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Am J Physiol Gastrointest Liver Physiol 277: G709-G716, 1999;
0193-1857/99 $5.00
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Vol. 277, Issue 3, G709-G716, September 1999

CFTR-mediated inhibition of epithelial Na+ conductance in human colon is defective in cystic fibrosis

M. Mall1,2, M. Bleich2, J. Kuehr1, M. Brandis1, R. Greger2, and K. Kunzelmann2

1 University Children's Hospital, Albert-Ludwigs-University Freiburg, 79106 Freiburg; and 2 Institute of Physiology, Albert-Ludwigs-University Freiburg, 79104 Freiburg, Germany


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cystic fibrosis (CF) patients show characteristic defects in epithelial ion transport, such as failure in cAMP-dependent Cl- secretion. Because the cystic fibrosis transmembrane conductance regulator (CFTR) also functions as a downregulator of epithelial Na+ channels (ENaC), enhanced Na+ conductance was found in the airways of CF patients. Here, we examined whether enhanced epithelial Na+ conductance is also present in the colonic epithelium of CF patients and examined the underlying mechanisms. Thus transepithelial voltages were measured, and equivalent short-circuit currents (Isc-eq) were determined by means of a novel type of Ussing chamber. Non-CF tissues demonstrated cAMP-dependent Cl- secretion that was absent in biopsies of CF patients. Correspondingly, Isc-eq was inhibited in non-CF but not in CF epithelia when synthesis of endogenous prostaglandins was blocked by indomethacin. In the presence of indomethacin, a larger portion of amiloride-sensitive Isc-eq was detected in CF tissues, suggesting enhanced ENaC conductance in colonic mucosa of CF patients. Increase of intracellular cAMP by forskolin and IBMX inhibited amiloride-sensitive ENaC currents in non-CF tissues but not in CF biopsies. Therefore, enhanced epithelial Na+ conductance is present in the CF colon and is probably due to missing downregulation by CFTR.

cystic fibrosis transmembrane conductance regulator; epithelial transport; amiloride-sensitive epithelial sodium channels; Ussing chamber; transepithelial voltage; colonic sodium absorption


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

CYSTIC FIBROSIS (CF) is a common inherited disease that is characterized by a defective cAMP-regulated Cl- conductance due to impaired function of the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR has been studied and characterized intensively, and its function as a cAMP-regulated Cl- channel has been assessed in several previous reports (34). Apart from the defect in cAMP activated Cl- conductance, it has been known for several years that amiloride-inhibited Na+ reabsorption is enhanced in the airways of CF patients (3, 4). Only recently, after cloning of the amiloride-sensitive epithelial Na+ channel (ENaC) (8), it became obvious that enhanced reabsorption of fluid and electrolytes in the respiratory tract of CF patients is due to enhanced ENaC currents (20, 26). It was demonstrated in several previous studies that ENaCs, when coexpressed, e.g., in oocytes of Xenopus laevis, with wild-type CFTR but not with Delta F508-CFTR, are inhibited during stimulation by agonists raising intracellular cAMP (28, 36). This mechanism also takes place in cells expressing both proteins endogenously (11, 22) and was shown in normal human airways but not in airways of CF patients (26). These recent findings may explain the enhanced amiloride-sensitive Na+ conductance and increased reabsorption of electrolytes in CF airways that lead to highly viscous mucus and reduced mucociliary clearance (39).

CFTR probably is the only apical Cl- conductance in the human intestinal mucosa; therefore, it is not surprising that altered cAMP-dependent and cholinergic Cl- conductance has been reported in CF (1, 12, 17, 27, 31, 38). Thus intestinal Cl- secretion relies entirely on luminal CFTR Cl- channels. This abnormality in intestinal electrolyte transport may be essential for the pathogenesis of meconium impactions as well as for other gastrointestinal complications in CF like the distal intestinal obstruction syndrome (1, 24, 29). In this respect, murine and rat colonic epithelia reflect much of the properties of the human colon (2, 15, 25). Altered cholinergic and cAMP-dependent intestinal Cl- transport was demonstrated in CFTR (-/-) knockout mice (9, 10). Moreover, these animals display many features common to CF patients, like failure to thrive and meconium ileus, and typically the animals die from intestinal obstruction during the first month (9, 35).

Apart from this basic defect in CFTR Cl- conductance, enhanced amiloride-sensitive Na+ conductance was reported for the intestinal epithelium of CFTR (-/-) knockout mice (14), which was, however, questioned by the results of another group (10). It was suggested that differences in amiloride-sensitive Na+ conductance between normal and CF mice only become evident when these mice were placed on a low-Na+ diet or when treated with aldosterone (15). Furthermore, conflicting results have also been obtained in previous studies, when transrectal potential differences were measured in vivo in CF patients and healthy volunteers or when ion transport was measured on rectal biopsies from CF patients and controls. Although greater amiloride-sensitive transport was found in the CF intestine in some studies (13, 32), it was not detected in others (12, 17).

Because it is not clear whether amiloride-sensitive Na+ conductance is enhanced in the human intestinal epithelium, we performed the present experiments on human forceps biopsies and measured ion transport under well-controlled conditions in a novel type of Ussing chamber. The data presented here indicate enhanced amiloride-sensitive Na+ conductance in CF intestinal epithelium that is caused by a lack of CFTR-dependent inhibition of the ENaC.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Patients. Tissue biopsies were obtained from 34 non-CF patients with a mean age of 25.4 ± 4.3 yr (ranging from 1 mo to 88 yr) and 14 CF patients with a mean age of 22.0 ± 2.4 yr (ranging from 6 mo to 35 yr). Six of the Non-CF patients underwent routine surgical procedures of the distal colon at the University Hospital Freiburg. Immediately after the operation, small forceps biopsies were taken from tissue not affected by the primary disease that called for the surgical intervention. In the remaining 28 non-CF patients and in all 14 CF patients, small tissue biopsies of ~2-3 mm diameter were obtained by rectoscopy and forceps biopsy was performed at the University Children's Hospital Freiburg. The study was approved by the ethical committee, and the patients had given their written informed consent. For children under the age of 18 yr, parents obtained detailed information and gave their signed informed consent.

Histology. To delineate the contribution of different tissue layers to the transepithelial resistance (Rte), histology was performed on some of the tissue biopsies used for Ussing chamber experiments. Forceps biopsies were fixed in a 4% formalin solution and embedded in paraffin, and sections were stained with hematoxylin-eosin.

Ussing chamber experiments. Tissue biopsies of ~2-3 mm diameter were immediately put into an ice-cold buffer solution of the following composition (mmol/l): 127 NaCl, 5 KCl, 5 D-glucose, 1 MgCl2, 5 sodium pyruvate, 10 HEPES, 1.25 CaCl2, and albumin (10 g/l). The biopsies were mounted into a modified Ussing chamber with a circular aperture of 0.95 mm2. The luminal and basolateral sides of the epithelium were perfused continuously at a rate of 15 ml/min (chamber volume of 1 ml), allowing for the paired examination of the effects of amiloride in the absence and presence of cAMP stimulation. The bath solution had the following composition (mmol/l): 145 NaCl, 0.4 KH2PO4, 1.6 K2HPO4, 5 D-glucose, 1 MgCl2, and 1.3 calcium gluconate. The pH was adjusted to 7.4. Bath solutions were heated by a water jacket to 37°C. Experiments were carried out under open-circuit conditions. This was done by determining the Rte by applying short (1 s) current pulses (Delta I = 0.5 µA) and by recording the corresponding changes in transepithelial voltage (Delta Vte) as well as the basal Vte continuously. Values for Vte were referred to the serosal side of the epithelium. Resistance of the empty chamber was 9.5 Omega  · cm2, and the voltage deflections obtained under conditions without the mucosa present (Delta V'te) were subtracted from those obtained in the presence of the tissues. Rte was calculated according to Ohms law (Rte = Delta Vte - Delta V'te/Delta I). Tissue preparations were only accepted if Rte values exceeded those obtained for an empty chamber by at least a factor of 2. The equivalent short-circuit current (Isc-eq) was determined from Vte and Rte, i.e., Isc-eq = Vte/Rte. From each of patients 1-6, in most cases four biopsies were examined.

Experimental protocols. Typically, an equilibration period of 20-30 min was allowed for stabilization of basal Vte and Rte. It has been shown that endogenously produced PGE2 is a strong activator of cAMP-dependent Cl- secretion in distal colon and that the effect of PGE2 can be inhibited by the cyclooxygenase inhibitor indomethacin (6, 7, 30). To suppress the influence of cAMP-dependent luminal Cl- channels on lumen-negative Vte, indomethacin (10 µmol/l) was added to the basolateral side of the colonic mucosa for 40-60 min. After inhibition of endogenous prostaglandins and intracellular cAMP, the effect of amiloride (10 µmol/l), added to the luminal side of the colonic mucosa, was examined. Subsequently, in the presence of indomethacin (10 µmol/l, basolateral solution) and amiloride (10 µmol/l, luminal solution), we examined the effects of activators of the intracellular cAMP pathway on Cl- secretion by adding IBMX (100 µmol/l) and forskolin (1 µmol/l) to the basolateral side of the mucosa. In a subset of experiments, the effect of amiloride (10 µmol/l) was studied under three different conditions in a strictly paired fashion: 1) under control conditions, 2) in the presence of indomethacin, and 3) after cAMP activation with IBMX and forskolin. Between each step, amiloride was washed out for 20-30 min. The whole protocol typically took 3-4 h, and stable recordings with relatively large Isc-eq were obtained during this time period. In four CF rectal tissue biopsies, the amiloride effect was examined at five different concentrations (0.01, 0.1, 1, 10, and 100 µmol/l) and a concentration-response curve was constructed. The data were fitted with a fit routine using the equation I = Imax/[1 + (IC50/C)n], were I is the measured equivalent short-circuit current, Imax is the maximum equivalent short-circuit current, C is the concentration of amiloride used, IC50 is the concentration of amiloride required to achieve half-maximal inhibition, and n is the degree of cooperativity (19). The IC50 value for inhibition by amiloride was obtained during the fit routine.

Compounds and analysis. Amiloride, indomethacin, tetraethylammonium, Ba2+, and IBMX were all obtained from Sigma and Merck (Deisenhofen and Darmstadt, Germany). Forskolin was obtained from Hoechst (Frankfurt, Germany). All chemicals used were of the highest grade of purity available. Data are shown as individual recordings or as means ± SE (n = number of tissue samples). Statistical analysis was performed using paired Student's t-test. Data obtained from CF and non-CF tissues were compared by the unpaired Student's t-test. If not stated otherwise, P values < 0.05 were accepted to indicate statistical significance.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Contribution of Na+ conductance to ion transport in non-CF and CF rectal mucosa. After equilibration for 20-30 min in the Ussing chamber, non-CF tissues had a lumen-negative Isc-eq of -33.4 ± 2.2 µA/cm2 (Vte = -0.8 ± 0.1 mV; Rte = 23.6 ± 1.4 Omega  · cm2; n = 80). In CF tissues (n = 34), Isc-eq was significantly smaller (-15.0 ± 3.9 µA/cm2, P < 0.0001), Vte was significantly lower (-0.5 ± 0.1 mV, P < 0.03), and Rte was significantly increased (33.4 ± 2.9 Omega  · cm2; P < 0.001) compared with non-CF tissues (see Figs. 2, A and B, and 4). A histological section of a biopsy specimen used for Ussing chamber experiments is shown in Fig. 1. Superficial forceps biopsies contained mucosa with surface epithelium and crypts. Occasionally, small islets of muscularis mucosae, but no continuous muscle layer, were detected in these tissues, demonstrating that muscularis propria did not contribute to the formation of Rte.


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  		Fig. 1.   Histological section (hematoxylin-eosin staining) of a typical biopsy specimen used for Ussing chamber experiments. Rectal mucosa is shown with surface epithelium and crypts. As shown here, small islets of muscularis mucosae, but no continuous muscle layer, were occasionally detected in these tissues, demonstrating that muscularis propria did not contribute to the formation of transepithelial resistance (Rte).

cAMP-dependent luminal Cl- conductance was blocked by incubating the tissues with the cyclooxygenase inhibitor indomethacin (10 µmol/l) (6, 7, 30). In non-CF tissues, a significant decrease in lumen-negative Vte (Delta Vte = 0.4 ± 0.1 mV) was observed and Rte was significantly increased by 4.5 ± 0.7 Omega  · cm2. Thus, due to inhibition of Cl- secretion, Isc-eq was significantly attenuated by 19.9 ± 1.9 µA/cm2 (n = 80). In CF tissues, indomethacin did not inhibit Cl- secretion and had inverse effects on Vte and Isc-eq: Vte and Isc-eq significantly increased by -0.3 ± 0.1 mV and -5.7 ± 2.4 µA/cm2, respectively, and Rte was significantly increased by 3.9 ± 1.3 Omega  · cm2 (n = 34) (Figs. 2B and 4).



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  		Fig. 2.   Effects of indomethacin (10 µmol/l) and amiloride (10 µmol/l) on transepithelial voltage (Vte) and Rte of non-cystic fibrosis (CF) (A) and CF (B) human colonic epithelia. Rte was determined from the Vte downward deflections obtained by pulsed current injection. A: spontaneous lumen-negative Vte of a non-CF epithelium was attenuated by perfusion of the bath with indomethacin and was completely abolished when amiloride was applied to the luminal compartment in the presence of indomethacin. Subsequent stimulation with IBMX and forskolin (IBMX/Fors) (100 and 1 µmol/l, respectively) induced lumen-negative Vte, which is caused by activation of Cl- secretion. Time gaps between records were 30 and 10 min, respectively. B: spontaneous lumen-negative Vte of a CF epithelium was not compromised by indomethacin but was completely abolished by amiloride. Subsequent addition of IBMX and forskolin failed to induce Cl- secretion but induced a slightly positive Vte probably caused by activation of K+ secretion. Time gaps between records were 20 and 10 min, respectively. Con, control.

After inhibition of prostaglandin synthesis and in the presence of indomethacin, the magnitude of the epithelial Na+ conductance was assessed by applying amiloride (10 µmol/l) to the luminal side of the epithelium. Inhibition of Isc-eq by amiloride was concentration dependent and entirely reversible on washout. The IC50 for the inhibitory effects of amiloride on Isc-eq was obtained by fitting the concentration response curve (Fig. 3) and was 0.617 µmol/l, indicating the presence of high-affinity Na+ channels (ENaC) in human colonic and rectal mucosa (n = 4). In non-CF tissues, amiloride significantly reduced lumen-negative Vte from -0.4 ± 0.0 mV to -0.1 ± 0.0 mV and increased Rte significantly from 22.7 ± 1.5 to 28.7 ± 1.5 Omega  · cm2. Thus Isc-eq was significantly decreased from -13.3 ± 1.2 to -3.0 ± 0.6 µA/cm2 (Delta Isc-eq = 10.4 ± 1.1 µA/cm2, n = 80; P < 0.0001). The effects on rectal biopsies from CF patients were more pronounced compared with non-CF tissues. Amiloride significantly reduced Vte from -0.8 ± 0.2 to 0.0 ± 0.0 mV. Rte was significantly increased from 37.4 ± 2.5 to 38.1 ± 2.6 Omega  · cm2, and Isc-eq was significantly inhibited from -17.4 ± 3.8 to -0.4 ± 0.9 µA/cm2 (Delta Isc-eq = 17.0 ± 3.6 µA/cm2, n = 34; P < 0.0001). Thus the amiloride-sensitive Isc for CF tissues was significantly increased compared with that for non-CF tissues (P < 0.03). These results demonstrate enhanced amiloride-sensitive Na+ transport in the rectal mucosa of CF patients (Figs. 2, 4, and 6), similar to what has been described already for the respiratory tract of CF patients (26).


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  		Fig. 3.   Concentration response curve for the inhibition of equivalent short-circuit current (Isc-eq) by amiloride in CF mucosal epithelia. IC50 was 0.617 µmol/l, indicating inhibition of amiloride-sensitive epithelial Na+ channel (ENaC). Number in parentheses is the number of tissue samples. Here, Isc-eq is indicated by the abbreviation Isc and Isc max is the maximum Isc-eq.

cAMP-dependent Cl- conductance in non-CF and CF rectal mucosa. We further examined the ability of both non-CF and CF colonic mucosa to secrete Cl- in response to stimulation with agonists increasing intracellular cAMP. In the presence of amiloride and indomethacin, cAMP-dependent Cl- secretion was induced in non-CF tissues (Delta Vte = -1.1 ± 0.1 mV, Delta Rte = -3.1 ± 0.7 Omega  · cm2, Delta Isc-eq = -47.4 ± 4.2 µA/cm2, n = 80) by stimulating the tissues with IBMX (100 µmol/l) and forskolin (1 µmol/l), similar to what has been reported previously (27). However, in CF tissues, the effects were reversed compared with those in non-CF tissues. In contrast to normal tissues, IBMX and forskolin significantly increased lumen-positive Vte by 0.2 ± 0.0 mV and Rte significantly decreased by 2.7 ± 1.1 Omega  · cm2. Accordingly, a significant lumen-positive Isc-eq of 3.4 ± 1.6 µA/cm2 was activated (n = 34). Thus Cl- secretion was activated in non-CF colonic mucosa by cAMP-dependent stimulation, whereas this was not observed in CF tissues. The lumen-positive Isc-eq activated by IBMX and forskolin in CF colonic epithelia is most likely caused by activation of a K+ secretion, which remains intact in CF intestine (27) (Figs. 2 and 4).


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  		Fig. 4.   Summary of the Isc-eq data calculated from experiments shown in Fig. 2A (non-CF) and Fig. 2B (CF). In non-CF tissue, indomethacin significantly inhibited lumen-negative Isc-eq, which was further inhibited by addition of amiloride. Subsequent application of IBMX and forskolin activated a Cl- secretion and thus enhanced lumen-negative Isc-eq. In CF tissues, lumen-negative Isc-eq was significantly lower. Indomethacin did not block lumen-negative Isc-eq but had an inverse effect. A significantly larger portion of Isc-eq was blocked by amiloride compared with the non-CF mucosa. No Cl- secretion was activated by IBMX and forskolin in the CF epithelium. Instead, a small lumen-positive response was observed, indicating activation of K+ secretion. Left bars for non-CF and CF data are controls. * Statistical significance (paired t-test). § Statistical significance compared with the experiments performed in non-CF tissues (unpaired t-test). n = Number of tissue samples (shown in parentheses).

Inhibition of epithelial Na+ conductance by CFTR in non-CF but not in CF rectal mucosa. We further investigated the mechanism responsible for enhanced Na+ absorption in CF colon. In particular, we were interested in a possible impact of CFTR function on the activity of ENaC. To this end, in a subset of experiments, the effects of amiloride were additionally examined in CF and non-CF tissues under basal conditions and subsequently after cAMP-dependent stimulation. In non-CF tissues, amiloride, under basal conditions, significantly reduced Vte from -0.8 ± 0.1 to -0.6 ± 0.1 mV and Rte was significantly increased from 22.9 ± 1.4 to 24.1 ± 1.5 Omega  · cm2. Accordingly, Isc-eq was significantly inhibited from -32.3 ± 2.3 to -25.8 ± 2.1 µA/cm2, corresponding to Delta Isc-eq = 6.5 ± 1.2 µA/cm2 (n = 70; P < 0.0001) (Figs. 5A and 6). Thus the amount of amiloride-sensitive Isc-eq was significantly smaller compared with that observed in the presence of indomethacin (P < 0.002) in strictly paired experiments, i.e., after blocking endogenous prostaglandin synthesis and thus reducing CFTR activity (Delta Isc-eq = 10.9 ± 1.2 µA/cm2; n = 70; P < 0.0001). In addition, amiloride-sensitive Isc-eq was even further and significantly attenuated after stimulation of the tissues with IBMX and forskolin (P < 0.0001). Under these conditions, amiloride inhibited Isc-eq only slightly but significantly from -53.5 ± 5.0 to -50.5 ± 4.6 µA/cm2 (Delta Isc-eq = 3.0 ± 0.7 µA/cm2; n = 70; P < 0.0001), with only minimal changes of Vte and Rte (Delta Vte = 0.1 ± 0.0 mV, Delta Rte = 0.1 ± 0.1 Omega  · cm2) (Figs. 5A and 6).



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  		Fig. 5.   Effects of amiloride under various experimental conditions indicate inhibition of amiloride-sensitive Na+ transport by IBMX and forskolin in non-CF (A) but not in CF (B) intestinal biopsies. A: a small but significant inhibition of lumen-negative Vte was observed in non-CF biopsies under basal conditions. After incubation with indomethacin (10 µmol/l), Vte was attenuated and the effect of amiloride was augmented. The opposite, namely, an increase in lumen-negative Vte and small effects of amiloride, was observed when the epithelium was stimulated with IBMX and forskolin. Time gaps between records were 30 min. B: in CF mucosa, neither lumen-negative Vte nor inhibition by amiloride was essentially changed by either incubation with indomethacin or stimulation with IBMX and forskolin. Time gaps between records were 30 min.

Different observations were made for the CF intestinal epithelium. Under basal conditions, lumen-negative Vte was significantly reduced from -0.5 ± 0.2 to 0.2 ± 0.1 mV and Rte significantly increased from 35.7 ± 3.0 to 38.0 ± 3.2 Omega  · cm2. Isc-eq was inhibited from -14.2 ± 3.4 to 4.9 ± 1.4 µA/cm2 (Delta Isc-eq = 19.1 ± 3.7 µA/cm2; n = 30; P < 0.0001) (Figs. 5B and 6). Incubation with indomethacin did not change amiloride-sensitive Isc-eq (Delta Isc-eq = 19.2 ± 4.0 µA/cm2; n = 30; P < 0.0001) in CF tissues. After stimulation with IBMX and forskolin, amiloride significantly attenuated Vte from -0.6 ± 0.1 to +0.2 ± 0.1 mV and significantly increased Rte from 37.4 ± 2.0 to 38.1 ± 2.1 Omega  · cm2. Isc-eq was significantly inhibited from -14.8 ± 3.0 to 5.0 ± 1.4 µA/cm2. However, the amiloride-sensitive Isc-eq (Delta Isc-eq = 19.8 ± 3.0 µA/cm2; n = 30; P < 0.001) remained unchanged compared with control conditions or incubation with indomethacin. When the results from non-CF and CF tissues (Fig. 6) are compared, it becomes obvious that 1) amiloride-sensitive Isc-eq is significantly enhanced in CF independent of the absence (P < 0.0001) or presence of either indomethacin (P < 0.03) or IBMX and forskolin (P < 0.0001) and 2) amiloride-sensitive Na+ currents are inhibited by activation of CFTR only in non-CF but not in CF tissues. These results demonstrate enhanced amiloride-sensitive Na+ conductance that is caused by a lack of CFTR-dependent regulation of ENaC in CF (Figs. 5A and 6).


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  		Fig. 6.   Summary of the amiloride-sensitive Isc-eq as calculated from data obtained in experiments shown in Fig. 5A (non-CF) and Fig. 5B (CF). Left: in non-CF tissue, amiloride-sensitive Isc-eq was significantly enhanced after incubation with indomethacin ($ P < 0.002, paired t-test) and was significantly attenuated after stimulation with IBMX and forskolin (# P < 0.0001, paired t-test). Right: in CF tissue, amiloride-sensitive Isc-eq was not affected by either indomethacin or stimulation with IBMX and forskolin. However, when compared with non-CF tissues, the amiloride-sensitive Isc-eq was significantly enhanced in CF under basal conditions (§ P < 0.0001, unpaired t-test), in the presence of indomethacin ( P < 0.03, unpaired t-test) and in the presence of IBMX and forskolin (§). * Statistical significant effect of amiloride (P < 0.0001, paired t-test). n = Number of tissue samples, shown in parentheses.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The present study compares ion transport in human rectal and colonic mucosae from normal individuals with those from CF patients. To be able to examine small human tissues biopsies, we developed a novel type of micro-Ussing chamber, with a very small exposed tissue area of <1 mm2. This allows for stable and continuous measurement of Vte and Rte for the whole course of the experiments, which typically took 3-4 h. No continuous muscle layers were found in histological sections of biopsies used in this study. Only small islets of muscularis mucosae were present in superficial forceps biopsies (Fig. 1). Therefore, muscle layers did not contribute essentially to the formation of Rte. Continuous bath exchange enables paired experiments that are essential for detection of regulatory mechanisms of electrolyte transport. The tissue biopsies were examined under open-circuit conditions, in principle resembling the in vivo situation. Similar Isc-eq recordings were taken from different sections of the descending colon and rectal mucosae. Due to imperfect edge sealing, the absolute magnitude of measured Rte was decreased and therefore Vte was certainly underestimated compared with conditions in vivo (13, 33).

Analysis of Cl- transport confirms missing cAMP-dependent secretory responses in the CF intestine that was reported in previous studies (1, 12, 17, 38). Both cholinergic and cAMP-activated Cl- secretion depend on luminal CFTR Cl- channels, and both are defective in CF (27, 38). Accordingly, in non-CF tissue, lumen-negative Isc-eq was significantly inhibited when prostaglandin production, and thus cAMP synthesis, was blocked with the cyclooxygenase inhibitor indomethacin (6, 7, 30). In CF tissues, inverse changes of Isc-eq were observed when exposed to either indomethacin or IBMX and forskolin. Activation of a lumen-positive Isc-eq by IBMX and forskolin in CF was most probably due to activation of a luminal K+ conductance that was reversibly blocked by Ba2+ (5 mmol/l) added to the luminal side of the mucosa (data not shown). However, the present experiments also demonstrate an enhanced amiloride-sensitive Na+ conductance that exists in the CF intestine. This was suggested in a previous report on the basis of in vivo-measured potential differences but was not confirmed by a subsequent study that showed Ussing chamber recordings of CF and non-CF rectal mucosa (17, 33). This discrepancy is probably caused by the different experimental conditions used in both studies.

In the present experiments, the effects of amiloride on Vte and Rte were examined under control conditions, in the absence and in the presence of cAMP activation, which had a large influence in non-CF but no influence in CF tissues. As described previously for transrectal measurements in vivo (13, 33), amiloride significantly inhibited lumen-negative Vte in non-CF and CF rectal and colonic tissues. Independent of cAMP-dependent stimulation, the effects of amiloride were significantly enhanced in CF. However, in vivo potential difference measurements do not allow for quantitative assessment of the contribution of different membrane conductances to ion transport. To be able to quantify transepithelial membrane currents, Rte was recorded continuously, allowing the determination of the Isc-eq. Although compromised by edge leak conductance, inhibition of Vte by amiloride was accompanied by an increase in Rte as expected for ENaC inhibition. In the present study, we demonstrated that absolute values for amiloride-sensitive Isc-eq were significantly increased in CF and downregulated by cAMP activation in non-CF tissues, thereby unmasking the cause for enhanced Na+ transport in CF intestine. Enhanced intestinal electrogenic Na+ transport has also been demonstrated in the meantime for CFTR (-/-) knockout mice (14, 16).

The present results suggest that CFTR-dependent regulation of ENaC exists in human intestine. Very similar results have been previously reported for rat colonic epithelial cells (11). The mechanisms of CFTR-dependent regulation of ENaC are not very well understood and are currently under investigation. It was initially observed in Madin-Darby canine kidney cells expressing both ENaC and CFTR and was subsequently demonstrated in Xenopus oocytes (28, 36). Additional evidence for the inhibitory impact of CFTR on ENaC came from experiments on mouse kidney cells, rat colonic epithelial cells, and Xenopus A6 cells (11, 22, 23). A rather close regulatory relationship of both proteins is likely, since inhibition of ENaC by CFTR was identified in excised membrane patches and planar lipid bilayers carrying both proteins, and direct interaction was shown in one study (18, 21, 37). Moreover, Cl- flux through CFTR probably triggers inhibition of ENaC (5). Subsequent experiments are required to demonstrate whether additional proteins are involved in the regulatory cascade conferring inhibitory effects of CFTR on ENaC.

The possible pathophysiological implications for missing downregulation of ENaC by CFTR and consecutive increase in colonic Na+ absorption are obvious. In neonates suffering from CF, this perturbation of Na+ transport may contribute to alteration of mucous properties, hyperabsorption, obstruction, and meconium ileus. In older CF patients, enhanced Na+ absorption may cause the meconium ileus equivalent termed distal intestinal obstruction syndrome (24, 29). Therefore, the potential therapeutic benefit of amiloride should be evaluated. For this, the CF mouse would serve as an excellent model to examine effects of amiloride or related compounds such as phenamil or benzamil on the observed intestinal alterations in these animals.


    ACKNOWLEDGEMENTS

We gratefully thank Dr. P. Greiner at the Children's Hospital, University of Freiburg, for performing rectoscopy procedures and Dr. M. Kleinschmidt at the Institute of Pathology, University of Freiburg, for performing histology on tissue biopsies. We further acknowledge the expert technical assistance by S. Hirtz and C. Hodler.


    FOOTNOTES

This work was supported by Deutscheforschungsgemeinschaft (DFG) Ku756/2-3, DFG Gr480/11, Zentrum Klinische Forschung 1, and Fritz Thyssen Stiftung.

K. Kunzelmann is supported by a Heisenberg fellowship.

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 and other correspondence: M. Mall, Univ. Children's Hospital, Albert-Ludwigs-Univ. Freiburg, Mathildenstrasse 1, 79106 Freiburg, Germany (E-mail: mall{at}ruf.unifreiburg.de).

Received 1 March 1999; accepted in final form 21 June 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Am J Physiol Gastroint Liver Physiol 277(3):G709-G716
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society


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