Intestinal NaCl transport in NHE2 and NHE3 knockout mice

Lara R. Gawenis1, Xavier Stien1, Gary E. Shull2, Patrick J. Schultheis2, Alison L. Woo2, Nancy M. Walker1, and Lane L. Clarke1

1 Dalton Cardiovascular Research Center and Department of Biomedical Sciences, University of Missouri-Columbia, Columbia, Missouri 65211; and 2 Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Sodium/proton exchangers [Na+/H+ (NHEs)] play an important role in salt and water absorption from the intestinal tract. To investigate the contribution of the apical membrane NHEs, NHE2 and NHE3, to electroneutral NaCl absorption, we measured radioisotopic Na+ and Cl- flux across isolated jejuna from wild-type [NHE(+)], NHE2 knockout [NHE2(-)], and NHE3 knockout [NHE3(-)] mice. Under basal conditions, NHE(+) and NHE2(-) jejuna had similar rates of net Na+ (~6 µeq/cm2 · h) and Cl- (~3 µeq/cm2 · h) absorption. In contrast, NHE3(-) jejuna had reduced net Na+ absorption (~2 µeq/cm2 · h) but absorbed Cl- at rates similar to NHE(+) and NHE2(-) jejuna. Treatment with 100 µM 5-(N-ethyl-N-isopropyl) amiloride (EIPA) completely inhibited net Na+ and Cl- absorption in all genotypes. Studies of the Na+ absorptive flux (J<UP><SUB>ms</SUB><SUP>Na<SUP>+</SUP></SUP></UP>) indicated that J<UP><SUB>ms</SUB><SUP>Na<SUP>+</SUP></SUP></UP> in NHE(+) jejunum was not sensitive to 1 µM EIPA, whereas J<UP><SUB>ms</SUB><SUP>Na<SUP>+</SUP></SUP></UP> in NHE3(-) jejunum was equally sensitive to 1 and 100 µM EIPA. Treatment with forskolin/IBMX to increase intracellular cAMP (cAMPi) abolished net NaCl absorption and stimulated electrogenic Cl- secretion in all three genotypes. Quantitative RT-PCR of epithelia from NHE2(-) and NHE3(-) jejuna did not reveal differences in mRNA expression of NHE3 and NHE2, respectively, when compared with jejunal epithelia from NHE(+) siblings. We conclude that 1) NHE3 is the dominant NHE involved in small intestinal Na+ absorption; 2) an amiloride-sensitive Na+ transporter partially compensates for Na+ absorption in NHE3(-) jejunum; 3) cAMPi stimulation abolishes net Na+ absorption in NHE(+), NHE2(-), and NHE3(-) jejunum; and 4) electroneutral Cl- absorption is not directly dependent on either NHE2 or NHE3.

sodium; chloride; cystic fibrosis transmembrane conductance regulator; adenosine 3',5'-cyclic monophosphate; chloride-bicarbonate exchanger; sodium-hydrogen exchange; jejunum; small intestine; cystic fibrosis


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SODIUM/PROTON EXCHANGE ACROSS the apical membrane of small intestinal epithelia is required for electroneutral NaCl absorption from the intestinal lumen, a process that involves coupling with Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (or OH-) exchange (19, 25, 49, 54). Regulation of electroneutral NaCl absorption in the intestine is critical to salt and water homeostasis, as shown by the contrasting disease states of secretory diarrhea and cystic fibrosis. In secretory diarrhea, enterotoxin stimulation of intracellular cyclic nucleotide activity causes fulminate diarrhea by the combined effect of inhibiting electroneutral NaCl absorption while increasing anion secretion mediated by the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel (17). In cystic fibrosis, loss of CFTR activity causes intestinal obstruction by a combination of normal (or increased) electroneutral NaCl absorption and deficient anion secretion during stimulation of intracellular cyclic nucleotide activity (3, 39).

To date, DNA cloning experiments have identified five plasma membrane Na+/H+ exchanger (NHE) isoforms; three of the isoforms (NHE1-3) are prominently expressed in intestinal epithelia (18, 21). NHE1 plays an important role in intracellular pH regulation and has been immunolocalized to the basolateral membrane (1, 52). NHE2 and NHE3 are typically coexpressed in intestinal epithelium and have been localized to the apical membrane (18, 22, 53). Despite relatively high expression levels of NHE2 in both small and large bowel, little is known about the physiological function of NHE2 in the intestine. NHE2 knockout mice have a progressive loss of parietal cell viability, consistent with expression in murine gastric mucosa, but do not demonstrate overt intestinal dysfunction (46). In contrast, NHE3 is critical to electroneutral NaCl absorption across the small and large intestine (33, 34). Mice homozygous for knockout of NHE3 demonstrate a disease phenotype of chronic diarrhea and altered salt and water homeostasis (47). NHE4 expression has also been reported in intestinal epithelium, but the level of expression is either very low or variable, and detection of its mRNA is confounded by cross-hybridization with NHE2 (5, 14, 40).

Functional studies of recombinant NHE2 and NHE3 expressed in heterologous cell systems have shown differences in amiloride sensitivity and intracellular cAMP (cAMPi) regulation between the two isoforms. In media containing physiological Na+ concentrations, NHE2 is a relatively amiloride-sensitive isoform [5-(N-ethyl-N-isopropyl) amiloride (EIPA) IC50 = 0.076 µM], whereas NHE3 is more resistant to inhibition by amiloride and its analogs (EIPA IC50 = 6.25 µM) (42). The two isoforms also differ in their response to cAMPi/protein kinase A (PKA) stimulation. NHE2 is either unaffected or activated by cAMPi stimulation (23). NHE3, on the other hand, is inhibited (25-60%) by cAMPi/PKA when expressed in AP1-CHO cells or PS120 fibroblasts that coexpress the regulatory phosphoproteins E3KARP or NHE-RF (6, 23, 28, 36). However, analysis of PKA phosphorylation of NHE3 indicates that only a fraction of cAMPi inhibition is attributable to direct phosphorylation (15). Rather, cAMPi regulation of NHE3 may also depend on indirect cellular mechanisms such as internalization of the protein or changes in the cytoskeleton (16, 27). It is clear from these studies that cell type greatly influences regulation of the exchangers, thereby necessitating evaluation of function and regulation in the tissue of interest. In the present study, intact jejunal mucosa was used in evaluating basal activity and cAMPi regulation of Na+ and Cl- flux across the intestinal epithelia from wild-type, NHE2(-), and NHE3(-) mice.


    MATERIALS AND METHODS
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MATERIALS AND METHODS
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Animals

The development of mouse lines with targeted disruption (knockout) of genes encoding the protein products NHE2 (Slc9a2) and NHE3 (Slc9a3) was described in previous studies (46, 47). Heterozygous breeding pairs were maintained on a Black Swiss background by periodically backcrossing to avoid impaired viability due to inbreeding (30). The mice were fed a standard laboratory chow [Formulab 5008 Rodent chow (Ralston Purina, St. Louis, MO) or Teklad Autoclavable diet], and water was provided ad libitum. At 3-6 wk of age, all offspring were genotyped using specific primers for murine NHE2, NHE3, and the disrupted alleles as previously reported (11, 46, 47). The mice were identified as wild-type [NHE(+)] for Slc9a2+/+ or Slc9a3+/+, [NHE2(-)] for Slc9a2-/-, or [NHE3(-)] for Slc9a3-/- offspring, respectively. Experiments were performed on intestinal tissues obtained from weaned mice at 2-4 mo of age. Before each experiment, the mice were fasted overnight but provided with water ad libitum. All experimental protocols were approved by the Univ. of Missouri Animal Care and Use Committee.

In Vitro Bioelectric and Radioisotope Flux Measurements

Animals were killed by asphyxiation in a 100% CO2 atmosphere followed by a surgical thoracotomy to induce pneumothorax. Approximately 5 cm of proximal jejunum was removed via an abdominal incision and immediately placed in oxygenated, ice-cold Ringer solution. The jejunal segment was opened along the mesenteric border using sharp dissection and divided into sections for mounting in standard Ussing chambers (0.238-cm2 exposed surface area). To minimize edge damage to the tissue where it was secured, Parafilm O-rings were used between the chamber halves.

The mucosal and serosal surfaces of the unstripped (smooth muscle layers intact) intestinal sections mounted in Ussing chambers were independently bathed with 4 ml of a Krebs-bicarbonate-Ringer solution (KBR) with the following composition (in mM): 115 NaCl, 25 NaHCO3, 5 KCl, 1.2 MgCl2, and 1.2 CaCl2, pH 7.4. Glucose (10 mM) was added to the serosal bath; mannitol (10 mM) was substituted for glucose in the mucosal bath to avoid an inward current due to Na+-coupled glucose cotransport (12). Both mucosal and serosal bathing solutions were gassed with 95% O2 and 5% CO2 throughout the experiment in a gas-lift recirculation system and warmed to 37°C by water-jacketed reservoirs. Indomethacin (1 µM) was added to the KBR solution for both tissue dissection and the duration of all experiments to minimize tissue exposure to endogenously generated prostanoids resulting from manipulation of the intestinal sections (7, 10). After being mounted in the Ussing chambers, intestinal tissues were immediately treated with TTX (0.1 µM) in the serosal bath to minimize variation in the intrinsic neural tone (50).

Transepithelial short-circuit current (Isc; reported as µeq/cm2 · h) was measured via an automatic voltage clamp (VCC-600, Physiologic Instruments, San Diego, CA) and calomel electrodes connected to the chamber baths with 4% agar-3 M KCl bridges, as previously described (12). The Isc was applied through Ag-AgCl electrodes that were in contact with the chamber baths via 4% agar-KBR bridges. At 5-min intervals during the experiment, a 5-mV pulse was passed across the tissue to determine the total tissue conductance (Gt; mS/cm2 tissue surface area). Gt was determined by measuring the magnitude of the resulting current deflections and applying Ohm's law. All experiments were carried out under short-circuited conditions with the serosal bath serving as ground.

Radioisotopic Fluxes

Unidirectional mucosal-to-serosal (Jms), serosal-to-mucosal (Jsm), and net fluxes (Jnet = Jms - Jsm) of 22Na and/or 36Cl were calculated from triplicate aliquots (250 µl) taken at the beginning and end of each flux period as previously described (10). Thirty-five minutes before the first flux period, an aliquot (250 µl) was taken from the designated "sink" bathing medium to determine the background radioactivity of each chamber, after which 2 µCi of 22Na and 3 µCi of 36Cl were added to the opposite ("source") bathing medium. Thirty minutes were allowed for equilibration of the isotope before the start of the first flux period in either direction. During this time and at the end of the experiment, triplicate 4-µl aliquots were taken from the source bathing medium for determination of specific activity. Bioelectric and isotope measurements were taken during two sequential 30-min flux periods. The first 30 min served as a control (basal) period that was compared with a subsequent 30-min treatment period. To stimulate cAMPi, forskolin (10 µM) and IBMX (100 µM) were added at the end of the first flux period to both the mucosal and serosal baths 30 min before the start of the second flux period. For both flux periods, triplicate samples from the sink bathing medium were replaced by an equal volume of the appropriate KBR solution. Jejunal tissues from each mouse were paired by Gt (within 25%) at the conclusion of the experiment for calculation of Jnet. In separate experiments, unidirectional Jms 22Na of paired jejunal tissues were measured in the presence and absence (+vehicle) of EIPA. EIPA-sensitive fluxes were calculated using the formula EIPA-sensitive J<UP><SUB>ms</SUB><SUP>Na<SUP>+</SUP></SUP></UP> = vehicle J<UP><SUB>ms</SUB><SUP>Na<SUP>+</SUP></SUP></UP> - EIPA J<UP><SUB>ms</SUB><SUP>Na<SUP>+</SUP></SUP></UP>.

Samples were analyzed for 22Na and 36Cl activity by counting for 5 min in a gamma radiation counter (Packard Instruments, Meriden, CT) and 5 min in a liquid-scintillation system (Packard Instruments). 36Cl radioactivity was calculated after adjusting for the 22Na spillover. Separate standards of 22Na and 36Cl were used to estimate the counting efficiencies for each isotope and the 22Na spillover ratio. The rate of radioisotope flux was calculated as previously described (48). For net fluxes, a positive sign indicates net absorption (mucosal to serosal) and a negative sign indicates net secretion (serosal to mucosal).

Real-Time Quantitative RT-PCR

Freshly excised jejunum (~3 cm) was removed and flushed with ice-cold PBS. The jejunal segment was filled with citrate buffer and incubated for 5 min at 37°C. After being incubated, the segment was filled with EDTA buffer and the incubation continued for 10 min. The epithelial cells were then rinsed free of the surrounding tissue with freshly made ice-cold PBS spiked with 4 mM CaCl2 and pelleted at 600 g for 5 min before being frozen in liquid N2.

RNA was extracted using an RNeasy maxi kit (Qiagen, Valencia, CA) according to the manufacturer recommendations. Reverse transcription was performed using Superscript II RT (GIBCO, Grand Island, NY) and 1.5 µg of total RNA (determined by spectrophotometry). FastStart DNA Master SYBR Green I was used in a LightCycler (Roche, Indianapolis, IN) according to the manufacturer's directions. The primers for NHE3 were 5' GGC CTT CAT TCG CTC CCC AAG and 3' ATG CTT GTA CTC CTG CCG AGG; for NHE2, 5' GTG AAG ACT GGG ATT GAA GAT G and 3' TCG GGA GGT TGA AGT AGA AGC; and for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 5' TAT GAC TCC ACT CAC GGC AAA T and 3' TGC TTC ACC ACC TTC TTG ATG T. The cycle profiles were 5 s at 95°C, 5 s at 55°C, 20 s at 72°C, and 10 s at 81-86°C to allow for measurement of the double-stranded PCR products by fluorescence (SYBR green dye binding). The temperature setting for the final cycle was chosen after completion of a melting curve on the PCR product (to eliminate primer dimers or nonspecific PCR products). After each PCR, a melting curve analysis was performed to ensure the specificity of quantification.

After completion of the PCR reaction, the cycle number (CT) required for each sample to cross a fluorescence threshold value (set in the exponential amplification portion of the reaction) was determined. The absolute concentration of cDNA in the reverse-transcribed samples was calculated from the CT value of cDNA standards run in a parallel amplification. The concentration for each cDNA was normalized to the concentration of GAPDH cDNA in each sample to control for differences in cell number and the quality of the mRNA preparations.

Statistics

Data within genotype groups were compared using a two-tailed unpaired Student's t-test assuming equal variances. Data from more than two groups were compared using a one-way ANOVA with a post hoc Tukey's t-test. A P value <0.05 was considered statistically significant. All values represent means ± SE for n intestinal preparations (with no more than 2 preparations per animal).

Materials

Radioisotopes were obtained from Amersham (22Na; Arlington Heights, IL) and New England Nuclear (36Cl and 32P-dCTP; Boston, MA). EIPA was obtained from RBI/Sigma (Natick, MA). All other reagents were obtained from either Sigma Chemical (St. Louis, MO) or Fisher Scientific (Springfield, NJ). TTX was dissolved in 0.2% acetic acid at a stock concentration of 100 µM. Indomethacin and forskolin were dissolved in DMSO at stock concentrations of 10 mM. IBMX was dissolved in sterile water at a stock concentration of 10 mM. EIPA was dissolved in DMSO at stock concentrations of either 1 or 100 mM.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Isotopic Fluxes of 22Na and 36Cl

Basal. Under baseline conditions, jejuna from NHE(+) mice absorbed Na+ and Cl- at net rates of ~6 and ~3 µeq/cm2 · h, respectively (see Fig. 1). Given the possibility that the Isc of these preparations represents electrogenic Cl- secretion, total Cl- absorption may actually exceed the net Cl- absorption by ~1 µeq/cm2 · h. Overall, the rate estimates of net NaCl absorption agree reasonably well with previous measurements of 22Na and 36Cl flux across wild-type murine jejunum (8, 13, 50). Furthermore, the relatively high rates of NaCl absorption compared with the basal Isc are consistent with an electroneutral process of absorption. The unidirectional rates of isotope flux, Jms and Jsm, are given in the corresponding table (Table 1). Note that the unidirectional fluxes of Na+ and Cl- are large relative to the Jnet rates. These measurements reflect a high paracellular conductance, which accounts for >90% of Gt in the jejunum (43, 44).


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Fig. 1.   Bioelectric parameters and net 22Na and 36Cl flux across jejunum from Na+/H+ exchange (NHE) (+), NHE2(-), and NHE3(-) mice under basal conditions. J<UP><SUB>net</SUB><SUP>Na<SUP>+</SUP></SUP></UP>, net Na+ flux; J<UP><SUB>net</SUB><SUP>Cl<SUP>−</SUP></SUP></UP>, net Cl- flux; Isc, short-circuit current; Gt, total tissue conductance. See Table 1 for unidirectional fluxes from these experiments. * Significantly different from NHE(+) (P < 0.05).


                              
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Table 1.   Unidirectional and net JNa+ and JCl-, Isc, and Gt in NHE(+), NHE2(-), and NHE3(-) jejuna under basal and cAMPi-stimulated conditions

Interestingly, the unidirectional and net NaCl fluxes, Gt, and Isc of the NHE2(-) jejunum were essentially identical to the findings in the NHE(+) mice (Fig. 1). In contrast, NHE3(-) jejuna had a significantly reduced rate of net Na+ absorption but apparently normal net Cl- absorption (Fig. 1). Note that Gt in NHE3(-) jejuna was significantly less than in the other groups, and this was reflected by significantly reduced unidirectional flux rates for both Na+ and Cl- (Table 1). The large difference in NHE3(-) unidirectional flux rates complicated the question of whether the reduced rate of net Na+ absorption resulted from a decrease in the J<UP><SUB>ms</SUB><SUP>Na<SUP>+</SUP></SUP></UP>. To facilitate comparison, we normalized the unidirectional Na+ fluxes of the NHE3(-) jejunum to those of the NHE(+) jejunum by multiplying by the ratio of NHE3(-) J<UP><SUB>sm</SUB><SUP>Na<SUP>+</SUP></SUP></UP> to NHE(+) J<UP><SUB>sm</SUB><SUP>Na<SUP>+</SUP></SUP></UP> (= 1.4). This normalization method is based on the assumption that the J<UP><SUB>sm</SUB><SUP>Na<SUP>+</SUP></SUP></UP> is entirely passive and therefore a direct index of the paracellular Na+ movement (20). The mean unidirectional Na+ fluxes of the NHE3(-) jejunum normalized by this method were J<UP><SUB>ms</SUB><SUP>Na<SUP>+</SUP></SUP></UP> = 21.9 and 18.4 µeq/cm2 · h, which yields a J<UP><SUB>net</SUB><SUP>Na<SUP>+</SUP></SUP></UP> of 3.5 µeq/cm2 · h. Compared with the Na+ fluxes of the NHE(+) jejunum, the normalized J<UP><SUB>net</SUB><SUP>Na<SUP>+</SUP></SUP></UP> of the NHE3(-) jejunum was reduced by 2.9 µeq/cm2 · h and matched by an equivalent decrease in the normalized J<UP><SUB>ms</SUB><SUP>Na<SUP>+</SUP></SUP></UP> of 2.9 µeq/cm2 · h.

cAMPi. Stimulation of jejunal tissues with forskolin/IBMX to increase intracellular levels of cAMP abolished net Na+ absorption in all groups (Fig. 2). Net Na+ transport was not significantly different from zero in the NHE(+) and NHE3(-) jejuna, but J<UP><SUB>net</SUB><SUP>Na<SUP>+</SUP></SUP></UP> in the NHE2(-) jejunum was in the direction of net secretion. Net Cl- absorption in all groups was also abolished by cAMPi stimulation and reversed to net Cl- secretion, a change that was matched by an increase in the Isc (Fig. 2). Interestingly, the NHE3(-) jejunum had significantly reduced net Cl- secretion in response to cAMPi stimulation compared with the NHE(+) intestine. Although the differences in mean Isc of the second flux period did not attain statistical significance, the reduced Cl- secretory response in the NHE3(-) jejunum was shown by a lower peak Delta Isc during the 30 min immediately following forskolin/IBMX. In contrast, the NHE2(-) jejuna had a slightly greater peak Delta Isc compared with NHE(+) jejuna [NHE(+) Delta Isc = 132.5 ± 3.5 µA/cm2, n = 93; NHE2(-) Delta Isc = 150.3 ± 5.4 µA/cm2, n = 48; NHE3(-) Delta Isc = 82.6 ± 4.0 µA/cm2, n = 65; P < 0.05 for all pairwise comparisons].


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Fig. 2.   Bioelectric parameters and net 22Na and 36Cl fluxes across jejunum from NHE(+), NHE2(-), and NHE3(-) mice following treatment with 10 µM forskolin/100 µM IBMX (cAMP). All values were significantly different from basal within the same genotype (P < 0.05). * Significantly different from NHE(+) (P < 0.05). EIPA, ethylisopropylamiloride; J<UP><SUB>ms</SUB><SUP>Na<SUP>+</SUP></SUP></UP>, Na+ absorptive flux.

As previously reported (13), cAMPi stimulation resulted in a decrease in Gt for all genotypes. Despite the tightening induced by cAMPi stimulation, the Gt in NHE3(-) jejuna remained significantly less than in the NHE(+) and NHE2(-) jejuna. The cAMPi-induced decrease in Gt reduced the rates of unidirectional Na+ and Cl- fluxes in all mouse groups (Table 1); however, it is clear that the decrease in net NaCl absorption after cAMPi stimulation resulted from a larger decrease in Jms than Jsm for both Na+ and Cl-.

EIPA. To estimate the contribution of NHE activity to basal Na+ absorption, jejunal tissues from NHE(+), NHE2(-), and NHE3(-) mice were treated with 100 µM EIPA in the mucosal bath during a single flux period. Previous studies have shown that this concentration of EIPA inhibits NHE2 and NHE3 activity in native rat epithelia bathed in media containing physiological concentrations of Na+ (42). As shown in Table 2, 100 µM EIPA completely inhibited net Na+ absorption in all mouse groups. Compared with the unidirectional flux data given in Table 1, it is apparent that the significant decrease in J<UP><SUB>net</SUB><SUP>Na<SUP>+</SUP></SUP></UP> induced by EIPA resulted from a significant decrease in J<UP><SUB>ms</SUB><SUP>Na<SUP>+</SUP></SUP></UP> rather than in J<UP><SUB>sm</SUB><SUP>Na<SUP>+</SUP></SUP></UP>. Because EIPA only had a minor effect on the basal Isc, it is unlikely that electrogenic Na+ transport contributes significantly to net Na+ absorption. These findings are consistent with inhibition of NHE activity by EIPA in all groups of mice. Interestingly, 100 µM EIPA treatment also inhibited net Cl- absorption across the jejunum from all mouse groups, indicating inhibition of a coupled NaCl absorptive process.

                              
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Table 2.   Effect of 100 µM EIPA on Na+ and Cl- fluxes across NHE(+), NHE2(-), and NHE3(-) jejuna

EIPA-Sensitive J<UP><SUB>ms</SUB><SUP>Na<SUP>+</SUP></SUP></UP> in NHE3(-) Jejunum

The preceding studies raised the question of the role of NHE2 in transepithelial Na+ absorption in the jejunum. Whereas the Na+ flux studies on NHE2(-) jejunum indicated that NHE2 does not significantly contribute to net Na+ absorption under basal conditions, it was also apparent that a finite amount of net Na+ absorption was present in the NHE3(-) jejunum. To characterize the Na+/H+ transport process in the NHE3(-) intestine, two different concentrations of EIPA (1 and 100 µM) were used to distinguish between amiloride-sensitive isoforms (e.g., NHE2) and amiloride-insensitive isoforms (e.g., NHE3) involved in the Na+ absorptive flux (42). As shown in Fig. 3, 1 µM EIPA had essentially no effect on J<UP><SUB>ms</SUB><SUP>Na<SUP>+</SUP></SUP></UP> in NHE(+) jejuna, whereas 100 µM EIPA inhibited J<UP><SUB>ms</SUB><SUP>Na<SUP>+</SUP></SUP></UP> by ~6 µeq/cm2 · h, a rate identical to net transepithelial Na+ absorption (see Fig. 1 for comparison). In contrast, the residual J<UP><SUB>ms</SUB><SUP>Na<SUP>+</SUP></SUP></UP> in the NHE3(-) jejunum was inhibited completely by both 1 and 100 µM EIPA. Treatment of either NHE(+) or NHE3(-) jejuna with forskolin/IBMX did not further increase the inhibition resulting from EIPA (-0.2 ± 0.4 and -0.1 ± 0.3 µeq/cm2 · h, respectively; not significant). Additionally, EIPA-sensitive J<UP><SUB>ms</SUB><SUP>Na<SUP>+</SUP></SUP></UP> was not different between NHE(+) and NHE2(-) jejuna for either 1 or 100 µM EIPA [1 µM EIPA: NHE(+) J<UP><SUB>EIPA-sensitive</SUB><SUP>Na<SUP>+</SUP></SUP></UP> -0.6 ± 0.6 µeq/cm2 · h (n = 6) vs. NHE2(-) J<UP><SUB>EIPA-sensitive</SUB><SUP>Na<SUP>+</SUP></SUP></UP> = 0.5 ± 0.6 µeq/cm2 · h (n = 10); 100 µM EIPA: NHE(+) J<UP><SUB>EIPA-sensitive</SUB><SUP>Na<SUP>+</SUP></SUP></UP> = 5.8 ± 0.6 µeq/cm2 · h (n = 6) vs. NHE2(-) J<UP><SUB>EIPA-sensitive</SUB><SUP>Na<SUP>+</SUP></SUP></UP> = 5.1 ± 0.8 µeq/cm2 · h (n = 6), not significant].


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Fig. 3.   Inhibition of J<UP><SUB>ms</SUB><SUP>Na<SUP>+</SUP></SUP></UP> across NHE(+) and NHE3(-) jejunum. Bars represent mean change in J<UP><SUB>ms</SUB><SUP>Na<SUP>+</SUP></SUP></UP> after treatment with 1 or 100 µM EIPA. * Significantly different from 1 µM EIPA treatment (P < 0.05). +Significantly different from NHE(+) (P < 0.05).

Expression of NHE2 and NHE3

Results from the 22Na flux experiments indicated that net Na+ absorption was not reduced in the absence of NHE2 [i.e., NHE2(-) vs. NHE(+) jejuna], whereas an amiloride-sensitive transporter may provide Na+ absorption in the absence of NHE3. To determine whether these differences resulted from altered expression of NHE2 or NHE3 in two NHE knockout mice, we compared mRNA expression of NHE2 and NHE3 using real-time quantitative RT-PCR in jejunal epithelial cells isolated from NHE2(-), NHE3(-), and wild-type sibling mice. In NHE2(-) jejunum, expression of NHE3 mRNA was not increased but, in fact, was slightly decreased relative to NHE2(+) jejunum (Fig. 4A). In the NHE3(-) jejunum, NHE2 mRNA expression was essentially unchanged relative to the NHE3(+) jejunum. The absence of significant changes in the mRNA expression of NHE2 or NHE3 in the various mouse genotypes confirms recent findings by Northern blot analyses (30).


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Fig. 4.   Expression of NHE2 and NHE3 transcripts in jejunal epithelium from NHE2(-), NHE2(+), NHE3(-), and NHE3(+) mice using real-time quantitative RT-PCR. A: comparison of NHE3 mRNA expression between NHE2(-) and NHE2(+) siblings (age and gender matched). B: comparison of NHE2 mRNA expression between NHE3(-) and NHE3(+) siblings (age and gender matched). Bars represent the mean quantity of NHE cDNA relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA (n = 5).


    DISCUSSION
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ABSTRACT
INTRODUCTION
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Recent information regarding the molecular physiology of epithelial NHEs requires integration with the function of NaCl and water absorption by the intestine. Mice with gene-targeted disruption of murine NHE2 or NHE3 provide powerful tools for studying the activity of the apical NHE isoforms in an intact intestinal epithelium. With the use of these animal models, we confirm earlier reports that NHE3 is the dominant NHE involved in vectorial Na+ transport across murine jejunum. In the absence of NHE3 activity, an "amiloride-sensitive" electroneutral process incompletely compensates for loss of Na+ absorption. In contrast, the absence of NHE2 activity in the intestine of NHE2(-) mice or in NHE(+) intestine treated with 1 µM EIPA did not affect the overall rate of net Na+ absorption. Interestingly, the net rate of transepithelial Cl- absorption, presumably mediated by anion exchange, was not affected by the loss of either NHE2 or NHE3.

Previous studies have established that in the absence of luminal nutrient solutes, the major mechanism of transepithelial Na+ absorption across the murine jejunum is electroneutral NHE activity (13). Present findings that support this supposition include 1) the net rate of Na+ absorption did not correlate with the Isc; 2) the amiloride analog EIPA did not significantly affect the Isc; and 3) EIPA, at a concentration (100 µM) known to inhibit both NHE2 and NHE3 in media containing physiological Na+ content (42), abolished net Na+ absorption and reduced the unidirectional absorptive flux of Na+ (J<UP><SUB>ms</SUB><SUP>Na<SUP>+</SUP></SUP></UP>) by an equivalent amount. Together with the demonstration that deletion of NHE3 greatly reduces net Na+ absorption, these observations indicate that the electroneutral process of Na+ absorption across murine jejunum is largely mediated by NHE activity.

The major disease phenotype of NHE3(-) mice is chronic diarrhea and sodium depletion (47); however, our in vitro studies demonstrate that the jejunum of NHE3(-) mice is able to absorb Na+ at ~30% of the net rate measured across NHE(+) jejunum. The residual Na+ absorption is likely an important factor modifying the survival of the NHE3(-) mice (47). The NHE3(-) intestine also demonstrated a significantly reduced transepithelial conductance under both basal and cAMPi-stimulated conditions compared with intestine from normal littermates. The reduced Gt may be secondary to Na+ malabsorption and reduced fluid accumulation within the lateral intercellular spaces (4, 26). Although the reduced Gt confounded analysis of the unidirectional Na+ fluxes, normalization to the estimated rate of paracellular Na+ movement indicated that the lower rate of net Na+ absorption in the NHE3(-) jejuna was matched exactly by a decrease in the unidirectional absorptive flux of Na+ (i.e., J<UP><SUB>ms</SUB><SUP>Na<SUP>+</SUP></SUP></UP>).

The residual Na+ absorption observed in the NHE3(-) intestine is of further interest because it may indicate a role for NHE2 in the absorption of salt and water by the intestine. Although NHE2 is expressed at high levels in the intestine (18, 40, 46) and has been localized to the brush border membrane (18, 22), the physiological role of NHE2 remains elusive. Evidence that favors a compensatory role for NHE2 includes the observations that the residual Na+ absorption in the NHE3(-) intestine was electroneutral and sensitive to 1 µM EIPA. Less likely are contributions by NHE1, which has been localized to the basolateral membrane of intestinal epithelium, and NHE4, an NHE isoform reported to be highly insensitive to EIPA (9). If NHE2 is the isoform responsible for residual Na+ absorption in the NHE3(-) jejunum, then the compensation apparently does not result from increased mRNA expression of the transporter (see Fig. 4). However, increased expression or activity of the protein might be expected if NHE2 is required to maintain intracellular pH or to offset the alkaline luminal conditions in the NHE3(-) intestine (47).

Nonetheless, the evidence suggesting that NHE2 contributes to intestinal Na+ absorption must be viewed with caution for a number of reasons. First, it is noteworthy that the compensatory Na+ absorption in the NHE3(-) intestine occurred under extreme physiological circumstances. The preponderance of evidence suggests that NHE2 contributes very little to transepithelial Na+ absorption across NHE3-expressing jejunum. Second, studies of other NHE2(-) epithelia, including the renal tubules (29), pancreatic ducts (31), and parotid gland (41), have shown little or no role for NHE2 in Na+ or fluid absorption despite its location in the apical membrane. Third, there is evidence of intestinal NHE activity that cannot be ascribed to the known NHEs. Congenital Na+ diarrhea is an inherited disease of defective NHE activity in the intestine but is not caused by mutations in NHE1-3 or 5 (37). Studies of colonic crypt epithelium by Rajendran et al. (45) have also shown functional evidence of a novel Cl--dependent NHE that is moderately sensitive to EIPA (IC50 1.1 µM). Fourth, recent studies of pancreatic and submandibular salivary duct epithelia from NHE-deficient mice have revealed an EIPA-sensitive Na+ absorptive mechanism (IC50 = 1.3 µM) that is not NHE2 or NHE3 but rather may be the activity of an electroneutral NaHCO3 cotransporter at the apical membrane (31, 32). The IC50 of both the Cl-dependent NHE and the NaHCO3 cotransporter are very similar to those (between 0.05 and 1 µM) reported for NHE2 (42, 45). Together, these observations raise questions regarding the role of NHE2 in intestinal Na+ absorption and highlight the need for additional investigations of compensatory Na+ transport mechanisms in the NHE3(-) intestine.

Previous studies have shown that electroneutral NaCl absorption across the small intestine often involves coupling of NHE activity with Cl- absorption via Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchangers (19, 25, 49, 54). The data in Table 1 show that 40-60% of net Na+ absorption could be coupled to Cl- absorption across the NHE(+) intestine. Surprisingly, knockout of either NHE2 or NHE3 did not affect the rate of net Cl- absorption, indicating that the processes of Cl- absorption are not strictly linked to the presence of either NHE. However, a requirement for simultaneous Na+ absorption with Cl- absorption could not be ruled out because a finite amount of Na+ absorption was present even in the NHE3(-) intestine. Furthermore, acute inhibition of Na+ transport activity with 100 µM EIPA abolished Cl- absorption in all genotypes. This finding is consistent with pharmacological studies that have used amiloride inhibition of NaCl absorption as evidence that intestinal Na+/H+ and Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchangers are functionally coupled by intracellular pH (17). The caveat is that these studies cannot discriminate between amiloride-sensitive NHEs and amiloride-sensitive NaHCO3 cotransporters. Thus the apparent inconsistency between the flux data on the NHE knockout intestine and the EIPA studies can be reconciled by postulating that net Cl- absorption may be functionally coupled to an Na+-dependent HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-loading mechanism (i.e., either NHE or NaHCO3 cotransport) that is required to drive apical membrane Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange.

Net Na+ absorption across NHE(+), NHE2(-), or NHE3(-) intestine was completely inhibited by acute treatment with cAMPi agonists, consistent with previous reports of murine intestine and polarized C2 intestinal epithelial cell monolayers (13, 35). However, these findings contrast with studies of recombinant NHE2 and NHE3 expressed in heterologous cell systems. The activity of recombinant NHE2 is either unaffected or increased after cAMPi stimulation (23), whereas the activity of recombinant NHE3 is incompletely inhibited (25-60% inhibited) by cAMPi/PKA treatment, an effect that may depend on coexpression with NHE-RF (55, 56). The present findings suggest that complete cAMPi inhibition of NHE (or apical membrane NaHCO3 cotransport) activity in intestinal mucosa may require tissue-specific mechanisms such as regulated membrane recycling and/or cytoskeletal associations (16, 24, 27, 38). Additionally, we have previously shown that complete inhibition of net Na+ absorption in murine intestine requires expression of CFTR (13). Interdependence between CFTR and NHE3 was also evident in the present study as shown by the reduced Isc response to forskolin in the NHE3(-) intestine. Thus additional studies of intact intestine will be necessary to elucidate the interaction between CFTR and inhibition of Na+ absorption during increased cAMPi.

From these studies, we confirm previous evidence that NHE3 is the dominant NHE responsible for transepithelial Na+ absorption across the murine jejunum. It was also shown that an electroneutral amiloride-sensitive mechanism exhibits a limited ability to compensate for loss of Na+ absorption in the isolated NHE3(-) jejunum. Although NHE2 would be an obvious candidate for compensation of Na+ absorption, mRNA expression of NHE2 was not altered in the NHE3(-) jejunum and NHE2 did not contribute to electroneutral Na+ absorption in NHE3(+) jejunum. Other candidate mechanisms for compensation would include either a novel NHE protein or an amiloride-sensitive NaHCO3 cotransport protein (whose activity would be indistinguishable from NHE by the methods used in the present study). Interestingly, 36Cl flux measurements indicated that neither NHE2 nor NHE3 were required for net Cl- absorption but that apical membrane treatment with EIPA abolished net Cl- absorption. Thus coupling between apical NHE and the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (or OH-) exchange appears to be more functional than isoform dependent. Stimulation of cAMPi also completely inhibited net NaCl absorption in both NHE2(-) and NHE3(-) jejuna. However, this finding contrasts somewhat with the reported effect of cAMPi on recombinant NHE2 and NHE3 in cell expression systems, suggesting that additional mechanisms of inhibition exist in the intact intestine. It is noteworthy therefore, that cAMPi-induced inhibition of Na+ absorption in the intact intestine occurs concurrently with activation of CFTR-mediated anion secretion. Activation of CFTR expressed in the villus epithelium (2, 51) may induce cellular changes that play a role in cAMPi inhibition of intestinal NaCl absorption.


    ACKNOWLEDGEMENTS

The authors gratefully acknowledge the expert technical assistance of M. Harline and T. Parks.


    FOOTNOTES

The study was supported by grants from the Univ. of Missouri College of Veterinary Medicine Committee on Research, National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-48816 (to L. L. Clarke) and DK-50594 (to G. E. Shull), and the American Heart Association fellowship 9910104Z (to L. R. Gawenis).

Address for reprint requests and other correspondence: L. L. Clarke, 324D Dalton Cardiovascular Research Center, Research Park, Univ. of Missouri-Columbia, Columbia, MO 65211 (E-mail: clarkel{at}missouri.edu).

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.

10.1152/ajpgi.00297.2001

Received 6 July 2001; accepted in final form 26 November 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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