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Am J Physiol Gastrointest Liver Physiol 291: G868-G876, 2006. First published July 6, 2006; doi:10.1152/ajpgi.00023.2006
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LIVER AND BILIARY TRACT

Impaired intestinal NHE3 activity in the PDK1 hypomorphic mouse

Ciprian Sandu,1 Ferruh Artunc,1 Monica Palmada,1 Rexhep Rexhepaj,1 Florian Grahammer,1 Azeemudeen Hussain,1 Chris Yun,2 Dario R. Alessi,3 and Florian Lang1

1Department of Physiology I, University of Tübingen, Germany; 2Division of Digestive Diseases, Departments of Medicine and Physiology, Emory University School of Medicine, Atlanta, Georgia; and 3MRC Phosphorylation Unit, University of Dundee, Dundee, Scotland

Submitted 17 January 2006 ; accepted in final form 31 May 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In vitro experiments have demonstrated the stimulating effect of serum- and glucocorticoid-inducible kinase (SGK)1 on the activity of the Na+/H+ exchanger (NHE3). SGK1 requires activation by phosphoinositide-dependent kinase (PDK)1, which may thus similarly play a role in the regulation of NHE3-dependent epithelial electrolyte transport. The present study was performed to explore the role of PDK1 in the regulation of NHE3 activity. Because mice completely lacking functional PDK1 are not viable, hypomorphic mice expressing ~20% of PDK1 (pdk1hm) were compared with their wild-type littermates (pdk1wt). NHE3 activity in the intestine and PDK1-overexpressing HEK-293 cells was estimated by utilizing 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein fluorescence for the determination of intracellular pH. NHE activity was reflected by the Na+-dependent pH recovery from an ammonium prepulse ({Delta}pHNHE). The pH changes after an ammonium pulse allowed the calculation of cellular buffer capacity, which was not significantly different between pdk1hm and pdk1wt mice. {Delta}pHNHE was in pdk1hm mice, only 30 ± 6% of the value obtained in pdk1wt mice. Conversely, {Delta}pHNHE was 32 ± 7% larger in PDK1-overexpressing HEK-293 cells than in HEK-293 cells expressing the empty vector. The difference between pdk1hm and pdk1wt mice and between PDK1-overexpressing and empty vector-transfected HEK cells, respectively, was completely abolished in the presence of the NHE3 inhibitor S3226 (10 µM). In conclusion, defective PDK1 expression leads to significant impairment of NHE3 activity in the intestine, pointing to a role of PDK1-dependent signaling in the regulation of NHE-mediated electrolyte transport.

phosphatidylinositol 3-kinase; transport regulation; Na+/H+ exchanger; pH regulation

THE NA/H+ EXCHANGER (NHE3) is expressed in the apical cell membrane of several epithelia including the intestine (54, 77), stomach (58), pancreatic duct (1), kidney (11), epididymis (36, 72), and airways (20). In those epithelia, it accomplishes luminal acidification and/or reabsorption of Na+ and HCO3– (76). Tight regulation of the carrier allows the adjustment of epithelial transport to the respective functional requirements (29, 39, 43, 50, 51, 60). Beyond that, NHE3 serves in the regulation of cytosolic pH in several nonpolarized cells (19, 34, 37, 71).

Signaling molecules regulating NHE3 activity include serum- and glucocorticoid-inducible kinase (SGK)1, which has originally been cloned as a glucocorticoid-inducible gene (23, 68, 69). The human kinase has been cloned as a cell volume-regulated gene (65). According to in vitro experiments, SGK1 enhances NHE3 activity by direct phosphorylation of the carrier protein and by increasing the protein abundance at the cell membrane (66, 73, 74). The effect requires the presence of NHE regulatory factor 2 (NHERF2) (73, 74).

Homology screening led to the discovery of the SGK isoforms SGK2 (40) and SGK3 (40). All three kinases stimulate a variety of channels and transporters (46). All three kinases are activated by the phosphoinositide-dependent kinase 1 (PDK1) (2, 3, 18, 25, 41, 56). Thus lack of PDK1 is expected to impair the function of all three kinases. The PDK1 knockout mouse is not viable (48), pointing to the functional importance of this kinase.

The present experiments were performed in mice with suppressed PDK1 activity to some 20% (pdk1hm) and in their wild-type littermates (pdk1wt) (48) to explore whether PDK1 is indeed required for the function of intestinal NHE3.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
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The generation and basic properties of PDK1 hypomorphic mice have been described earlier (48). Genotyping was made by PCR on tail DNA using PDK1 and neo-R-specific primers as previously described (48). Mice had free access to standard mouse diet (Altromin, Langen, Germany) and tap water. All animal experiments were conducted according to the guidelines of the American Physiological Society and the German law for the welfare of animals and were approved by the local animal committee.

Intestinal NHE3 activity. For isolation of ileal villi, animals were fasted for 6 h before experiments. After the death of the animals, the terminal 2 cm of the ileum was removed and cut longitudinally. After being washed with standard HEPES solution, the intestine was sliced into 0.3-cm2 sections. The tissues were transferred onto the cooled stage of a dissecting microscope, and individual villi were detached from the intestine by snapping off the ileal base with sharpened microdissection tweezers. Care was taken not to damage the apical part of the villi. The villi were attached to a glass coverslip precoated with Cell-Tak adhesive (BD Biosciences, Heidelberg, Germany).

For quantitative digital imaging of intracellular pH (pHi), isolated individual villi were incubated in a HEPES-buffered Ringer solution containing 10 µM 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-AM (Molecular Probes, Leiden, The Netherlands) for 15 min at 37°C. After being loaded, the chamber was flushed for 5 min with Ringer solution to remove any deesterified dye sticking to the outside of the villi. The perfusion chamber was mounted on the stage of an inverted microscope (Zeiss Axiovert 135, Oberkochen, Germany), which was used in the epifluorescence mode with a x40 oil-immersion objective (Zeiss Neoplan, Oberkochen, Germany). BCECF was successively excited at 490 ± 10 and 440 ± 10 nm, and the resultant fluorescent signal was monitored at 535 ± 10 nm via an intensified charge-coupled device camera (Proxitronic, Bensheim, Germany) and specialized computer software (Metafluor, Universal Imaging, Downingtown, PA). Individual regions of interest at the tip of the villi were outlined and monitored during the course of the measurement. Intensity ratio data (490/440) were converted into pH values by the high-K+/nigericin calibration technique (26).

In a second series of experiments, human embryonic kidney (HEK-293) cells were seeded at a density of 1.5 x 105 cells on polylysine-coated coverslips in 35-mm cell culture dishes in DMEM medium containing 10% fetal calf serum under standard culture conditions (37°C, 5% CO2). One day after being seeded, the cells were transfected with empty vector pIRES2-EGFP (Invitrogen, Karlsruhe) or a PDK1 construct (pIRES-EGFPPDK1) (40) using Lipofectamine (Invitrogen, Karlsruhe, Germany) (12). Briefly, 4 µl of Lipofectamine were added to 2 µg of plasmid DNA in 200 µl Optimem (Invitrogen), and the mixture was allowed to stand at room temperature for 20 min. The transfection mixture was diluted to 2 ml by adding serum-free DMEM (Invitrogen), the mixture then was added dropwise to HEK293 cells, and cells were returned to the incubator. Transfection efficiency was determined 24 h later by fluorescence microscopy to visualize the EGFP protein expression.

For digital imaging of HEK293 cells, the coverslips with transfected cells were mounted in the perfusion chamber, washed with standard HEPES solution, and monitored for enhanced green fluorescent protein (EGFP) fluorescence. Transfected cells (cells showing EGFP fluorescence) were marked by drawing regions in the acquisition window and were then incubated with 10 µM BCECF for 15 min. After incubation, to eliminate the EGFP fluorescence excitation light, intensity was decreased at least 10 times by using neutral density filters, and only the previously marked cells (expressing EGFP) were taken into consideration.

The solutions, flow lines, and perfusion chamber were maintained at 37°C by a thermostatically controlled heating system. The volume of the perfusion chamber was 600 µl and the flow rate was 4 ml/min for all solutions. For acid loading, cells were transiently exposed to a solution containing 20 mM NH4Cl leading to marked initial alkalinization of pHi owing to entry of NH3 and binding of H+ to form NH4+ (57). The acidification of pHi upon removal of ammonia allowed us to calculate the mean intrinsic buffering power (beta) of the cells (57), assuming that NH4+ and NH3 are in equilibrium in cytosolic and extracellular fluid and that ammonia leaves the cells as NH3:

Formula
where {Delta}pHi is the decrease of pHi after ammonia removal and {Delta}[NH4+]i is the decrease of cytosolic NH4+ concentration, which is identical to the concentration of NH4+ immediately before the removal of ammonia. Given the pK for NH4+/NH3 of 8.9 (9), an extracellular pH (pH0) of 7.4 and an NH4+ concentration in extracellular fluid ([NH4+]0) of 19.37 [20/(1 + 10pHo – pK)], then [NH4+]i = 19.37 x 10pHo – pK.

For some experiments, the selective NHE3 inhibitor S3226 (Aventis, Frankfurt, Germany) was added to all solutions at a concentration of 10 µM.

The solutions were composed of the following (in mM): for standard HEPES, 115 NaCl, 5 KCl, 1 CaCl2, 1.2 MgSO4, 2 NaH2PO4, 10 glucose and 32.2 HEPES; for sodium-free HEPES, 132.8 NMDG, 3 KCl, 1 CaCl2, 1.2 MgSO4, 2 KH2PO4, 32 HEPES, 10 mannitol, and 10 glucose; for sodium-free ammonium chloride, 15 NMDG-Cl and 10 mannitol were replaced by 20 mM NH4Cl; and for calibration, 105 KCl, 1 CaCl2, 1.2 MgSO4, 32.2 HEPES, and 10 mannitol (5 µM nigericin). The pH of the solutions was titrated to 7.4 or 7.0 with HCl-NaOH, HCl-NMDG, and HCl-KOH, respectively, at 37°C.

All substances were from Sigma (Taufkirchen, Germany) or Roth (Karlsruhe, Germany).

SDS-PAGE and Western blot analysis. For brush-border membrane preparations, the small intestine was collected and rinsed with ice-cold saline solution and then opened longitudinally, and the mucosa was scraped off with a glass slide in a buffer containing (in mM) 250 sucrose, 20 Tris (pH 7.5), 5 EGTA, and a protease inhibitor cocktail at the concentration suggested by the manufacturer (Roche, Mannheim, Germany). The suspension was homogenized for 2 min on ice. MgCl2 was added to the homogenate to a final concentration of 10 mM. The suspension was then centrifuged at 1,600 g for 15 min. The plasma membranes retained in the supernatant were collected by centrifugation at 20,000 g for 30 min. The resultant pellet was suspended in pH 7.4 buffer consisting of (in mM) 125 sucrose, 10 Tris (pH 7.5), 2.5 EGTA, and 2.5 MgSO4 and centrifuged at 20,000 g for 30 min. The final pellet, containing the purified brush-border membrane vesicles (BBMVs), was homogenized by passing the suspension through 25- and 28-gauge needles and solubilized. All the steps were carried out at 4°C. After the final suspension, samples were frozen at –20°C for later use. Membrane protein was assessed by the Bradford method.

Similar amounts of BBMV protein (40 µg) were solubilized in Laemmli sample buffer and resolved by 8% SDS-PAGE. Proteins were electrotransferred onto a nitrocellulose membrane and blocked for 1 h in 5% nonfat milk in PBS-0.15% Tween 20 at room temperature. The membrane was incubated with 1:500 anti-rat NHE3 (Alpha Diagnostics, San Antonio, TX) overnight at 4°C followed by goat anti-rabbit IgG coupled to horseradish peroxidase (1:1,000, Amersham Biosciences, Freiburg, Germany). After each incubation, the membrane was washed extensively with PBS-0.15% Tween 20, and then visualization with ECL was achieved following the manufacturer's instructions (Amersham). Densitometric analysis of NHE3 was performed using Bio-Rad Quantity One software (Bio-Rad, Munich, Germany).

Ussing chamber experiments. Epithelial Na+ channel (ENaC) activity was estimated from the amiloride-sensitive potential difference across the colonic epithelium. After removal of outer serosal and muscular layers of the distal colon under a microscope, tissues were mounted onto a custom-made mini-Ussing chamber with an opening diameter of 0.99 mm and an opening area of 0.00769 cm2. Transepithelial potential difference (Vte) was determined continuously and transepithelial resistance (Rte) was estimated from the voltage deflections ({Delta}Vte) elicited by imposing rectangular test currents of 1 µA and 1.2 s of duration at a rate of 8/min. Rte was 16.8 ± 0.8 {Omega} · cm2 (n = 7) in pdk1wt mice and 16.6 ± 0.8 {Omega} · cm2 (n = 7) in pdk1hm mice, which may, however, be an underestimate due to leakage at the tissue borders. The serosal and luminal perfusate contained (in mM) 145 NaCl, 1 MgCl2, 2.6 Ca-gluconate, 0.4 KH2PO4, 1.6 K2HPO4, and 5 glucose. To assess ENaC-mediated transport, 50 µM amiloride (Sigma, Taufkirchen, Germany) in ethanol was added to the luminal perfusate. In all experiments, two adjacent segments of the distal colon were studied simultaneously in two separate Ussing chambers. The average of each result was taken for analysis.

Collection and preparation of feces and determination of fecal Na+. Matched pairs of mice were placed in individual metabolic cages (Tecniplast, Hohenpeißenberg, Germany) on a standard diet with a Na+ content of 120 µmol/g (C1000, Altromin, Langen, Germany) and tap water (1.41 mM/l measured sodium content). After a training period of 2 days, food and fluid intake as well as fecal output were determined over two consecutive 24-h periods, and results were averaged for each animal. Fecal dry weight was taken after drying the samples at 80°C for ~3 h. The feces were prepared for the determination of the electrolyte content: after being dissolved in 5 ml of 0.75 M HNO3, the samples were shaken 48 h to yield a homogenous creamy mass. The mixture was centrifuged at 2,500 g for 15 min, 1 ml of the supernatant were collected and centrifuged again at 10,000 g in a small Eppendorf centrifuge for 5 min, and the Na+ concentration in the supernatant was determined by flame photometry (AFM 5051, Eppendorf).

Statistical analysis. Data are provided as means ± SE; n represents the number of independent experiments. All data were tested for significance by the Student's t-test or the nonparametric Mann-Whitney test as applicable, and only results with P < 0.05 were considered statistically significant.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Intestinal NHE3 activity. On the basis of the measurement of pHi using the BCECF fluorescence dye, the pHi was similar in the ileum from PDK1 hypomorphic (pdk1hm) mice (7.220 ± 0.002, n = 7 experiments in 122 cells) and their wild-type littermates (pdk1wt) (7.188 ± 0.015, n = 6 experiments in 84 cells).

Ammonium pulses were utilized to load the cells with H+ (57). The superfusion with 20 mM NH4Cl and simultaneous removal of Na+ (replacement with NMDG+) was followed by slight alkalinization (Fig. 1). The subsequent replacement of NH4Cl with NMDG+ Cl– in the continued absence of Na+ led to a sharp acidification due to exit of NH3 and retention of H+ within the cells. The acidification allowed the calculation of an apparent cellular buffer capacity (see METHODS), which was similar in the ileum from pdk1wt mice (26.5 ± 1.0 mM/pH unit, n = 7 experiments in 122 cells) and pdk1hm mice (28.4 ± 3.7 mM/pH unit, n = 6 experiments in 84 cells).


Figure 1
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  		Fig. 1. pH recovery in ileum after an ammonium pulse in phosphoinositide-dependent kinase 1 (PDK1) hypomorphic mice (pdk1hm) and their wild-type littermates (pdk1wt). Alterations of cytosolic pH ({Delta}pHi) in ileum after an ammonium prepulse was shown. To load the cells with H+, 20 mM NH4Cl was added and Na+ was removed (replaced by NMDG+) in a 1st step, NH4Cl was removed in a 2nd step, Na+ was added in a 3rd step, and nigericin (Nig) applied in a 4th step to calibrate each individual experiment. A: original tracings illustrating alterations of pHi in typical experiments on ileum from pdk1wt mice (left) or pdk1hm mice (right). B: arithmetic means ± SE of {Delta}pHi in ileum from pdk1wt mice (solid bars, n = 7 experiments in 122 cells) and pdk1hm mice (open bars, n = 7 experiments in 84 cells) before (left) and after (right) addition of Na+. *Statistically significant difference (P < 0.05) between pdk1wt and pdk1hm mice.

 
pHi recovery was minimal in the absence of Na+ in the ileum from pdk1wt and pdk1hm mice (Table 1) but markedly accelerated by subsequent replacement of NMDG+ with Na+ (Fig. 1). The Na+-dependent realkalinization ({Delta}pHNHE) was significantly more rapid in pdk1wt than in pdk1hm mice (Fig. 1).


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  		Table 1. NHE activity in pdk1hm mice, pdk1wt mice, HEK293PDK1 cells, and HEK293vector cells

 
The specific NHE3 inhibitor S3226 significantly decreased {Delta}pHNHE in pdk1wt and pdk1hm mice (Fig. 2). In the presence of S3226, {Delta}pHNHE was not significantly different between pdk1wt and pdk1hm mice, indicating that only S3226-sensitive realkalinization was affected by PDK1 (Fig. 2).


Figure 2
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  		Fig. 2. Effect of NHE3 inhibitor S3226 on pH recovery in ileum after an ammonium pulse in pdk1hm and pdk1wt mice. {Delta}pH in ileum after an ammonium prepulse was determined as described in Fig. 1. A: original tracings illustrating alterations of pH in typical experiments in pdk1wt mice in the absence (left) and presence (right) of S3226. B: arithmetic means ± SE of {Delta}pH in the presence of S3226 in ileum from pdk1wt mice (solid bars, n = 6 experiments in 70 cells) and pdk1hm mice (open bars, n = 6 experiments in 78 cells) before (left) and after (right) addition of Na+. C: arithmetic means ± SE of S3226-sensitive {Delta}pH in ileum from pdk1wt mice (solid bar) and pdk1hm mice (open bar). *Statistically significant difference (P < 0.05) between pdk1wt and pdk1hm mice.

 
To confirm the effects of PDK1 on NHE3 activity, we overexpressed PDK1 in HEK-293 cells, which endogenously express NHE3 (Fig. 3). pHi was similar in PDK1-overexpressing (7.163 ± 0.007, n = 8 experiments in 142 cells) and vector-transfected HEK-293 cells (7.182 ± 0.009, n = 8 experiments in 136 cells). Internal buffer capacity was also similar in PDK1-transfected cells (28.1 ± 1.9 mM/pH unit, n = 8 experiments in 142 cells) and cells expressing vector alone (25.3 ± 1.7 mM/pH unit, n = 8 experiments in 136 cells). Sodium-independent pH recovery was also not significantly different between PDK1-transfected HEK-293 cells and cells expressing vector alone (see Table 1). In the presence of Na+, however, the rate of pH recovery was significantly larger in PDK1-transfected cells. In the presence of S3226, sodium-dependent pH recovery was not significantly different between PDK1- and vector alone-transfected cells, indicating that only the S3226-sensitive fraction of the realkalinization was affected by overexpression of PDK1 (Fig. 4).


Figure 3
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  		Fig. 3. pH recovery in PDK1 and vector-transfected HEK-293 cells after an ammonium pulse. Alterations of {Delta}pH in transfected HEK-293 cells after an ammonium prepulse were determined as described in Fig. 1. A: original tracings illustrating alterations of pH in typical experiments in PDK1- (left) and vector alone- (right) transfected HEK-293 cells. B: arithmetic means ± SE of {Delta}pH in PDK1 (solid bars, n = 8 experiments in 142 cells) and vector-transfected cells (open bars, n = 8 experiments in 136 cells) before (left) and after (right) addition of Na+. *Statistically significant difference (P < 0.05) between PDK1- and vector alone-transfected HEK-293 cells.

 

Figure 4
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  		Fig. 4. Effect of NHE3 inhibitor S3226 on pH recovery in transfected HEK-293 cells after an ammonium pulse. Alterations of {Delta}pH in transfected HEK-293 cells after an ammonium prepulse were determined as described in Fig. 1. A: original tracings illustrating alterations of pH in typical experiments in PDK1-transfected cells in the absence (left) and presence (right) of NHE3 inhibitor S3226. B: arithmetic means ± SE of {Delta}pH in the presence of S3226 in PDK1-transfected (solid bars, n = 6 experiments in 85 cells) and vector alone-transfected HEK-293 cells (open bars, n = 6 experiments in 89 cells) before (left) and after (right) addition of Na+. C: arithmetic means ± SE of S3226-sensitive {Delta}pH in PDK1-transfected (solid bar) and vector alone-transfected HEK-293 cells (open bar). *Statistically significant difference (P < 0.005) between PDK1- and vector alone-expressing cells.

 
PDK1 could regulate sodium transport by enhancing the expression and/or by increasing the activity of expressed protein. To explore the effect of PDK1 on NHE3 expression, immunoblotting was performed in brush-border membranes of the intestine isolated from pdk1hm and pdk1wt mice (Fig. 5). In contrast to the reduced NHE3 activity observed, no significant difference in NHE3 protein abundance could be detected in the brush-border membrane fraction obtained from the intestines of pdk1hm and pdk1wt mice (n = 6 for each genotype).


Figure 5
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  		Fig. 5. NHE3 transporter abundance in intestinal brush-border membrane vesicles (BBMVs). NHE3 expression was assessed by Western blotting of intestinal BBMVs isolated from pdk1wt and pdk1hm mice. A: original blot. B: arithmetic means ± SE (n = 6 each) of NHE3 band intensities normalized to the value of NHE3 intensity in BBMVs isolated from pdk1wt mice.

 
Colonic ENaC activity and fecal Na+ excretion. To explore whether impaired intestinal Na+ transport affects transport proteins other than NHE3, additional experiments were performed to elucidate colonic ENaC activity in pdk1wt and pdk1hm mice (Fig. 6). The Vte across the colonic epithelium was slightly higher in pdk1wt mice (–4.6 ± 0.5 mV) than in pdk1hm mice (–3.5 ± 0.5 mV, n = 7). Addition of the Na+ channel blocker amiloride (50 µM) decreased Vte to –1.2 ± 0.3 mV in pdk1wt mice and to –1.3 ± 0.1 mV in pdk1hm mice without altering Rte. Thus Vte is fully amiloride sensitive and presumably due to ENaC activity. Vte and Rte allowed the calculation of the amiloride-sensitive equivalent short-circuit current in the distal colon, which was significantly larger in pdk1wt mice (–203 ± 21 µA/cm, n = 7) than in pdk1hm mice (–133 ± 22 µA/cm2, n = 7). As illustrated in Fig. 7, the fecal Na+ excretion was significantly higher in pdk1hm mice (138.8 ± 12.9 µmol/24 h, n = 10) than in pdk1wt mice (101.1 ± 10.8 µmol/24 h, n = 10). Thus intestinal absorption of Na+ was indeed impaired by partial deficiency of PDK1.


Figure 6
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  		Fig. 6. Amiloride-sensitive equivalent short-circuit current (Iamil) across colonic epithelium. A: representative original tracings of the transepithelial colonic potential difference (Vte) in pdk1wt (top trace) and pdk1hm (bottom trace) mice before and after addition of 50 µM amiloride (open arrow). B: arithmetic means ± SE (n = 7 each) of Iamil in distal colon of pdk1wt and pdk1hm mice as measured by Ussing chamber experiments. *Statistically significant difference (P < 0.05) between pdk1wt and pdk1hm mice.

 

Figure 7
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  		Fig. 7. Fecal sodium excretion. Arithmetic means ± SE (n = 10 each) of fecal sodium excretion in pdk1wt and pdk1hm mice (in µmol/24 h) are shown. *Statistically significant difference (P < 0.05) between pdk1wt and pdk1hm mice.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present experiments reveal a potent influence of PDK1 on intestinal NHE3 activity. Even though the hypomorphic PDK1 mouse still expresses residual PDK1, the NHE3 activity is markedly decreased.

Because SGK1 is downstream of PDK1, the decreased NHE3 activity may have been due to reduced activation of this kinase. In vitro observations indeed documented the ability of SGK1 to profoundly enhance NHE3 activity (66, 73, 74). SGK1 could particularly contribute to the stimulation of NHE3 by glucocorticoids, which stimulate SGK1 transcription (23, 68, 69) and, on the other hand, are known to upregulate NHE3 (70). Glucocorticoids increase NHE3 abundance in part by stimulation of expression (10, 35, 75), an effect presumably not requiring the participation of SGK1. SGK1 rather affects the trafficking of NHE3 to the cell membrane, an effect requiring the participation of the scaffold protein NHERF2 (73, 74). The regulation of NHE3 by glucocorticoids might lead to the sensitization against the effects of insulin (38). Along those lines, the stimulation of NHE3 activity by glucocorticoids is largely abolished in gene-targeted mice lacking SGK1 (28). However, in the absence of dexamethasone treatment, intestinal NHE3 activity is not significantly different between SGK1 knockout mice and their wild-type littermates, indicating that SGK1 is not required to maintain the basal activity of NHE3 (28). Thus the defective NHE3 activity in PDK1 hypomorphic mice points to further kinases downstream of PDK1 that are important for the regulation of NHE3.

Candidate kinases possibly replacing SGK1 include the isoforms SGK2 and SGK3 and the related kinase PKB. The kinases share the putative phosphorylation site of RxRxxS/T (16, 55), where R, S, T, and X stand for arginine, serine, threonine, and any amino acid, respectively, which is present in several substrates (21, 27, 33, 47) including NHE (66).

The protein abundance of NHE3 in intestinal brush-border membranes was not significantly altered in pdk1hm mice. Thus PDK1 may not be required for normal expression of NHE3 or the residual PDK1 activity in pdk1hm mice is sufficient to maintain NHE3 expression. Moreover, a minor decrease of NHE3 protein expression may escape detection by Western blotting. Nevertheless, the observations suggest that PDK1 is effective at least in part by activating expressed NHE3 protein.

PDK1 may not only be important for the regulation of NHE3. Similar to SGK1, SGK2 and SGK3 enhance the protein abundance and/or activity of several channels and transporters (46). PKB has previously been shown to enhance the abundance of several glucose transporters in the cell membrane (17, 32, 42, 62). Moreover, SGK1 has been shown to interact with ENaC (67) and to enhance the activity of ENaC heterologously expressed in Xenopus oocytes (5, 8, 13, 14, 47, 52, 63, 64), cortical collecting duct cells (30, 53), and A6 cells (6, 7, 22). The effect of SGK1 is shared by SGK2 and SGK3 (24). The present observations indeed disclose a slight decline of amiloride-sensitive Vte, presumably reflecting slightly decreased ENaC activity. The SGK isoforms have further been shown to enhance the activity of Na+-K+-ATPase (31, 59, 61, 78), an effect maintaining the chemical driving force for Na+ entry through NHE3.

In addition, constitutively active PKB has been shown to increase NHE3 activity by facilitating trafficking of NHE3 into the plasma membrane (49). In contrast to SGK1, there is no published evidence for transcriptional regulation of SGK2 and SGK3 by hormones (45). Thus it appears that SGK2, SGK3, and PKB are constitutively expressed. All three SGK isoforms and PKB require activation by insulin and growth factors including IGF-I (45). Thus all these kinases may participate in the known stimulating effect of IGF on intestinal transport (4, 15, 44).

In summary, we showed that deficiency in PDK1 expression significantly compromises NHE3 activity in the intestine. The experiments thus disclose a novel element in the regulation of NHE3 activity.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Bundesministerium für Bildung und Forschung (to F. Lang).


   FOOTNOTES
 

Address for reprint requests and other correspondence: F. Lang, Dept. of Physiology, Univ. of Tübingen, Gmelinstrasse 5, Tübingen D-72076, Germany (e-mail: florian.lang{at}uni-tuebingen.de)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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