by fluorometric measurements with BCECF (2, 3) and pharmacologically differentiated the activities of the NHE1 and NHE2 isoforms by using HOE-642, which has been shown to sequentially block NHE12 in a dose-dependent manner with a smaller overlap between the isoforms than other inhibitors (28), and assessed additional NHE3 activity by inhibiting it with 700 µM DMA (21). This approach is only valid if no other Na+/H+ exchangers are contributing to acid-, hyperosmolarity-, and forskolin-activated Na+/H+ exchange in colonic crypts. Because this is the critical issue in our experiments, we have performed a number of experiments to ascertain the fact that we really do differentiate between NHE1, -2, and -3. We have performed concentration-response curves, which clearly show that the chosen HOE-642 concentrations do not have an overlapping effect on NHE1 and NHE2 or on NHE2 and NHE3 (or some other NHE) under our experimental conditions. By using NHE2-deficient mice, we also ascertained that the NHE2-inhibitory concentration of HOE-642 does not inhibit something else. We ruled out the presence of NHE4 (Rossmann, unpublished observation) and of a Cl-dependent, amiloride-sensitive Na+/H+ exchanger as found in the rat distal colon (24, 26). Because we measure Na+-dependent changes in pHi to assess NHE activity and not 22Na+ uptake, the presence of Na+ conductances does not interfere with our measurements.
Of interest was the fact that Cl removal did not seem to affect Na+/H+ exchange rates at the pHi that we acidified the colonic crypts to. Aharonovitz et al. (1) had recently shown that in NHE13-transfected AP1 cells (NHE-deficient Chinese hamster ovary cells), Cl replacement by nitrate or isethionate caused a decrease in the exchange rates of all three isoforms. On the other hand, other groups had found maximal Na+/H+ exchange rates to be independent of Cl in membrane preparations (e.g., Ref. 19). The reason for the discrepancy is not clear, but one possibility is that not all Cl replacements are inert. Another one is that the volume changes caused by Cl removal affect different cell types differently, and a third is that coupled Cl/anion exchange may be necessary for intracellular gradient dissipation in some cells.
When heterologously expressed in PS120 fibroblasts, NHE13 proteins have different half-lives in the plasma membrane, as recently reported by Cavet et al. (7). The half-life of NHE1 was almost twice as long as that of NHE3, and NHE2 had the shortest. When we assume that NHE13 in native cells have similar differences in their half-lives [which needs not be the case, as PS120 fibroblasts have been found to lack important PDZ-domain adapter proteins for these transporters (36) which may influence localization of these NHEs in the membrane], this would explain why NHE1 expression levels, which are only one-third to one-fourth of those found for NHE2, are associated with a similar acid-activated transport activity. In addition, activation of NHE3 by pHi has been found to be slower than for NHE1 and NHE2, possibly explaining a relatively minor role in cryptal pHi regulation when the other NHEs are highly active (14). Taking all this into account, there is a surprisingly good correlation between the relative expression levels of the different NHEs and their respective activity in acid-activated Na+/H+ exchange in surface and crypts. In summary, our findings suggest that the determination of the NHE2-mediated part of Na+/H+ exchange is reliable using the pharmacological tools that we have.
With this approach, we found similarities between NHE2 and NHE1, in that they are both activated by low pHi, hyperosmolarity, and cAMP. This has been observed for NHE1 and, more so, NHE2, heterologously expressed in the Na+/H+-deficient Chinese hamster ovary cell line AP1 (1, 18), and for NHE2 in LLC-PK1 cells (8), whereas an inhibition of NHE2 by hyperosmolarity has been described in PS120 cells (23) and by cAMP in the Caco-2 subclone C2bbe (5). Another recent publication studied the ability of NHE1-, NHE2-, and NHE3-transfected PS120 cells to perform a regulatory volume increase after hyperosmolar shrinkage (34). They found that only NHE1, not NHE2 and NHE3, could mediate this effect and that a point mutation in the first extracellular loop renders NHE2 sensitive to volume changes. This resembles the situation found for the small intestinal sodium channel hINaC, where a point mutation in an extracellular domain results in an enormous increase in channel activity and amiloride sensitivity (27), suggesting, in the author's opinion, the existence of an unknown physiological regulator of this channel. The reason(s) for these discrepancies are completely unclear at present and may be cell-type related. For example, NHE1 is activated by cAMP in a number of cell types and not affected in others. When transfected into OK cells, it is sorted to the basolateral membrane, but inhibited by cAMP, just like the endogenous amiloride-resistant apical Na+/H+ exchanger (15). A similar situation may exist for NHE2 expressed in different tissues or species.
The high distal ileal and colonic expression levels and the apical location suggested a possible involvement in electroneutral NaCl absorption, similar to NHE3 (35), and several studies are in accordance with this hypothesis (11, 35). However, NHE2-deficient intestine displays neither gross morphological changes nor an obvious absorptive defect (29). Additional disruption of NHE2 in NHE3-deficient mice does not lead to any worsening of the diarrheal state (20), and NHE2 (/) mice were found to have similar small intestinal sodium absorption rates as controls (13). Gawenis et al. (13) did, however, find a residual highly EIPA-sensitive Na+ absorptive rate in NHE3 (/) jejunum, which was abolished by forskolin stimulation. The stimulatory rather than inhibitory effect of cAMP on NHE2 (found in this study) makes it unlikely that NHE2 is the residual Na+ absorptive mechanism in NHE3-deficient intestine.
To get further insight into the physiological role of colonic NHE2, we studied potential pHi-regulatory defects in NHE2 (/) crypts, as well as the compensatory mechanisms that may prevent them. Acid extrusion rates after intracellular acidification were normal in NHE2-deficient crypts, the part of pHi recovery apparently mediated by NHE1 was larger, and interestingly, there was clearly also a higher percentage of non-NHE1 (and non-NHE2), DMA-sensitive Na+/H+ exchange activity. We therefore measured NHE3 expression levels in NHE2 (+/+) and (/) crypts and indeed found significantly higher NHE3 expression levels but unchanged NHE1 expression levels in NHE2 (/) crypts. Obviously, NHE1 is not operating at its maximal capacity during pHi recovery from pHi 6.4 and can increase transport rate without a need for an increase in transcriptional activity. The interesting question is, why is NHE3 upregulated in NHE2-deficient colon? When NHE2 is heterologously expressed in acid-suicide-selected LLC-PK1 cells, endogenous NHE3 expression is inhibited, and this precedes functional NHE2 expression (32). So this would suggest that some function of NHE2, possibly control of local Na+ concentration, or something else, influences NHE3 expression in a reciprocal fashion. It is, of course, tempting to speculate that this upregulation of NHE3 may compensate an Na+-absorptive defect in NHE2 (/) colon. Due to restricted breeding facilities for NHE2 (/) mice and the difficulty of selectively blocking NHE3 in intact mucosa, we have not been able to perform sufficient experiments to rule in or out an increase in NHE3-mediated Na+ absorption in NHE2 (/) murine colon.
In summary, our data suggest that NHE2 is strongly expressed in murine colonic crypts where it is activated by low pHi, hyperosmolarity, and an increase in intracellular cAMP levels. In the absence of NHE2 expression, other NHEs compensate for NHE2-mediated pHi recovery in NHE2-deficient crypts, and both NHE3 expression and participation in pHi recovery are upregulated. Further studies will have to clarify the exact membrane location and provide detailed insight into the cellular functions of NHE2 in secretory epithelia.
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GRANTS |
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ACKNOWLEDGMENTS |
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Present address of P. J. Schultheis: Dept of Biol. Sciences, Northern Kentucky Univ., Highland Heights, KY.
Present address of H. Rossman: Dept of Clinical Chemistry, University of Heidelberg, Heidelberg, Germany.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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