Independence of apical Cl-/HCO3- exchange and anion conductance in duodenal HCO3- secretion

S. Spiegel,1 M. Phillipper,1 H. Rossmann,1 B. Riederer,1,2 M. Gregor,1 and U. Seidler1,2

1First Department of Medicine, Eberhard-Karls-Universität, 72076 Tübingen; and 2Zentrum Innere Medizin, Abteilung VI, Medizinische Hochschule Hannover, 30625 Hannover, Germany

Submitted 19 February 2003 ; accepted in final form 26 June 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Reduced gastrointestinal secretion contributes to malabsorption and obstructive syndromes in cystic fibrosis. The apical transport pathways in these organs have not been defined. We therefore assessed the involvement of apical Cl-/ exchangers and anion conductances in basal and cAMP-stimulated duodenal secretion. Muscle-stripped rat and rabbit proximal duodena were mounted in Ussing chambers, and electrical parameters, secretion rates, and 36Cl-, 22Na+, and 3H+ mannitol fluxes were assessed. mRNA expression levels were measured by a quantitative PCR technique. Removal of Cl- from or addition of 1 mM DIDS to the luminal perfusate markedly decreased basal secretion but did not influence the secretory response to 8-bromo-cAMP, which was inhibited by luminal 5-nitro-2-(3-phenylpropylamino)-benzoate. Bidirectional 22Na+ and 36Cl- flux measurements demonstrated an inhibition rather than a stimulation of apical anion exchange during cAMP-stimulated secretion. The ratio of Cl- to in the anion secretory response was compatible with both Cl- and being secreted via the CFTR anion channel. CFTR expression was very high in the duodenal mucosa of both species. We conclude that in rat and rabbit duodena, an apical Cl-/ exchanger mediates a significant part of basal secretion but is not involved in the secretory response to cAMP analogs. The inhibitor profile, the strong predominance of Cl- over in the anion secretory response, and the high duodenal CFTR expression levels suggest that a major portion of cAMP-stimulated duodenal secretion is directly mediated by CFTR.

cystic fibrosis transmembrane conductance regulator; 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; 5-nitro-2-(3-phenylpropylamino)-benzoate; bicarbonate; cystic fibrosis; intestine


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Table 1.
 

IT HAS LONG BEEN RECOGNIZED that -secreting organs such as pancreas, small intestine, bile ducts, and epididymal duct display reduced secretory rates in patients with cystic fibrosis (CF) and are severely affected by the disease (24, 34, 44, 47). Thus a role for a CF-related anion channel in epithelial secretion was envisioned long before the cftr gene was cloned and the CFTR protein expressed and found to be an anion channel (2).

Because of the relatively low conductance of all apical anion channels found in CFTR-expressing cell lines (11, 25, 35), theoretical considerations suggested that the CFTR channel itself or other CFTR-dependent anion channels cannot mediate the high secretory rates found in several gastrointestinal organs. Alternatively, it has been proposed that secretion across the apical membrane in gastrointestinal organs occurs by parallel operation of CFTR Cl- channels and Cl-/ exchangers. In this model, the Cl- channel provides luminal Cl- and acts as a Cl- leak pathway to prevent intracellular Cl- accumulation as the exchanger cycles (15, 31, 38, 45). This model gained acceptance because of its plausibility and because a luminal Cl-/ exchange process has been functionally described in several CFTR-expressing epithelia, such as gallbladder, small and large intestine, and the pancreatic duct system (4, 7, 39).

Because modes to stimulate secretion in CF epithelia could be of considerable advantage for CF patients, the scientific interest in secretion in CF epithelia has strongly increased (6, 37, 43, 45). Several years ago, we observed in isolated rat duodenal mucosa that the secretory response to guanylin was unaltered in the absence of luminal Cl- (13). Further experiments in isolated mouse intestinal mucosa revealed that cAMP, cGMP, and Ca2+-dependent agonists lost their secretory potential in the absence of CFTR expression but that apical anion exchange activity was unaltered (42). Thus CFTR channel activation was essential for all forms of agonist-stimulated secretion, but the role of an apical anion exchanger was less evident.

The present study was undertaken to test the basic concept of a coupling between a decrease in intracellular Cl- concentration and stimulation of apical Cl-/ exchange in the small intestinal epithelium and to investigate whether this mechanism is indeed involved in agonist-stimulated duodenal secretion in rat, a relatively low secretor, or rabbit, a high secretor (possibly because of the contribution by Brunner's glands). We also tested whether the CFTR anion channel itself is the likely transport pathway for agonist-stimulated secretion.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Chemicals and Solutions

H36Cl and 22NaCl were obtained from Amersham (Braunschweig, Germany); scintillation cocktail was from Packard (Frankfurt am Main, Germany); Rompun was from Bayer, and Ketanest was from Parke-Davis. Unless otherwise specified, all other reagents were purchased from Sigma-Aldrich (Deisenhofen, Germany) [including agarose type III high EEO, DIDS, bumetanide, forskolin, indomethacin, TTX, 8-bromo-cAMP (8-Br-cAMP), and ouabain], Fluka (Deisenhofen, Germany), Roth (Karlsruhe, Germany), or Merck (Darmstadt, Germany) at tissue culture grade, molecular biology grade, or the highest grade available.

The luminal solution contained 154 mmol/l NaCl, gassed with 100% O2, and the nutrient solution contained (in mmol/l) 140.5 Na+, 4.5 K+, 2 Ca2+, 1.3 Mg2+, 126 Cl-, 1.3 , 20 , 1.5 , 11.9 dextrose, and 10 sodium pyruvate plus 3·10-5 mol/l indomethacin and 10-6 mol/l TTX, gassed with 95% O2-5% CO2, pH 7.4 ± 0.03, both at 37°C. In the case of the Cl--free studies, Cl- was replaced by gluconate. When voltage clamp was applied, the luminal and serosal solutions were identical and were both gassed by 5% CO2, except that glucose was replaced by mannitol in the luminal bath. The electrodes contained 3 M KCl agarose or, in the case of substitution of Cl-, 3 M potassium nitrate agarose.

Experimental Methods

Animals. Male New Zealand White rabbits weighing 2,500-3,000 g and female Wistar rats weighing 280-350 g were maintained under standard temperature (21-22°C) and light conditions (12:12-h light-dark cycle). Animals had access to tap water and pelleted food ad libitum.

Duodenal isolation. For rat duodenal isolation, the procedure has been described (13). Rabbits were preanesthetized by an intramuscular injection of 100 mg/kg ketamine, 10 mg/kg dihydroxylidin, and 0.2 mg/kg atropine. The abdomen was opened, a 3-cm-long segment of the duodenum was excised, and the animals were killed with an overdose of phenobarbital. The proximal part of the duodenum was stripped of external serosal and muscle layers (effectively removing the Brunner's glands as well) with fine forceps under the stereomicroscope, mounted between two Lucite half-chambers of 0.636-cm2 exposed area, and placed in an Ussing chamber. TTX (10-6 mmol/l, to abolish the influence the submucosal plexus) and indomethacin (3 x 10-5 mol/l, to prevent endogenous prostaglandin production) were administered to the basolateral solution. Drugs were administered at the indicated times. Ouabain was applied as a standard test for active secretion and viability (although it does not appropriately reflect these parameters when strong transepithelial gradients are present; see DISCUSSION).

Electrophysiology. The open-circuit transepithelial electrical potential difference (PD) was recorded (DVC-1000 dual voltage clamp; World Precision Instruments, Sarasota, FL) via agar 3 mol/l KCl bridges. Before the tissue was placed into the chamber, the series resistances of the solutions etc. were assessed, and a fluid resistance compensation was performed before each experiment. The direct-current electrical resistance was determined from the change in PD after sending a current of ~40 µA/cm2 through the mucosa in either direction in a 200-ms interval. The open-circuit condition was chosen because to measure secretion, no CO2 or may be in the luminal solution. This results in the presence of a concentration gradient, which should not be present under voltage-clamp conditions. In addition, the open-circuit condition is closer to the physiological situation. Under open-circuit conditions, short-circuit current (Isc) is calculated from PD and R, and is, of course, not a true short-circuit current, because the transepithelial PD is a driving force for passive ion movement. To assess whether changes in "calculated Isc" can be used as an approximate measurement for changes in true Isc, we applied voltage-clamp conditions to a complete set of experiments (including flux rates) for the control conditions. In all experiments, a positive PD or a positive Isc reflects a net anion movement from the serosal to the mucosal side. Because we often compare secretion with changes in Isc, anion movement into the luminal bath is defined as positive in both cases.

In vitro determination of duodenal secretion. Luminal pH was maintained at 7.4 by a continuous pH stat titration method (Radiometer, Copenhagen, Denmark), and the rate of alkalization was calculated from the consumption volume of the HCl- or -containing titrant solution and is given as micromoles per hour per square centimeter.

Isotope flux studies. 36Cl--, 22Na+-, and 3H+ mannitol flux studies were performed in the open-circuit mode and during voltage clamp to 0 PD. 74 kBq/ml H36Cl for 22Na+ and 62 kBq/ml for 3H+-mannitol (2 mmol/l), respectively, were added either to the serosal or the mucosal solution after it had reached stable flux () and electrical parameters. For each isotope, neighboring pieces of rabbit duodenum and identical sections of age-, weight-, and sex-matched inbred rats were used. After a 30-min period of equilibration, aliquots were taken in 20-min intervals for rat and 15-min intervals for rabbit, radioactivity was determined in a liquid scintillation counter, and the bidirectional flux rates for the respective substance was calculated. The values for Isc and J represent the average values of the 20- and 15-min periods, respectively.

RNA isolation and semiquantitative RT-PCR. The RNA isolation method, the semiquantitative RT-PCR procedure, and the sequence information of histone 3.3a and 18s rRNA primers were described in detail previously (1, 41). Homologous primers for rabbit (forward: 5'-ACCAAATCCATCAAACCATCC-3'; reverse: 5'-CATTGCCTCTATCCTGTGTTC-3') and rat (forward: 5'-CTGAACTCAAAGTCTACGTCC-3'; reverse: 5'-CCACCTCAACCAGAAAAACC-3') CFTR were deduced from published sequence information (GenBank accession nos. AF189720 [GenBank] and M89906 [GenBank] ). The obtained PCR fragments displayed the expected size (696 bp for rabbit and 717 bp for rat CFTR). The identity of the amplimers was confirmed by restriction analysis.

Statistics

All results are expressed as means ± SE; n is the number of separate experiments. Error bars are not shown when included within the symbol. All examinations were performed at least in triplicate. If applicable, P values were determined by using the Student's t-test.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Secretion, Isc, and Bilateral Cl-, Na+, and 3H-Mannitol Fluxes Under Basal and cAMP-Stimulated Conditions

After stable tissue parameters were reached, basal secretion was 1.02 ± 0.2 and mean Isc was 1.65 ± 0.16 µeq·cm-2·h-1 in rat duodenum (Fig. 1A). The basal secretory rate of rabbit duodenal mucosa was 3.75 ± 0.12; mean Isc was 1.08 ± 0.16 (Fig. 1B). 8-Br-cAMP (1 mmol/l) added to the basolateral perfusate increased secretory rate ({Delta}0.66 in rat and {Delta}1.73 µeq·cm-2·h-1 in rabbit) and Isc ({Delta}3.16 in rat and {Delta}2.12 µeq·cm-2·h-1 in rabbit). To determine the true Cl-/ relationship in the stimulated anion secretion under open-circuit conditions, serosal-to-mucosal and mucosal-to-serosal flux rates for Cl- and Na+ were measured by isotope flux measurements (Fig. 2, A and B). Despite a very different Isc/ ratio in rat and rabbit duodenal mucosa, the increase in serosal-to-mucosal Cl- flux was relatively similar in rat and rabbit, and in both species the calculated Isc increase during stimulation approximated the sum of {Delta}Cl- and {Delta} minus {Delta}Na+ (Fig. 2B). The relationship of {Delta}Cl- to {Delta} during the first 15 min of stimulation was 11:1 in rat and 5:1 in rabbit duodenum. Thus, even with a strong concentration gradient from cell to lumen for and an opposite gradient for Cl-, Cl- strongly predominated in the anion secretory response. Moreover, the relationship of secreted to Cl- found in these experiments is well within the values for the CFTR anion conductance in patch-clamp experiments (11, 35).



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Fig. 1. secretory rate (, right axis) and calculated short-circuit current (Isc; left axis) of rat (A) and rabbit (B) isolated duodenum before and after stimulation with 8-bromo-cAMP (8-Br-cAMP). The data demonstrate the very different basal and stimulated short-circuit current (Isc) and secretory rates in rat and rabbit duodenum. Values are means ± SE; n = 5 for both species.

 


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Fig. 2. Serosal-to-mucosal (s > m; A and C) and mucosal-to-serosal (m > s; B and D) Na+ and Cl- fluxes, averaged for 20 min in rat (A and B) and 15 min in rabbit (C and D) before and after stimulation with 8-Br-cAMP. It is clear that 8-Br-cAMP increases and Isc as well as serosal-to-mucosal Na+ and Cl- fluxes while inhibiting mucosal-to-serosal Na+ and Cl- flux strongly in rat (A and B; all changes are significant at P < 0.05 or 0.01; n = 6 for rat and 5 for rabbit) and weakly in rabbit duodenum (C and D; not significant). Mucosal-to-serosal Cl- flux is to a large extent DIDS sensitive and therefore likely mediated by apical Cl-/ exchange, and its inhibition is consistent with the current model of cAMP inhibition of coupled Na+/H+ and Cl-/ exchange. We found no stimulation of apical Cl-/ exchange during cAMP-mediated anion secretion, as would have to be expected if a coupling of these 2 pathways was mandatory for secretion. E and F: 8-Br-cAMP-induced increase in serosal-to-mucosal flux of Cl- and Na+, as well as and Isc, all averaged over the fist 15 min after stimulation, in rat (E) and rabbit (F) duodenum. Interestingly, the increase in serosal-to-mucosal Cl- flux by the cAMP analog (significant for P < 0.05) was relatively similar in rat and rabbit duodenum, despite a very different ratio. The ratio of change in Cl- flux to was 11:1 in rat and 5:1 in rabbit anion secretory response.

 

If an apical Cl-/ exchanger was the transporter for cAMP-induced secretion, it should be stimulated during cAMP-stimulated anion secretion. We therefore measured bilateral flux rates for Cl- and Na+ under open-circuit conditions (Fig. 2B) and searched for an increase in mucosal-to-serosal Cl- flux. However, we found a marked decrease in the mucosal-to-serosal Cl- flux rate after stimulation with a cAMP analog in the rat duodenum and a very small decrease in rabbit duodenum (Fig. 2B). In both species, serosal-to-mucosal Cl- fluxes strongly increased. The Cl- movements were paralleled by Na+ movements, albeit not in a one-to-one fashion. This is most likely due to the open-circuit conditions, with a lumen-negative PD and the unequal ion concentrations in the luminal and serosal perfusate as additional driving forces for ion movement. The decrease in mucosal-to-serosal Cl- and Na+ flux is most likely due to the known inhibitory effect of cAMP on electroneutral Na+Cl- absorption mediated by Na+/H+ exchanger NHE3 and an anion exchanger whose molecular identity in the duodenum is under debate. These data demonstrate that under our experimental conditions, apical anion exchange is inhibited (in rat) or unchanged (in rabbit) rather than stimulated during cAMP-mediated electrogenic anion secretion in rat and rabbit duodenum.

3H+ mannitol flux experiments were performed to assess changes in paracellular flux under conditions of the experiments. A net mucosal-to-serosal flux was found for mannitol, and 8-Br-cAMP elicited no significant changes (data not shown). Thus cAMP-induced changes in paracellular flux cannot explain the observed changes in anion secretory rate.

We then performed the same experiments under short-circuit conditions and bilateral identical CO2/-containing solutions in rabbit duodenum (n = 4). Under these circumstances, secretion cannot be measured. The mean Isc was 0.15 ± 0.18 under resting-state conditions (compared with the calculated Isc of 1.08 ± 0.16 during open-circuit conditions), and the {Delta}Isc after 8-Br-cAMP was 2.55 ± 0.16 (compared with 2.12 ± 0.18), which decreased to 0.25 ± 0.15 (compared with -0.9 ± 0.22) 1 h after ouabain application. For Na+ and Cl-, the mucosal-to-serosal flux rates were slightly lower than during open-circuit conditions (possibly due to lack of concentration gradient) and did not significantly change after 8-Br-cAMP application (data not shown), the serosal-to-mucosal Cl- flux was identical under open and short-circuit conditions, and the Cl- secretory response was similar (9.3 ± 3.5 vs. 7.1 ± 2.8). The serosal-to-mucosal Na+ flux was lower (16.8 ± 1.3 vs. 21.5 ± 0.5), and 8-Br-cAMP caused a markedly lower rise in serosal-to-mucosal Na+ flux (18.1 ± 3.7). The data demonstrate that both the measured {Delta}Isc under short-circuit conditions and the calculated {Delta}Isc under open-circuit conditions are relatively close, and both underrepresent the cAMP-induced anion secretion. They further demonstrate that the likely driving force for the strong serosal-to-mucosal Na+ flux under open-circuit conditions is the strong increase in PD. Third, they show that the Cl- secretory response in the presence of 5% CO2/24 mM in the luminal bath is identical to if not enhanced from that in its complete absence. These data suggest that concentrations that are likely to occur physiologically within the intestinal lumen do not compromise the Cl- secretory response [contrasting the recent report of a strong inhibition of the CFTR Cl- conductance by external in a cultured cell line (32)].

Ion Substitution Experiments

If cAMP-stimulated duodenal secretion is primarily mediated by an apical Cl-/ exchange process, removal of Cl- should strongly inhibit this process. Therefore, we first studied the effect of complete Cl- removal from the system. In bilateral Cl--free conditions, basal secretion decreased in both species to the same values as found in the absence of luminal Cl-, and 8-Br-cAMP elicited a secretory response that was markedly higher than in the presence of Cl- (Fig. 3). This rules out an essential role of Cl-/ exchange in cAMP-stimulated duodenal secretion.



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Fig. 3. (right axis) and Isc (left axis) in rat (A) and rabbit (B) isolated duodenum before and after complete removal of Cl- from both perfusates and the tissue as well as subsequent stimulation with 8-Br-cAMP (forskolin in the rat). Basal secretion was reduced in Cl--free conditions, but the secretory response was larger than during control conditions (P < 0.05 for both parameters), clearly ruling out a Cl-/ exchanger as the essential export mechanism in the duodenum.

 

It could be argued that the CFTR channel is permeable for in the absence but not in the presence of intracellular Cl-. Therefore, we next tested the effect of selective removal of luminal Cl-. The luminal substitution of Cl- by the gluconate ion increased Isc in both epithelia but reduced basal ouabain-sensitive (as determined in the experiments of Fig. 1) secretory rate by ~40% in rat (Fig. 4A) and rabbit duodenum (Fig. 4B), suggesting that a substantial part of basal secretion was mediated by luminal Cl-/ exchange. However, 8-Br-cAMP led to an increase of secretion that was even somewhat higher (~20% in both epithelia) than after stimulation in the presence of luminal Cl-. In the absence of luminal Cl-, ouabain does not inhibit Isc. This could be either due to the fact that in the absence of luminal Cl-, a negative membrane potential (which would be generated by basolateral K+ channel and Na+-K+-ATPase activity) is not required for Cl- flux via apical Cl- channels.



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Fig. 4. (right axis) and Isc (left axis) in rat (A) and rabbit (B) isolated duodenum before and after removal of Cl- from the luminal perfusate and subsequent stimulation with 8-Br-cAMP. Luminal Cl- removal resulted in a significant reduction of the basal secretory rate (P < 0.05; n = 5), suggesting that a substantial part of basal secretion is mediated by apical Cl-/ exchange. The secretory response to 8-Br-cAMP was even somewhat larger than in the control (not significant for n = 5).

 

Because Cl- is transported across the epithelium during luminal Cl--free conditions, and this Cl- could potentially be exchanged for intracellular via apical Cl-/ exchange, we measured the actual amount of Cl- that is transported or leaks across the mucosa into the luminal compartment. The rate of serosal-to-mucosal Cl- movement in the absence of luminal Cl- was 10.3 µmol·cm-2·h-1; thus the Cl- concentration in the luminal bulk solution was ~2 µM at the time of 8-Br-cAMP addition. This value is far lower than any Kd value for external Cl- reported for any apical anion exchange process within the intestine or for any cloned and expressed anion exchanger protein studies so far. This makes it unlikely that an apical Cl-/ is not inhibited by luminal Cl- substitution.

Effect of Bumetanide in the Presence and Absence of Luminal Cl-

Bumetanide inhibits the basolateral Na+-K+-2Cl- cotransporter, which could lead to a decrease in intracellular Cl- concentration ([Cl-]i) and an increase in Cl- import via the apical Cl-/ exchanger, in exchange for intracellular . Indeed, bumetanide in the serosal bath resulted in a decrease in Isc, an increase in secretion, and an increase in Cl- (and Na+) absorption in rat duodenum (Fig. 5A), and the most likely explanation is that bumetanide results in a decrease in [Cl-]i, which in turn stimulates apical Cl-/ exchange. Interestingly, in the rabbit duodenum, bumetanide also resulted in an increase in Na+ and Cl- reabsorption (data not shown) but not in a fall in Isc or an increase in secretion.



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Fig. 5. (right axis) and Isc (left axis) in rat isolated duodenum before (A) and after (B) removal of Cl- from the luminal perfusate and subsequent application of bumetanide (10-4 M) followed by stimulation with 8-Br-cAMP. A: as expected of an agent that blocks Na+-K+-2Cl- cotransport, bumetanide application resulted in a decrease in basal Isc and a reduced Isc response. secretion, however, increased after bumetanide application, both in the basal state and when applied during the plateau phase after 8-Br-cAMP addition (data not shown). This suggested that an inhibition of basolateral Cl- influx stimulates apical Cl- uptake via Cl-/ exchange. B: in the absence of Cl- in the luminal bath, no increase in secretion was observed after bumetanide application, suggesting that luminal Cl- removal is effective in reducing Cl- concentration near the apical membrane of the enterocytes to values low enough to inhibit the apical Cl-/ exchange. 8-Br-cAMP results in a significantly reduced Isc (P < 0.01) but normal secretory response (not significant) in both conditions, compared with the stimulation in the absence of bumetanide.

 

Thus the use of bumetanide allowed us to test whether secretion mediated by the apical Cl-/ exchanger is indeed inhibited by removal of Cl- from the luminal bath. Of course, this hypothesis could only be tested in the rat duodenum. The increase in basal secretion by bumetanide was completely inhibited by removal of luminal Cl-, whereas the subsequent secretory response to 8-Br-cAMP was slightly increased, as seen during substitution of luminal Cl- in the absence of bumetanide (Fig. 5B). The most likely explanation for this experiment is that the increased activity of apical anion exchange, induced by inhibition of basolateral Cl- uptake, is inhibited by luminal Cl- substitution. This demonstrates that luminal Cl- substitution is indeed effectively inhibiting apical Cl-/ exchange.

DIDS and 5-nitro-2-(3-phenylpropylamino)-benzoate Experiments

The brush-border membrane Cl-/ exchanger in rat and rabbit duodenum is inhibited by the stilbene derivative DIDS. The luminal application of DIDS in a concentration of 1 mmol/l approximately halved basal ouabain-sensitive secretion in rat and rabbit; but again, the 8-Br-cAMP-induced secretory response was slightly higher than in the absence of luminal DIDS (Fig. 6A for rabbit duodenum only, but



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Fig. 6. (right axis) and Isc (left axis) in rabbit duodenum after the luminal application of 1 mM DIDS, followed by stimulation with 8-Br-cAMP. The application of 1 mM DIDS resulted in a strong decrease in the ouabain-sensitive basal secretory rate (P < 0.05 and 0.01, respectively; n = 5), but the secretory response to 8-Br-cAMP was even slightly larger than under control conditions (P < 0.05; A). To test whether the major effect of luminal DIDS application was really an inhibition of apical Cl/ exchange, we also applied luminal DIDS in the complete absence of Cl in the system. In the absence of Cl, luminal DIDS had only a minimal effect on secretion (0.3 vs. 2.2 µeq·cm—2·h—1 in the presence of Cl; B).

 

rat yielded qualitatively similar results). To test whether DIDS exerts its effect on basal secretion primarily via inhibition of apical anion exchange or via inhibition of a DIDS-sensitive anion conductance or the basolateral - cotransporter, we then tested the effect of luminal DIDS on basal and stimulated secretion in the complete absence of Cl in the system (Fig. 6B). In the absence of Cl, luminal DIDS had virtually no effect on basal or stimulated secretion, demonstrating that the majority of luminal DIDS action is likely due to inhibition of the same pathway (apical Cl/ exchange) as that which is inhibited by removal of luminal Cl.

When 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), another Cl channel blocker with a known inhibitory effect on CFTR anion channels, was applied luminally, a reduction in basal Isc and a very strong inhibition of both the magnitude and the duration of 8-Br-cAMP-induced Isc and secretory response was observed ({Delta}Isc = 0.9 for rat and 0.34 for rabbit and = 0.18 for rat and 0.52 for rabbit). These results suggest that an NPPB-sensitive, DIDS-insensitive anion conductance is responsible for a major part of cAMP-stimulated secretion. The similar NPPB sensitivity of both Isc and secretory response is compatible with, but certainly does not prove, secretion via CFTR.

CFTR mRNA Expression in Rat and Rabbit Duodenum

It is known that the conductivity of the CFTR channel is not very high (3, 33), and therefore, it has been suggested that CFTR-dependent anion secretion may occur via CFTR-regulated channels or transporters rather than via the CFTR channel itself. We studied CFTR mRNA expression in rat and rabbit duodenum and compared it to the expression of other organs with a known, and high, CFTR expression, such as the pancreas or the colon. Both rat and rabbit duodenum had similarly high or higher CFTR expression levels in relationship to the other tested organs (Fig. 7). Moreover, comparing the CFTR expression levels with those for ion transport proteins such as the electroneutral Cl/ exchanger downregulated in adenoma (DRA) in the brush-border membrane of duodenal enterocytes (19), the - cotransporter NBC1 (18), or the anion exchanger AE2 in the basolateral membrane (19), we found expression levels in the same order of magnitude. Even though protein abundance and/or function may not always correlate with mRNA levels (for example, mutated CFTR protein gets degraded quickly, whereas mRNA expression levels remain unchanged), we believe that the extremely high CFTR expression levels (for a channel protein) will easily balance the relatively low conductance of each individual CFTR channel protein.



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Fig. 7. Semiquantitative RT-PCR analysis of CFTR mRNA expression levels in different segments of the gastrointestinal tract in rat (B) and rabbit (C) intestine. Exemplary for all PCR experiments, Fig. 7A shows similar amplification efficiency of the gene of interest (CFTR) and the control gene (histone 3.3a) from rabbit duodenal mucosa. The bars depict relative expression levels of rat CFTR vs. 18S RNA (B) and rabbit CFTR vs. histone 3.3a (C) (n = 3). In rabbit, the expression levels are also given (as ODI ratios) in stomach and kidney, organs that are also known to express CFTR. By comparison, duodenal CFTR expression is high.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
This study investigates the involvement of an apical Cl/ exchange process in basal and cAMP-stimulated duodenal secretion. It was found that although a Cl/ exchanger mediates an important part of the basal secretory rate, its activity is neither necessary for, nor stimulated during, cAMP-induced secretion. This finding was observed in rats, a species with rather low pancreatic and duodenal secretory rates, and rabbits, one with high rates comparable with humans. In both species, a DIDS-insensitive, NPPB-sensitive, cAMP-activated anion conductance mediates a secretory response with a strong predominance of Cl over in the secreted anions.

Gastrointestinal and hepatobiliary secretion has been recognized as a crucial transport process for the maintenance of mucosal integrity, enzymatic digestion, and Cl absorption (8, 9, 29, 31). All -secreting epithelia in CF patients show abnormal function, likely related to pH changes of their secretions. Recent evidence suggests that CFTR mutations with a strong secretory defect are associated with severe pancreatic and intestinal disease (6). Thus the question of whether the transport of occurs via the CFTR protein itself or via a CFTR-regulated anion transporter may have considerable clinical implications. In the first instance, no option exists to restore secretory function other than a repair of the defect in the CFTR protein itself. If another protein is responsible for transport, then the potential exists to activate this transport process independently of CFTR activation.

A number of theoretical considerations has suggested that the high agonist-induced secretory rates found in duodenal or pancreatic epithelium cannot be transported by the CFTR protein. Apical anion channels in epithelial cell lines with properties of the CFTR anion conductance, as well as CFTR-like channels in CFTR expression systems, had -to- permeability ratios between 1:3 and 1:8 (11, 16, 35). By extrapolating intracellular anion concentrations and membrane potentials that had been measured in primitive epithelial cell lines to the conditions in native epithelia, it was calculated that under "physiological" driving forces these apical anion conductances would secrete Cl rather than (12, 25). The actual intestinal and pancreatic transport pathways were therefore thought to be CFTR-dependent anion channels with a higher conductivity and permeability than CFTR channels or Cl/ exchangers coupled to CFTR channel activity via the intra- or extracellular Cl concentration. But these hypotheses were neither substantiated nor ruled out.

The most attractive hypothesis is a coupling between an apical anion exchanger and the CFTR anion channel. Both DRA, a recently identified intestinal anion transport protein whose mutational defects are the molecular basis for the Finnish familial chloride diarrhea, and putative anion transporter (PAT)1, a recently identified anion transport protein from the same gene family, are apically expressed in gastrointestinal and pancreatic epithelia (19, 46), and recent studies suggest both structural (27, 28) and functional interaction of CFTR and DRA/PAT1 (23).

Effective removal of Cl from the lumen inhibits any apical Cl/ exchange process, and the stilbene DIDS has been shown to inhibit DRA and PAT1. Both Cl removal from and the addition of DIDS to the luminal perfusate inhibited a substantial part of the ouabain-sensitive basal secretory rate despite having completely different effects on PD and R. This suggests that an apical Cl/ exchanger mediates a significant part of basal secretion in the duodenum of rat and rabbit and makes it unlikely that the observed changes in basal secretion were secondary to changes in paracellular movement. The application of luminal DIDS has a minimal effect on the residual secretion after complete removal of Cl, demonstrating that indeed the same transport pathway is inhibited by these maneuvers, namely, an apical anion exchange process. It rules out a substantial contribution of the highly DIDS-sensitive outwardly rectifying Cl- channel (ORCC) (10), a strong inhibition of the basolateral Na+- cotransporter by luminal DIDS application, or a major effect of external DIDS on the conductivity of the CFTR channel.

In contrast, the cAMP-induced secretory response was unaffected by Cl- removal or luminal DIDS. The strong secretory response in the complete absence of Cl- was also seen in the duodenum of CFTR +/+ but not of -/- mice (I. Blumenstein, unpublished data). The absence of in the luminal solution also makes an electrogenic 1 -to-2 exchange unlikely (23). Together, the data argue against the concept that Cl-/ exchange is involved in cAMP-dependent duodenal secretion.

A lack of effect of luminal Cl- removal on intraluminal alkalinization has been observed in isolated pancreatic and epididymal ducts (5, 17). However, it has been argued that movement of serosal Cl- through the epithelium may cause Cl- concentrations near the brush-border membrane sufficiently high to allow un-inhibited operation of an apical anion exchanger. We therefore sought for a way to stimulate the apical anion exchanger. In rat duodenum, bumetanide inhibits basolateral Na+-K+-2Cl- cotransport and reduces Isc but stimulates secretion. This stimulation is completely dependent on the presence of Cl- in the luminal bath, whereas subsequent stimulation by cAMP analogs is unaffected. The likely explanation is that the inhibition of basolateral Na+-K+-2Cl- cotransport results in a decrease in [Cl-]i, which in turn stimulates apical Cl- uptake by Cl-/ exchange. Thus the basic principle of apical Cl-/ exchange activation by low [Cl-]i is applicable. Cl- removal from the luminal bath appears to effectively inhibit apical Cl-/ exchange.

What then is the likely mechanism for agonist-stimulated duodenal secretion? Its complete absence in CFTR -/- mice is indicative of a CFTR-dependent mechanism (42). The involvement of ORCCs seems improbable because of their DIDS sensitivity (10, 21, 22). Purinergic, receptor-activated, Ca2+-dependent channels of the CaCC family are inhibited by CFTR activation (26, 48). Cl- channel ClC-2 expression has also been found to be apically located in the intestine of normal and CFTR -/- mice (20), but these are activated by osmotic swelling and hyperpolarization (36, 40, 49), the opposite of which occurs during cAMP-dependent stimulation of enterocytes. Moreover, recently a tight-junctional localization of ClC-2 has been described in small intestine (14). Very recently, a ClC-4 channel has been colocalized with CFTR in mouse and human intestine (30), but again, its properties make it unlikely that this channel mediates the cAMP-stimulated current.

Thus none of the anion transport mechanisms discussed above appears to be a more likely candidate for agonist-stimulated duodenal secretion than the CFTR protein itself. The percentage of to Cl- in the rat and rabbit (as well as mouse; data not shown) duodenal secretory response is fully compatible with any /Cl- permeability ratio measured for CFTR expression systems. NPPB sensitivity is certainly not specific for the CFTR channel, but a lack of NPPB inhibition on Isc and secretion would have been noteworthy; however, this was not found. Duodenal CFTR mRNA expression levels were found to be in the range of those for electroneutral ion exchangers like DRA (19), which, given the high transport capacity of a channel compared with an exchanger, forms the basis for substantial intestinal anion flux via CFTR.

For the measurement of secretion, we studied the tissues in the open-circuit mode. In a leaky tissue like the duodenum, changes in PD could elicit considerable ion fluxes via the tight junctions. However, the marked increase in tissue resistance after the removal of Cl- did not decrease the secretory response to cAMP analogs. Because an increase in PD negativity would inhibit paracellular secretion, the observed increase is likely transcellular.

To better understand the observed increase in cAMP-induced Na+ flux under open-circuit conditions, we measured all parameters except secretion under voltage-clamp conditions in rabbit duodenum (in this instance with CO2/ on both sides of the epithelium). We found a markedly lower cAMP-induced serosal-to-mucosal Na+ efflux, demonstrating that the strong cAMP-induced serosal-to-mucosal Na+ efflux under open-circuit conditions is indeed due to the stimulation-associated increase in PD negativity and that short circuiting can actually be achieved quite successfully in rabbit duodenum. The Cl- secretory response was not significantly affected. The surprising finding was that the accompanying Isc increase still was markedly lower than the Cl- secretory response. We do not understand this difference. Possible explanations are a relatively large conductive pathway for K+ in the brush-border membrane or basolateral anion uptake via electrogenic pathways.

We conclude that an apical Cl-/ exchange process mediates a substantial part of basal duodenal secretion. Wherever present in the apical membrane, this exchanger will allow the generation of high luminal (or CO2 concentrations, if coupled with a Na+/H+ exchanger) and low luminal Cl- concentrations. The Cl-/ exchanger, however, is not the transport pathway for agonist-stimulated secretion. While stimulated by low [Cl-]i, it is not stimulated by an activation of electrogenic anion secretion, possibly because basolateral Cl- uptake mechanisms effectively prevent a secretion-associated decrease in [Cl-]i.


    DISCLOSURES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
This work was supported by Deutsche Forschungsgemeinschaft grants Se 460/13-1 and Se 460/13-2 and by a grant from the Interdisziplindres Zentrum für Klinische Forschung Tübingen (Project IIIC1).


    ACKNOWLEDGMENTS
 
We gratefully acknowledge the expert technical assistance of Christina Neff, the technical support of Drs. Markus Guba and Irina Blumenstein, and the constructive criticism of Prof. Dr. Michael Sessler and Dr. G. Lamprecht.

This article includes experimental work performed by Stefanie Spiegel and Michael Phillipper in fulfillment of the requirements for their doctoral theses.


    FOOTNOTES
 

Address for reprint requests and other correspondence: U. Seidler, Zentrum Innere Medizin, Abteilung IV, der Medizinischen Hochschule Hannover, Carl-Neuberg Str. 1, 30625 Hannover, Germany (E-mail: seidler.ursula{at}mh-hannover.de).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

  1. Alper SL, Rossmann H, Wilhelm S, Stuart-Tilley AK, Shmukler BE, and Seidler U. Expression of AE2 anion exchanger in mouse intestine. Am J Physiol Gastrointest Liver Physiol 277: G321-G332, 1999.[Abstract/Free Full Text]
  2. Anderson MP, Rich DP, Gregory RJ, Smith AE, and Welsh MJ. Generation of cAMP-activated chloride currents by expression of CFTR. Science 251: 679-682, 1991.[ISI][Medline]
  3. Bear CE, Duguay F, Naismith AL, Kartner N, Hanrahan JW, and Riordan JR. Cl- channel activity in Xenopus oocytes expressing the cystic fibrosis gene. J Biol Chem 266: 19142-19145, 1991.[Abstract/Free Full Text]
  4. Brown CD, Dunk CR, and Turnberg LA. Cl-HCO3 exchange and anion conductance in rat duodenal apical membrane vesicles. Am J Physiol Gastrointest Liver Physiol 257: G661-G667, 1989.[Abstract/Free Full Text]
  5. Chan HC, Ko WH, Zhao W, Fu WO, and Wong PY. Evidence for independent Cl- and secretion and involvement of an apical Na+- cotransporter in cultured rat epididymal epithelia. Exp Physiol 81: 515-524, 1996.[Abstract]
  6. Choi JY, Muallem D, Kiselyov K, Lee MG, Thomas PJ, and Muallem S. Aberrant CFTR-dependent transport in mutations associated with cystic fibrosis. Nature 410: 94-97, 2001.[ISI][Medline]
  7. Chow A, Dobbins JW, Aronson PS, and Igarashi P. cDNA cloning and localization of a band 3-related protein from ileum. Am J Physiol Gastrointest Liver Physiol 263: G345-G352, 1992.[Abstract/Free Full Text]
  8. Flemstrom G and Isenberg JI. Gastroduodenal mucosal alkaline secretion and mucosal protection. News Physiol Sci 16: 23-28, 2001.[Abstract/Free Full Text]
  9. Freedman SD and Scheele GA. Acid-base interactions during exocrine pancreatic secretion. Primary role for ductal bicarbonate in acinar lumen function. Ann NY Acad Sci 713: 199-206, 1994.[Abstract]
  10. Fuller CM and Benos DJ. CFTR! Am J Physiol Cell Physiol 263: C267-C286, 1992.[Abstract/Free Full Text]
  11. Gray MA, Plant S, and Argent BE. cAMP-regulated whole cell chloride currents in pancreatic duct cells. Am J Physiol Cell Physiol 264: C591-C602, 1993.[Abstract/Free Full Text]
  12. Greger R, Mall M, Bleich M, Ecke D, Warth R, Riedemann N, and Kunzelmann K. Regulation of epithelial ion channels by the cystic fibrosis transmembrane conductance regulator. J Mol Med 74: 527-534, 1996.[ISI][Medline]
  13. Guba M, Kuhn M, Forssmann WG, Classen M, Gregor M, and Seidler U. Guanylin strongly stimulates rat duodenal secretion: proposed mechanism and comparison with other secretagogues. Gastroenterology 111: 1558-1568, 1996.[ISI][Medline]
  14. Gyomorey K, Yeger H, Ackerley C, Garami E, and Bear CE. Expression of the chloride channel ClC-2 in the murine small intestine epithelium. Am J Physiol Cell Physiol 279: C1787-C1794, 2000.[Abstract/Free Full Text]
  15. Illek B, Fischer H, and Machen TE. Genetic disorders of membrane transport. II. Regulation of CFTR by small molecules including . Am J Physiol Gastrointest Liver Physiol 275: G1221-G1226, 1998.[Abstract/Free Full Text]
  16. Illek B, Tam AW, Fischer H, and Machen TE. Anion selectivity of apical membrane conductance of Calu 3 human airway epithelium. Pflügers Arch 437: 812-822, 1999.[ISI][Medline]
  17. Ishiguro H, Steward MC, Wilson RW, and Case RM. Bicarbonate secretion in interlobular ducts from guinea-pig pancreas. J Physiol 495: 179-191, 1996.[Abstract]
  18. Jacob P, Christiani S, Rossmann H, Lamprecht G, Vieillard-Baron D, Muller R, Gregor M, and Seidler U. Role of Na+ cotransporter NBC1, Na+/H+ exchanger NHE1, and carbonic anhydrase in rabbit duodenal bicarbonate secretion. Gastroenterology 119: 406-419, 2000.[ISI][Medline]
  19. Jacob P, Rossmann H, Lamprecht G, Kretz A, Neff C, Lin-Wu E, Gregor M, Groneberg DA, Kere J, and Seidler U. Down-regulated in adenoma mediates apical Cl-/ exchange in rabbit, rat, and human duodenum. Gastroenterology 122: 709-724, 2002.[ISI][Medline]
  20. Joo NS, Clarke LL, Han BH, Forte LR, and Kim HD. Cloning of ClC-2 chloride channel from murine duodenum and its presence in CFTR knockout mice. Biochim Biophys Acta 1446: 431-437, 1999.[ISI][Medline]
  21. Jovov B, Ismailov II, Berdiev BK, Fuller CM, Sorscher EJ, Dedman JR, Kaetzel MA, and Benos DJ. Interaction between cystic fibrosis transmembrane conductance regulator and outwardly rectified chloride channels. J. Biol Chem 270: 29194-29200, 1995.[Abstract/Free Full Text]
  22. Jovov B, Shlyonsky VG, Berdiev BK, Ismailov II, and Benos DJ. Purification and reconstitution of an outwardly rectified Cl- channel from tracheal epithelia. Am J Physiol Cell Physiol 275: C449-C458, 1998.[Abstract/Free Full Text]
  23. Ko SB, Shcheynikov N, Choi JY, Luo X, Ishibashi K, Thomas PJ, Kim JY, Kim KH, Lee MG, Naruse S, and Muallem S. A molecular mechanism for aberrant CFTR-dependent transport in cystic fibrosis. EMBO J 21: 5662-5672, 2002.[Abstract/Free Full Text]
  24. Kopelman H, Corey M, Gaskin K, Durie P, Weizman Z, and Forstner G. Impaired chloride secretion, as well as bicarbonate secretion, underlies the fluid secretory defect in the cystic fibrosis pancreas. Gastroenterology 95: 349-355, 1988.[ISI][Medline]
  25. Kunzelmann K, Gerlach L, Frobe U, and Greger R. Bicarbonate permeability of epithelial chloride channels. Pflügers Arch 417: 616-621, 1991.[ISI][Medline]
  26. Kunzelmann K, Mall M, Briel M, Hipper A, Nitschke R, Ricken S, and Greger R. The cystic fibrosis transmembrane conductance regulator attenuates the endogenous Ca2+ activated Cl- conductance of Xenopus oocytes. Pflügers Arch 435: 178-181, 1997.[ISI][Medline]
  27. Lamprecht G, Heil A, Baisch S, Lin-Wu E, Yun CC, Kalbacher H, Gregor M, and Seidler U. The down regulated in adenoma (dra) gene product binds to the second PDZ domain of the NHE3 kinase A regulatory protein (E3KARP), potentially linking intestinal Cl-/ exchange to Na+/H+ exchange. Biochemistry 41: 12336-12342, 2002.[ISI][Medline]
  28. Lohi H, Lamprecht G, Markovich D, Heil A, Kujala M, Seidler U, and Kere J. Isoforms of SLC26A6 mediate anion transport and have functional PDZ interaction domains. Am J Physiol Cell Physiol 284: C769-C779, 2003.[Abstract/Free Full Text]
  29. Lubcke R, Haag K, Berger E, Knauf H, and Gerok W. Ion transport in rat proximal colon in vivo. Am J Physiol Gastrointest Liver Physiol 251: G132-G139, 1986.[Abstract/Free Full Text]
  30. Mohammad-Panah R, Ackerley C, Rommens J, Choudhury M, Wang Y, and Bear CE. The chloride channel ClC-4 colocalizes with CFTR and may mediate chloride flux across the apical membrane of intestinal epithelia. J Biol Chem 277: 566-574, 2002.[Abstract/Free Full Text]
  31. Novak I. Keeping up with bicarbonate. J Physiol 528: 235, 2000.[Free Full Text]
  32. O'Reilly CM, Winpenny JP, Argent BE, and Gray MA. Cystic fibrosis transmembrane conductance regulator currents in guinea pig pancreatic duct cells: inhibition by bicarbonate ions. Gastroenterology 118: 1187-1196, 2000.[ISI][Medline]
  33. O'Riordan CR, Erickson A, Bear C, Li C, Manavalan P, Wang KX, Marshall J, Scheule RK, McPherson JM, and Cheng SH. Purification and characterization of recombinant cystic fibrosis transmembrane conductance regulator from Chinese hamster ovary and insect cells. J Biol Chem 270: 17033-17043, 1995.[Abstract/Free Full Text]
  34. Patrizio P, Ord T, Silber SJ, and Asch RH. Cystic fibrosis mutations impair the fertilization rate of epididymal sperm from men with congenital absence of the vas deferens. Hum Reprod 8: 1259-1263, 1993.[Abstract]
  35. Poulsen JH, Fischer H, Illek B, and Machen TE. Bicarbonate conductance and pH regulatory capability of cystic fibrosis transmembrane conductance regulator. Proc Natl Acad Sci USA 91: 5340-5344, 1994.[Abstract]
  36. Pusch M, Jordt SE, Stein V, and Jentsch TJ. Chloride dependence of hyperpolarization-activated chloride channel gates. J Physiol 515: 341-353, 1999.[Abstract/Free Full Text]
  37. Quinton PM. The neglected ion: . Nat Med 7: 292-293, 2001.[ISI][Medline]
  38. Raeder MG. The origin of and subcellular mechanisms causing pancreatic bicarbonate secretion. Gastroenterology 103: 1674-1684, 1992.[ISI][Medline]
  39. Reuss L. Cyclic AMP inhibits Cl-/ exchange at the apical membrane of Necturus gallbladder epithelium. J Gen Physiol 90: 173-196, 1987.[Abstract]
  40. Roman RM, Smith RL, Feranchak AP, Clayton GH, Doctor RB, and Fitz JG. ClC-2 chloride channels contribute to HTC cell volume homeostasis. Am J Physiol Gastrointest Liver Physiol 280: G344-G353, 2001.[Abstract/Free Full Text]
  41. Rossmann H, Bachmann O, Vieillard-Baron D, Gregor M, and Seidler U. Na+/ cotransport and expression of NBC1 and NBC2 in rabbit gastric parietal and mucous cells. Gastroenterology 116: 1389-1398, 1999.[ISI][Medline]
  42. Seidler U, Blumenstein I, Kretz A, Viellard BD, Rossmann H, Colledge WH, Evans M, Ratcliff R, and Gregor M. A functional CFTR protein is required for mouse intestinal cAMP-, cGMP- and Ca2+-dependent secretion. J Physiol 505: 411-423, 1997.[Abstract]
  43. Soleimani M and Ulrich CD. How cystic fibrosis affects pancreatic ductal bicarbonate secretion. Med Clin North Am 84: 641-655, 2000.[ISI][Medline]
  44. Sorrell VF and Becroft DM. The late intestinal and hepatic complications of fibrocystic disease of the pancreas (mucoviscidosis). Aust NZ J Surg 37: 217-222, 1968.[Medline]
  45. Ulrich CD. Bicarbonate secretion and CFTR: continuing the paradigm shift. Gastroenterology 118: 1258-1261, 2000.[ISI][Medline]
  46. Wang Z, Petrovic S, Mann E, and Soleimani M. Identification of an apical Cl-/ exchanger in the small intestine. Am J Physiol Gastrointest Liver Physiol 282: G573-G579, 2002.[Abstract/Free Full Text]
  47. Warren LC. Manifestations of cystic fibrosis. Prim Care 4: 705-720, 1977.[Medline]
  48. Wei L, Vankeerberghen A, Cuppens H, Eggermont J, Cassiman JJ, Droogmans G, and Nilius B. Interaction between calcium-activated chloride channels and the cystic fibrosis transmembrane conductance regulator. Pflügers Arch 438: 635-641, 1999.[ISI][Medline]
  49. Xiong H, Li C, Garami E, Wang Y, Ramjeesingh M, Galley K, and Bear CE. ClC-2 activation modulates regulatory volume decrease. J Membr Biol 167: 215-221, 1999.[ISI][Medline]