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
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ABSTRACT |
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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
) 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
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INTRODUCTION |
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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
) 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.
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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(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 = JmsSamples 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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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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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
) 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
Isc during the 30 min immediately following
forskolin/IBMX. In contrast, the NHE2(
) jejuna had a slightly greater
peak
Isc compared with NHE(+) jejuna [NHE(+)
Isc = 132.5 ± 3.5 µA/cm2, n = 93; NHE2(
)
Isc = 150.3 ± 5.4 µA/cm2, n = 48; NHE3(
)
Isc = 82.6 ± 4.0 µA/cm2, n = 65; P < 0.05 for all pairwise comparisons].
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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
absorption across the jejunum from all mouse groups,
indicating inhibition of a coupled NaCl absorptive process.
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EIPA-Sensitive J)
Jejunum
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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(
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DISCUSSION |
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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
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
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
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
absorption may be functionally coupled to an
Na+-dependent HCO
/HCO
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
) 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.
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ACKNOWLEDGEMENTS |
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The authors gratefully acknowledge the expert technical assistance of M. Harline and T. Parks.
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FOOTNOTES |
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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.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Akhter, S,
Cavet ME,
Tse CM,
and
Donowitz M.
C-terminal domains of Na+/H+ exchanger isoform 3 are involved in the basal and serum-stimulated membrane trafficking of the exchanger.
Biochemistry
39:
1990-2000,
2000[ISI][Medline].
2.
Ameen, NA,
Ardito T,
Kashgarian M,
and
Marino CR.
A unique subset of rat and human intestinal villus cells express the cystic fibrosis transmembrane conductance regulator.
Gastroenterology
108:
1016-1023,
1995[ISI][Medline].
3.
Berschneider, HM,
Knowles MR,
Azizkhan RG,
Boucher RC,
Tobey NA,
Orlando RC,
and
Powell DW.
Altered intestinal chloride transport in cystic fibrosis.
FASEB J
2:
2625-2629,
1988
4.
Blikslager, AT,
Roberts MC,
and
Argenzio RA.
Prostaglandin-induced recovery of barrier function in porcine ileum is triggered by chloride secretion.
Am J Physiol Gastrointest Liver Physiol
276:
G28-G36,
1999
5.
Bookstein, C,
Xie Y,
Rabenau K,
Musch MW,
McSwine RL,
Rao MC,
and
Chang EB.
Tissue distribution of Na+/H+ exchanger isoforms NHE2 and NHE4 in rat intestine and kidney.
Am J Physiol Cell Physiol
273:
C1496-C1505,
1997[ISI][Medline].
6.
Brant, SR,
Yun CHC,
Donowitz M,
and
Tse CM.
Cloning, tissue distribution, and functional analysis of the human Na+/H+ exchanger isoform, NHE3.
Am J Physiol Cell Physiol
269:
C198-C206,
1995
7.
Bukhave, K,
and
Rask-Madsen J.
Saturation kinetics applied to in vitro effects of low prostaglandin E2 and F2 concentrations on ion transport across human jejunal mucosa.
Gastroenterology
78:
32-42,
1980[ISI][Medline].
8.
Carey, HV,
and
Cooke HJ.
Neuromodulation of intestinal transport in the suckling mouse.
Am J Physiol Regulatory Integrative Comp Physiol
256:
R481-R486,
1989
9.
Chambrey, R,
Achard JM,
and
Warnock D.
Heterologous expression of rat NHE4: a highly amiloride-resistant Na+/H+ exchanger isoform.
Am J Physiol Cell Physiol
272:
C90-C98,
1997
10.
Clarke, LL,
and
Argenzio RA.
NaCl transport across equine proximal colon and the effect of endogenous prostenoids.
Am J Physiol Gastrointest Liver Physiol
259:
G62-G69,
1990
11.
Clarke, LL,
Gawenis LR,
Franklin C,
and
Harline MC.
Increased survival of CFTR knockout mice with an oral osmotic laxative.
Lab Anim Sci
46:
612-618,
1996[Medline].
12.
Clarke, LL,
Grubb BR,
Gabriel SE,
Smithies O,
Coller BH,
and
Boucher RC.
Defective epithelial chloride transport in a gene-targeted mouse model of cystic fibrosis.
Science
257:
1125-1128,
1992[ISI][Medline].
13.
Clarke, LL,
and
Harline MC.
CFTR is required for cAMP inhibition of intestinal Na+ absorption in a cystic fibrosis mouse model.
Am J Physiol Gastrointest Liver Physiol
270:
G259-G267,
1996
14.
Collins, JF,
Xu H,
Kiela PR,
Zeng J,
and
Gishan FK.
Ontogeny of basolateral membrane sodium-hydrogen exchange (NHE) activity and mRNA expression of NHE-1 and NHE-4 in rat kidney and jejunum.
Biochim Biophys Acta
1369:
247-258,
1998[ISI][Medline].
15.
Counillon, L,
and
Pouyssegur J.
The expanding family of eucaryotic Na+/H+ exchangers.
J Biol Chem
275:
1-4,
2000
16.
D'souza, S,
Garcia-Cabado A,
Yu F,
Teter K,
Lukacs G,
Skorecki K,
Moore HP,
Orlowski J,
and
Grinstein S.
The epithelial sodium-hydrogen antiporter Na+/H+ exchanger 3 accumulates and is functional in recycling endosomes.
J Biol Chem
273:
2035-2043,
1998
17.
Donowitz, M,
and
Welsh MJ.
Regulation of mammalian small intestinal electrolyte secretion.
In: Physiology of the Gastrointestinal Tract, edited by Johnson LR.. New York: Raven, 1987, p. 1351-1388.
18.
Dudeja, PK,
Rao D,
Syed I,
Joshi V,
Dahdal RY,
Gardner C,
Risk MC,
Schmidt L,
Bavishi D,
Kim KE,
Harig JM,
Goldstein JL,
Layden TJ,
and
Ramaswamy K.
Intestinal distribution of human Na+/H+ exchanger isoforms NHE-1, NHE-2, and NHE-3 mRNA.
Am J Physiol Gastrointest Liver Physiol
271:
G483-G493,
1996
19.
Field, M,
Fromm M,
and
McColl I.
Ion transport in rabbit ileal mucosa. I. Na and Cl fluxes and short-circuit current.
Am J Physiol
220:
1388-1396,
1971[ISI][Medline].
20.
Frizzell, RA,
and
Schultz SG.
Ionic conductances of extracellular shunt pathway in rabbit ileum: influence of shunt on transmural sodium transport and electrical potential differences.
J Gen Physiol
59:
318-337,
1972
21.
Ghishan, FK,
Knobel S,
Barnard JA,
and
Breyer M.
Expression of a novel sodium-hydrogen exchanger in the gastrointestinal tract and kidney.
J Membr Biol
144:
267-271,
1995[ISI][Medline].
22.
Hoogerwerf, WA,
Tsao SC,
Devuyst O,
Levine SA,
Yun CHC,
Yip JW,
Cohen ME,
Wilson PD,
Lazenby AJ,
Tse CM,
and
Donowitz M.
NHE2 and NHE3 are human and rabbit intestinal brush-border proteins.
Am J Physiol Gastrointest Liver Physiol
270:
G29-G41,
1996
23.
Kandasamy, R,
Yu F,
Harris R,
Boucher A,
Hanrahan JW,
and
Orlowski J.
Plasma membrane Na+/H+ exchanger isoforms (NHE-1, -2, and -3) are differentially responsive to second messenger agonists of the protein kinase A and C pathways.
J Biol Chem
270:
29209-29216,
1995
24.
Khurana, S,
Arpin M,
Patterson R,
and
Donowitz M.
Ileal microvillar protein villin is tyrosine-phosphroylated and associates with PLC-1.
J Biol Chem
272:
30115-30121,
1997
25.
Knickelbein, R,
Aronson PS,
Schron CM,
Seifter J,
and
Dobbins JW.
Sodium and chloride transport across rabbit ileal brush border. II. Evidence for Cl/HCO3 exchange and mechanism of coupling.
Am J Physiol Gastrointest Liver Physiol
249:
G236-G245,
1985
26.
Kottra, G,
Haase W,
and
Fromter E.
Tight-junction tightness of Necturus gall bladder epithelium is not regulated by cAMP or intracellular Ca2+. I. Microscopic and general electrophysiological observations.
Pflügers Arch
425:
528-534,
1993[ISI][Medline].
27.
Kurashima, K,
D'souza S,
Szaszi K,
Ramjeesingh R,
Orlowski J,
and
Grinstein S.
The apical membrane Na+/H+ exchanger isoform NHE3 is regulated by the actin cytoskeleton.
J Biol Chem
274:
29843-29849,
1999
28.
Kurashima, K,
Yu F,
Cabado AG,
Szabo EZ,
Grinstein S,
and
Orlowski J.
Identification of sites required for down-regulation of Na+/H+ exchanger NHE3 activity by cAMP-dependent protein kinase.
J Biol Chem
272:
28672-28679,
1997
29.
Ledoussal, C,
Lorenz JN,
Nieman ML,
Soleimani M,
Schultheis PJ,
and
Shull GE.
Renal salt-wasting in mice lacking the NHE3 Na+/H+ exchanger, but not in mice lacking NHE2.
Am J Physiol Renal Physiol
281:
F718-F727,
2001
30.
Ledoussal, C,
Woo AL,
Miller ML,
and
Shull GE.
Loss of the NHE2 Na+/H+ exchanger has no apparent effect on the diarrheal state of NHE3-deficient mice.
Am J Physiol Gastrointest Liver Physiol
281:
G1385-G1396,
2001
31.
Lee, MG,
Ahn W,
Choi JY,
Luo X,
Seo JT,
Schultheis PJ,
Shull GE,
Kim KH,
and
Muallem S.
Na+-dependent transporters mediate HCO
32.
Luo, X,
Choi JY,
Ko SBH,
Pushkin A,
Kurtz I,
Ahn W,
Lee MG,
and
Muallem S.
HCO
33.
Maher, MM,
Gontarek JD,
Bess RS,
Donowitz M,
and
Yeo CJ.
The Na+/H+ exchange isoform NHE3 regulates basal canine ileal sodium absorption in vivo.
Gastroenterology
112:
174-183,
1997[ISI][Medline].
34.
Maher, MM,
Gontarek JD,
Jimenez RE,
Donowitz M,
and
Yeo CJ.
Role of brush border Na+/H+ exchange in canine ileal absorption.
Dig Dis Sci
41:
651-659,
1996[ISI][Medline].
35.
McSwine, RL,
Musch MW,
Bookstein C,
Xie Y,
Rao M,
and
Chang EB.
Regulation of apical membrane Na+/H+ exchangers NHE2 and NHE3 in intestinal epithelial cell line C2/bbe.
Am J Physiol Cell Physiol
275:
C693-C701,
1998[Abstract].
36.
Moe, OW,
Amemiya M,
and
Yamaji Y.
Activation of protein kinase A acutely inhibits and phosphorylates Na/H exchanger NHE3.
J Clin Invest
92:
2187-2194,
1995.
37.
Muller, T,
Wijmenga C,
Phillips AD,
Janecke A,
Houwen RHJ,
Fischer H,
Ellemunter H,
Fruhwirth M,
Offner F,
Hofer S,
Muller W,
Booth IW,
and
Heinz-Erian P.
Congenital sodium diarrhea is an autosomal recessive disorder of sodium/proton exchange but unrelated to known candidate genes.
Gastroenterology
119:
1506-1513,
2000[ISI][Medline].
38.
Nath, SK,
Hang CY,
Levine SA,
Yun CHC,
Montrose MH,
Donowitz M,
and
Tse CM.
Hyperosmolarity inhibits the Na+/H+ exchanger isoforms NHE2 and NHE3: an effect opposite to that on NHE1.
Am J Physiol Gastrointest Liver Physiol
270:
G431-G441,
1996
39.
O'Loughlin, EV,
Hunt DM,
Gaskin KJ,
Stiel D,
Bruzuszcak LM,
Martine HCO,
Bambrach C,
and
Smith A.
Abnormal epithelial transport in cystic fibrosis jejunum.
Am J Physiol Gastrointest Liver Physiol
260:
G758-G763,
1991
40.
Orlowski, J,
Kandasamy RA,
and
Shull GE.
Molecular cloning of putative members of the Na/H exchanger gene family.
J Biol Chem
267:
9331-9339,
1992
41.
Park, K,
Evans RL,
Watson GE,
Nehrke K,
Richardson L,
Bell SM,
Schultheis PJ,
Hand AR,
Shull GE,
and
Melvin JE.
Defective fluid secretion and NaCl absorption in the parotid glands of Na+/H+ exchanger-deficient mice.
Am J Physiol Cell Physiol
274:
C1261-C1272,
1998
42.
Park, K,
Olschowsk JA,
Richardson LA,
Bookstein C,
Chang EB,
and
Melvin JE.
Expression of multiple Na+/H+ exchanger isoforms in rat parotid acinar and ductal cells.
Am J Physiol Gastrointest Liver Physiol
276:
G470-G478,
1999
43.
Powell, DW.
Intestinal conductance and permselectivity changes with theophylline and choleragen.
Am J Physiol
227:
1436-1443,
1974[ISI][Medline].
44.
Powell, DW.
Intestinal water and electrolyte transport.
In: Physiology of the Gastrointestinal Tract, edited by Johnson LR.. New York: Raven, 1987, p. 1267-1306.
45.
Rajendran, VM,
Geibel JP,
and
Binder HJ.
Characterization of apical membrane Cl-dependent Na/H exchange in crypt cells of rat distal colon.
Am J Physiol Gastrointest Liver Physiol
280:
G400-G405,
2001
46.
Schultheis, PJ,
Clarke LL,
Meneton P,
Harline MC,
Boivin GP,
Stemmermann G,
Duffy JJ,
Doetschman T,
Miller ML,
and
Shull GE.
Targeted disruption of the murine Na+/H+ exchanger isoform 2 gene causes reduced viability of gastric parietal cells and loss of net acid secretion.
J Clin Invest
101:
1243-1253,
1998
47.
Schultheis, PJ,
Clarke LL,
Meneton P,
Miller ML,
Soleimani M,
Gawenis LR,
Riddle TM,
Duffy JJ,
Doetschman T,
Wang T,
Giebisch G,
Aronson PS,
Lorenz JN,
and
Shull GE.
Renal and intestinal absorptive defects in mice lacking the NHE3 Na+/H+ exchanger.
Nat Genet
19:
282-285,
1998[ISI][Medline].
48.
Schultz, SG,
and
Zalusky R.
Ion transport in isolated rabbit ileum. I. Short-circuit current and Na fluxes.
J Gen Physiol
47:
567-584,
1964
49.
Sellin, JS,
and
Desoignie R.
Rabbit proximal colon: a distinct transport epithelium.
Am J Physiol Gastrointest Liver Physiol
246:
G603-G610,
1984
50.
Sheldon, RJ,
Malarchik ME,
Fox DA,
Burks TF,
and
Porreca F.
Pharmacological characterization of neural mechanisms regulating mucosal ion transport in mouse jejunum.
J Pharmacol Exp Ther
249:
572-582,
1988[Abstract].
51.
Strong, TV,
Boehm K,
and
Collins FS.
Localization of cystic fibrosis transmembrane conductance regulator mRNA in the human gastrointestinal tract by in situ hybridization.
J Clin Invest
93:
347-354,
1994[ISI][Medline].
52.
Trezise, AE,
and
Buchwald M.
In vivo cell-specific expression of the cystic fibrosis transmembrane conductance regulator.
Nature
353:
434-437,
1991[ISI][Medline].
53.
Tse, CM,
Ma AI,
Yang VW,
Watson AJM,
Levine SA,
Montrose MH,
Potter J,
Sardet C,
Pouyssegur J,
and
Donowitz M.
Cloning and sequencing of a rabbit cDNA encoding an intestinal and kidney-specific Na+/H+ exchanger isoform (NHE-3).
J Biol Chem
267:
9340-9346,
1992
54.
Turnberg, LA,
Bieberdorf FA,
Morawski SG,
and
Fordtran J.
Interrelationship of chloride, bicarbonate, sodium and hydrogen transport in human ileum.
J Clin Invest
49:
557-567,
1970[Medline].
55.
Yun, CHC,
Oh S,
Zizak M,
Steplock D,
Tsao SC,
Tse CM,
Weinman EJ,
and
Donowitz M.
cAMP-mediated inhibition of the epithelial brush border Na+/H+ exchanger, NHE3, requires an associated regulatory protein.
Proc Natl Acad Sci USA
94:
3010-3015,
1997
56.
Zizak, M,
Lamprecht G,
Steplock D,
Tariq N,
Shenolikar S,
Donowitz M,
Yun CHC,
and
Weinman EJ.
cAMP-induced phosphorylation and inhibition of Na+/H+ exchanger 3 (NHE3) are dependent on the presence but not the phosphorylation of NHE regulatory factor.
J Biol Chem
274:
24753-24758,
1999