Nitric oxide inhibits potassium transport in the rat distal colon

Roman Aizman, Hjalmar Brismar, and Gianni Celsi

Department of Woman and Child Health, Astrid Lindgren Children's Hospital, Karolinska Institutet, S-17176 Stockholm, Sweden


    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

The effect of the nitric oxide (NO) pathway on K+ (measured using 86Rb) transport in adult rat distal colon was investigated in muscle-stripped segments of colons mounted in Ussing chambers. When added to the mucosal solution, the endogenous precursor of NO, L-arginine (30 mM), inhibited both mucosal-to-serosal and serosal-to-mucosal 86Rb fluxes and caused a prolonged decrease of short-circuit current (Isc). This effect was significantly reduced by the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) but not by D-NAME. Mucosal application of S-nitroso-N-acetyl-penicillamine (SNAP) inhibited mucosal-to-serosal 86Rb flux without affecting serosal-to-mucosal transport. Serosal addition of two different exogenous NO donors, sodium nitroprusside (0.1 mM) and SNAP (0.2 mM), decreased serosal-to-mucosal 86Rb flux, whereas Isc increased. The SNAP-induced decrease in 86Rb flux was abolished by 1H-(1,2,4)oxodiazolo(4,3-a)quinoxalin-1-one (0.2 mM), a selective inhibitor of NO-stimulated soluble guanylyl cyclase, and by methylene blue (0.01 mM). Addition of 8-bromo-cGMP (2 × 10-4 M) in the presence of an inhibitor of cGMP-specific phosphodiesterase mimicked the effects of NO-donating compounds. This study provides evidence that NO inhibits K+ transport in the rat distal colon via a cGMP-dependent pathway. The effect on net K+ transport may depend on the side of NO action.

nitric oxide donors; guanosine 3',5'-cyclic monophosphate pathway


    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

NITRIC OXIDE (NO) is an important mediator of several physiological processes in the gastrointestinal tract (35). Endogenous NO is derived from enzymatic conversion of L-arginine by NO synthase (NOS), a family of isoenzymes (22). In the gut, NOS is expressed throughout the intestinal wall in neurons (10), blood vessels (25), and epithelia (40). Because NO breaks down rapidly under aerobic conditions (11), its effect may be limited to the local region of production. NO modulates and influences both gut motility (6) and mucosal blood flow (26). Moreover, elevated levels of NOS, NO, and products of NO oxidation have been found in patients with active ulcerative colitis and gastroenteritis (9) and in an experimental model of hypoxia-induced colonic dysfunction (5), suggesting a role for NO also in pathological situations.

In addition, it has been suggested that NO directly regulates intestinal electrolyte and fluid transport, although the effect of NO on ion and water transport is far from being completely elucidated. Although some studies have suggested that NO produces a net secretory effect (18, 24, 39, 42), other studies have shown a net absorptive effect (15, 29, 33). The conclusions concerning the NO effects on ion transport are mainly based on electrophysiological recordings of short-circuit current (Isc) and transmural potential difference (PD) without direct measurements of unidirectional ion fluxes (18, 28, 34, 39). There are only a few studies directly indicating that NO donors stimulate electrogenic Cl- secretion and inhibit Na+ and Cl- absorption in the intestine (18, 42). Moreover, it has been suggested that the effect of NO on net water and ion transport in the intestine may depend on the side of application of the NO-donating compounds (24, 34).

Scanty information is available on the effect of NO on K+ transport in the distal colon. Indeed, the distal part of the gastrointestinal tract plays an important role in K+ balance by regulating fecal K+ excretion (3). It is well documented that K+ excretion in the gut is the vectorial sum of two opposite fluxes, secretion and absorption, which are mediated by different K+ pumps and transporters, located in both basolateral and apical membranes of colonocytes (1, 2, 7, 17, 30).

Therefore, the main aim of this study was to determine if NO regulates K+ transport in the rat distal colon and if the effect on absorptive or secretory pathways depends on the side of NO application.


    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Animals. Adult male Sprague-Dawley rats (65-70 days old, 250-300 g) fed standard rodent laboratory chow and water ad libitum were used in all studies. Animals were maintained in a 12:12-h light-dark environment. All experiments were carried out during the light phase. The protocols used in the present studies were approved by the Swedish National Board for Animal Research.

Preparation of intestinal tissue. The rats were anesthetized intraperitoneally with thiobutabarbital (6 mg/100 g body wt). Thereafter, the distal colon (one-third from the colorectal junction) was quickly removed, cut open, emptied of feces, and bathed in ice-cold physiological saline solution (PSS; in mM: 118 NaCl, 4.5 KCl, 0.54 MgCl2, 2.5 CaCl2, 1.2 NaH2PO4, 25 NaHCO3, and 10 D-glucose, pH 7.4) constantly gassed with 95% O2 and 5% CO2. The tissue was stretched and carefully stripped of its serosal and muscular layers by blunt dissection. Colonic sheets (0.725-cm2 exposed surface area) were mounted into Ussing chambers (WPI, New Haven, CT) and bathed on both sides with 10 ml of PSS, which was circulated by a peristaltic pump. The solution was constantly gassed (95% O2-5% CO2) and maintained at 37°C by a water-jacketed bath.

Bioelectrical and unidirectional flux measurements. Transmural PD (mV) and Isc (µA/cm2) were measured with the aid of an automatic voltage clamp (EVC-1000 voltage/current clamp; WPI) using Ag-AgCl electrodes connected to the bathing solution via agar bridges. The tissues were continuously short-circuited to zero PD except for brief intervals of 4 s every 1 min, at which time the open-circuit PD was read. Tissue conductance (Gt, in mS/cm2) was calculated using the formula Isc/PD. Tissue PD, Isc, and Gt were continuously monitored with the help of a personal computer equipped with a digital input/output and analog-digital converter card (Advantech PCL-711B). After tissues were mounted, they were allowed to equilibrate for at least 30 min to achieve stable PD and Isc before any drug addition and unidirectional flux measurements. Tissues with baseline Gt higher than 25 mS/cm2 were excluded from the study, since higher conductances might be related to tissue damage or transepithelial leakage (8, 27, 30). For the same reason, tissues having Gt values that significantly changed during the study were excluded from unidirectional K+ flux determinations.

Unidirectional K+ fluxes were measured by a sample-and-replace protocol (12) across short-circuited colonic mucosa using 86Rb as a marker of K+ (43). After equilibration, 1 µCi/ml 86Rb was added to the serosal or mucosal "hot" side. Samples (1 ml) were collected from the corresponding "cold" bathing solution at 10-min intervals and counted in a liquid scintillation counter (LKB). Immediately after collection, 1 ml of prewarmed bathing solution was replaced on the "cold" side.

After four control periods, the mean of which was calculated and used as the baseline, drugs were added in small volumes from concentrated stocks to solutions bathing either the serosal or the mucosal side, and drug-induced fluxes were measured during five additional periods. In all studies in which the drug was added to the "cold" side, the replacement buffer contained the same drug to maintain the appropriate concentration in the bathing solution during the whole experiment.

Mucosal-to-serosal (Jmright-arrow s) and serosal-to-mucosal (Jsright-arrow m) fluxes were measured in different tissues. To calculate net 86Rb flux (Jnet = Jmright-arrow s - Jsright-arrow m), tissues were paired on the basis of a conductance difference of <20% (24, 42, 43). A positive Jnet indicated 86Rb absorption, and a negative Jnet represented 86Rb secretion.

Materials. S-nitroso-N-acetyl-penicillamine (SNAP), sodium nitroprusside (SNP), L-arginine, N G-nitro-L-arginine methyl ester (L-NAME), D-NAME, 8-bromo-cGMP, and methylene blue were obtained from Sigma Chemical; the specific inhibitor of cGMP phosphodiesterase, 4-{[3',4'-(methylenedioxy)benzyl]amino}-6-methoxyquinazoline, was obtained from Calbiochem; 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ) was purchased from Tocris Cookson; and 86Rb was from Amersham.

Statistics. Data are presented as means ± SE, with n indicating the number of rats. The paired Student's t-test and the two-way ANOVA (when indicated) were used to determine statistical difference. A P value of <0.05 was considered significant.


    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

To evaluate if colonic K+ transport is regulated by NO production, segments of the rat distal colon were incubated with the natural precursor of NO, i.e., L-arginine. Addition of L-arginine (30 mM) to the mucosal bathing solution produced a sustained decrease in Isc and PD without significant changes in Gt (Table 1). A statistical difference in Isc and PD was observed after 20 min. To study the effect of L-arginine on K+ transport, we measured the unidirectional K+ fluxes using 86Rb as a marker for K+. Unidirectional K+ fluxes were rapidly affected by L-arginine (Fig. 1). In basal conditions, there was a negative Jnet, i.e., a 86Rb flux from the serosal to mucosal side representing secretion. Both Jmright-arrow s and Jsright-arrow m were inhibited by L-arginine, although the time of onset was different. The Jsright-arrow m was inhibited at 10 min, whereas the effect on Jmright-arrow s was observed after 20 min. Thereafter, the fluxes were inhibited almost to the same extent. Therefore, during the first 10 min, we observed a net K+ absorption, whereas after 20 min the net K+ transport did not differ from basal values. At lower concentrations (20 mM) L-arginine did not affect fluxes, whereas at higher concentrations (40-50 mM) it produced marked changes in tissue electrical parameters (>2- to 3-fold elevation in Gt), most likely reflecting changes in transepithelial permeability and/or tissue damage (data not shown). Serosal application of L-arginine (30 mM) did not alter Jsright-arrow m or Jmright-arrow s (Table 2).

                              
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Table 1.   Effect of mucosal L-arginine (30 mM) on electrical parameters of rat distal colon



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Fig. 1.   Unidirectional [mucosal-to-serosal (Jmright-arrow s) and serosal-to-mucosal (Jsright-arrow m)] and net (Jnet) 86Rb fluxes (in µM · cm-2 · h-1) in rat distal colon following mucosal addition of L-arginine (30 mM). Here and in Figs. 2-7, arrows indicate addition of drugs. *Significantly different from baseline; n (no. of rats) = 4 in each group.

                              
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Table 2.   Effect of serosal L-arginine (30 mM) on unidirectional 86Rb fluxes in rat distal colon

To evaluate if the effect of L-arginine on K+ transport is mimicked by NO, we studied the effect of mucosal SNAP, an exogenous NO donor. Mucosal SNAP (0.2 mM) evoked a rapid and prolonged increase of Isc and PD without affecting Gt (Fig. 2A). Mucosal SNAP did not affect Jsright-arrow m but significantly decreased Jmright-arrow s after 20 min, thus resulting in increased K+ secretion compared with baseline (Fig. 3A).


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Fig. 2.   Time course of short-circuit current (Isc), potential difference (PD), and tissue conductance (Gt) responses to mucosal (A) or serosal (B) addition of 0.2 mM S-nitroso-N-acetylpenicillamine (SNAP) in rat distal colon. Isc and PD are presented as difference from baseline; Gt is presented in absolute values. *Significantly different from baseline; n = 4 in each group.


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Fig. 3.   Unidirectional and net K+ (86Rb) fluxes in rat distal colon following mucosal (A) or serosal (B) addition of 0.2 mM SNAP. Data are presented in absolute values, in µM · cm-2 · h-1. *Significantly different from baseline; n = 4 for each group.

To estimate if the NO-induced alteration in K+ transport depended on the side of application, the effect of SNAP added from the mucosal side was compared with the response from the serosal side. Serosal addition of SNAP (0.2 mM) rapidly increased Isc and PD (Fig. 2B) without affecting Gt. In contrast to mucosal addition, serosal SNAP did not alter Jmright-arrow s but significantly decreased Jsright-arrow m after 20 min, thus resulting in increased K+ absorption compared with baseline (Fig. 3B). The effect of serosal SNAP was determined also at different concentrations (Table 3). At lower doses (0.04 mM), the effect of SNAP on Jsright-arrow m was relatively small and therefore the mean Jnet did not differ from basal values. At higher concentrations (1.0 mM), Jsright-arrow m was affected approximately to the same extent as with 0.2 mM SNAP; therefore, the mean Jnet reversed from secretion to absorption. However, we observed that 1.0 mM SNAP affected baseline Gt (the mean value increased from 13.2 ± 2.0 to 17.2 ± 2.5 mS/cm2; n = 4).

                              
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Table 3.   Dose-dependent effect of serosal SNAP on unidirectional 86Rb fluxes in rat distal colon

To evaluate if the effect of SNAP is due to the release of NO, we also determined the response to another NO donor, SNP, which is structurally unrelated to SNAP. Serosal addition of SNP (0.1 mM) produced a rapid and transient increase in Isc and PD without affecting Gt (Fig. 4A). SNP also significantly decreased Jsright-arrow m (Fig. 4B) but did not alter Jmright-arrow s. Therefore, Jnet was reversed from secretion to absorption within 30 min after exposure to serosal SNP.


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Fig. 4.   Time course of Isc, PD, and Gt (A) and unidirectional and net K+ (86Rb) fluxes (in µM · cm-2 · h-1) (B) in rat distal colon following serosal addition of 0.1 mM sodium nitroprusside (SNP). *Significantly different from baseline; n = 4 in each group.

In the following protocols, we further characterized the NO-dependent intracellular pathway inhibiting K+ fluxes in rat distal colon.

The inhibitory effects of L-arginine on Jmright-arrow s were significantly abolished by the mucosal addition of the NOS inhibitor, L-NAME (7 mM), 5 min before L-ar-ginine, whereas the inactive compound, D-NAME (7 mM), did not alter the L-arginine-induced decrease in Jmright-arrow s (Fig. 5). L-NAME alone did not significantly change Jmright-arrow s (basal value of 0.52 ± 0.01, 0.49 ± 0.02 after 10 min, 0.53 ± 0.03 after 20 min, and 0.48 ± 0.04 µM · cm-2 · h-1 after 30 min; n = 4 at each time point).


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Fig. 5.   86Rb Jmright-arrow s in rat distal colon after mucosal addition of L-arginine (30 mM) + N G-nitro-L-arginine methyl ester (L-NAME; 7 mM) or L-arginine + D-NAME (7 mM). Statistical changes in Jmright-arrow s following mucosal addition of L-arginine (30 mM) alone are presented in Fig. 1 and are indicated here only for comparison. +Significantly different from L-arginine alone (2-way ANOVA); n = 4-5 for each group.

To evaluate if the NO-induced inhibition of K+ transport is mediated by the cGMP pathway, the colonic mucosa was incubated with SNAP in the presence of guanylyl cyclase inhibitors. The selective inhibitor of NO-stimulated soluble guanylyl cyclase, ODQ (0.2 mM), added to the mucosal (Fig. 6A) or serosal (Fig. 6B) side, blocked the SNAP-induced inhibition of unidirectional 86Rb fluxes and significantly reduced the SNAP-mediated elevation of Isc and PD (data not shown). Methylene blue (10 µM), a less selective inhibitor of guanylyl cyclase, was also used. As expected, methylene blue, added to the serosal side, prevented the decrease of K+ flux (Jsright-arrow m) caused by SNAP alone (Fig. 7).


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Fig. 6.   Unidirectional K+ fluxes in rat distal colon after mucosal (A) or serosal (B) addition of SNAP (0.2 mM) and 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ) (0.2 mM). Data are presented in absolute values, in µM · cm-2 · h-1. *Significantly different from baseline; +significantly different from SNAP alone (2-way ANOVA); n = 4-5 for each group.


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Fig. 7.   Effect of serosal methylene blue (10 µM) on serosal SNAP (0.2 mM)-induced inhibition of 86Rb Jsright-arrow m. Data are presented in absolute values, in µM · cm-2 · h-1. *Significantly different from baseline; +significantly different from SNAP alone (2-way ANOVA); n = 3-4 for each group.

In the last protocol, the colon was treated with exogenous 8-bromo-cGMP (2 × 10-4 M) in the presence of the cGMP-specific phosphodiesterase inhibitor, methoxyquinazoline (5 × 10-7 M) (38). Both drugs were added to the mucosal side. Application of these drugs produced a sustained increase in Isc and PD without affecting Gt. Moreover, these drugs caused a significant decline in Jmright-arrow s, similar to the response induced by mucosal SNAP or L-arginine (Table 4).

                              
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Table 4.   Effect of 8-bromo-cGMP in presence of cGMP-specific phosphodiesterase inhibitor on 86Rb Jmright-arrow s and on electrical parameters of rat distal colon


    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

The present study demonstrates that K+ transport in the rat distal colon is regulated by an NO-dependent pathway and suggests that this regulation may be of physiological relevance. These conclusions are based on the following results.

The natural precursor of NO, i.e., L-arginine, affected K+ transport, and this effect was abolished by the inhibitor of NOS, L-NAME, but not by the inactive compound D-NAME. It is therefore likely that the effect of L-arginine is mediated by an NOS-dependent production of NO. Indeed, both the constitutive and the inducible isoforms of NOS are expressed in the rat colon and may therefore convert L-arginine to NO (14).

Two different NO donors (SNP and SNAP) added to the serosal side induced similar effects on K+ transport. It has been suggested that the action of NO donors may be partially explained by the release of other substances unrelated to NO (13, 36). The finding that SNP and SNAP had similar effects on 86Rb fluxes tends to exclude this possibility. In this regard, SNAP, a nitrosothiol that is structurally unrelated to SNP, produces fewer byproducts (13) and may be more identical to NO in its actions than SNP (34). Furthermore, the dose-dependent response to SNAP suggests that toxicity does not contribute to the NO donor response. In this study, the effect of NO donors on K+ transport was observed at doses that did not cause epithelial leakage, as determined by a stable electrical conductance (Gt). Higher doses of L-arginine (40-50 mM) and SNAP (1 mM) increased electrical conductance, indicating that cell damage and/or increased epithelial permeability may occur (4, 16).

The intracellular pathway mediating the intestinal effects of NO is still disputed. Most studies found that NO stimulates intestinal cGMP production (18, 22, 39, 41). However, some studies deny or question the role of cGMP in intestinal NO action (32, 33). The results of the present study strongly indicate that cGMP mediates the effect of NO on colonic K+ transport (Fig. 8). ODQ, a specific inhibitor of NO-activated soluble guanylyl cyclase, which does not affect NOS or NO (23), completely prevented the effect of SNAP on K+ fluxes. Moreover, a general inhibitor of guanylyl cyclase, methylene blue, significantly attenuated the inhibitory effect of NO on K+ transport. In addition, application of 8-bromo-cGMP in the presence of the cGMP-specific phosphodiesterase inhibitor mimicked the effect of NO donors on K+ fluxes.


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Fig. 8.   Scheme of nitric oxide (NO) production and NO-activated signaling pathways.

The mechanisms responsible for K+ transport in the large intestine consist of two opposite fluxes: secretion and absorption (3). K+ secretion is mediated by Na+-K+-ATPase and Na+-K+-2Cl- cotransporter, both located in the basolateral membrane of colonocytes. These enzymes stimulate K+ uptake across the basolateral membrane, leading to K+ secretion across the apical membrane through K+ channels (30). K+ absorption occurs via K+-dependent ATPases located in the apical membrane, mainly in the distal colon. Several lines of evidence suggest that there are at least two K+-absorptive pumps, namely, an ouabain-insensitive H+-K+-ATPase and an ouabain-sensitive Na+-independent K+-ATPase (1, 7, 17). Besides transcellular K+ transport, paracellular pathways can also contribute to net K+ flux, but the colonic paracellular route is believed to have low conductance and be anion selective; therefore, its contribution to K+ transport under normal conditions may be neglected (20). In this study, the finding that the effect of different NO donors on K+ transport was observed in the absence of changes in transepithelial conductance strongly supports the concept that K+ is mainly transported transcellularly. However, the effect of NO on individual K+ transporters remains to be evaluated.

In this study we observed that mucosal L-arginine inhibited both serosal-to-mucosal and mucosal-to-serosal fluxes, whereas serosal addition of L-arginine did not alter ion fluxes. On the other hand, SNAP inhibited unidirectional fluxes, depending on the side of addition. Indeed, it has been shown that L-arginine affects intestinal water and ion transport following intraluminal infusion but not when infused intraperitoneally (19, 24). We may assume that L-arginine added to the mucosal side is converted by NOS to NO, which inhibits both apical and basolateral K+ transporters. Although NO is lipophilic and should easily traverse a plasma membrane, our results may suggest that NO produced by SNAP/SNP acts near the site of its production, due to its rapid inactivation under aerobic conditions (11). Therefore, NO produced in the lumen could affect only apical K+ transporters, whereas NO produced at the serosal side could affect only basolateral K+ transporters. Further in vitro studies are required to evaluate this hypothesis.

The effects of L-arginine and NO donors (SNAP and SNP) on the electrophysiological parameters (Isc and PD) are controversial. Similar opposite changes in Isc following SNAP/SNP or L-arginine addition to the intestine have been reported (18, 28, 29, 34, 37, 39, 42), but the ionic mechanisms for these responses are not fully elucidated. It has been suggested that Cl- is the primary ion involved in NO-induced changes of Isc, although alterations in Na+ flux may also affect this value (18, 42). In our study, we found a discrepancy between changes in electrical parameters and K+ fluxes following NO addition. Changes in Isc occured within 20 min, whereas the maximal inhibition of Jsright-arrow m and Jmright-arrow s was observed after 30 min of treatment. Moreover, Isc and PD changed in opposite directions after L-arginine or SNAP/SNP, whereas K+ fluxes were inhibited by all NO donors. Therefore, a detailed evaluation of the effects of NO precursors on different ion fluxes and of their relative contribution to Isc is probably needed to address these controversial findings.

It has been shown that NOS is increased in active idiopathic ulcerative colitis (21), suggesting a role for NO in the pathogenesis of this condition. NO is generated by a number of immunologic cell types that have been shown to be present in increased quantities in the lamina propria and the lumen of the inflamed bowel. Moreover, in vivo studies have indicated NO involvement in the pathophysiology of secretion induced by Escherichia coli heat stable enterotoxin (31). Our results demonstrate that K+ absorption and/or secretion in the rat distal colon can be regulated by locally produced NO and that this effect may depend on the side of NO action. Taken together, these results suggest that the NO system is an important regulator of K+ transport during normal and pathological conditions.


    ACKNOWLEDGEMENTS

This work was supported by the Swedish Medical Research Council (project no. 11555) and by the Wiberg Foundation. R. Aizman was supported by a scholarship from the Karolinska Institute.


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests: G. Celsi, Research Laboratory, Astrid Lindgren Children's Hospital, S-17176 Stockholm, Sweden.

Received 10 June 1998; accepted in final form 7 October 1998.


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Top
Abstract
Introduction
Methods
Results
Discussion
References

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Am J Physiol Gastroint Liver Physiol 276(1):G146-G154
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