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Logo of jphysiolThe Journal of Physiology SiteMembershipSubmissionJ Physiol
J Physiol. Jun 15, 2007; 581(Pt 3): 1235–1246.
Published online Mar 29, 2007. doi:  10.1113/jphysiol.2007.131722
PMCID: PMC2170846

NHE3 inhibition by cAMP and Ca2+ is abolished in PDZ-domain protein PDZK1-deficient murine enterocytes

Abstract

The PDZ-binding protein PDZK1 (NHERF3/CAP70/PDZ-dc-1) in vitro binds to NHE3, but its role in the regulation of NHE3 activity in native enterocytes is unknown. This study was undertaken to understand the physiological role of PDZK1 in regulating NHE3 activity in native murine colonic enterocytes. NHE3 transport rates were assessed fluorometrically in BCECF-loaded colonic crypts in the NHE3-expressing cryptal openings by measuring acid-activated, Na+-dependent, Hoe 642-insensitive proton efflux rates. NHE3 mRNA expression levels and NHE3 total enterocyte and brush border membrane (BBM) protein abundance were determined by quantitative PCR and Western analysis and immunohistochemistry. In pdzk1 −/− colonic surface cells, acid-activated NHE3 transport rates were strongly reduced, and the inhibitory effect of forskolin and ionomcyin was virtually abolished. Hyperosmolarity, on the other hand, still had an inhibitory effect. In addition, the NHE3-selective inhibitor S1611 inhibited acid-activated NHE3 activity in pdzk1 −/− and +/+ mice, suggesting that functional NHE3 is present in pdzk1-deficient colonocytes. NHE1 and NHE2 activity was not altered in pdzk1 −/− colonic crypts. Immunohistochemistry revealed apical NHE3 staining in pdzk1 −/− and +/+ proximal colon, and Western blot analysis revealed no difference in NHE3 abundance in colonic enterocyte homogenate as well as brush border membrane. Lack of the PDZ-adaptor protein PDZK1 in murine proximal colonic enterocytes does not influence NHE3 abundance or targeting to the apical membrane, but abolishes NHE3 regulation by cAMPergic and Ca2+ -dependent pathways. It leaves NHE3 inhibition by hyperosmolarity intact, suggesting an important and selective role for PDZK1 in the agonist-mediated regulation of intestinal NHE3 activity.

In the intestinal tract, NHE3, coupled to one or several of the SLC26 family of anion exchangers, is the most important transport protein for electroneutral NaCl absorption (Zachos et al. 2005). A common pathophysiological event in secretory diarrhoea, elicited by a large variety of enterotoxins, hormones, drugs and other exogenous and endogenous substances, appears to be the inhibition of electroneutral salt absorption. Many of these substances act by receptor-mediated stimulation of intracellular signalling pathways, such as an increase of intracellular cAMP, cGMP or Ca2+ levels (Field, 2003).

Elegant heterologous expression studies have suggested that PDZ-domain proteins of the NHERF family, NHERF1 and NHERF2, bind to NHE3 and are required for the cAMP- and Ca2+-mediated inhibition of the Na+/H+ exchanger isoform NHE3 (Yun et al. 1997; Lamprecht et al. 1998; Weinman et al. 1998; Zizak et al. 1999; Weinman et al. 2000a,Weinman 2000b). PDZK1 is another member of the NHERF family that also binds to NHE3 (Gisler et al. 2003b), and it was therefore suggested to call it NHERF3. It is a multifunctional PDZ-domain protein with high expression levels in kidney, where it is involved in the scaffolding and regulation of a variety of membrane transporters (Gisler et al. 2003a,Gisler 2003b; Thomson et al. 2005b; Kato et al. 2006), the liver, where it regulates the membrane abundance of the scavenger receptor (Kocher et al. 2003b; Yesilaltay et al. 2006) and in the intestine. We had previously studied electroneutral sodium absorption in the small intestine of pdzk1 +/+ and −/− mice, and found a significantly reduced basal Na+ absorptive rate in the latter, and a strongly delayed and incomplete time course of its inhibition by forskolin (Cinar et al. 2005). We also found similar NHE3 protein abundance in small intestinal enterocyte brush border membranes and cytoplasm of pdzk1 +/+ and −/− intestine, but an up-regulation of NHE3 mRNA expression, which suggested to us an increase in NHE3 turnover, possibly indicative of decreased NHE3 stability in the membrane (Hillesheim et al. 2007).

To investigate the observed Na+-absorptive defect on a molecular level, we developed a method that allowed a direct measurement of NHE3 transport activity in colonic surface enterocytes within intact epithelial structures, and applied this method to pdzk1 +/+ and −/− enterocytes. Surprisingly, acid-activated NHE3 activity was strongly reduced in the absence of PDZK1, and the inhibition of NHE3 by cAMP or by ionomycin was virtually absent. On the other hand, no indication was found for a reduced abundance of NHE3 in the enterocyte or the brush border membrane, or a change in distribution of NHE3 across the brush border membrane. Hyperosmolarity or the NHE3-selective blocker S1611 also inhibited NHE3, and this inhibition was preserved in the absence of PDZK1, suggesting a differential loss of certain NHE3 regulatory pathways in the absence of PDZK1.

Methods

Chemicals

If not indicated in the next sections, chemicals were either obtained from Sigma (Deisenhofen, Germany) or Merck (Darmstadt, Germany) at tissue culture grade or the highest grade available. Hoe 642 and S1611 were kindly provided by Sanofi-Aventis, Frankfurt, Germany. Nigericin and 2′,7′-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) were purchased from Molecular Probes, Leiden, the Netherlands.

Animal breeding

The PDZK1-deficient mouse strain was generated in the Department of Pathology, Beth Israel Deaconess Medical Center as described before (Kocher et al. 2003a). These mice have normal body electrolyte concentration and normal intestinal mucosal histology (Kocher et al. 2003a). pdzk1 −/− and +/+ mice were bred on a 129SvEv background in the animal facility of the Hannover Medical School under standardized light and climate conditions and had access to water and chow ad libitum. They were used at 6 weeks to 12 months of age, and for each set of experiments, age- and sex-matched littermates or cousins were used. Nhe3 +/+ and −/− mice had been originally generated in the laboratory of Gary Shull and were kept for many generations on the original background (Schultheis et al. 1998). In order to reduce morbidity and mortality in the nhe3 −/− mice, they were given an oral rehydration solution, and most mice survived to fairly old age. Animal experiments followed approved protocols at the Hannover Medical School and the local authorities for the regulation of animal welfare (Regierungspräsidium).

Preparation of colonic crypts

Murine colonic crypts were isolated as previously described (Bachmann et al. 2003). Briefly, after CO2 narcosis and cervical dislocation, the proximal colon (first 3–4 cm of murine colon) was excised, washed through thoroughly with ice-cold buffer A (composition, mm: 120 NaCl, 14 Hepes, 7 Tris, 3 KH2PO4, 2 K2HPO4, 1.2 MgSO4, 1.2 calcium gluconate, 20 glucose, pH 7.4) gassed with 100% O2, everted, filled with EDTA-containing solution (composition, mm: 127 NaCl, 5 KCl, 1 MgCl2, 5 sodium pyruvate, 10 Hepes, 5 EDTA, 1% BSA, pH 7.4), and incubated in the same solution at 37°C with gentle mixing. After 15 min, crypts were isolated by gentle shaking, harvested by low-speed centrifugation, immersed in ice-cold buffer A, gassed with 100% O2 and kept on ice until use.

pHi measurements

Crypts were loaded for approx. 15 min with 5 μm BCECF in buffer A at room temperature followed by a 10 min washout period in buffer A. Subsequently, 50 μl of the crypt suspension was pipetted on a glass coverslip. After 2–3 min of sedimentation, crypts were fixed on the coverslip with a polycarbonate membrane (25 mm diameter, pore size 3 μm, Osmonics, Minnetonka, MN, USA) in a custom-made perfusion chamber, mounted onto the heated (37°C) stage of an inverted microscope (Zeiss Axiovert 200, Carl Zeiss AG, Jena, Germany) and perfused with prewarmed (37°C) O2-gassed buffer A for 20 min for a stable baseline reading. Crypts were acidified using an ammonium prepulse (40 mm NH4Cl isotonically replacing NaCl) for 5 min, then perfused for 5 min with a Na+-free buffer (TMA+ isotonically replacing Na+), until pHi reached its lowest value plateau. Subsequently, 50 μm Hoe 642 and, if appropriate, 10−5m forskolin or 2 μm ionomycin was added to the Na+-free buffer. Hoe 642 at 50 μm had previously been shown to completely inhibit NHE1 and NHE2 (Bachmann et al. 2004). After a further 2–3 min, the buffer was switched to buffer A, containing 50 μm Hoe 642 and forskolin, ionomyin, or mannitol. If appropriate, images were digitized every 1 (phase of rapid pHi recovery) to 30 s (plateau phases) with a cooled CCD camera (CoolSnap ES, Roper Scientific, Ottobrunn, Germany) using the Metafluor software (Universal Imaging Corporation, Downington, PA, USA) during exposure of cells to alternating 440 and 495 nm light from a monochromator (Visichrome, Visitron Systems, Puchheim, Germany) with a 515 nm DCXR dichroic mirror and a 535 nm barrier filter (Chroma Technology Corp, Rockingham, VT, USA) in the emission pathway. Calibration of the 440/495 ratio was performed as previously described (Bachmann et al. 2003) for the pH values 6.6 and 7.4 using the high K+–nigericin method (solution composition, mm: 100 potassium gluconate, 40 KCl, 7 Hepes, 1.2 calcium gluconate, 1.2 MgSO4, 20 glucose, 10 μm nigericin, pH 7.4 or 6.6). Regions of interest (ROIs) were selected in the apical and basal part of the crypt, as illustrated in Fig. 2A. One region of interest was chosen in the apical and one in the basal part of the crypt and was large enough to include a representative area of the whole crypt region. Approx. five to eight crypts were measured per experiment, and the results of these flux measurements averaged for each mouse. n is the number of mice that were used for each individual data bar, usually indicating the pairs of +/+ and −/− mice used for each experiment. The 440/495 time course was reproduced from the stored images after background subtraction, followed by conversion to pHi values using Microsoft Excel. Proton fluxes were calculated by multiplying the initial steep pHi slope after readdition of sodium, which was determined by regression analysis, with the total buffering capacity at the initial pHi, including the intrinsic βi and, in addition, the CO2-dependent buffering capacity for CO2/HCO3-containing solutions, as described in Bachmann et al. (2003). Intrinsic buffering capacity was determined for isolated murine colonic crypts of both pdzk1 +/+ and −/− mice using the protocol as described by Boyarsky et al. (1988a). βi did not differ significantly between +/+ and −/− mice and was not significantly different from that published in graphic form before (Bachmann et al. 2003).

Figure 2
Western analysis of brush border membranes and enterocyte homogenate for NHE3 abundance isolated from pdzk1 +/+ and −/− proximal colonic mucosa The integral brush border protein villin was taken as an internal control. A, the blots from ...

Preparation of brush border membranes from murine colonic mucosa and Western analysis

Mouse BBM vesicles from scrapings of large intestinal epithelium were prepared as previously described (Walker et al. 2003). Twenty micrograms of purified brush border membranes or 40 μg of homogenate were size fractionated on 10% SDS polyacrylamide minigels (Amersham, Biosciences) under denaturing conditions, transferred to PVDF membranes (Hybond-P, Amersham Biosciences), and blocked with 5% non-fat dry milk in TBS-Tween. Blots were probed with anti-NHE3 antibody, stripped and probed again with anti-villin antibody. Affinity purified anti-rat-NHE3 IgG (Alpha Diagnostics International, San Antonio, TX, USA) was used at a concentration of 0.1 μg ml−1 in TBS-Tween overnight at 4°C. Anti-villin antibody (HCT-8, BD biosciences, Pharmingen, Germany) was used in TBS-Tween at a concentration of 25 ng ml−1. The secondary antibody (goat anti-rabbit IgG conjugated to horseradish peroxidase, KPL) was diluted 1 : 10 000 in TBS-Tween and incubated for 1 h at room temperature. The antigen-antibody complexes on the PVDF membranes were visualized by chemiluminescence (ECL Western blotting detection reagents, Amersham Pharmacia Biotech) and the image captured on light sensitive imaging film (Hyperfilm ECL, Amersham Biosciences).

Immunofluorescence staining

Mouse colon was rinsed with ice-cold phosphate-buffered saline (PBS) and fixed for 2 h at 4°C with 2% paraformaldehyd in PBS. Fixed tissue was rinsed with PBS and transferred to 30% sucrose in PBS overnight. The tissue was embedded with tissue-freezing medium (TissueTec O.C.T., Sakura). Cryosectioning was done with a microtome cryostat at −20°C and 10 μm thick sections were collected on microscope slides (SuperFrost Plus, Menzel-Gläser, Germany). Sections were treated sequentially with PBS for 5 min, washing buffer of PBS with 50 mm NH4Cl two times for 10 min each, background reducing buffer (Dako) for 20 min for blocking and the first antibody, affinity purified anti-rat-NHE3-IgG (Alpha Diagnostics International) at a concentration of 1.7 μg ml−1 in blocking buffer. Washing four times for 5 min in washing buffer was followed by blocking for 20 min in 3% goat serum in PBS and two times washing for 5 min each. Secondary antibody (Alexa Fluor 488-labelled goat anti rabbit IgG, Molecular Probes) was incubated for 1 h at room temperature in a concentration of 2 μg ml−1 in background reducing buffer. After two washes for 5 min each, a nuclear staining with 2 μg ml−1 propidium iodide in PBS for 5 min and another two washes followed. Cover slides were imaged on the confocal microscope (Leica TCS SP2). Samples were excited with 488 nm and emission was collected at 500–550 nm (Alexa Fluor) and 600–680 nm (propidium iodide). Sections from pdzk1 −/− and +/+ colon were imaged with identical confocal settings. For analysing the distribution of the NHE3 signal, a rectangular plot profile with vertical averaging over at least 20 pixels (for noise reduction) was placed perpendicularly over the band-shaped NHE3 signal using ImageJ (NIH, Bethesda, MD, USA, http://rsb.info.nih.gov/ij/). The pixel intensity distribution was normalized to 100% and plotted against the distance after aligning the peak intensities.

Statistics and calculation of proton fluxes

Results are given as means ± s.e.m. Statistical significance was determined using ANOVA with Tukey's HSD test as a post hoc test for multiple comparisons and Student's t test in its unpaired form for pairwise comparisons. For s.e.m. calculation and statistics, n = 1 was defined as the mean of values from one mouse, per experimental setting (with each treatment, or gene deficiency, being one experimental setting). For example, n = x means that x pairs of +/+ and −/− mice were used. For fluorometry, this means that the numbers of pHi traces were averaged for each mouse, and for mRNA or BBM preparations, one preparation equalled one pair of +/+ and −/− mice.

Results

Immunohistochemical staining of proximal colonic NHE3

Immunohistochemistry revealed apical staining in both pdzk1 −/− and +/+ colon and small intestine (Fig. 1A). This demonstrates that NHE3 traffics to the correct location. We estimated the relative distribution of fluorescence across the apical region of the cell and found no difference in the distribution between pdzk1 +/+ and −/− apical membrane region (Fig. 1B). However, this method is not quantitative and unable to pick up subtle decreases in overall NHE3 BBM abundance.

Figure 1
NHE3 immunohistochemistry in murine colon A, NHE3 immunostaining is detected in the apical region of the surface and upper crypt cells in the proximal colon of pdzk1 +/+ and −/− mice. B, the shape of the distribution curve of NHE3 fluorescence ...

Western blot analysis of NHE3 abundance in colonic enterocyte homogenate and brush border membranes

We next investigated whether there is a change in NHE3 protein abundance in colonic enterocyte homogenate and in the colonic brush border membrane. Western analysis revealed no difference in NHE3 abundance in either homogenate or BBM in relation to villin, a structural protein of the microvillus brush border (Fig. 2A and B). Thus, the above results demonstrated that NHE3 abundance was not changed in a major fashion in the absence of PDZK1 in the colon.

NHE3 transport activity in surface enterocytes of nhe3 +/+ and −/− mice

In order to quantify NHE3 exchange activity in native NHE3-expressing enterocytes, we developed a technique to directly measure NHE3 activity in the enterocytes within the very upper part of proximal colonic crypts, which strongly express NHE3. We loaded isolated colonic crypts with the pH-sensitive dye BCECF and measured acid-activated NHE activity in the upper and the basal portions of the glands in the absence and presence of 50 μm Hoe 642, which, at this concentration, inhibits NHE1 and NHE2 but not NHE3 activity (Bachmann et al. 2004). As shown elsewhere (Bachmann et al. 2004), and in Fig. 8A, 50 μm Hoe 642 reduced NHE activity in the cryptal region by ~95%, but only by approx. 30% in the surface cell region (Bachmann et al. 2004).

Figure 8
Total, Hoe 642-insensitive, and NHE2-mediated proton flux rates in pdzk1 +/+ and −/− colonic crypts A, in the absence of Hoe 642, acid-activated, Na+-dependent proton flux rates are high both in the surface and basal regions of the crypts, ...

Figure 3A shows the false colours that represent the pHi distribution along the crypt length (red alkaline, blue acidic), in the presence of 50 μm Hoe 642, and shortly after the readdition of Na+. Figure 3B shows the pHi traces of the ROIs in the apical and basal portions of the crypts measured in a typical experiment. When NHE1 and NHE2 activity are inhibited by Hoe 642, Na+-dependent pHi recovery is fast near the crypt opening and very slow in the base of the crypt. In the absence of Hoe 642, Na+-dependent pHi recovery is fast in both basal and apical regions of the crypt (Figs 3B and 8A and B).

Figure 3
Determination of NHE3 transport rates in the surface enterocytes of isolated colonic crypts A, the BCECF-loaded colonic crypts in false colours (blue is acidic, red is alkaline) during the pHi recovery phase in the presence of HOE 642 and after the addition ...

To test whether the Hoe 642-insensitive Na+/H+ exchange activity in the surface cell/apical crypt portion is indeed NHE3 activity, we studied Hoe 642-insensitive, Na+-dependent proton flux rates in colonic crypts from nhe3 +/+ and −/− mice (Fig. 4). A strong reduction of acid activated proton flux was observed in the apical crypt regions, and the remaining flux was forskolin insensitive. The data show that in colonic surface cells, NHE3 is the only Na+-dependent, Hoe 642-insensitive transport pathway that is inhibited by cAMP.

Figure 4
NHE3 transport activity in surface enterocytes of nhe3 +/+ and −/− mice Hoe 642-insensitive proton flux rate after an intracellular acid load in surface enterocytes and basal cryptal cells of nhe3 +/+ and −/− colon. It ...

Acid-activated NHE3 activity in pdzk1 +/+ and −/− enterocytes and effect of the NHE3 inhibitor S1611

We next investigated whether acid activation of NHE3 is compromized in the absence of PDZK1 expression. In the apical regions of pdzk1 −/− colonic crypts, acid-activated NHE3 activity was strongly decreased (Fig. 5A and B). The pHi to which the cells had been acidified was not significantly different (6.76 ± 0.3 versus 6.78 ± 0.3). Because this strong decrease was surprising to us, we investigated whether NHE3 participated at all in the acid-activated pHi recovery. Ten micromolar of the selective NHE3 inhibitor S1611 was added shortly prior to Na+ readdition. Figure 5A and B shows that 10 μm S1611 inhibits approx. 56% of acid-activated, Hoe 642-insensitive Na+/H+ exchange in the pdzk1 +/+ enterocytes and 41% of that in pdzk1 −/− enterocytes. If one subtracts the acid-activated, Na+-dependent, non-NHE3-mediated proton flux in the colonic surface cells that becomes evident during the experiments with NHE3-deficient mice (Fig. 4) from the values in Fig. 5A and B, the inhibition of NHE3 by S1611 is not reduced in PDZK1-deficient enterocytes. This demonstrates that acid-activated NHE3 activity is present, but reduced, in the absence of PDZK1.

Figure 5
Acid-activated NHE3 activity in pdzk1 +/+ and −/− surface enterocytes A, In pdzk1 +/+ surface colonocytes, acid-activated Hoe 642-insensitive proton flux was significantly inhibited by 10 μm of the NHE3-selective inhibitor S1611. ...

cAMP-, cGMP- and Ca2+-sensitive inhibition of NHE3 activity in pdzk1 +/+ and −/− enterocytes

In order to study the involvement of PDZK1 in the regulation of NHE3 activity, we next studied the effect of forskolin and of the Ca2+ ionophore ionomycin on acid-activated NHE3 activity in pdzk1 +/+ and −/− enterocytes. Forskolin and 8-Br-cGMP were applied 2 min and ionomycin approx. 5 min before addition of Na+, and the initial pHi recovery during the first minute after Na+ readdition was used to calculate proton-flux rates. The results are shown in Fig. 6A and B. They demonstrate that in the absence of PDZK1, forskolin and ionomycin have no significant inhibitory effect on acid-activated NHE3 activity. The lack of inhibitory effect by both cAMP and Ca2+ suggests a severe functional defect in NHE3 regulation.

Figure 6
Effect of cAMP and Ca2+ on NHE3 activity in pdzk1 +/+ and −/− surface enterocytes A, forskolin (10−5m) caused a significant inhibition of acid-activated Hoe 642-insensitive proton flux rates in pdzk1 +/+ enterocytes, whereas no ...

Effect of hyperosmolarity on NHE3 activity in pdzk1 +/+ and −/− enterocytes

Another important aspect of NHE3 regulation in heterologous expression systems is its inhibition by hyperosmolarity (Kapus et al. 1994; Nath et al. 1996). We therefore investigated the effect of hyperosmolar solutions on acid-activated NHE3 activity in native colonic enterocytes. This was done by elevating the osmolarity of the Na+-containing solutions to 320 and 400 mosmol l−1 by mannitol addition. (Fig. 7A and B). Since even the percentage inhibition seen with 400 mosmol l−1 on NHE3 activity in WT colonic enterocytes was weaker than that seen with forskolin, we used the latter concentration to study the effect of PDZK1 knockout on hyperosmolarity-mediated NHE3 inhibition. The results are shown in Fig. 7B and demonstrate that hyperosmolarity-induced inhibition of NHE3 is not dependent on the presence of PDZK1.

Figure 7
Inhibition of NHE3 activity by hyperosmolarity in pdzk1 +/+ and −/− surface enterocytes A, in WT colon, acid-activated Hoe 642-insensitive proton flux was not significantly different in 320 versus 290 mosmol l−1, but was significantly ...

NHE1 and NHE2 exchange activity is not significantly altered in pdzk1 −/− enterocytes

The strong reduction of NHE3 activity raised the question whether the activity of other NHEs were also affected. We therefore performed experiments in the apical and basal portions of pdzk1 +/+ and −/− crypts in the presence and absence of 1 μm Hoe 642 (to selectively inhibit NHE1) and 50 μm Hoe 642 (to inhibit NHE1 and NHE2).

Figure 8A shows the total (mediated primarily by NHE1, NHE2 and NHE3; Bachmann et al. 2004) and the Hoe 642-insensitive Na+-dependent proton flux (mediated predominantly by NHE3) in the upper part (surface region), as well as the total (mediated predominantly by NHE1 and NHE2) and Hoe 642-insensitive proton flux in the basal part of the crypts (basal region). Figure 8A demonstrates that the lack of PDZK1 expression reduced total, as well as Hoe 642-insensitive proton flux rate in the apical portions of the glands. In the basal parts, however, the lack of PDZK1 had no significant influence on total proton efflux rate. This suggests that the Hoe 642-sensitive NHE exchangers are not affected by lack of PDZK1, and that the reduction in NHE3 activity is not likely due to a reduced driving force for Na+/H+ exchange.

To further clarify the question whether the activity of NHE2, which is also localized in the brush border membrane, but with a crypt-predominent expression (Chu et al. 2002; Bachmann et al. 2004), is altered in the absence of PDZK1, we performed the experiments in which we selectively inhibited NHE1 by 1 μm Hoe 642 and looked at the acid-activated, Na+-dependent proton efflux rates (Fig. 8B) specifically at the base of the glands, where Na+/H+ exchange is completely sensitive to 50 μm Hoe 642. Hoe 642 at 1 μm reduced acid-activated Na+/H+ activity to the same degree in pdzk1 +/+ and −/− crypts, and the remaining flux, which is > 90% NHE2-mediated, was similar. These data suggest, with the certainty that we can experimentally attain, that acid-activated NHE2 activity is not significantly altered in the absence of PDZK1.

Discussion

The present study reveals an important role for the PDZ-domain adaptor protein PDZK1 in the regulation of enterocyte NHE3 activity. Acid-activation of NHE3 was markedly reduced in the absence of PDZK1, and the inhibition by a rise in intracellular cAMP and Ca2+ was abolished. On the other hand, hyperosmolarity and the NHE3-specific inhibitor S1611 inhibited NHE3. No evidence was found for a difference in NHE3 distribution or abundance in the proximal colonic brush border membrane or the colonic enterocyte homogenate, suggesting that the lack of PDZK1 results in a functional defect of NHE3 regulation.

We have previously found that intestinal Na+ absorption was reduced in pdzk1-deficient mice, and its regulation was altered (Cinar et al. 2005; Hillesheim et al. 2007). However, it was not possible to clarify the molecular nature of the observed defect. We therefore tested various techniques to directly measure NHE3 activity in native enterocytes, and measuring NHE3 activity in the very upper cells of crypts isolated from the proximal colon gave the highest acid-activated NHE3 activity rates, the best signal over noise ratio and superior agonist-mediated regulation. The surface cells in the opening of the crypts to the lumen strongly express NHE3 protein, and isolated crypts load well with the pH-sensitive dye BCECF. These cells also express NHE1 and NHE2, but these two NHE exchangers can be blocked by appropriate concentrations of Hoe 642 without affecting NHE3 activity (Bachmann et al. 2004). Standard techniques were applied for the assessment of Na+-dependent pHi recovery from intracellular acidification and the calculation of proton fluxes (Boyarsky et al. 1988a,Boyarsky 1988b). While NHE exchange rates were high in both the crypt and surface cell regions in the absence of Hoe 642, this substance inhibited acid-activated NHE activity virtually completely only in the cryptal region, while approx 70% of acid-activated NHE activity in the apical region was still present after Hoe 642 application.

In order to further validate the technique, we studied acid-activated, Hoe 642-insensitive, Na+-dependent proton flux in the surface and basal region of proximal colonic crypts isolated from nhe3 +/+ and −/− mice. We observed a more than 85% reduction in Hoe 642-insensitive proton flux in the surface region in the absence of NHE3 expression. Therefore, this method seemed suitable to study NHE3 activity and regulation in the intestine on a cellular basis.

We found a strong reduction of acid-activated NHE3 activity in proximal colonic surface cells in the absence of PDZK1, and a complete loss of NHE3 inhibition by forskolin and ionomycin. In these experiments, Na+ was added shortly after maximal cellular acidification had occurred, and in the experiments with forskolin and ionomycin, the substance was added shortly before the addition of Na+. Therefore, we only assessed early, not late, events that occur during acid activation and forskolin or ionomycin inhibition of NHE3.

Our results were highly surprising to us in several aspects. Firstly, the lack of cAMP- and Ca2+-induced inhibition of NHE3 in the absence of PDZK1 was unexpected. As mentioned above, NHERF1,2 and PDZK1 bind to NHE3 in vitro (Lamprecht et al. 1998; Yun et al. 1998; Thomson et al. 2005a), and NHERF1 and NHERF2 were shown to mediate the inhibition of NHE3 by cAMP when coexpressed with NHE3 in PS120 fibroblasts (Lamprecht et al. 1998; Yun et al. 1998). Based upon these findings, a model has been created whereby NHERF1 or NHERF2 provides a link between the PKA anchor protein ezrin and NHE3 and this the close spatial association between NHE3 and PKA necessary for phosphorylation. This model gained further acceptance from the finding that in renal brush border membranes from nherf1 −/− mice, cAMP inhibition of proton-driven sodium uptake, also mediated by NHE3, is abolished (Weinman et al. 2003). In contrast, in the intestinal surface cells of nherf1 −/− mice, cAMP-mediated inhibition of NHE3 is still observed (Murtazina et al. 2005; Cinar et al. unpublished observations). Similar to the proximal tubule, both PDZK1 and NHERF1 are expressed in the brush border membrane of enterocytes (Rossmann et al. 2005). Thus, the surprising finding of this study is the inability of NHERF1 to substitute for PDZK1 in the mediation of cAMP inhibition of NHE3 in murine intestine, and the other way round in the proximal tubule. Similarly, NHERF2 but not NHERF1 is involved in the mediation of ionomycin-induced inhibition of NHE3 in PS120 fibroblasts (Kim et al. 2002; Lee-Kwon et al. 2003), and the molecular model that has been created operates without the involvement of PDZK1. This suggests that in the native epithelium as yet unrecognized components may play important roles in these signalling complexes. From the literature, some speculations as to what such components might be come to mind.

(1) One such component could be a PDZK1-mediated interaction of NHE3 with additional transport proteins such as CFTR or SLC26A3. Both have been shown to bind to PDZK1 (Lamprecht et al. 2002; Lohi et al. 2003; Rossmann et al. 2005). Recently, Lee et al. (2001) have provided evidence for a direct interaction of NHE3 and CFTR, with an augmentation of cAMP-mediated NHE3 inhibition by the presence of CFTR. NHE3 was coprecipitated with CFTR but not with a mutant CFTR lacking the C-terminal PDZ-domain binding motif. However, CFTR expression is very low if present at all in colonic surface epithelium. On the other hand, the expression of SLC26A3 is very high. CFTR has been shown to physically interact with members of the SLC26 family via cytoplasmic domains of both proteins in a PDZ-protein dependent fashion (Ko et al. 2004), which results in a reciprocal augmentation of ion transport activity through each protein. A similar situation could exist in the intestinal brush border membrane, where NHE3 and SLC26A3 might be partners in the signalling complex that mediates NHE3 inhibition by cAMP. For this interaction, PDZK1 may be necessary.

(2) Another important recent finding is that NHERF1 and PDZK1 display widely differing affinities for the PKA-anchoring proteins ezrin and D-ACAP-2, which are both expressed in the renal proximal tubule (Gisler et al. 2003a,Gisler 2003b). Cell type-specific expression levels of two different PKA-anchor proteins with different affinities from the two PDZ-proteins, if also present in intestinal cells, could well result in a cell type-specific predominance in the mediation of cAMP action on NHE3 of one PDZ-protein over the other.

(3) Recent data have shown that NHE3 inhibition in the kidney is mediated not only by PKA, but also by the cAMP exchange protein EPAC (Honegger et al. 2006). Other epithelia in which this PKA-independent process of NHE3 plays a role, and potential additional proteins necessary for it, and are currently being searched for.

(4) NHERF1 and PDZK1 have been shown to bind to each other (Gisler et al. 2003b). The biological function of this interaction is unclear, but it could be important in a situation where the availability of one or several players in the formation of signalling complexes is either limited or made scarce though competition with the members of another complex.

While the lack of agonist inhibition of NHE3 in the absence of PDZK1 is still reconcilable with the concept that PDZ proteins are necessary for the formation of multiprotein complexes in which regulation of transport activity occurs (Weinman et al. 2000a: Zachos et al. 2005), a more surprising finding was the reduced rate of acid-activated NHE3 activity. We first suspected this to be due to a reduced NHE3 protein content in the enterocyte brush border membrane. But both in the cytoplasm and the isolated brush border membrane, this could not be detected. Also, the distribution of NHE3 at the BBM was not altered, as measured with the techniques that were available to us. Nevertheless, the up-regulation of NHE3 mRNA (Hillesheim et al. 2007) points to an increased turnover of NHE3, likely to be due to a reduced membrane retention time of NHE3 in the absence of PDZK1. To our knowledge it is unknown, but feasible, whether stability in the membrane affects the regulation of a transport protein. Future studies employing biotinylation of NHE3 in native intestine may provide a clearer picture of the trafficking of NHE3 in the native intestine of pdzk1 +/+ and −/− mice.

Since there was no indication that a decrease of NHE3 could explain this finding, the most likely explanation is that acid activation of NHE3 in the native intestine also requires PDZ proteins. The early theory that the H+ ion binding to an internal modifier site activates NHEs has recently become more elaborate. Acid-regulated signalling pathways involving activation of c-Src, ERK, increases in c-fos, c-jun, junB, egr-1 expression and Pyk-2 phosphorylation have been shown to be necessary for the acid activation of NHE3 (Li et al. 2004; Tsuganezawa et al. 2002; Yang et al. 2000). In addition, Charney et al. (2002) published that increasing and lowering PCO2 results in a trafficking of NHE3-containing vesicles from a subapical vesicle pool into the membrane and vice versa. Thus, it is likely that low pHi activates NHE3 by a signalling mechanism involving PDZK1 in addition to the above-mentioned proteins.

In order to test whether the low acid-activated rates and loss of inhibition of NHE3 activity was due to an absence of NHE3 in the membrane, we tested the effect of the NHE3-specific inhibitor S1611. In WT mice, 10 μm S1611 inhibited approx. 60% of acid-activated, Hoe 642-insensitive proton flux, as compared to approx. 85% by NHE3 knockout. Possibly, the lesser effect of S1611 was due to an incomplete penetration of the rather lipophilic substance into the crypt lumen. In pdzk1 −/− crypts, S1611 still inhibited > 40% of acid-activated Hoe 642-insensitive proton flux. After subtraction of the proton flux in the surface region that was not mediated by NHE1, 2 or 3 (that value of around 4 mm min−1 became evident during experiments with NHE knockout mice (Fig. 4) and the combination of 50 μm Hoe 642 and 20 μm S1611, data not shown), the inhibitory effect of S1611 was similar in pdzk1 +/+ and −/− crypts. This demonstrates that the lack of inhibition by forskolin or ionomycin is not due to an absence of inhibitable NHE3 in the brush border membrane.

Inhibition by hyperosmolarity is another physiologically relevant but incompletely understood mode of NHE3 regulation (Kapus et al. 1994; Nath et al. 1996; Watts et al. 1998; Soleimani et al. 1998; Doble et al. 2000; Bachmann et al. 2004). We therefore tested its effect on acid-activated NHE3 activity in pdzk1 +/+ and −/− colonic crypts. Interestingly, inhibition by hyperosmolarity was also preserved. Recent data from heterologous expression systems suggest that changes in membrane convexity as induced by cell swelling may influence acid-activation of NHE3 (Alexander et al. 2007), and a similar situation may exist for hyperosmolarity-induced inhibition. Similarly, the inhibition of NHE3 by S1611 is likely to be a direct inhibition at the NHE3 molecule, and independent of protein–protein interactions, because it also occurs in isolated membranes (H. J. Lang, formerly of Hoechst AG, personal communication). Thus, the lack of PDZK1 may selectively interfere with NHE3 inhibition through cellular signal transduction mechanisms, in congruency with the evolving concepts of PDZ protein-mediated multiprotein signal complex formation.

In search of a potential defect in exchange activity of other NHE isoforms, or a reduced driving force (defective Na+/K+-ATPase activity, for example) for Na+/H+ exchange, we measured acid-activated total and Hoe 642-inhibitable Na+/H+ exchange rates in the surface region and the basal parts of the crypts. The experiments demonstrated that both in the surface and the cryptal region, NHE1 and NHE2 activity is not influenced by the absence of PDZK1, which makes a lack of driving force for Na+/H+ exchange an unlikely explanation for the defect in NHE3 activity. In addition, we investigated whether NHE2, another NHE isoform residing in the apical membrane with predominant cryptal expression (Chu et al. 2002), was also dysregulated in the absence of PDZK1, but we found no evidence for NHE2 dysfunction.

In summary, our data demonstrate a prominent role for the PDZ adaptor protein PDZK1 in the regulation of intestinal NHE3 activity. In addition, the different roles of individual NHERF proteins in regulating the same physiological process in different organs suggests that NHE3-binding PDZ adaptor proteins display a cell-type specificity in native tissue. This suggests that the multiprotein signalling complexes that regulate the activity of NHE3 in the different organs are more complex than previously thought.

Acknowledgments

We acknowledge the help of many colleagues during the process of establishing the methods, in particular Prof Christian Lytle, UC Riverside. In particular, we thank Prof Mark Donowitz for his crucial advice in the compilation of the data for the manuscript. The work was supported by the DFG grants Se 460/13-1/2, DFG Se 460/17-1, and Sonderforschungsbereich SFB 621/project C9.

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