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Departments of Medicine and Physiology, GI Division, John Hopkins University School of Medicine, Baltimore, MD 21205, USA
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Abstract |
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-actinin-4 and protein kinase C were among those NHE3-associated proteins because they were more efficiently coimmunoprecipitated from total BBM after carbachol treatment. Moreover, Src was involved in the carbachol-mediated inhibition since: (1) c-Src was rapidly activated in the detergent-resistant membranes by carbachol; and (2) carbachol inhibition of ileal Na+ absorption was completely abolished by the Src family inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Moreover, the carbachol-induced increase in the size of NHE3-containing complexes was reversed by PP2. These data demonstrate that regulation of NHE3 activity by carbachol can be achieved at several interrelated levels: (1) the subcellular level, at which NHE3 is rapidly endocytosed from BBM to endocytic vesicles upon treatment with carbachol; (2) multiple BBM pools, in which carbachol selectively decreases the amount of NHE3 in the BBM detergent-soluble fraction but not the detergent-resistant membrane; and (3) the molecular level, at which NHE3 complex-associated proteins can be changed upon carbachol treatment, with carbachol leading to larger BBM NHE3 complexes and increased co-IP of NHERF2 with -actinin-4 and activated PKC. The study further describes NHE3 presence simultaneously in multiple dynamic BBM pools in which NHE3 distribution and associated proteins are altered as part of carbachol-induced and Src-mediated rapid signal transduction, which decreases the amount of BBM NHE3 and thus inhibits NHE3 activity.
(Received 9 January 2004;
accepted after revision 15 February 2004;
first published online 20 February 2004)
Corresponding author M. Donowitz: John Hopkins University School of Medicine, 925 Ross Research Bldg, 720 Rutland Ave., Baltimore, MD 21205-2195, USA. Email: mdonowit{at}jhmi.edu
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Introduction |
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Intestinal NaCl absorption and BBM Na+–H+ exchange are inhibited by elevation of Ca2+ as induced by multiple physiological and pathological agonists. These include carbachol, serotonin, heat stable E. coli toxin B, and the rotavirus enterotoxin, NSP5 (Goyal et al. 1989; Cohen et al. 1991; Morris & Estes, 2001). This inhibitory effect on NHE3 by elevation of Ca2+ can also be observed in Caco-2 cells (McSwine et al. 1998). However, elevating Ca2+ had no effect on NHE3 activity in PS120 and AP-1 fibroblasts (Wakabayashi et al. 1995). We recently reported that this discrepancy in Ca2+ regulation between cell lines resulted from the fact that the PS120 and AP-1 fibroblasts lack NHERF2, a PDZ domain-containing protein that is required for Ca2+-dependent inhibition of NHE3 (Kim et al. 2002).
NHE3 appears to exist simultaneously in several pools in the plasma membrane, at least some of which change in size and/or composition with signal transduction that regulates NHE3 activity (Li et al. 2001; Akhter et al. 2002). Expressed stably in PS120 cells and occurring endogenously in the polarized epithelial cell lines Caco-2 and OK cells and in native rabbit ileal villus Na+-absorptive cells, NHE3: (1) is present in both detergent-soluble (DS) and detergent-resistant membranes (DRM), as separated by solubility in 1% Triton X-100; (2) the DRM fraction can be further divided into that which sediments with high and low density by density gradient centrifugation, with the latter shifting to higher density when cholesterol is removed or masked by exposure to methyl ß-cyclodextrin, fillipin, or cholesterol oxidase (all tested in PS120 cells; data not shown). This fits the definition of a pool of NHE3 being present in lipid rafts in the BBM/plasma membrane. The amount and percentage of total cell NHE3 in lipid rafts increases when growth factors (EGF) and activation of guanine nucleotide-binding protein-coupled receptors (2-adrenergic agonist, clonidine) rapidly (within minutes) stimulate NHE3 activity by increasing the amount of NHE3 in the BBM (Li et al. 2001). Density gradient sedimentation of ileal Na+-absorptive cell BBM and plasma membranes from Caco-2, OK and PS120 cells showed that NHE3 was simultaneously present in many sized complexes, up to 900 kDa (Akhter et al. 2002; X. Li and M. Donowitz, unpublished observations).
The most detailed study of changes in pools of NHE3 occurring in a single plasma membrane as part of signal transduction has been with elevated Ca2+ inhibition of NHE3 in PS120 fibroblasts stably expressing NHERF2 (Kim et al. 2002). Elevating Ca2+ in these cells inhibited NHE3 and decreased the amount of plasma membrane NHE3. This inhibition of NHE3 required NHERF2 and was preceeded by formation of increased amounts of NHE3 oligomers and a decrease in NHE3 monomers. This Ca2+ effect was associated with formation of plasma membrane NHE3 complexes that included NHERF2, -actinin-4 and PKC. In addition, NHERF2 coprecipitated PKC and -actinin-4 in a Ca2+-dependent manner (more co-IP with elevated Ca2+; Kim et al. 2002; Lee-Kwon et al. 2003).
Thus it appears that NHE3 may exist in several plasma membrane complexes simultaneously. The idea that a single transport protein can exist in several pools at the same time in a single plasma membrane, including the apical membrane in polarized epithelial cells, is a new concept, still to be confirmed in intact small intestine. Moreover, EGF stimulates NHE3 activity in ileal BBM by increasing the DRM NHE3 pool without altering the NHE3 in the BBM DS pool (Li et al. 2001). Thus regulation of NHE3 appears to be able to occur via one specific pool and does not necessarily involve all the BBM NHE3 pools simultaneously.
The present study explores in more depth the contribution of BBM NHE3-containing complexes and the DS and DRM pools of small intestinal (ileal) BBM NHE3 in carbachol-elevated Ca2+ regulation of NHE3. Here, we demonstrate that NHE3 inhibition by carbachol is achieved at multiple levels. These include specifically decreasing the NHE3 amount in the BBM by decreasing that in the DS BBM pool, while increasing NHE3 in the endosomal pool, and increasing the BBM NHE3 complex size by promoting formation of NHE3 complexes with -actinin-4 and activated protein kinase C. In addition, we show that carbachol inhibition of NHE3 is associated with and dependent on activation of c-Src, with c-Src activation being required for the increase in size of the NHE3 containing complexes.
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Methods |
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Male New Zealand White rabbits weighing 2.5–5 kg were killed by overdose of i.v. Nembutal (according to John Hopkins University School of Medicine-approved animal protocol), and ileum was obtained. Treatment of ileum with muscle layers intact in vitro with different reagents was as previously described (Li et al. 2001) and specified in the figure legends. NHE3-specific polyclonal antibodies (Pabs, 1381) and NHERF2-specific Pabs were previously described (Hoogerwerf et al. 1996; Yun et al. 1997). The latter were a gift from C. Yun, Emory University, GI division, School of Medicine. Pabs to phosphorylated conventional protein kinase C (p-PKC) and to phosphorylated c-Src (p-Src) were purchased from Cell Signalling Technology, (Beverly, MA, USA) respectively. Monoclonal antibodies (mAb) to c-Src and polyclonal antibodies to c-Fyn and c-Yes were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody to early endosome autoantigen 1 (EEA1) was from Transduction Laboratories, (West Grove, PA, USA). Pabs to -actinin-4 and to sucrase and mAbs to Na+–K+-ATPase were generous gifts from Dr T. Yamada (National Cancer Center Research Institute, Japan), Dr H. Poutalakis (Harvard Medical School), and Dr D. Fambrough (Johns Hopkins University), respectively.
Isolation of ileum, ileal villus cells and preparation of ileal BBM and total cell membranes
All procedures have been described previously (Li et al. 2001; Hoogerwerf et al. 1996).
Preparation of DS and DRM fractions
All steps were done at 4°C. The DS and DRM fractions were prepared from ileal total membranes or BBM, as we described previously using solubilization in 1% Triton X-100 (Li et al. 2001).
Fractionation of total membrane vesicles by OptiPrep step gradient
Total membrane vesicles (1–1.5 mg protein) were isolated from ileal villus cells by centrifugation, as described previously (Li et al. 2001). Briefly, total membranes were loaded onto the top of an 11-step OptiPrep gradient, which consisted of 30, 27.5, 25, 22.5, 20, 17.5, 15, 12.5 and 10% OptiPrep. Each step gradient was prepared with OptiPrep and Hepes buffer (20 mM, pH 7.2) containing 150 mM NaCl, 1 mM Na3VO4 and 50 mM NaF. Centrifugation was done in a Beckman SW 40 rotor at 23 000 r.p.m. for 90 min at 4°C. Membrane vesicles were sedimented to different fractions according to their densities. Twenty fractions were collected from the bottom of each centrifuge tube. One-sixth of each fraction was analysed with SDS-PAGE and Western blotting.
Separation of protein complexes: size fractionation by sucrose density gradients
Either the DS fraction or the total lysate of ileal BBM (1–1.5 mg protein) exposed to Triton X-100 (1%) was overlaid on top of an 11-step discontinuous sucrose gradient (5–30%) and centrifuged at 40 000 r.p.m. in a Beckman SW 40Ti rotor for 16 h (Li et al. 2001). Twenty fractions were collected from the bottom of each gradient. One-eighth of each fraction was analysed by SDS-PAGE and Western blotting.
Immunoprecipitation (IP)
NHERF2 or -actinin-4 were immunoprecipitated from the total lysate of ileal BBM (in the presence of 1% Triton X-100). All IPs were done at 4°C with constant mixing on a rotary shaker. Briefly, each sample was first precleared with Protein-A-Sepharose beads (Sigma) for 1 h. The precleared lysate was then incubated with 4 µg of antibodies to NHERF2, -actinin-4 or preimmune serum (control) for 1 h. Protein A-Sepharose B beads were then added to each IP mixture and incubation was continued for another 1 h. The beads were washed 4 times with PBS buffer containing 0.1% Tween-20 (Sigma). The IP pellets were analysed by SDS-PAGE and Western blotted with corresponding antibodies.
SDS-PAGE, Western blotting and chemiluminescence detection
Proteins were separated on 10% SDS polyacrylamide gels using Bio-Rad Protein xii gel system and blotted onto nitrocellulose (NC) membranes. Detection of each protein using corresponding Ab was done using Renaissance Enhanced Luminol Reagent (NENTM, Dupont NEN, Boston, MA, USA) and HyperfilmTM MP film (Amersham).
Ileal water absorption
Full thickness ileal loops 
10 cm long were prepared immediately after rabbits were killed. The ileal loops were incubated for 30 min in Ringer-HCO3, pH 7.4 at 37°C gassed with 95% O2–5% CO2 containing 10 mMD-glucose plus 50 µM bumetanide with or without 10 µM carbachol. Luminal fluid (4 ml) was instilled, consisting of Ringer-HCO3, 14C-polyethyleneglycol (PEG), MW 8000 (10 000 d.p.m. ml–1 in 0.5 mM PEG) with or without the Src family kinase inhibitor 20 µM PP2 (Cal Biochemical). Net water transport was determined by comparing the PEG (d.p.m. ml–1) in the fluid placed in the loop and that remaining after 30 min. Water transport rate was determined in microlitres per 30 min per milligram dry tissue (tissue heated at 130°C for 18 h).
Confocal imaging of p-Src in ileal villus cells
Ileum exposed in vitro to carbachol/control for 10 min was fixed in 3% paraformaldehyde, then 10 µm cryostat sections were cut and blocked and permeabilized at room temperature in PBS containing 0.1% saponin, 2% BSA and 15% fetal bovin serum (FBS) for 2 h. Primary anti-p-Src Pabs (1:50 in PBS) was exposed for 2 h and secondary antibody (Alexa Fluor 568 (Molecular Probes, Inc.) 1:100 for 1 h. Images were taken on a Zeiss LSM-410 (100x, ZSESS Plan-Apochromat, 1.4 na objective), with excitation via a helium–neon laser (543 nm), with detection by a long-pass 570 nm filter. Fifteen serial 1.5 µm sections were taken and merged to produce a single image of a representative sample. The signal intensity was quantified with Metamorph software of fifteen 44 x 44 pixel boxes along the surface of the villi with thresholding and avoidance of lamina propria signal. Grey scale values were compared by Student's unpaired t tests.
Results shown are means ±S.D. if the number of experiments was fewer than six and means ±S.E.M. if greater than or equal to six.
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Results |
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Although carbachol inhibition of ileal NaCl absorption and BBM NHE3 activity is known to be mediated through the phospholipases, PLC
, PLD and PKC (Khurana et al. 1997; Cohen et al. 1991; M. Cohen and M. Donowitz, unpublished observations), the mechanism of this inhibition is not fully understood. Therefore, we determined whether carbachol inhibition of NHE3 occurs by a reduction in the amount of NHE3 in the BBM and which BBM pool(s) was involved. This was done by determining separately the changes in the amount of NHE3 in ileal BBM DS and DRM pools, prepared after in vitro exposure of ileum to carbachol. As shown in Fig. 1A, carbachol treatment decreased the DS-associated BBM NHE3. There was no effect of carbachol on the amount of NHE3 in the DRM pool. Carbachol decreased the amount of total BBM NHE3 by 24 ± 3% when changes of total BBM were calculated from three independent experiments (P < 0.04; Fig. 1B). This change is in good agreement with our previous Na+ transport data, in which ileal Na+ absorption and BB Na+–H+ exchange were decreased after carbachol and/or elevated Ca2+ (Emmer et al. 1989; Khurana et al. 1997).
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To determine whether inhibition of NHE3 and the decrease in BBM NHE3 by carbachol are due to an alteration of NHE3 trafficking between the ileal endosomal compartments and BBM, we analysed the effects of carbachol treatment on the distribution of NHE3 among different ileal membrane vesicles, representing multiple organelles and membrane compartments. Membrane vesicle preparation was done by OptiPrep density gradient fractionation, as previously described (Li et al. 2001), starting with total membranes. OptiPrep was used because it causes less damage to vesicles from hyperosmolarity compared to sucrose gradient centrifugation (Li et al. 2001). Four marker proteins were used to show that different vesicle populations were well separated on these gradients (Fig. 2Aa–d). These were EEA1 and ß-adaptin (for endosomal vesicles), sucrase (for BBM) and Na+–K+ ATPase (for basal lateral membranes). The ileal NHE3 peaks at two distinctly separate vesicle populations, the endosomal compartments and BBM (Fig. 2Ae and f, also see Li et al. 2001). Under basal conditions, 20% of total NHE3 in the ileal epithelial cells was localized in the endosomal compartments and 80% at the BBM. Carbachol treatment increased the amount of NHE3 in the endosomal compartments from 21.7 ± 1.1 to 38.2 ± 2.1% of total NHE3 (P < 0.03; Fig. 2B) and concomitantly decreased the amount of NHE3 in the BBM from 69.2 ± 2.6 to 49.6 ± 6.2% (Fig. 2Ae and f).
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-actinin-4 containing complexes.
This occurs in the DRM and is accompanied by increased association of NHE3 with -actinin-4 and protein
kinase C. We have previously shown that elevation of Ca2+ promotes internalization of NHE3 in PS120 cells stably expressing NHERF2 through formation of large NHE3–NHERF2–-actinin-4–PKC complexes (Kim et al. 2002; Lee-Kwon et al. 2003). To test the relevance of these findings to ileal BBM NHE3, we first examined the size of NHE3 complexes in ileal BBM by sucrose density gradient fractionation (modified from Li et al. 2001; Li & Sze, 1999). As shown in Fig. 3Aa, upper panel, under basal conditions (no carbachol), NHE3 appears to be mostly present in total BBM in complexes of 400 kDa (fraction numbers 11–14) with a small percentage of larger complexes of 500–1000 kDa (fraction numbers 1–10, more visible after longer exposure). After 10 min of carbachol treatment, the amount of larger NHE3 complexes (500–1000 kDa) was increased (Fig. 3Aa, lower panel, fraction numbers 1–10). This shift of NHE3 by carbachol to larger complexes closely resembled the shift of -actinin-4 (Fig. 3Ab) in the ileal BBM. Since both NHERF2 and -actinin-4 are known to interact with the actin cytoskeleton, the formation of the NHE3–NHERF2–-actinin-4 complex was predicted to be restricted to the DRM, with no change in the DS expected. To test this assumption, the BBM DS fraction was isolated and subjected to the same sucrose gradient fractionation. Carbachol treatment did not change the size of NHE3 complexes in the DS pool (decreased size by 1 fraction; Fig. 3B). These results support the suggestion that the increased size of NHE3 complexes occurred entirely in the BBM DRM.
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-actinin-4 in total BBM strongly suggests that carbachol induces complex formation that directly or indirectly involves NHE3 and -actinin-4. This observation agrees with our previous data from PS120/NHE3/NHERF2 cells in which elevation of Ca2+ stimulated formation of complexes that contain NHE3 and -actinin-4 which were formed by scaffolding through NHERF2 (Kim et al. 2002). Does NHERF2, which is known to bind both NHE3 and -actinin-4 (Yun et al. 1997; Kim et al. 2002), mediate the carbachol-activated enlarged complex formation? To begin answering this question, we tested whether the association of NHERF2 and -actinin-4 in ileal BBM was altered by Ca2+ elevation by carbachol using coimmunoprecipitation. Since carbachol- and elevated Ca2+-inhibition of NHE3 is known to be mediated by PKC (Cohen et al. 1991; Janecki et al. 1998), we also examined whether PKC is involved in the carbachol-induced complex formation. As shown in Fig. 4, even at basal Ca2+ conditions, NHERF2, -actinin-4 and activated PKC (phosphorylated conventional PKC) could all be coprecipitated from the ileal BB, suggesting that they all are part of a large complex. Carbachol treatment enhanced the association among the three proteins, resulting in more -actinin-4 and p-PKC Co-IP with NHERF2 from BBM. The total amount of BBM -actinin-4 IP also appeared to be elevated by carbachol exposure (Fig. 4).
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It has been shown in the Cl– secretory T84 cell line that carbachol not only causes Cl– secretion but also stimulates a time-dependent and tyrosine kinase-dependent pathway that limits the Cl– secretion (Keely & Barrett, 2000). c-Src is activated in T84 cells by carbachol (Keely et al. 2000; McCole et al. 2002). Carbachol-activated Src appears to be an intermediate in transactivation of the EGFR which is involved in turning off carbachol activated Cl– secretion. Since there is often coordinated regulation of intestinal Na absorption and Cl– secretion, we determined whether Src family kinases were involved in carbachol inhibition of ileal absorption and whether carbachol altered Src family kinase activity. To determine whether Src family tyrosine kinases were involved in carbachol inhibition of ileal Na+ absorption, a model for carbachol inhibition of rabbit ileal water transport was standardized and the effect of pretreatment with the Src family kinase inhibitor, PP2, on carbachol effects determined. Exposure of ileal loops to serosal carbachol (10 µM, 37°C for 30 min) converted water absorption present in control conditions to water secretion (Fig. 5). The presence of the Src family tyrosine kinase inhibitor PP2 (20 µM) on the luminal surface for 30 min did not alter basal water absorption compared to untreated controls, but did prevent the carbachol-induced decrease in water absorption. These studies were performed in the presence of bumetanide (50 µM) on the ileal serosal surface to inhibit Cl– secretion. These findings in the presence of bumetanide suggest that the changes in water transport in Fig. 5 were due to changes in Na+ absorption. These results are consistent with Src family tyrosine kinase(s) being necessary for carbachol inhibition of ileal Na+ absorption.
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-actinin-4 BBM complexes was determined. Ileum was exposed to PP2 under conditions which prevented the carbachol effect on ileal Na+ absorption (Fig. 5). PP2 prevented carbachol activation of ileal membrane c-Src without affecting the total amount of Src (Fig. 8A). In fact, PP2 decreased p-Src activity in carbachol-exposed ileum to a level that was lower than that under basal conditions (control). Further, to determine whether the carbachol-activated c-Src was involved in regulation of the NHE3–-actinin-4 complexes, sucrose gradient centrifugation was used to study whether PP2 reversed the carbachol-induced increase in NHE3–-actinin-4 complex size. As shown in Fig. 8B, PP2 treatment prevented carbachol from increasing the size of NHE3 and - actinin-4 complexes in ileal BBM membranes. Thus the increase in NHE3–-actinin-4 complexes caused by carbachol are dependent on c-Src activation, and the changes in the size of NHE3–-actinin-4 complexes occur in parallel with carbachol treatment and both fail to occur when carbachol activation of c-Src is prevented.
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Confocal microscopy after immunofluorescent labelling of anti p-Src antibody was used to determine which pools of Src were activated in ileal villi. As shown in Fig. 9, 10 min after carbachol exposure increased p-Src was detected in villus epithelial cells, particularly at the BBM, although in addition cytosolic p-Src increased compared to untreated controls. The lamina propria had a large amount of p-Src staining under control conditions and this did not appear to change with carbachol treatment.
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Discussion |
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(1) Elevated Ca2+ (carbachol, 10 min) lowers BBM NHE3 in amounts similar to the magnitude of the decrease in Na+ absorption previously reported (Emmer et al. 1989; Cohen et al. 1991). The elevated Ca2+ is associated with an increased percentage of total NHE3 in endosomes along with a decreased percentage of the NHE3 in the BBM.
(2) Carbachol-elevated Ca2+ increases the size of NHE3 complexes in the BBM. This occurs in the BBM DRM pool. With elevated Ca2+ there is increased colocalization of NHE3 with -actinin-4 in the larger BBM complexes. Interestingly, NHE3 complexes present in the DS pool are not significantly changed in size after carbachol treatment.
(3) Elevated Ca2+ is associated with changes in specific signalling molecules in the BBM DRM with more coprecipitation of BBM NHERF2 with -actinin-4 and active PKC. There is also more BBM -actinin-4, the only molecule which shows changes in amount in the BBM along with the decrease in the amount of NHE3. These findings suggest that elevated Ca2+ leads to larger BBM NHE3 complexes that include NHERF2, -actinin-4 and activated PKC.
The increase in size of NHE3 complexes as part of signal transduction with elevated Ca2+ was first suggested based on studies using PS120/NHERF2/NHE3 cells, a NHE null fibroblast, stably transfected with NHE3 and NHERF2 (Kim et al. 2002). In these cells, elevating Ca2+ increased the appearance of NHE3 in oligomers and decreased NHE3 in monomers and increased the appearance of NHE3–NHERF2–-actinin-4–PKC complexes studied by coprecipitation and confocal microscopy. Since both NHE3 and NHERF2 were overexpressed in these cells, the NHE3 complexes could have represented artifacts from overexpression. The demonstration here of similar dynamic complexes in cells with only endogenous NHE3 and NHERF2 shows that carbachol-elevated Ca2+-induced dynamic NHE3–NHERF2 complex formation is not due to overexpression artifacts and occurs in normal ileal BBM.
In addition, these ileal BBM studies increase our understanding of NHE3 presence in the BBM in multiple pools, which go beyond the findings in PS120 fibroblasts. By dividing NHE3 in the BBM into DS and DRM compartments, we found that in spite of NHE3 being associated with the cytoskeleton (Reczek et al. 1997; Yun et al. 1998), there was a large DS component. In fact, when BBM was exposed to 0.5% Triton X-100, NHE3 was distributed almost equally in DS and DRM. However, given the method of separating these two pools, it is possible that treatment with Triton X-100 plus mechanical disruption of cells artificially destroys components of the cytoskeleton and thus appears to increase NHE3 in the DS fraction. The present study shows that changes in NHE3 in multiple BBM pools occur as part of Ca2+-related signal transduction and demonstrate that the presence of NHE3 in these complexes is dynamic. Changes in the size of NHE3 complexes are consistent with there being changes in the nature and/or amount of associating proteins. What is not yet known is how the stoichiometry of NHE3 and its associating proteins change with signal transduction (i.e. ratio of NHE3 to NHERF2 to -actinin-4, to p-PKC in NHE3-containing complexes).
Several other findings remain incompletely understood from these studies. These include the fate and role of the enlarged BBM DRM complexes that contain NHE3 after carbachol and the fate of the decreased BBM DS pool of NHE3. The amount of NHE3 in the DS pool decreases. The simplest interpretation is that it is the DS NHE3 which is endocytosed and accounts for the decrease the amount of NHE3 in BBM. The latter could be explained by association of NHE3 in the DS with clathrin-coated vesicles. It has been reported that NHE3 associates with clathrin-coated vesicles in fibroblasts (Chow et al. 1999). However, association with the endocytic machinery in the ileal villus cells, including with clathrin-coated vesicles, would be expected to increase the size of the NHE3 complexes, while what is observed is no change or a small decrease in size of this pool of NHE3 complexes. Thus it seems more likely that with Ca2+ elevation the DS pool of NHE3 initially is shifted to the DRM pool (perhaps with removal of some associated proteins). In the DRM, NHE3 directly or indirectly associates with other regulatory proteins (-actinin-4, activated PKC, BBM PDZ domain-containing proteins, including but not necessarily limited to NHERF2 and NHERF1), which form the larger NHE3 complexes. This pool would then be endocytosed.
Also unexplained is what becomes of the larger BBM DRM NHE3 complexes. In addition to the increase in size of NHE3 complex, carbachol treatment results in rapid changes in DRM in signalling molecules such as activated PKC and activated c-Src. These data suggest that DRM is the major pool through which carbachol regulates NHE3. In PS120 cells, elevated Ca2+ produced large NHE3-containing complexes which could be demonstrated by cell surface biotinylation and confocal microscopy (Kim et al. 2002). The fact that the same complex components, including NHE3, NHERF2, -actinin-4 and p-PKC have been shown to occur in both fibroblast plasma membranes and ileal BBM suggests that some signalling mechanisms in a nonpolarized cell have been adapted to act in a localized manner in polarized epithelial cells. What becomes of these large complexes in ileal Na+ absorptive cells is not yet known. In PS120 cells, the timing of formation of the oligomers of NHE3 comes before the decrease in surface NHE3 complexes as determined by surface biotinylation, which suggests that the complexes were formed before internalization occurred (Kim et al. 2002). However, there was such a large amount of internalized NHE3 complexes by confocal microscopy that it was necessary to consider initial intracellular complex formation as well. In ileum, since there is no change in the amount of DRM NHE3, we cannot assume that this is the internalization pool and this raises the question of what the functional consequences of NHE3 complex formation might be. It is still possible that large complexes in the DRM are the pool which is endocytosed, with a similar rate of the DS NHE3 joining the DRM to the rate of endocytosis of DRM NHE3. Because this is a dynamic process, the amount of NHE3 in the BBM DRM would not change even though the rate of NHE3 endocytosis increases.
The dynamic changes in large NHE3 complexes in the ileal BBM and the amount of NHE3 colocalizing with endosomal markers indicates that molecular physiological studies from cell culture models appear to be duplicating what occurs in intact tissue and encourages further dissection of mechanisms of NHE3 regulation by comparing both models. For instance, these ileal studies suggest that carbachol inhibition of NHE3 is associated with increased endocytosis. In fact, either stimulation of endocytosis or inhibition of exocytosis of NHE3 is consistent with the findings presented. Determination of whether inhibition of BBM NHE3 is due to one or the other or both processes needs to be made and is probably most easily carried out in the cell culture models. In addition, understanding the relationship among the NHE3 pools as affected by signal transduction (changes in stoichiometry, identification of further proteins in the NHE3 complexes, understanding of the relationship between the DS and DRM pools as affected by elevated Ca2+) is necessary for understanding of how Ca2+ decreases BBM NHE3. A combination of studies in ileal Na+ absorptive cells and cell culture models, especially model polarized epithelial cells, has the best chance of giving insights into details of this process which is part of digestive physiology and is critical to the pathobiology of diarrhoeal diseases.
The demonstration of a role for the c-Src family in carbachol inhibition of NHE3 is not surprising given the previous demonstration that Src is an intermediate in carbachol regulation of Cl– secretion in T84 cells (Barrett, 1993; Keely et al. 2000; McCole et al. 2002; Chow et al. 2003). In addition, parallel and opposite changes in regulation of intestinal Na absorption and Cl– secretion have been demonstrated in the past (for instance, stimulation of Na absorption and inhibition of Cl– secretion by activation of PI 3-kinase), which would lead to coordinated, rather than opposing, changes in water transport. The model we used here (Fig. 5) almost certainly represents changes in Na absorption, since we used conditions that are known to inhibit Cl– secretion (serosal bumetanide), although we cannot be certain whether all Cl– secretion (basal and carbachol stimulated) was totally inhibited. The involvement of c-Src in carbachol inhibition of Na absorption should be interpreted recognizing that in T84 cells, c-Src is activated by carbachol where it appears to be involved in transactivation of the EGFR, which is involved in turning off the Ca2+ activated Cl– secretion (Keely et al. 2000; McCole et al. 2002). The role of c-Src needs more characterization in the regulation of NHE3 before parallels can be made between its effects in intestinal Na+ absorptive and Cl– secretory cells, although we have established that activated c-Src is also necessary as an intermediate in the carbachol-induced formation of large NHE3 and -actinin-4-containing BBM complexes. Moreover, we demonstrated that it is c-Src in the DRM pool of BBM which is involved, further supporting the DRM as the critical pool of NHE3 in carbachol inhibition. Which kinase activates BBM c-Src, as well as the relevant c-Src substrate involved in NHE3 complex formation and/or decrease in surface NHE3, are also not known.
This study strongly supports the hypothesis that -actinin-4 associates in BBM with NHE3 in a Ca2+-dependent manner. Until this study the involvement of -actinin-4 in Ca2+ regulation of NHE3 came from proteomic studies in which lysates of intestine bound -actinin-4. This was followed up with studies in PS120 fibroblasts which showed that the coprecipitation of NHERF2 and -actinin-4 occurred in a Ca2+-dependent manner with evidence of involvement functionally based on the EF hands domains of -actinin-4 acting as a dominant-negative regulator to prevent Ca2+ inhibition of NHE3. The present studies in intestinal Na+ absorptive cells show that BBM -actinin-4 coprecipitates with NHERF2 in a Ca2+-dependent manner and that there is parallelism between the distribution of NHE3 and -actinin-4 in large BBM complexes, with increase in size with Ca2+ elevation and the changes of both being dependent on c-Src activation. The functional role in intestinal Na+ absorptive cells of -actinin-4 has not been established, but given the parallels with regulation of NHE3 in fibroblasts, we hypothesize that the role of -actinin-4 in Ca2+ regulation of NHE3 is in forming the large NHE3 complexes and in increasing endocytosis of NHE3 from these complexes.
In addition, elevated Ca2+ is associated with more BBM -actinin-4. The source of the increased BBM -actinin-4 could be from the terminal web and/or tight junctions, which are known to be the major BBM pools of -actinin-4 (Geiger et al. 1979; Craig & Lancashire, 1980; Mooseker & Stephens, 1980). The importance of this observation is that its mechanism must be explored to understand the role of cytoskeleton or tight junctional changes in elevated Ca2+ inhibition of NHE3.
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), in comparison with the untreated control (
), which was set as 100%. Results were calculated from three independent experiments similar to that shown in A (means ±S.D.). P values were in comparison to untreated controls (Student's paired t test).
