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. 2010 Oct 20;588(Pt 24):5049–5063. doi: 10.1113/jphysiol.2010.198721

Loss of PDZ-adaptor protein NHERF2 affects membrane localization and cGMP- and [Ca2+]- but not cAMP-dependent regulation of Na+/H+ exchanger 3 in murine intestine

Mingmin Chen 1, Ayesha Sultan 1, Ayhan Cinar 1, Sunil Yeruva 1, Brigitte Riederer 1, Anurag Kumar Singh 1, Junhua Li 1,2, Janina Bonhagen 1, Gang Chen 2, Chris Yun 3, Mark Donowitz 4, Boris Hogema 5, Hugo deJonge 5, Ursula Seidler 1
PMCID: PMC3036196  PMID: 20962002

Abstract

Trafficking and regulation of the epithelial brush border membrane (BBM) Na+/H+ exchanger 3 (NHE3) in the intestine involves interaction with four different members of the NHERF family in a signal-dependent and possibly segment-specific fashion. The aim of this research was to study the role of NHERF2 (E3KARP) in intestinal NHE3 BBM localization and second messenger-mediated and receptor-mediated inhibition of NHE3. Immunolocalization of NHE3 in WT mice revealed predominant microvillar localization in jejunum and colon, a mixed distribution in the proximal ileum but localization near the terminal web in the distal ileum. The terminal web localization of NHE3 in the distal ileum correlated with reduced acid-activated NHE3 activity (fluorometrically assessed). NHERF2 ablation resulted in a shift of NHE3 to the microvilli and higher basal fluid absorption rates in the ileum, but no change in overall NHE3 protein or mRNA expression. Forskolin-induced NHE3 inhibition was preserved in the absence of NHERF2, whereas Ca2+ ionophore- or carbachol-mediated inhibition was abolished. Likewise, Escherichia coli heat stable enterotoxin peptide (STp) lost its inhibitory effect on intestinal NHE3. It is concluded that in native murine intestine, the NHE3 adaptor protein NHERF2 plays important roles in tethering NHE3 to a position near the terminal web and in second messenger inhibition of NHE3 in a signal- and segment-specific fashion, and is therefore an important regulator of intestinal fluid transport.

Introduction

The Na+/H+ exchanger isoform 3 (NHE3) is responsible for a major part of intestinal NaCl absorption (Zachos et al. 2005). When it was cloned from rabbit intestine and expressed in the Na+/H+ exchanger-deficient fibroblast cell line PS120, cAMP analogues failed to inhibit NHE3 transport rate (Tse et al. 1991). Subsequently, a PDZ-adaptor protein interacting with NHE3 was cloned from a human lung library (Yun et al. 1997). This protein was called NHE3 kinase A regulatory protein (E3KARP), because coexpression with NHE3 restored the cAMP-mediated NHE3 inhibition (Yun et al. 1997; Lamprecht et al. 1998; Zizak et al. 1999). The same effect was seen when the highly similar PDZ-protein NHERF1 was coexpressed with NHE3. E3KARP was found to be identical to the NHERF1 homologue NHERF2 (Hall et al. 1998). NHERF2, but not NHERF1, was later demonstrated to be essential for Ca2+- and cGMP-mediated inhibition of NHE3 by facilitating the formation of the NHE3–NHERF2–α-actinin-4 complex that promotes [Ca2+]i-dependent NHE3 oligomerization and endocytosis (Kim et al. 2002), and by bringing cGMP-dependent protein kinase type II (cGKII) into close proximity to NHE3, which is necessary for cGMP-dependent NHE3 inhibition (Cha et al. 2005).

Investigating the recently generated NHERF1 deficient mice resulted in the surprising finding that whereas NHERF1 proved essential for cAMP-mediated NHE3 inhibition in the kidney, this function was not affected in the NHERF1 deficient intestine (Murtazina et al. 2007; Broere et al. 2009). In contrast, another member of the NHERF family, PDZK1, was shown to be of particular importance for cAMP-mediated inhibition in the murine intestine (Cinar et al. 2007; Hillesheim et al. 2007; Broere et al. 2009). Thus, it appeared that the action of the NHERFs on NHE3 may be cell type as well as signal specific. These findings prompted us to study the localization and agonist-mediated regulation of NHE3 in the different intestinal segments of the recently generated NHERF2 deficient mouse model (Broere et al. 2007).

Methods

Chemicals

Chemicals were obtained from Sigma (Deisenhofen, Germany) or Merck (Darmstadt, Germany) at tissue culture grade or the highest grade available, unless indicated otherwise. Hoe 642 and S1611 were kindly provided by Sanofi-Aventis (Frankfurt, Germany). 4-Bromo-A23187 was purchased from Biomol (Hamburg, Germany) and E. coli enterotoxin STp was from Bachem (Weil am Rhein, Germany). Nigericin and 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) were purchased from Molecular Probes (Leiden, the Netherlands). The antibodies used are specified in the appropriate Methods section.

Animal breeding

Nherf2−/− mice were originally generated from Lexicon genetics clone OST2298 (Broere et al. 2007) and were bred into a congenic FVB/N background in the animal facility of the Hannover Medical School under standardized light and climate conditions with access to water and chow ad libitum. Age- and sex-matched wild-type littermates were used as controls. The genotype was determined by PCR as previously described (Singh et al. 2009). The NHE3-deficient mouse strain had originally been generated by Schultheis et al. (1998) and had been bred for =10 generations at Hannover Medical School. Animal experiments followed approved protocols at the Hannover Medical School and were reviewed and approved by an independent committee on animal rights assembled by the local authorities for the regulation of animal welfare.

mRNA isolation and quantitative real-time PCR

mRNA was isolated from the different segments of murine intestine that were also functionally studied (see below). Isolation method, primers and PCR procedure have been described previously (Hillesheim et al. 2007; Broere et al. 2009).

In vivo intestinal fluid transport measurements

The measurements of fluid absorptive rates were performed via a single pass method, as described by us for the jejunum (Broere et al. 2009; Chen et al. 2010; Singh et al. 2010). The mice were anaesthetised by isoflurane inhalation, and blood pressure and ventilatory rate were continuously monitored using a PowerLab II-26 (ADInstruments, Spechbach, Germany). For jejunal perfusion, the inlet tube was inserted approx. 5–8 cm distal to the pylorus and a 5–7 cm segment was perfused; for the ileal perfusion, 5 cm of the ileum was used, with the distal end of the perfused segment 1–2 cm proximal to the ileocaecal valve; and for colon perfusion, the second part of the colon (not the haustrated, wide part of the proximal colon) was perfused with the distal end about 1 cm proximal to the anus. For colon perfusion, a higher perfusion speed (30 ml h−1) was chosen to prevent clogging of mucous material, and the fluid was recirculated with a precision pump (Minipuls Evolution; Gilson, Villiers, France) and constantly kept at 37°C in a water bath. At the end of the experiment, the mouse was killed by cervical dislocation while still under anaesthesia. Since the incision is made as small as possible, and exact determination of the length and the distance to pylorus of the perfused jejunal segment could only be made after killing of the mouse and excision of the gut, and therefore varies somewhat between experiments. S1611 was used to inhibit NHE3-mediated fluid absorption (Schwark et al. 1998; Bachmann et al. 2004; Cinar et al. 2007; Singh et al. 2010); in addition to the experiments performed in the cited studies, it was tested by us for potential inhibition of ENaC in distal colon of mice on a low salt diet, and NHE2 WT and KO mice, and found not to do so (X, Fang & J, Li, unpublished observations).

Ligated loop experiments in different segments of the small intestine

While the above described single pass perfusion techniques allow better control of acid/base balance during anaesthesia, a sequential assessment of basal and inhibitor-treated absorptive rates, and do not alter luminal fluid pressure, they are less well suited to perform simultaneous fluid absorption measurements in different intestinal segments in the same mouse. Therefore, classic ligated loop experiments were also performed as previously described (Broere et al. 2009). In these experiments, the mice were anaesthetized with ketamine/xylazine (100 and 20 mg kg−1, respectively, i.p.); 10–12 cm of proximal (approx. 5 cm from pylorus) and distal (approx. 17–20 cm from pylorus) and ileal (last 10–12 cm of small intestine) segments were ligated, and defined amounts of isotonic saline were injected into the ligated intestinal loops; the abdomen was closed, and the mice were kept in a humidified 37°C atmosphere for 1 h. Thereafter the mice were killed, the segments were removed, the fluid was removed and weighed, and the segment length was measured.

NHE3 distribution relative to the brush border membrane

Stained sections were imaged on the confocal microscope (Leica DM IRB with a TCS SP2 AOBS scan head equipped with a 405 nm laser for excitation of blue dyes). Sections from Nherf2+/+ and Nherf2−/− jejunum, ileum and colon were imaged with identical confocal settings. A detailed method description is given in online Supplemental Material. In this paper, we call the location of the peak of the actin signal which is also the innermost part of the apical actin stain the ‘terminal web’, because PEPT1 is seen in the complete microvillar region but not in this part (Chen et al. 2010), whereas NHE3 can be found in the innermost part of the actin band (likely to be in recycling endosomes within the terminal web; Hansen et al. 2009). We did not distinguish technically between the base of the microvilli and the terminal web, but looking at the localization of NHE3 in relation to F-actin, and comparing that to the ultrastructural architecture of the microvilli and the terminal web, we have to conclude that most likely a part of NHE3 is in the terminal web region and not just at the base of the microvilli (Hirokawa et al. 1982). However, since this was not the focus of the study (since a difference between NHE3 at the base of the microvilli or the recycling endosomes would not have an impact on the interpretation of our current data), a more detailed ultrastructural localization of NHE3 was not attempted.

Intestinal brush border membrane preparation

The preparation of jejunal and ileal brush border membranes (BBMs) was performed by the divalent cation precipitation technique similar to previously published protocols (Hillesheim et al. 2007), with modifications as described in the Supplemental Material.

Immunoblot analysis

BBM was denatured in sample buffer at 65°C for 10 min, resolved on a 10% SDS-polyacrylamide gel and transferred to PVDF membrane (GE Healthcare, Little Chalfont, UK). Blots were blocked with 5% skimmed milk and first incubated with rabbit anti-rat NHE3 (Alpha Diagnostic, San Antonio, TX, USA, NHE31-A; 1:2500) or rabbit anti-rat villin (H-60, Santa Cruz Biotechnology, Santa, Cruz, CA, USA; 1:2500) for 2 h at room temperature (RT) followed by horseradish peroxidase (HRP) conjugated anti-rabbit antibody (KPL, Inc., Gaithersburg, MD, USA; 1:10,000) for 1 h at RT. Blots were subsequently developed by using ECL (GE Healthcare) and the signal was quantified by TotalLab software (TotalLab Ltd, Newcastle upon Tyne, UK). In case of excessive villin signal the membrane was stripped by incubating with stripping buffer (62 mm Tris, pH 6.8, 2% SDS, 0.7%β-mercaptoethanol) at 50°C for 30 min, washed and re-blocked with 5% skimmed milk, followed by the above specified Western protocol.

Preparation of isolated intestinal villi for fluorometry

For measuring pHi in isolated jejunal and ileal villi, the proximal jejunum (approximately 6 cm distal to the pylorus), the ileum (approx. 8 cm proximal to the ileocaecal junction) and the distal ileum (approx. 1 cm proximal to the ileocaecal junction) were removed and immediately placed in ice-cold Ringer solution (solution composition in mm: 147 NaCl, 4 KCl, 2.2 CaCl2 in 500 μm DTT to prevent mucus clogging of the villi, pH 7.4). The method is described in detail in Chen et al. (2010).

Fluorometric pHi measurements in intact villi

The method for pHi measurement and assessment of acid-activated NHE3 activity in intact microdissected jejunal villi has recently been described in Chen et al. (2010) and was performed identically here. Intrinsic buffering capacity was determined in the villous enterocytes of each studied intestinal segment and the curves are given in the Supplemental Material. Calibration of the 440/495 ratio was performed as described in Hegyi et al. (2004). For the calculation of proton extrusion rates, the intrinsic buffer capacity was determined for jejunal (shown in Chen et al. 2010), and ileal (see Suppl. Fig. 2) enterocytes within microdissected villi, as described in Chen et al. (2010). The buffer capacity of colonic crypts had previously been reported in Bachmann et al. (2003). Although we did not construct a full buffer curve for NHERF2 knockout enterocytes of the different intestinal segments, we did perform individual buffer capacity measurements and these values were in the same range as those of the WT mice.

Preparation of colonic crypts and fluorometric pHi measurements

The isolation of murine colonic crypts and pHi recovery from an ammonium prepulse-induced acidification were performed exactly as previously described in Cinar et al. (2007) and Broere et al. (2009).

Statistics

Data are presented as means ±s.e.m. The Mann–Whitney rank-sum, Student's t test and ANOVA were used for statistics, where appropriate, and values of P < 0.05 were considered significant.

Results

In vivo fluid absorption in the different segments of the intestine of anaesthetized NHERF2-deficient and WT mice

NHE3 is involved in electroneutral NaCl uptake, resulting in fluid absorption. To obtain an indication as to whether NHE3 regulation is altered in NHERF2-deficient mice, we first measured fluid absorption by single pass experiments in the jejunum, ileum and mid colon of anaesthetized NHERF2 KO and WT mice (segments defined in Methods). Interestingly, NHERF2 ablation resulted in significantly increased absorptive rates in the ileum (Fig. 1A and C), but not the jejunum and colon (Fig. 1A and C). The NHE3 inhibitor S1611 almost fully inhibited fluid absorption in all segments, indicating that NHE3 is the major Na+ absorptive transporter in the absence of nutrients in the murine small and large colon (Fig. 1B).

Figure 1. in vivo fluid absorption in jejunum, mid ileum and colon of NHERF2-deficient and WT mice.

Figure 1

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A, no significant differences were observed in fluid absorptive rates in jejunum and colon of NHERF2-deficient mice and WT littermates except in the ileum, in which the fluid absorptive rate was significantly higher in the NHERF2-deficient mice. B, 20 μm of S1611, which inhibits NHE3, strongly reduced fluid absorptive rates in all intestinal segments of FVB/N mice, demonstrating that fluid absorptive rates adequately reflect NHE3 activity in vivo. C, simultaneous fluid absorption measurements in ligated loops of proximal and distal jejunum and ileum of NHERF2-deficient and WT mice. The bars indicate the fluid absorption in NHERF2-deficient and WT ligated loops from different parts of the intestine in the basal state. Basal absorptive rate was similar or higher in the NHERF2-deficient intestine (significant increase in ileum). n= 6–9. *P < 0.05, **P < 0.01.

Fluid absorption was also studied simultaneously in the same mouse (ligated loop experiments) in proximal and distal jejunal, as well as ileal loops of NHERF2 WT and KO mice (Fig. 1C, segments defined in Methods). The increased fluid absorption under basal conditions in the ileum but not jejunum of NHERF2 KO mice was confirmed in these experiments (Fig. 1C).

NHE3 mRNA expression throughout the intestine was not changed by NHERF2 ablation

No difference was seen in the NHE3 mRNA levels between NHERF2-deficient and WT mice in any of the intestinal segments studied (Fig. 2A). We normalized NHE3 mRNA expression to villin, which is a structural BBM component with a similar expression pattern to NHE3, and to RPS9, which is an indicator of overall RNA content, but has the disadvantage that changes in a non-epithelial component of the intestine may skew the results.

Figure 2. mRNA and protein expression levels of NHE3 in the mucosa of different intestinal segments in NHERF2-deficient mice and WT littermates.

Figure 2

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A, mRNA expression in scraped mucosa of jejunal (8–10 cm from pylorus), proximal (8–12 cm form ileocaecal junction) and distal (0.5–1 cm from ileocaecal junction) ileal, and colonic mucosa (1 cm distal from the caecocolonic junction to 1 cm proximal to the anus) relative to the ribosomal protein 9 (RPS9), as a control for overall cellular content (left panel), and to the brush border marker gene villin (right panel), which is a expressed in the villus enterocytes. No significant differences were observed in the expression of NHE3 in any intestinal segment. n= 5 for each segment and mouse genotype. B and C, immunoblot and densitometric analysis of scraped jejunal (B) and ileal BBM (C) for NHE3 content, normalized to the BBM protein villin. No significant difference was observed in small intestinal NHE3 BBM content between NHERF2 KO and WT mice. n= 4 for each segment and genotype.

NHE3 protein in the BBM was not different in NHERF2 KO and WT small intestine

Because the increase in fluid absorptive rates in the ileum of NHERF2-deficient mice was not explained by an increase in the NHE3 mRNA expression, we isolated the BBM from the small intestine of NHERF2 KO and WT mice, and quantified NHE3 BBM protein content in relation to the structural brush border membrane protein villin. Supplementary Fig. 1 demonstrates the experiment from three pooled NHERF2 WT and KO small intestines, where assessment of enrichment against villin is outlined. Figure 2B shows four individual preparations from four matched pairs of WT and KO mouse jejuni and Fig. 2C shows the same for ilei. No significant difference was seen between NHE3 BBM content in the jejunal as well as the ileal BBM, although variability was stronger in the ileal samples.

NHERF1/2 mRNA expression throughout the intestine was not changed by NHERF2 ablation

Because basal small intestinal Na+ or fluid absorptive rates had been found to be reduced in NHERF-1 KO, NHERF1/PDZK1 double KO (Broere et al. 2009) and PDZK1-deficient mice, the latter even in the absence of a decreased BBM NHE3 content (Hillesheim et al. 2007), we also assessed the mRNA expression levels of the two other NHERF family members, NHERF1 (Fig. 3A–C, left panel) and PDZK1 (Fig. 3A–C, right panel), which play a role in agonist-mediated NHE3 inhibition (Yun et al. 1997, 1998; Zizak et al. 1999; Cinar et al. 2007; Zachos et al. 2008; Broere et al. 2009). NHERF1 mRNA expression was unaltered in all segments; PDZK1 expression on the other hand was significantly reduced in the distal ileum and showed a trend towards lower PDZK1 expression levels in the colon (Fig. 3).

Figure 3. mRNA expression levels of NHERF1 and PDZK1 in jejunal, distal ileal and colonic mucosa in NHERF2-deficient mice and WT littermates.

Figure 3

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No significant differences were seen in the mRNA expression of two other members of the NHERF family that are involved in NHE3 inhibition in the intestine, except for a trend towards reduced PDZK1 expression in the colon of the NHERF2-deficient mice, and a significantly reduced PDZK1 expression in the distal ileum when standardized against villin. n= 4–6. The left panels show the mRNA expression levels for NHERF1 relative to RPS9 and villin; the right panels show the mRNA expression of PDZK1. *P < 0.05.

Subcellular NHE3 distribution was altered in NHERF-2 KO mice

Isolated intestinal BBMs contain the microvillar membrane as well as adherent cytoskeletal elements (as they are strongly enriched both for membrane-resident proteins and villin, but to a much lesser extent also for F-actin and the NHERF proteins which associate with membrane transporters but are not membrane-resident proteins). Since a different location of NHE3 along the proximal tubule BBM has recently beeen implicated with different transport activity (Riquier et al. 2009, 2010), we analysed the distribution of NHE3 along the microvillar axis in jejunum, mid- and distal-ileum, and colon in NHERF2 KO and WT littermates (Fig. 4 and Supplementary Fig. 3 display method). The ‘terminal web’ region was defined as the region towards the innermost part of the BBM, where the actin signal is particularly strong and which does not stain with the nutrient absorptive transporter PEPT1 (Chen et al. 2010). In the jejunum, approximately two-thirds of the overall NHE3 immunoreactivity was in the ‘microvillar tip’ (upper half of the microvilli) region (Figs 4 and 5, upper panels), whereas in the mid-ileum, approximately two-thirds of NHE3 was in the terminal web region (not shown). In the distal ileum, NHE3 was very strongly terminal web located (Figs 4 and 5, middle panels), and in the colon, NHE3 was predominantly in the microvilli (Figs 4 and 5). There was a tendency for more microvillar NHE3 in all parts of the NHERF2 KO small intestine, but the effect was stronger in the ileum, and particularly strong in distal ileum (Figs 4B and 5, middle panels), i.e. in the areas with more NHE3 in the terminal web region in the WT mice. Since a microvillar location of NHE3 appears to be associated with more NHE3 transport function (Fig. 6, and Riquier et al. 2010), the higher fluid absorptive rate in the ileum of NHERF2 KO compared to WT mice (Fig. 1A and C) may be explained by the higher percentage of NHE3 in the microvillar region in these mice. However, given the short segment of intestine that is affected, this is not likely to result in major differences in overall intestinal fluid transport in these mice.

Figure 4. NHE3 subcellular distribution near the brush border membrane.

Figure 4

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Confocal section through the BBM of WT and NHERF2-deficient mice in jejunal enterocytes (A), distal ileal enterocytes (B), and colonocytes (C), with the apical F-actin cytoskeleton stained red and NHE3 stained green. To assess the relative distribution of NHE3 along the terminal web/microvillar axis, we measured the fluorescence distribution in a chosen area perpendicular to the BBM, which creates one distribution plot as shown in B. Five representative areas were selected from BBM of each segment of 3 WT and NHERF2-deficient mice. The fluorescence signal in the terminal web region was set to 1 for each of the 15 curves created for each segment, and relative distribution curves were created for F-actin and for NHE3 (see Fig. 5B, and Supplemental Material). Scale bars 10 μm.

Figure 5. NHERF2 influences the NHE3 distribution in the ileal BBM.

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A, lower magnification of confocal sections through the BBM of WT and NHERF2-deficient mice in jejunal enterocytes, distal ileal enterocytes, and colonocytes. B, relative distribution curves of F-actin and NHE3 along the BBM in jejunum, distal ileum and colon of WT and NHERF2-deficient mice. C, bar graphs representing the amount of NHE3 that is terminal web/intravillar cleft associated and the microvillus tip (upper part of the microvilli) associated for each of the studied segments. An exact method description is found in the Supplemental Material. It is evident that NHE3 distribution varies, with a predominant microvillar distribution in jejunum and colon, and predominant terminal web localization in the distal ileum. n= 3 for each segment and genotype. The scale bars represent 10 μm.

Figure 6. Effect of NHERF2 ablation on acid-activated NHE3 activity in the different intestinal segments.

Figure 6

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A–C, fluorometrically determined acid-activated NHE3 transport rates in the different segments of WT, NHERF2-deficient, NHE3-deficient and S1611-treated jejunal (A), mid-ileal (approx 4–6 cm proximal to the ileocaecal valve) (B), and distal ileal (last 1 cm before ileocaecal valve) (C) villi. D, NHE3 activity in the cryptal mouth of isolated colonic crypts of WT, NHERF2-, and NHE3-deficient and S1611-treated mice. Whereas no significant differences were observed in acid-activated NHE3 activity between WT and NHERF2 mice in jejunum, ileum and colon, there was a strikingly low acid-activated NHE3 activity in the distal ileum (C), which correlates with the predominant terminal web localization of NHE3 in this intestinal segment. D, NHE3 activity measurements in colonic crypts follow a different experimental protocol (see Methods and text) and can therefore not be compared directly with NHE3 activity measurements in small intestinal villi, but no difference in acid-activated NHE3 activity was observed between NHERF2-deficient and WT crypts. n= 4–7, *P < 0.05, **P < 0.01, ***P < 0.001.

No significant effect of NHERF2 ablation on acid-activated NHE3 activity in the different intestinal segments

The ammonium-prepulse technique was applied to measure acid-activated Hoe642-insensitive Na+/H+ exchange activity in BCECF-loaded colonic crypts or microdissected jejunal and ileal villi. The specificity of this technique for assessing acid-activated NHE3 activity has previously been described for colonic crypts (Cinar et al. 2007), and was even better for jejunal and ileal villi, as demonstrated by a near-total absence of acid-activated Hoe642-insensitive proton extrusion rate in NHE3-deficient jejunal and ileal enterocytes (Fig. 6A and B). In addition, 20 μm of the NHE3 inhibitor S1611 strongly inhibited acid-activated Hoe642-insensitive proton flux rates in small intestinal enterocytes (Fig. 6A and B). Similar experiments have been performed for the colon (Fig. 6D).

Acid-activated NHE3 activity varied significantly in the different segments of the WT small intestine, with much lower rates in the distal than the proximal ileum and the jejunum, despite identical conditions for the measurement technique and particular care to acidify enterocytes to a similarly low pHi (Fig. 6A–C). The NHE3 activity rates from the colonic crypts are not directly comparable to those obtained from the small intestine, because the acidification protocol is different, and because NHE3 activity is assessed in the enterocytes of the cryptal openings rather than in the very strongly NHE3 expressing surface enterocytes, which are lost during crypt isolation.

No significant differences were observed in NHE3 activity between NHERF2-deficient and WT enterocytes in any intestinal segment (Fig. 6A–D).

cAMP-induced inhibition of NHE3 activity does not require the presence of NHERF2 in murine jejunum, ileum and colon

NHERF2 coexpression conferred PKA inhibition of NHE3 in PS120 fibroblasts (Lamprecht et al. 1998; Zizak et al. 1999). We therefore assessed the inhibition of acid-activated NHE3 activity by 10−5m forskolin in different intestinal segments in NHERF2-deficient and WT mice. Forskolin application resulted in a similar degree of NHE3 inhibition in NHERF2-deficient and WT proximal ileum (Fig. 7A). Similar results were obtained in the colonocytes in the cryptal openings of isolated colonic crypts (Fig. 7B). The inhibitory effect of 10−5m forskolin on the percentage of acid-activated NHE3 activity in the jejunum was not different from that found in the proximal ileum both for NHERF2 KO and WT mice, but we did not repeat the experiments in this segment often enough for full statistical analysis.

Figure 7. cAMP-induced inhibition of acid-activated NHE3 activity in NHERF2-deficient and WT ileum (A) and in colonic crypts (B).

Figure 7

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Forskolin (10−5m)-mediated inhibition of acid-activated NHE3 activity in the mid (5 cm from ileocaecal junction) and the distal ileum (last cm before ileocaecal junction) of NHERF2 deficient and WT mice (A). No difference in the inhibitory effect of forskolin was seen in the mid ileum (A) or the colon (B). Forskolin inhibition in the distal ileum, which was weak in WT, was not observed in NHERF2-deficient terminal ileum. n= 4–8, *P < 0.05,**P < 0.01, ***P < 0.001.

Recent data obtained in proximal tubules suggested that NHE3 location in the villus cleft region is associated with reduced function (Riquier et al. 2009, 2010). Because we observed that NHE3 in the distal ileum is predominantly localized in a similar region (which we called the ‘terminal web’ region because it contains the highest amount of actin) (Figs 4 and 5), we also studied the effect of forskolin on acid-activated NHE3 activity in the very distal ileum. Interestingly, the distal ileum showed not only a strongly reduced acid-activated NHE3 activity, but also a low (non-significant) inhibitory effect of forskolin in the WT enterocytes (Fig. 7A). Likewise, no significant FSK inhibition was observed in the NHERF2 KO mice (Fig. 7A).

[Ca2+]i-induced NHE3 inhibition requires NHERF2 expression

In NHE3-expressing fibroblasts, [Ca2+]i-dependent NHE3 inhibition requires the expression of NHERF2 (Kim et al. 2002; Lee-Kwon et al. 2003). To investigate the role of NHERF2 in [Ca2+]i-induced inhibition of NHE3 activity in murine small and large intestine, we applied 10−3m carbachol or 20 μm 4-Br-A23187 5 min before Na+-dependent recovery to microdissected, BCECF-loaded jejunal NHERF2-deficient and WT villi. The jejunum was chosen for these experiments because carbachol elicited consistent inhibition of acid-activated NHE3 activity in the villi of this intestinal segment of WT mice. Carbachol and 4-Br-A23187 inhibited NHE3 activity in WT but not in NHERF2-deficient jejunal villi (Fig. 8A). Similar experiments were performed in BCECF-loaded colonic crypts, using preincubation with 2 mm ionomycin for 2 min (as described in the experiments with NHE3-expressing PS120 cells, Kim et al. 2002) (Fig. 8B), which resulted in significant inhibition of acid-activated NHE3 activity in WT but not NHERF2 KO colonic crypts. This indicates that NHERF2 plays a major role in [Ca2+]i-dependent NHE3 inhibition.

Figure 8. Ca2+-induced NHE3 inhibition requires NHERF2 expression.

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A, both carbachol (10−3m) and the Ca2+ionophore 4-BrA23187 inhibited acid-activated NHE3 activity in WT, but were without significant inhibitory effect in NHERF2-deficient jejunal villi. B, the same requirement for NHERF2 expression was found in colonic crypts, where 2 μm of the Ca2+-ionophore ionomycin was applied 3 min before adding back Na+. Thus, both receptor-dependent and -independent Ca2+-mediated NHE3 inhibition was abolished in the absence of NHERF-2 expression in small and large intestine. n= 4–9, *P < 0.05, **P < 0.01, ***P < 0.001.

cGMP-dependent inhibition of NHE3 activity requires NHERF2 in murine jejunum

Escherichia coli heat-stable enterotoxin (STp) binds to guanylate cyclase-C in the luminal enterocyte membrane and increases intracellular cGMP levels (Vaandrager et al. 1993). STp at 10−7m induced inhibition of acid-activated NHE3 activity in WT jejunal villi, whereas an increase of NHE3 activity after STp treatment was observed in the NHERF2 KO jejunal villi (Fig. 9). The effect of STa (these experiments were done with STa when it was still available from Sigma) on acid-activated NHE3 activity had also been studied in isolated colonic crypts, but no significant inhibition of NHE3 activity was seen, possibly because STa could not access its luminal receptors in this preparation (data not shown).

Figure 9. cGMP-dependent inhibition of NHE3 activity requires NHERF2 expression in jejunum.

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Application 10−7m of the Escherichia coli heat stable toxin peptide (STp) approx. 5 min before Na+ re-addition significantly inhibited acid-activated NHE3 activity in WT jejunal villi, whereas STp even increased acid-activated NHE3 activity in NHERF2-deficient villi. n= 4–7, *P < 0.05, **P < 0.01***P < 0.001.

The reason for the increase in NHE3 activity after STp application in the NHERF2-deficient small intestinal villi is at present unknown. However, the data unequivocally demonstrate that NHERF2 is necessary for cGMP-mediated inhibition of NHE3 activity in the small intestine.

Discussion

The goal of the current study was to examine NHE3 expression, membrane localization, and in particular, agonist-mediated NHE3 transport regulation in different intestinal segments of NHERF2-deficient mice and WT littermates.

The first finding was an unexpected increase in the basal fluid absorption rates in the ileum of anaesthetized NHERF-2 deficient mice compared to WT littermates, and no significant difference in fluid absorption in the other studied intestinal segments. In search of an explanation we observed (1) no difference in NHE3 mRNA expression levels, (2) no difference in NHE3 protein content in isolated BBM vesicles (which contain the complete BBM plus subapical cytoskeletal structures), but (3) significant differences in the relative distribution of NHE3 in the microvillar tip region versus the terminal web region along the small intestine of WT mice. NHE3 was found in a predominantly microvillar location in the proximal jejunum, and a gradual shift toward a higher percentage of terminal web NHE3 in the ileum was observed. A relatively higher percentage of NHE3 in the microvilli was associated with higher NHE3 transport rates within the different small intestinal segments (Fig. 6). NHERF2 deficiency affected this distribution, in that a more diffuse distribution with more NHE3 in the microvilli was observed in the ileum of NHERF2-deficient mice. This may explain the higher fluid absorption rates in the NHERF2 KO ileum. In the proximal tubule, NHE3 transport activity has recently been demonstrated to be regulated by trafficking from the intervillar clefts into the microvilli (Riquier et al. 2009, 2010). Movement of NHE3 from the tip of the microvilli to the intervillar cleft was associated with reduced transport activity. This observation resembles somewhat that seen in the murine ileum. However, we believe that a part of the ileal NHE3 was likely to have been localized in recycling endosomes in the terminal web rather than the microvillar base, because most NHE3-mediated immunofluorescence could be assessed at the innermost part of the F-actin band. Danielsen & Hansen (2003) describe apical membrane invaginations or ‘deep apical tubules’ that extend up to 0.5–1 μm into the underlying terminal web region in small intestinal brush border, so we cannot differentiate between a recycling endosome or this deep apical tubule localization of NHE3. However, this localization was associated with reduced acid-activated NHE3 activity and an almost complete loss of inhibition by forskolin, resembling the situation in the kidney. Accepting the concept that a microvillar tip location of NHE3 correlates with higher NHE3 transport rates, the increased fluid absorptive rates in the distal ileum of NHERF2 KO mice may be explained by the finding of a higher percentage of overall NHE3 in the microvillar tip compared to the terminal web region. These findings are the first hint that NHERF2 is somehow involved in membrane tethering of NHE3 within a specific region of the intestinal microvilli. An endogenous NHE3 inhibitor may keep NHE3 in the ‘terminal web’ location in the anaesthetized mice in a NHERF2-dependent fashion, and this ‘endogenous inhibition’ may be absent or reduced in the NHERF2 KO ileum.

The next goal of this study was therefore to assess the importance of NHERF2 expression for agonist-mediated NHE3 inhibition in the murine intestine. PS120/NHE3V cells require the expression of NHERF2 or NHERF1 for inhibition of NHE3 by cAMP analogues (Zizak et al. 1999). Likewise, the PKA-induced, CFTR-mediated inhibition of NHE3 in the Xenopus A9 cells required NHERF2 expression (Bagorda et al. 2002). However, NHERF1 was previously found to be important for PKA-mediated NHE3 inhibition in the kidney, but not in the intestine (Weinman et al. 2003, 2005; Murtazina et al. 2007; Broere et al. 2009), demonstrating the cell-specificity of NHERF1-mediated NHE3 regulation. Likewise, NHERF2 expression appeared not to be necessary for cAMP-mediated inhibition of NHE3 in any of the segments of the murine intestine except possibly the distal ileum. The latter segment is characterized by a low NHE3 activity, strongly reduced inhibitor action of forskolin (even in the WT mice), and a predominant terminal web/intervillar cleft localization of NHE3. It is possible that endogenous factors inhibit NHE3 in this narrow part of the murine gut to prevent blockage by mucus/luminal contents (with a possible role of NHERF2 in this endogenous inhibition; see above). However, this suggested that this segment of the gut is not optimal to study the molecular regulation of inhibitory NHE3 signals.

Interestingly, forskolin-induced inhibition of NHE3 in the jejunum or proximal ileum was rapid (we added forskolin only 2 min prior to re-addition of Na+) and was not associated with a detectable retrieval of NHE3 from its microvillar location up to 20 min after forskolin application in either WT or NHERF2-deficient jejunum (data not shown). This is in concordance with studies in PS120/NHE3/NHERF1 or NHERF2 cells, where no change in membrane abundance of NHE3 was seen despite transport inhibition by cAMP (Lamprecht et al. 1998).

Consistent with the findings in heterologous expression systems, NHERF2 ablation abolished both receptor-dependent and receptor-independent Ca2+-mediated NHE3 inhibition in both murine small intestine and colon. We could not, however, detect a carbachol-induced change of NHE3 membrane localization (e.g. retrieval to the terminal web or complete exocytosis from the membrane), as was the predicted mechanism for Ca2+-mediated NHE3 inhibition in heterologous expression systems (Kim et al. 2002). Even at concentrations sufficient for full muscarinic receptor activation, carbachol inhibits only a fraction of the acid-activated NHE3 activity that is inhibited by the NHE3 inhibitor S1611. It is therefore possible that the signalling events following muscarinergic receptor activation acts on a subset of BBM-resident NHE3 only. It is a challenge for the future to characterize such subpools of NHE3 and find out what may determine the agonist targeting of such subpools of NHE3. The first steps toward delineation of different pools of NHE3 are under way (Alexander et al. 2005; Alexander & Grinstein, 2009).

Ca2+-mediated NHE3 inhibition was also abolished in the PDZK1 deficient murine colon (Cinar et al. 2007), and in NHE3-expressing Caco-2bbe cells after siRNA-mediated PDZK1 silencing (Zachos et al. 2009). Zachos et al. (2009) searched for the molecular role of PDZK1 in Ca2+-mediated NHE3 inhibition and observed strongly reduced NHE3 membrane abundance in Caco-2bbe cells after PDZK1 silencing. In the PDZK1-deficient colon, NHE3 membrane abundance was normal but NHE3 mRNA was strongly increased, suggesting that increased production may compensate for the reduced membrane retention time (Cinar et al. 2007). We therefore wondered if, similar to the results in PDZK1-deficient mice, NHE3 mRNA or expression of the other NHE3-inhibitory PDZ-adaptor proteins was altered in the NHERF2-deficient intestine. No significant change in mRNA expression for NHE3, NHERF1 or PDZK1 was found, except the small reduction of PDZK1 mRNA when normalized to villin in the distal ileum, whose significance is uncertain at present. Thus, it appears that both NHERF2 and PDZK1 are required for Ca2+-mediated NHE3 inhibition, but that the two proteins have different molecular tasks in this process, explaining that both proteins cannot substitute for each other and both are essential.

The guanylins and their bacterial analogue heat-stable Eschericha coli enterotoxin (STa or STp) inhibit NHE3 via an intracellular cGMP increase (Donowitz et al. 2005). Vaandrager et al. (2000) provided evidence that whereas cGMP inhibits salt absorption in both the small and the large intestine, the involved signalling pathways are different. In the small intestine, but not the colon, expression of the cGMP-dependent protein kinase II is necessary for inhibition of NHE3 by STa, whereas in the colon, cGMP-mediated inhibition of cAMP-specific phosphodiesterases may result in an increase in PKA activity, which also results in NHE3 inhibition. Cha et al. (2005) provided biochemical evidence for a binding of NHERF2, but not NHERF1, to cGKII, and only a coexpression of both NHERF2 and cGKII with NHE3 in PS120 cells resulted in NHE3 inhibition by cGMP analogues. Thus, the lack of inhibition of NHE3 by the heat-stable E.coli toxin peptide STp in the small intestine in the absence of NHERF2 agrees well with the data from the heterologous expression system. What was totally surprising, however, was the significant stimulation of NHE3 activity by STp in NHERF2-deficient small intestinal villi. It is known that STp stimulates enterocyte cGMP production via binding to the transmembrane receptor enzyme guanylate cyclase-C (GCC) (Vaandrager, 2002), which is expressed not only in the crypt but also in the villus enterocyte apical membrane and therefore in part shows a similar cellular distribution to NHE3 (Swenson et al. 1996). Using the C-terminus of the GCC as bait in yeast two-hybrid screening, Scott et al. (2002) identified a PDZ protein of the NHERF family, which is now called NHERF4 (Thelin et al. 2005). NHERF4 also binds to NHE3 but stimulates its activity in a Ca2+-dependent fashion when co-expressed with NHE3 in PS120 cells (Zachos et al. 2008). It is possible, but as yet speculative, that in the absence of NHERF2, the NHERF4 clusters to GCC upon STp binding and activates NHE3. In any case the findings do underline how crucial each member of the NHERF family is for receptor-mediated regulation of ion transporters, similar to what we found earlier for CFTR regulation (Singh et al. 2009).

In summary, our study demonstrates that the expression of the PDZ-adaptor NHERF2 plays a highly selective, signal- and segment-specific role in NHE3 activity regulation in the intestine. A major function of intestinal NHERF2 may lie in conferring inhibitory (as well as stimulatory, Lin et al. 2010) signals to NHE3. A loss of Ca2+-mediated NHE3 inhibition may confer increased resistence to rotavirus enterotoxins (Morris et al. 1999; Lorrot & Vasseur, 2007) as well as enterotoxin-released neurotransmittors such as serotonin (Lundgren et al. 2002; Gershon & Tack, 2007; Xue et al. 2007), or to ATP released from the cells during stress (Xue et al. 2007). A loss of STa-mediated inhibition may increase intestinal resistance to the most common form of traveller's diarrhoea (Fleckenstein et al. 2010). In addition to its prominent role in agonist regulation of NHE3, NHERF2 appears to be involved in the tethering of NHE3 to the terminal web/intravillar cleft region and releasing it from this location in an agonist-specific fashion. We could only study this phenomenon in the ileum, because that was the only segment that displayed a NHERF2-dependent segregation of NHE3 towards the terminal web region (this paper, and Lin et al. 2010). However, a recent publication confirmed this function of NHERF2 in heterologous expression models (Cha et al. 2010). While more work is needed to understand the concerted action of several members of the NHERF family in agonist-specific NHE3 regulation, it has become clear that NHERF2 may be a major player in NHE3 regulation as well as an interesting drug target in diarrhoeal diseases.

Acknowledgments

This work was supported by Deutsche Forschungsgemeinschaft grant nos SFB621-C9, Se460/9-5 and Se460/9-6 (to U.S.), the Volkswagen Vorab-Stiftung, the Hannover Biomedical Research School and the PhD program ‘Molecular Medicine’ with a stipend to M.C., A.S. and A.S. We thank Regina Engelhardt, Brigitte Rausch and Natascha Cirpka for their help with the animal breeding and genotyping.

Glossary

Abbreviations

BBM

brush border membrane

NHE3

Na+/H+ exchanger isoform 3

Author contributions

Conception and design of the experiments: M.C., A. S., A.C., S.Y., B.R., AK.S, J.L. B.H. and U.S.; collection, analysis and interpretation of data: M.C, A.C., S.Y., B.R., AK.S., J.L., A.S., J.B., B.H., U.S.; drafting the article or revising it critically for important intellectual content M.C., A.S., G.C., CC.Y, M.D., B.H. H.J. U.S. All authors approved the final version of the manuscript. The authors have no conflict of interest to disclose.

Author's present address

B. Hogema: The Sanquin blood supply foundation, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands.

Supplemental material

Supplementary Figure 1

Supplementary Figure 2

Supplementary Figure 3

tjp0588-5049-SD1.pdf (195.4KB, pdf)

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