1 Department of Molecular Genetics, Biochemistry, and Microbiology and 2 Department of Environmental Health, University of Cincinnati College of Medicine, Cincinnati, Ohio, 45267; and 3 Department of Biological Sciences, Northern Kentucky University, Highland Heights, Kentucky 41099
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ABSTRACT |
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Absorption and
secretion of ions in gastrointestinal and other epithelial tissues
require the concerted activities of ion pumps, channels, symporters,
and exchangers, which operate in coupled systems to mediate
transepithelial transport. Our understanding of the identities,
membrane locations, and biochemical activities of epithelial ion
transporters has advanced significantly in recent years, but major gaps
and uncertainties remain in our understanding of their physiological
functions. Increasingly, this problem is being addressed by the
analysis of mutant mouse models developed by gene targeting. In this
review, we discuss gene knockout studies of the secretory isoform of
the Na+-K+-2Cl
cotransporter, isoforms 1, 2, and 3 of the
Na+/H+ exchanger, and the colonic
H+-K+-ATPase. This approach is leading to a
clearer understanding of the functions of these transporters in the
living animal.
embryonic stem cells; acid secretion; chloride secretion; sodium absorption; potassium absorption
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INTRODUCTION |
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THE PROPER FUNCTIONING of the gastrointestinal tract is critically dependent on the concerted activities of ion transport proteins located on apical and basolateral membranes of gastric and intestinal epithelial cells. In the stomach, these transporters mediate HCl and KCl secretion by the gastric parietal cell and contribute to the maintenance of epithelial cell viability (18, 19, 22). In the small intestine they maintain the appropriate fluidity and composition of the intestinal contents necessary for nutrient absorption, and in the colon they function in the recovery of fluid, electrolytes, and acid-base equivalents (12). A major research challenge is to determine the identities, in vivo physiological roles, and functional coupling of the apical and basolateral transporters that mediate absorption and secretion of ions.
For many years, the principal means of studying epithelial ion transport was to examine cells, tissues, and the intact animal using a variety of physiological procedures. These studies, in conjunction with biochemical analyses, have led to important insights about the relevant ion transport proteins. With the development of molecular biological techniques, major efforts were directed toward the task of cloning and characterizing these transporters. This approach yielded their primary structures, often revealed the existence of multiple isoforms, and provided the necessary reagents for further studies of their biochemical characteristics, tissue distributions, and membrane locations. More recently, the use of gene targeting technology has made it possible to directly test hypotheses regarding the physiological functions of these transporters in vivo.
The first of the epithelial ion transporters for which a gene knockout
model was prepared was the cystic fibrosis transmembrane conductance
regulator (CFTR) (27). As a result of many studies using CFTR-deficient
mice, a great deal is known about its physiological role and its
relative importance within the larger ensemble of apical and
basolateral transporters of which it is a part. During the past two
years, gene knockout models have been prepared for several additional
transporters that are thought to play direct or indirect roles in
absorption and secretion of ions in the gastrointestinal tract. These
include the secretory isoform of the
Na+-K+-2Cl cotransporter
(NKCC1) (6, 8), isoforms 1, 2, and 3 of the Na+/H+ exchanger (NHE1, NHE2, and NHE3) (1, 4,
24, 25), and the colonic H+-K+-ATPase (cHKA)
(16). Here we review the results of these recent gene knockout studies
and discuss the insights that have been gained regarding the functions
of these transporters in absorption and secretion in the
gastrointestinal tract.
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SECRETORY NA+-K+-2CL![]() |
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NKCC1, the basolateral or secretory isoform of the
Na+-K+-2Cl cotransporter, is
a member of a family of cation-coupled Cl
cotransporters that includes the thiazide-sensitive
Na+-Cl
cotransporter, the apical
Na+-K+-2Cl
cotransporter of
the renal thick ascending limb, and several K+-Cl
cotransporters (17). Typically
viewed as a basolateral Cl
uptake system involved in
Cl
secretion in the intestine (3), stomach (14), and
other organs (Fig. 1), NKCC1 also provides
a leak pathway for Na+ needed to drive the
Na+-K+-ATPase activity that maintains the
electrical driving force for anion secretion. In addition, there is
evidence that K+ uptake via NKCC1 may contribute to
K+ secretion into the endolymph in the inner ear (6, 8) and that it might play a role in K+ secretion by a subset of
gastric parietal cells (14).
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Ussing chamber studies of intestinal segments and airway epithelial
cells of CFTR-deficient (Cftr/
) mice
showed that cAMP-stimulated short-circuit currents
(ISC) were absent (3), thereby demonstrating an
essential role for the CFTR in anion secretion. Bumetanide, an
inhibitor of NKCC1, significantly reduced the cAMP-stimulated
ISC in wild-type tissues but had no effect in
tissues from Cftr
/
mice. This
indicated that NKCC1 is an important basolateral component of the ion
transport pathways mediating cAMP-stimulated secretion via the apical CFTR.
To study the physiological functions of NKCC1 in more detail, null
mutant (Nkcc1/
) mice were prepared
and analyzed (6, 8). Nkcc1
/
mice
exhibited growth retardation before weaning, and ~25-30% of the
mutants died around the time of weaning. The cause of death was
unclear, although there was a high incidence of bleeding in the
intestine and a few cases of intestinal blockage were observed. Mutants
that survived beyond this critical period grew well and appeared to be
relatively healthy, although they had reduced blood pressure and a
balance disorder and an analysis of auditory brain stem responses
showed them to be profoundly deaf (8). Histological studies of the
inner ear revealed a collapse of the membranous labyrinth in both the
vestibular and auditory systems, indicating a severe impairment in the
generation of the K+-rich endolymph (6, 8). In the living
animal, the lack of NKCC1 did not cause a severe secretory defect in
the intestine such as that observed in
Cftr
/
mice (27), because newborn
Nkcc1
/
mice exhibited no evidence of
meconium ileus and older mutants did not show a high incidence of
intestinal blockage (8). Furthermore, treatment with the heat-stable
enterotoxin STa revealed a robust secretory response in the intestine
of 4- to 5-day-old mutant mice. A secretory deficit was, however,
demonstrated in Ussing chamber studies of jejunum, cecum, and cultured
tracheal epithelial cells from adult
Nkcc1
/
mice (8). Maximal
cAMP-stimulated ISC in
Nkcc1
/
tissues was reduced to ~50%
of that in wild-type controls; nevertheless, basal currents were
similar in Nkcc1
/
and wild-type
samples and a substantial level of cAMP-stimulated secretion did occur.
Finally, secretion of gastric acid in stomachs of
Nkcc1
/
mice was unimpaired and the
morphology of the parietal cell appeared normal (8).
The profound deafness and collapse of the membranous labyrinth in
Nkcc1/
mice provided strong support
for the hypothesis that NKCC1, coupled with
Na+-K+-ATPase, is necessary for K+
secretion by marginal cells of the stria vascularis. In contrast to the
apparently essential role of NKCC1 in K+ secretion in the
inner ear, NKCC1 appears to serve an important, but less critical,
function in anion secretion in intestinal and tracheal epithelium. As
illustrated in Fig. 1A, Cl
uptake via NKCC1
on the basolateral membrane provides a substantial proportion of the
Cl
that is secreted by intestinal epithelial cells
during stimulation with cAMP. However, the high level of secretion that
remained in the absence of NKCC1 showed that other basolateral
transport mechanisms must contribute to cAMP-stimulated secretion.
These are likely to include coupled Na+/H+ and
Cl
/HCO
3 exchange
(Fig. 1A) and could also include Na+-HCO
3 cotransport if a
significant component of the anion current is due to transepithelial
HCO
3 transport, as suggested by a
recent study using Cftr
/
mice (11).
The role of NKCC1 in the gastric parietal cell (Fig. 1B)
remains unclear, but the lack of a deficit in acid secretion and the
unperturbed morphology are consistent with the view that NKCC1 is
expressed in older parietal cells located in deeper regions of the
gland, which secrete primarily KCl rather than HCl (14).
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NA+/H+ EXCHANGERS |
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Of the five known plasma membrane Na+/H+
exchangers, which mediate H+ efflux and Na+
influx, four (NHE1-NHE4) are present in epithelial tissues (21). NHE1 is expressed in all mammalian tissues and is located on the basolateral membranes of epithelial cells. NHE2 is expressed at high
levels in both the stomach, where it is probably restricted to
basolateral membranes, and in the intestinal tract, where it is
localized on apical membranes (9). NHE3 is abundant in the brush-border
membranes of both intestinal and renal proximal tubule epithelial cells
(2, 9). NHE4 is expressed at high levels in stomach and at low levels
in kidney and several other organs. The basolateral
Na+/H+ exchangers are generally thought to be
involved in intracellular pH and cell volume homeostasis and can also
function as components of the basolateral transport systems needed for
anion secretion (Fig. 1). The major physiological functions of apical
Na+/H+ exchangers are the absorption of
NaHCO3 and, when coupled with an apical
Cl/HCO
3 exchanger
(Fig. 2A), the absorption of NaCl.
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NHE1 knockout.
The phenotype of mice lacking NHE1 has been studied using both a
naturally occurring null mutant, termed swe for slow-wave epilepsy (4), and mice generated by gene targeting (1). NHE1-deficient
(Nhe1/
) mice were born in a normal
Mendelian ratio but exhibited ataxia and growth retardation, and
~
of the mutants died from epileptic seizures shortly
before or after weaning. The only indication of a perturbation of
gastrointestinal function was mild atrophy of the glandular mucosa and
a thickening of the lamina propria (1). This suggested either that NHE1
serves a relatively minor function in stomach or that NHE2 and/or NHE4 largely compensate for the absence of NHE1. The functions of NHE1 in
the stomach and intestine (Fig. 1), however, have not yet been rigorously examined using the Nhe1
/
mouse.
NHE2 knockout.
Because NHE2 is present at high levels in stomach, small intestine, and
colon, a major gastrointestinal phenotype was expected in
NHE2-deficient (Nhe2/
) mice. NHE2
colocalizes with NHE3 on brush-border membranes of intestinal
epithelium, suggesting that it might serve an absorptive role (9). In
stomach, NHE2 is thought to be expressed on basolateral membranes of
parietal, zymogenic, and mucous cells (discussed in Ref. 24). It seemed
possible that NHE2 might function in parietal cells as a component of
the coupled Na+/H+ and
Cl
/HCO
3 exchange
that has been postulated to provide much of the basolateral ion
transport activity required for acid secretion across the apical
membrane (18, 19, 22) and/or that it might function in the maintenance
of cell viability by regulating cell volume or pH homeostasis (Fig.
1B).
NHE3 knockout.
Before the development of the NHE3-deficient
(Nhe3/
) mouse, there was a
substantial body of information indicating that NHE3 was the major
absorptive Na+/H+ exchanger in both intestine
and kidney. Thus absorptive defects in the intestine and in the renal
proximal tubule of Nhe3
/
mice were
anticipated. Nevertheless, the relative importance of NHE3 was unclear
because NHE2 is also expressed in both organs and, as mentioned above,
in the intestine it colocalizes with NHE3 in brush-border membranes
(9). Thus when these studies were initiated it seemed possible that the
activity of either NHE2 or NHE3 might be sufficient to provide a
relatively normal absorptive capacity in the intestine.
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COLONIC H+-K+-ATPASE |
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Strong evidence for the existence of an H+-K+-ATPase that might mediate H+ secretion and K+ absorption in apical membranes of colonic epithelial cells came from Ussing chamber studies demonstrating the existence of K+-dependent H+ secretion in guinea pig distal colon that was Na+ independent and sensitive to both ouabain and vanadate (28). cDNA cloning studies revealed that cHKA is closely related to the gastric H+-K+-ATPase and Na+-K+-ATPase (5), and immunolocalization studies showed that it is expressed on apical membranes of surface epithelial cells in distal colon (10). cHKA mRNA is normally expressed at high levels in colon and very low levels in kidney but is sharply induced in kidney when animals are maintained on a K+-depleted diet (7). These and other considerations (discussed in Ref. 16) suggested that cHKA mediates K+ conservation in both colon and kidney during dietary K+ restriction.
cHKA-deficient (cHKA/
) mice developed
by gene targeting (16) were born in a normal Mendelian ratio, grew as
well as their heterozygous and wild-type littermates, and appeared
healthy. To test the hypothesis that cHKA plays a role in
K+ conservation in colon and kidney, wild-type and
cHKA
/
mice were fed control and
K+-depleted diets, and urine and fecal samples were
collected and analyzed (16). During the control diet period, excretion
of K+ in the urine was similar in mice of both genotypes.
When the mice were fed a K+-depleted diet, urinary
K+ excretion was reduced to very low levels in mice of both
genotypes and there was no significant urinary K+ loss in
cHKA
/
mice relative to wild-type
controls. These results indicated that cHKA does not play a major role
in urinary K+ conservation, at least under the conditions
employed in this study.
In contrast to the results with urinary K+ excretion, fecal
K+ excretion was twice as great in
cHKA/
mice as in the wild-type
controls when the mice were maintained on a normal diet. When they were
fed the K+-depleted diet, fecal K+ excretion
was reduced in both groups, but the relative difference in fecal
K+ losses became greater, with
cHKA
/
mice losing four times as much
K+ as wild-type mice. Because of the excess fecal
K+ excretion, hypokalemia was more severe in null mutant
mice, as indicated by lower K+ levels in both plasma and
muscle. These data demonstrated that cHKA in colon plays an important
role in the maintenance of K+ homeostasis in vivo during
dietary K+ depletion, because the greater degree of
hypokalemia observed in cHKA
/
mice
was due almost entirely to the reduced ability of the large intestine
to recover K+.
On the basis of these data, it is clear that the major physiological
function of cHKA in the colon is to recover K+ from the
intestinal contents. However, questions remain regarding the various
apical transporters with which it is coupled and the full range of its
physiological functions in colon (Fig. 2B). During
K+ depletion, the activity of cHKA alone on the apical
membrane would mediate K+ and
HCO3 absorption and, if coupled with
the apical Cl
/HCO
3
exchanger, cHKA would mediate KCl absorption. Kaunitz et al. (12)
suggested that a major function of cHKA might be K+
recycling during electrogenic Na+ reabsorption, in which
case it might be required to maintain efficient NaCl absorption during
dietary NaCl depletion. If so, then cHKA would probably be coupled with
the epithelial Na+ channel, an apical K+
channel, and the apical
Cl
/HCO
3 exchanger
(Fig. 2B). The observation that cHKA mRNA is upregulated in
colon by dietary Na+ depletion (23) is consistent with this
hypothesis. Finally, the massive induction of cHKA mRNA that was
observed in the colon of Nhe3
/
mice
(25) suggests that an additional function of cHKA is to serve as an
inducible system that can absorb HCO
3 (via H+ secretion) during diarrheal states. In such a
situation, functional coupling with a basolateral
HCO
3 extrusion system, such as the
AE2
Cl
/HCO
3 exchanger,
would be required.
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CONCLUDING REMARKS |
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As illustrated by the studies discussed here, the combined use of gene targeting and classical physiological techniques is significantly advancing our understanding of the in vivo functions of epithelial ion transporters. The phenotype of mice lacking a specific transporter provides an important experimental test of hypotheses and previous conclusions or assumptions regarding the physiological functions of the transporter. In addition, it often reveals compensatory mechanisms that may be highly informative about other proteins, including transporters, that contribute to the same physiological process or to another process that is affected by the loss of the transporter. The increase in the number of mouse models and advances in the sophistication of the procedures for assessing their phenotypes that will undoubtedly occur in the coming years will contribute to the development of a comprehensive understanding of ion absorption and secretion in the gastrointestinal tract and the mechanisms by which these processes are regulated.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institutes of Health Grants DK-50594, HL-41496, and ES 06096.
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FOOTNOTES |
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* Eighth in a series of invited articles on Lessons From Genetically Engineered Animal Models.
Address for reprint requests and other correspondence: G. E. Shull, Dept. of Molecular Genetics, Biochemistry, and Microbiology, Univ. of Cincinnati College of Medicine, 231 Bethesda Ave., ML 524, Cincinnati, OH 45267-0524 (E-mail: shullge{at}ucmail.uc.edu).
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