THEMES
Lessons from Genetically Engineered Animal Models
VIII. Absorption and secretion of ions in the gastrointestinal tract*

Gary E. Shull1, Marian L. Miller2, and Patrick J. Schultheis3

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
SECRETORY NA+-K+-2CL-...
NA+/H+ EXCHANGERS
COLONIC H+-K+-ATPASE
CONCLUDING REMARKS
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
SECRETORY NA+-K+-2CL-...
NA+/H+ EXCHANGERS
COLONIC H+-K+-ATPASE
CONCLUDING REMARKS
REFERENCES

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.


    SECRETORY NA+-K+-2CLminus COTRANSPORTER
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ABSTRACT
INTRODUCTION
SECRETORY NA+-K+-2CL-...
NA+/H+ EXCHANGERS
COLONIC H+-K+-ATPASE
CONCLUDING REMARKS
REFERENCES

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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Fig. 1.   Transporters required for ion secretion in gastrointestinal tract. A: cAMP-stimulated secretion of Cl- (and possibly HCO-3) by intestinal epithelial cells is mediated by cystic fibrosis transmembrane conductance regulator (CFTR). Na+-K+-2Cl- cotransporter NKCC1 mediates uptake of Cl-, K+, and Na+ across basolateral membrane. Na+-K+-ATPase extrudes Na+ and generates electrical potential needed for anion secretion. A basolateral K+ channel recycles K+. Additional basolateral uptake of anions and Na+ may occur via coupled Na+/H+ and Cl-/HCO-3 exchange and/or by Na+-HCO-3 cotransport. B: Secretion of H+, K+, and Cl- across apical membrane of gastric parietal cells is mediated by H+-K+-ATPase, a K+ channel, and a Cl- channel. Basolateral Cl-/HCO-3 exchange provides Cl- uptake and HCO-3 extrusion needed to balance HCl secretion. NKCC1 and coupled Na+/H+ and Cl-/HCO-3 exchange can mediate uptake of Na+ needed to drive Na+-K+-ATPase and additional Cl- needed for KCl secretion. Both Na+-K+-ATPase and NKCC1 can mediate uptake of K+ needed for secretion.

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).


    NA+/H+ EXCHANGERS
TOP
ABSTRACT
INTRODUCTION
SECRETORY NA+-K+-2CL-...
NA+/H+ EXCHANGERS
COLONIC H+-K+-ATPASE
CONCLUDING REMARKS
REFERENCES

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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Fig. 2.   Transporters required for ion absorption in intestinal tract. A: NaCl absorption across apical membranes in small intestine and colon is mediated by coupled Na+/H+ and Cl-/HCO-3 exchange. On basolateral membranes, Na+ is extruded by Na+-K+-ATPase and K+ and Cl- exit via channels. B: NaCl absorption across apical membranes in colon may be mediated in part by combined activities of epithelial Na+ channel, a K+ channel, colonic H+-K+-ATPase (cHKA), and a Cl-/HCO-3 exchanger. cHKA mediates K+ absorption directly and absorbs HCO-3 indirectly via H+ secretion. On basolateral membranes, Na+ is extruded by the Na+-K+-ATPase and K+ and Cl- exit via channels.

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 ~<FR><NU>2</NU><DE>3</DE></FR> 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).

Nhe2-/- mice were born in a normal Mendelian ratio and exhibited no evidence of diarrhea, which would have been a clear indication of an intestinal absorptive defect (24). In stomach, however, severe histopathology was observed in the gastric mucosa. By 17 days of age, the number of gastric parietal cells was sharply reduced in mutant mice, and it remained low throughout life. Despite the severe reduction in numbers, mature parietal cells were observed, and morphologically they appeared to be engaged in high levels of acid secretion. Other histological changes in NHE2-null mice were an increase in the number of inflammatory cells, hyperplasia of mucous cells in both corpus and antrum, an increased mitotic index, and a sharp increase in the number of degenerating parietal cells. Results from electron microscopic analyses and TUNEL assays indicated that parietal cell death was the result of necrosis rather than apoptosis.

To determine whether NHE2 is necessary for secretion of gastric acid, both the pH and acid-base equivalents in the stomach contents were measured after treatment with histamine, an acid secretogogue. In adult wild-type and heterozygous mice, gastric pH averaged ~3.1 and acid content averaged ~48 µeq/g wet wt, whereas gastric pH in homozygous mutants was ~7.8 and base content averaged 18 µeq/g wet wt. The pH of stomach contents from 18- to 19-day-old homozygous mutants, however, was only slightly greater (~4.1) than that of age-matched wild-type mice (~3.6). This result was surprising given the sharp reduction in the number of parietal cells and indicated that NHE2 is not strictly required for maintaining high levels of acid secretion by individual parietal cells. These data, however, do not rule out the possibility that NHE2 contributes to coupled Na+/H+ and Cl-/HCO-3 exchange needed for maximum acid secretion (Fig. 1B). Na+/H+ exchange on the basolateral membrane of the parietal cell may be due to the combined activities of NHE1, NHE2, and NHE4, with each contributing a portion of the activity needed for both acid secretion and maintenance of cell homeostasis. Thus the loss of any one isoform may perturb volume homeostasis but not be rate limiting for acid secretion, which is likely to be largely dependent on the H+-K+-ATPase. With the parietal cell in a volume-contracted state, upregulation of the other transporters may be sufficient to support high levels of acid secretion, although the viability of the cell might be severely perturbed.

The histological and ultrastructural analyses suggest that Nhe2-/- parietal cells develop normally but die shortly after they begin secreting acid, indicating that NHE2 is necessary for the long-term viability of the parietal cell. Interestingly, NHE2 has a high sensitivity to inhibition by extracellular protons and is upregulated by an increase in extracellular pH (30). Thus during acid secretion, when high levels of HCO-3 are being extruded across the basolateral membrane of the parietal cell, NHE2 should be upregulated. This unusual biochemical property of NHE2 would provide a powerful mechanism for maintaining volume homeostasis of the parietal cell during acid secretion and could contribute to the ion fluxes needed to maintain high levels of acid secretion. The observation that Na+/H+ exchange on the basolateral membrane of the parietal cell is upregulated by increased extracellular HCO-3 and pH (26) is consistent with this hypothesis. The increased alkalinity resulting from basolateral extrusion of HCO-3 by the acid-secreting parietal cell might also serve to upregulate NHE2 activity on the basolateral membrane of zymogenic and mucous cells, thereby protecting them from acid stress. However, because the lack of NHE2 leads to a sharp reduction in the number of parietal cells and achlorhydria, the knockout model did not allow a direct test of this hypothesis.

The hypothesis that NHE2 plays a major absorptive role in the intestine seems to have been negated, particularly in light of the severe intestinal absorptive defect in NHE3-deficient mice (25). Nevertheless, it is conceivable that NHE2 might serve an absorptive function under certain pathological conditions. As noted above, NHE2 is sensitive to extracellular pH and is upregulated by extracellular alkalinity (30). A consideration of these biochemical characteristics suggests that NHE2 would be relatively inactive at the extracellular pH occurring along the intestinal mucosa under normal conditions but might be activated under more basic conditions, such as those occurring in many diarrheal states. Because of its extracellular pH dependence, the activity of NHE2 should increase as the luminal contents become more alkaline. Although this would not prevent the diarrhea, it would lessen its severity, and the absorptive capacity provided by NHE2, which becomes fully activated at pH 9, would increase with the severity of disease.

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.

Nhe3-/- mice were born in a normal Mendelian ratio and grew well; however, they had a severe absorptive defect in the intestine, as indicated by diarrhea and an increase in the volume and alkalinity of the contents of all segments of the intestine (25). A number of apparent compensatory mechanisms were activated in the colon of NHE3-deficient mice. In distal colon, mRNAs encoding the beta - and gamma -subunits of the epithelial Na+ channel were upregulated and amiloride-sensitive Na+ currents, measured using voltage-clamped Ussing chambers, were sharply increased. cHKA mRNA was massively induced in both proximal and distal colon of NHE3-deficient mice (25), and mRNA encoding the downregulated in adenoma (DRA) protein, recently shown to be the apical Cl-/HCO-3 exchanger in the intestine, was moderately induced in colon (15).

Analysis of blood samples revealed that Nhe3-/- mice were slightly acidotic. This is consistent with a mild renal proximal tubular acidosis resulting from a deficit in HCO-3 reabsorption, although HCO-3 losses in the intestine would also contribute to the acidosis. Impaired reabsorption in the proximal tubule was confirmed by in situ microperfusion studies, which demonstrated that reabsorption of fluid and HCO-3 was reduced by ~60-70%. mRNA encoding the anion exchanger 1 (AE1) Cl-/HCO-3 exchanger was induced in kidney, suggesting that compensatory upregulation of HCO-3 reabsorption was occurring in the collecting duct. Although plasma Na+ concentrations were normal in Nhe3-/- mice, serum aldosterone levels and renin mRNA in kidney were sharply elevated and blood pressure was reduced. These data indicated that Na+-fluid volume homeostasis was impaired and that compensatory mechanisms that increase Na+ reabsorption were activated.

More detailed studies of the renal phenotype of NHE3-deficient mice have recently been reported (13, 20, 29). Micropuncture studies by Lorenz et al. (13) showed that proximal tubule fluid reabsorption is decreased not only in homozygous mutants but also in heterozygotes. Nevertheless, delivery of fluid to the early distal tubule was similar in mice of all three genotypes because of a compensatory reduction in the single-nephron glomerular filtration rate in Nhe3-/- mice and increased reabsorption in the loop segment in heterozygous mutants. Nakamura et al. (20) used isolated perfused tubules to show that compensatory upregulation of HCO-3 reabsorption occurred in both cortical and outer medullary collecting ducts of NHE3 null mutants. Using in situ microperfusion techniques, Wang et al. (29) showed that NHE3 is responsible for all of the detectable Na+/H+ exchanger-mediated HCO-3 reabsorption in the proximal convoluted tubule, thereby confirming its role as the major absorptive Na+/H+ exchanger of the renal nephron.

The occurrence of a severe intestinal absorptive defect in NHE3-deficient mice, but not in NHE2-deficient mice, demonstrated that the intestinal functions of these two isoforms are different. Thus it is now clear that NHE3 is responsible for most, if not all, of the apical Na+/H+ exchange activity on the apical membranes of the intestinal epithelium, which mediates NaCl absorption via coupling with the apical Cl-/HCO-3 exchanger (12) (Fig. 2A). In the absence of NHE3, increased activity of the epithelial Na+ channel, cHKA, and a K+ channel on the apical membrane of colonic epithelial cells (Fig. 2B), which in combination would be functionally equivalent to Na+/H+ exchange, appears to be the major compensatory mechanism.


    COLONIC H+-K+-ATPASE
TOP
ABSTRACT
INTRODUCTION
SECRETORY NA+-K+-2CL-...
NA+/H+ EXCHANGERS
COLONIC H+-K+-ATPASE
CONCLUDING REMARKS
REFERENCES

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 HCO-3 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.


    CONCLUDING REMARKS
TOP
ABSTRACT
INTRODUCTION
SECRETORY NA+-K+-2CL-...
NA+/H+ EXCHANGERS
COLONIC H+-K+-ATPASE
CONCLUDING REMARKS
REFERENCES

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.


    ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health Grants DK-50594, HL-41496, and ES 06096.


    FOOTNOTES

* 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).


    REFERENCES
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ABSTRACT
INTRODUCTION
SECRETORY NA+-K+-2CL-...
NA+/H+ EXCHANGERS
COLONIC H+-K+-ATPASE
CONCLUDING REMARKS
REFERENCES

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6.   Delpire, E., J. Lu, R. England, C. Dull, and T. Thorne. Deafness and imbalance associated with inactivation of the secretory Na-K-2Cl co-transporter. Nature Genet. 22: 192-195, 1999[ISI][Medline].

7.   DuBose, T. D., Jr., J. Codina, A. Burges, and T. A. Pressley. Regulation of H+-K+-ATPase expression in kidney. Am. J. Physiol. Renal Fluid Electrolyte Physiol. 269: F500-F507, 1995[Abstract/Free Full Text].

8.   Flagella, M., L. L. Clarke, M. L. Miller, L. C. Erway, R. A. Giannella, A. Andringa, L. R. Gawenis, J. Kramer, J. J. Duffy, T. Doetschman, J. N. Lorenz, E. N. Yamoah, E. L. Cardell, and G. E. Shull. Mice lacking the basolateral Na-K-2Cl cotransporter have impaired epithelial chloride secretion and are profoundly deaf. J. Biol. Chem. 274: 26946-26955, 1999[Abstract/Free Full Text].

9.   Hoogerwerf, W. A., S. C. Tsao, O. Devuyst, S. A. Levine, C. H. C. Yun, J. W. Yip, M. E. Cohen, P. D. Wilson, A. J. Lazenby, C.-M. Tse, and M. Donowitz. NHE2 and NHE3 are human and rabbit intestinal brush-border proteins. Am. J. Physiol. Gastrointest. Liver Physiol. 270: G29-G41, 1996[Abstract/Free Full Text].

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11.   Joo, N. S., R. M. London, H. D. Kim, L. R. Forte, and L. L. Clarke. Regulation of intestinal Cl- and HCO-3 secretion by uroguanylin. Am. J. Physiol. Gastrointest. Liver Physiol. 274: G633-G644, 1998[Abstract/Free Full Text].

12.   Kaunitz, J. D., K. E. Barrett, and J. A. McRoberts. Electrolyte secretion and absorption: small intestine and colon. In: Textbook of Gastroenterology (2nd ed.), edited by T. Yamada. Philadelphia, PA: J. B. Lippincott, 1995, p. 326-361.

13.   Lorenz, J. N., P. J. Schultheis, T. Traynor, G. E. Shull, and J. Schnermann. Micropuncture analysis of single nephron function in NHE3-deficient mice. Am. J. Physiol. Renal Physiol. 277: F447-F453, 1999[Abstract/Free Full Text].

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15.   Melvin, J. E., K. Park, L. Richardson, P. J. Schultheis, and G. E. Shull. Mouse down-regulated in adenoma (DRA) is a colonic C1-/HCO-3 exchanger and is upregulated in mice lacking the NHE3 Na+/H+ exchanger. J. Biol. Chem. 274: 22855-22861, 1999[Abstract/Free Full Text].

16.   Meneton, P., P. J. Schultheis, G. Greeb, M. L. Nieman, L. H. Liu, L. L. Clarke, J. J. Duffy, T. Doetschman, J. N. Lorenz, and G. E. Shull. Increased sensitivity to K+ deprivation in colonic H,K-ATPase-deficient mice. J. Clin. Invest. 101: 536-542, 1998[Abstract/Free Full Text].

17.   Mount, D. B., E. Delpire, G. Gamba, A. E. Hall, E. Poch, R. S. Hoover, Jr., and S. C. Hebert. The electroneutral cation-chloride cotransporters. J. Exp. Biol. 201: 2091-2102, 1998[Abstract/Free Full Text].

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20.   Nakamura, S., H. Amlal, P. J. Schultheis, J. H. Galla, G. E. Shull, and M. Soleimani. HCO-3 reabsorption in renal collecting duct of NHE-3-deficient mouse: a compensatory response. Am. J. Physiol. Renal Physiol. 276: F914-F921, 1999[Abstract/Free Full Text].

21.   Orlowski, J., and S. Grinstein. Na+/H+ exchangers of mammalian cells. J. Biol. Chem. 272: 22373-22376, 1997[Free Full Text].

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23.   Sangan, P., V. M. Rajendran, A. S. Mann, M. Kashgarian, and H. J. Binder. Regulation of colonic H-K-ATPase in large intestine and kidney by dietary Na depletion and dietary K depletion. Am. J. Physiol. Cell Physiol. 272: C685-C696, 1997[Abstract/Free Full Text].

24.   Schultheis, P. J., L. L. Clarke, P. Meneton, M. Harline, G. P. Boivin, G. Stemmermann, J. J. Duffy, T. Doetschman, M. L. Miller, and G. E. Shull. 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[Abstract/Free Full Text].

25.   Schultheis, P. J., L. L. Clarke, P. Meneton, M. L. Miller, M. Soleimani, L. R. Gawenis, T. M. Riddle, J. J. Duffy, T. Doetschman, T. Wang, G. Giebisch, P. S. Aronson, J. N. Lorenz, and G. E. Shull. Renal and intestinal absorptive defects in mice lacking the NHE3 Na+/H+ exchanger. Nature Genet. 19: 282-285, 1998[ISI][Medline].

26.   Seidler, U., P. Stumpf, and M. Classen. Interstitial buffer capacity influences Na+/H+ exchange kinetics and oxyntic pHi in intact frog gastric mucosa. Am. J. Physiol. Gastrointest. Liver Physiol. 268: G496-G504, 1995[Abstract/Free Full Text].

27.   Snouwaert, J. N., K. K. Brigman, A. M. Latour, N. N. Malouf, R. C. Boucher, O. Smithies, and B. H. Koller. An animal model for cystic fibrosis made by gene targeting. Science 257: 1083-1088, 1992[ISI][Medline].

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29.   Wang, T., C.-L. Yang, T. Abbiati, P. J. Schultheis, G. E. Shull, G. Giebisch, and P. S. Aronson. Mechanism of proximal tubule bicarbonate absorption in NHE3 null mice. Am. J. Physiol. Renal Physiol. 277: F298-F302, 1999[Abstract/Free Full Text].

30.   Yu, F. H., G. E. Shull, and J. Orlowski. Functional properties of the rat Na/H exchanger NHE-2 isoform expressed in Na/H exchanger-deficient Chinese hamster ovary cells. J. Biol. Chem. 268: 25536-25541, 1993[Abstract/Free Full Text].


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