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+-2CL COTRANSPORTER

TOP 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).

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Transporters required for ion secretion in gastrointestinal tract. A: cAMP-stimulated secretion of Cl (and possibly HCO3) 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/HCO3 exchange and/or by Na+-HCO3 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/HCO3 exchange provides Cl uptake and HCO3 extrusion needed to balance HCl secretion. NKCC1 and coupled Na+/H+ and Cl/HCO3 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 (I_SC) were absent (3), thereby demonstrating an essential role for the CFTR in anion secretion. Bumetanide, an inhibitor of NKCC1, significantly reduced the cAMP-stimulated I_SC 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 I_SC 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/HCO₃ exchange (Fig. 1A) and could also include Na⁺-HCO₃ cotransport if a significant component of the anion current is due to transepithelial HCO₃ 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/HCO₃ exchanger (Fig. 2A), the absorption of NaCl.

View larger version (25K): [in this window] [in a new window]   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/HCO3 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/HCO3 exchanger. cHKA mediates K+ absorption directly and absorbs HCO3 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 ~ 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/HCO₃ 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).

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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

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 HCO₃ absorption and, if coupled with the apical Cl/HCO₃ 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/HCO₃ 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 HCO₃ (via H⁺ secretion) during diarrheal states. In such a situation, functional coupling with a basolateral HCO₃ extrusion system, such as the AE2 Cl/HCO₃ 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.