Defective cholinergic Clminus secretion and detection of K+ secretion in rectal biopsies from cystic fibrosis patients

M. Mall1,2, A. Wissner2, H. H. Seydewitz1, J. Kuehr1, M. Brandis1, R. Greger2, and K. Kunzelmann3

1 Universitäts-Kinderklinik, Albert-Ludwigs-Universität Freiburg, 79106 Freiburg; 2 Physiologisches Institut, Albert-Ludwigs-Universität Freiburg, 79104 Freiburg, Germany; and 3 Department of Physiology, University of Sydney, Sydney NSW 2006, Australia


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Rectal biopsies from cystic fibrosis (CF) patients show defective cAMP-activated Cl- secretion and an inverse response of the short-circuit current (Isc) toward stimulation with carbachol (CCh). Alternative Cl- channels are found in airway epithelia and have been attributed to residual Cl- secretion in CF colon. The aim of the present study was to investigate ion conductances causing reversed Isc upon cholinergic stimulation. Furthermore, the putative role of an alternative Ca2+-dependent Cl- conductance in human distal colon was examined. Cholinergic ion secretion was assessed in the absence and presence of cAMP-dependent stimulation. Transepithelial voltage and Isc were measured in rectal biopsies from non-CF and CF individuals by means of a perfused micro-Ussing chamber. Under baseline conditions, CCh induced a positive Isc in CF rectal biopsies but caused a negative Isc in non-CF subjects. The CCh-induced negative Isc in non-CF biopsies was gradually reversed to a positive response by incubating the biopsies in indomethacin. The positive Isc was significantly enhanced in CF and was caused by activation of a luminal K+ conductance, as shown by the use of the K+ channel blockers Ba2+ and tetraethylammonium. Moreover, a cAMP-dependent luminal K+ conductance was detected in CF individuals. We conclude that the cystic fibrosis transmembrane conductance regulator is the predominant Cl- channel in human distal colon. Unlike human airways, no evidence was found for an alternative Cl- conductance in native tissues from CF patients. Furthermore, we demonstrated that both Ca2+- and cAMP-dependent K+ secretion are present in human distal colon, which are unmasked in rectal biopsies from CF patients.

cystic fibrosis transmembrane conductance regulator; human colon; epithelial transport; Ussing chamber


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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IN CYSTIC FIBROSIS (CF), the well-documented defect in cAMP-activated Cl- secretion affects epithelial tissues such as airways and intestine (4, 5). Alternative Ca2+-activated Cl- channels are present in CF airway epithelia (16, 21) and have been reported in several previous studies, some of which are recent, on cultured colonic carcinoma cells (1, 10, 22, 29). Furthermore, a Ca2+-dependent Cl- conductance has been suggested as an alternative Cl- secretory pathway in rectal biopsies of CF patients (34). From studies on native tissues, however, there is growing evidence that only one type of Cl- conductance is present in the luminal membrane of colonic epithelial cells that shares the properties of the cystic fibrosis transmembrane conductance regulator (CFTR) (11, 27, 32). It has also been shown that Ca2+-activated Cl- channels vanish and luminal membranes become dominated by cAMP-activated CFTR Cl- channels when these cells are grown as polarized monolayers (9). In highly differentiated tissues, Ca2+-activated Cl- channels may therefore be localized exclusively in the basolateral membrane. In addition, another recent study performed on native rat colonic epithelium demonstrated evidence for basolateral swelling-activated Cl- channels (31).

It has been shown previously that inactivation of luminal CFTR Cl- channels in native human colonic epithelium leads to complete disappearance of Ca2+-activated Cl- secretion (27). This and other reports (35) support the concept that Ca2+-activated Cl- secretion in human colonic epithelium is caused by activation of a basolateral K+ conductance increasing the electrical driving force for luminal Cl- exit via CFTR Cl- channels (11, 25, 27, 32). This was further confirmed by experiments on CFTR knockout mice showing a pathophysiology similar to that of CF patients (11, 15).

Besides the well-characterized defect in cAMP-dependent Cl- secretion, rectal biopsies from CF patients show a reversed lumen-positive response toward cholinergic stimulation. Here we examined the ion conductances underlying the inverse short-circuit current (Isc) response observed in rectal biopsies from CF subjects after stimulation with carbachol (CCh). We further investigated the possible role of Ca2+-dependent Cl- secretion that has been attributed to residual Cl- secretion in CF patients. The data show that a CF phenotype can be mimicked in non-CF patients when endogenous cAMP synthesis is inhibited by indomethacin treatment (6, 7). We further demonstrated that the inverse cholinergic Isc response, as observed in CF, is caused by K+ secretion. In future, detection of characteristic ion transport abnormalities in rectal biopsies could serve as a functional in vitro test for CF (30).


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INTRODUCTION
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Patients. Rectal biopsies were obtained from 26 non-CF volunteers with a mean age of 18.7 yr (ranging from 5 to 57 yr) and 8 CF patients with a mean age of 19.3 yr (ranging from 6 mo to 35 yr). All CF subjects fulfilled the diagnostic criteria for CF (30). Five CF patients were homozygous for the mutation Delta F508, and three were compound heterozygous for Delta F508 and the stop mutation R553X. In non-CF volunteers, in addition to functional studies on rectal biopsies, sweat tests were performed and DNA analysis was carried out for common CFTR mutations (Delta F508; R117H and S108F in exon 4; R347P, R347H, I336K, and T338I in exon 7; S549N, G551D, R553X, G542X, Q552X, and 1717-1 Gright-arrowA in exon 11; W1282X and 3905insT in exon 20; N1303K in exon 21; and 3849 + 10kB Cright-arrowT in intron 19) by PCR and single-strand conformation polymorphism analysis. The tests were unremarkable for all individuals studied. Superficial tissue biopsies ~2-3 mm in diameter were obtained by rectoscopy and forceps biopsy 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, parents obtained detailed information and gave their signed informed consent.

Ussing chamber experiments. Tissue biopsies were immediately stored in ice-cold buffer solution of the following composition (in mmol/l): 127 NaCl, 5 KCl, 5 D-glucose, 1 MgCl2, 5 sodium pyruvate, 10 HEPES, and 1.25 CaCl2; the solution also contained 10 g/l albumin. The biopsies were mounted in a modified Ussing chamber with a circular aperture of 0.95 mm2 as described previously (26). The luminal and basolateral sides of the epithelium were perfused continuously at a rate of 15 ml/min (chamber volume 1 ml), allowing for the paired examination of the effects of cholinergic activation in the absence or 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. Transepithelial resistance (Rte) was determined by applying short (1-s) current pulses (Delta I = 0.5 µA), and the corresponding changes in transepithelial voltage (Delta Vte) as well as the basal Vte were recorded continuously. Values for the Vte were referred to the serosal side of the epithelium. Voltage deflections obtained under conditions without the mucosa present (Delta Vte') were subtracted from those obtained in the presence of the tissues. Rte was calculated according to Ohm's law [Rte = (Delta Vte-Delta Vte')/Delta I]. The equivalent Isc was determined from Vte and Rte; i.e., Isc = Vte/Rte. After mounting the tissues in the Ussing chamber, an equilibration period of 30 min was allowed for stabilization of basal Vte and Rte. To inhibit electrogenic Na+ absorption, all experiments were performed in the presence of amiloride (10 µmol/l) added to the luminal side of the mucosa.

Compounds and analysis. Amiloride, indomethacin, tetraethylammonium (TEA+), Ba2+, and IBMX were all obtained from Sigma and Merck (Deisenhofen and Darmstadt, Germany). Forskolin was obtained from Hoechst (Frankfurt/Main, Germany). All chemicals used were of the highest grade of purity available. Several biopsies (1-6 samples) were obtained from single individuals. When multiple biopsy samples from one individual were studied by the same protocol, data were averaged to obtain a single value for each individual subject. Data are shown as individual recordings or as means ± SE (n = number of subjects studied). Data were analyzed using paired Student's t-test, and P < 0.05 was accepted to indicate statistical significance. Data obtained from CF and non-CF subjects were compared by unpaired t-test.


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Differential effects of cholinergic stimulation on rectal biopsies from CF and non-CF subjects. Baseline bioelectric measurements were taken following equilibration of rectal mucosa biopsies in the Ussing chamber. As reported previously, the amiloride-sensitive Isc was significantly increased in CF compared with non-CF tissues (28). In the presence of amiloride (10 µmol/l) non-CF rectal biopsies had a lumen-negative Vte of -0. 5 ± 0.1 mV. Isc was -25.2 ± 3.1 µA/cm2, and Rte was 21.9 ± 1.4 cm2. Addition of CCh (100 µmol/l) induced a Cl- secretory response and significantly increased lumen-negative Isc by -67.0 ± 5.5 µA/cm2 (n = 18). The effect of CCh was transient, and Isc returned to baseline values within 3-5 min. In CF subjects, Vte (0.1 ± 0.1 mV) and Isc (3.4 ± 1.8 µA/cm2) were significantly reduced, and Rte was significantly increased (36.3 ± 4.3 cm2) compared with non-CF subjects. In contrast to non-CF subjects, the response toward cholinergic stimulation was reversed in CF subjects and CCh increased lumen-positive Isc by 31.6 ± 10.4 µA/cm2 (n = 8) (Fig. 1 and Fig. 3).


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Fig. 1.   Effects of amiloride (10 µmol/l) and carbachol (CCh, 100 µmol/l) on transepithelial voltage and resistance (Vte and Rte) of human cystic fibrosis (CF; B) and non-CF (A) rectal biopsies. Rte was determined from Vte downward deflections obtained by pulsed-current injection. Under baseline conditions, CF tissues showed an enhanced response toward amiloride and a reversed, lumen-positive response toward stimulation with CCh.

Inhibition of the cAMP pathway mimics the CF phenotype in non-CF subjects. Endogenous PGE2 has been shown to act as one of the main agonists of cAMP-dependent Cl- secretion in distal colon (6, 7). In a previous study on a surgical specimen from non-CF colon, inhibition of PGE2 synthesis by the cyclooxygenase inhibitor indomethacin (10 µmol/l) antagonized cholinergic Cl- secretion (27), suggesting that CFTR acts as the predominant Cl- conductance in native human colonic tissue. In the present study, we investigated whether this mechanism was also present in non-CF rectal biopsies and how the change in CCh-induced Isc depends on the duration of indomethacin treatment. In 14 non-CF individuals, the effect of CCh was examined in a strictly paired fashion at intervals of 20 min for up to 1 h. It is shown that cholinergic Cl- secretion was gradually inhibited. After perfusion with the cyclooxygenase inhibitor indomethacin (10 µmol/l) for 60 min, a lumen-positive CCh response was detected in all non-CF subjects, thus mimicking the CF phenotype (Fig. 2). In another series of experiments on non-CF subjects (Fig. 3A), indomethacin significantly reduced Isc to -5.9 ± 1.0 µA/cm2 (n = 18). Subsequent addition of CCh induced a lumen-positive Isc (Delta Isc = 14.9 ± 2.9 µA/cm2) in all individuals. In 12 subjects (67%), the Isc response was biphasic and the initial lumen-positive deflection was followed by a largely diminished lumen-negative deflection (Fig. 3B). Thus CCh-induced Cl- secretion was almost abolished after incubation with indomethacin. Note that in some individuals (28%) an initial lumen-positive CCh response was already observed under baseline conditions (Fig. 3B). As shown previously for human colon, the inhibitory effect of indomethacin was entirely reversible after activation of the cAMP pathway. In the presence of indomethacin, stimulation with IBMX (100 µmol/l) and forskolin (1 µmol/l), as expected, induced Cl- secretion and increased lumen-negative Isc to -49.1 ± 7.1 µA/cm2 (n = 18). The effect of CCh (Delta Isc = -108.2 ± 13.7 µA/cm2) was significantly augmented under these conditions, demonstrating cooperativity of Ca2+- and cAMP-activated Cl- secretion in non-CF rectum (Fig. 3).



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Fig. 2.   Time course of CCh (100 µmol/l) response measured during 1 h of continuous perfusion with indomethacin (10 µmol/l) in strictly paired experiments. A: original recordings of Vte and Rte from a non-CF rectal biopsy. B: summary of CCh-induced Isc in non-CF subjects. C: summary of CCh-induced Isc in CF subjects. CCh was applied at intervals of 20 min. Lumen-negative response in non-CF tissues was gradually inhibited and reversed into a lumen-positive response. In CF biopsies, CCh response remained unchanged during incubation with indomethacin. t, Time. Numbers in parentheses indicate number of subjects.



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Fig. 3.   Effects of indomethacin (10 µmol/l), IBMX/forskolin (100 µmol/l and 1 µmol/l), and CCh (100 µmol/l) on rectal biopsies from non-CF (left) and CF (right) subjects. All experiments were performed in presence of amiloride (10 µmol/l). A: in non-CF subjects, Cl- secretion (lumen-negative Isc) was inhibited by indomethacin (60 min) and further increased by IBMX/forskolin. In CF subjects, reversed responses were observed: an initial lumen-positive Isc was inhibited by indomethacin and further increased by IBMX/forskolin. B: under baseline conditions, a lumen-negative CCh response was observed in most (72%) non-CF individuals. After incubation with indomethacin, CCh induced an initial lumen-positive Isc in all non-CF subjects studied. In CF subjects, CCh responses were exclusively lumen positive and did not depend on cAMP activation. Values are means ± SE. * Indicates statistical significance for effects of indomethacin, IBMX/forskolin, and CCh, # indicates significant difference from baseline conditions, $ indicates significant difference from indomethacin (paired t-test), and § indicates significant difference compared with non-CF subjects (unpaired t-test). Numbers in parentheses indicate number of subjects.

Lack of cholinergic and cAMP-dependent Cl- secretion in CF. In CF rectal biopsies, perfusion with indomethacin had an inverse effect and the lumen-positive Isc was abolished (Fig. 3A). It is shown that indomethacin treatment had no effect on CCh responses. In five CF subjects, CCh constantly induced a lumen-positive Isc response, irrespective of the duration of indomethacin perfusion (Fig. 2, B and C). In the summary of eight CF patients, it is shown that CCh significantly increased an inverse lumen-positive Isc by 30.9 ± 10.9 µA/cm2 (n = 8). After an initial peak, the response remained positive during a plateau phase of the CCh response. Interestingly, in the presence of indomethacin, the lumen-positive CCh-induced Isc was significantly increased in CF compared with non-CF subjects (Fig. 3). As expected, activation of the cAMP pathway failed to induce Cl- secretion in CF subjects. Surprisingly, stimulation with IBMX and forskolin induced an opposite effect, and Isc was increased to 8.2 ± 2.3 µA/cm2 (n = 8). The effect of CCh (Delta Isc = 21.3 ± 6.2 µA/cm2), however, remained unchanged after cAMP-dependent stimulation in CF subjects (n = 8) (Fig. 3). Together, these results demonstrate that Ca2+- and cAMP-activated Cl- secretion are both defective in CF rectum, indicating that functional CFTR is required for cholinergic Cl- secretion.

Cholinergic K+ secretion in CF. The reversed lumen-positive CCh responses observed in CF could be caused by either activation of a luminal K+ conductance or a basolateral Cl- conductance. To test for the first hypothesis, the effects of different K+-channel blockers were examined in the presence of amiloride and indomethacin. As shown in Fig. 4, CCh-induced lumen-positive Vte and Isc were reversibly inhibited when Ba2+ (5 mmol/l) and TEA+ (10 mmol/l) were added to the luminal side of the mucosa. Similar effects were observed on lumen-positive CCh responses in non-CF subjects in the presence of indomethacin. These data demonstrate the presence of K+ channels in the luminal membrane of CF and non-CF rectal tissues that are activated by cholinergic stimulation. To characterize this macroscopic K+ conductance further, several other K+-channel blockers were tested in non-CF tissues. As summarized in Fig. 4B, addition of TEA+ (10 mmol/l) alone was a less effective inhibitor of cholinergic K+ secretion. Clotrimazole (30 µmol/l), a blocker of small Ca2+-activated K+ channels (20), and the chromanol 293B (10 µmol/l), a specific inhibitor of KvLQT1 K+ channels in epithelia (3, 24), were both without effect on cholinergic K+ secretion.


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Fig. 4.   Inhibition of CCh-induced K+ secretion by different K+-channel blockers in CF and non-CF rectal biopsies. A: in presence of amiloride and indomethacin, CCh (100 µmol/l) induced a lumen-positive Vte that was reversibly blocked by luminal Ba2+ (5 mmol/l) and tetraethylammonium (TEA+) (10 mmol/l) in CF subjects. B: CCh-mediated lumen-positive Isc was significantly inhibited by Ba2+ and TEA+ in CF (left) and non-CF (right) subjects. Clotrimazole (30 µmol/l) and chromanol 293B (10 µmol/l) had no effect on CCh-activated luminal K+ conductance. Values are means ± SE. * Indicates statistical significance for effect of the K+-channel blocker (paired t-test). Numbers in parentheses indicate number of subjects.

The lumen-positive CCh-mediated Isc, reflecting K+ secretion, was significantly increased in CF compared with non-CF subjects (Fig. 3B). To examine a possible role of CFTR in K+-channel regulation, transepithelial Cl- transport was inhibited by bumetanide (50 µmol/l; basolateral) and cholinergic K+ secretion was studied in the absence and presence of cAMP activation. In the presence of indomethacin, the lumen-positive CCh response in non-CF subjects (Delta Isc = 38.9 ± 10.8 µA/cm2; n = 4) was not affected by bumetanide. When bumetanide was added in the presence of IBMX and forskolin, K+ secretion (Delta Isc = 10.1 ± 2.9 µA/cm2) was significantly decreased compared with experiments performed in the absence of cAMP activation (Fig. 5B). These results suggest inhibition of cholinergic K+ conductance during activation of CFTR, independent of Cl- transport.


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Fig. 5.   Effect of CFTR activation on cholinergic K+ secretion in non-CF subjects. Transepithelial Cl- transport was inhibited by bumetanide (50 µmol/l), and effect of CCh (100 µmol/l) was studied in absence and presence of IBMX/forskolin (100 µmol/l and 1 µmol/l). After cAMP activation and in presence of bumetanide, CCh induced lumen-positive responses of Vte (A) and Isc (B). This K+ secretory response was significantly decreased compared with experiments performed under similar conditions in absence of cAMP activation (B). * Indicates statistical significance for effects of indomethacin, IBMX/forskolin, bumetanide, and CCh, # indicates significant difference from IBMX/forskolin (paired t-test), and § indicates significant difference compared with experiments performed in absence of cAMP activation (unpaired t-test). Numbers in parentheses indicate number of subjects.

cAMP-dependent luminal K+ conductance in CF. As shown in Fig. 3A, a small lumen-positive Isc was observed in CF rectal biopsies under baseline conditions, which was largely inhibited by indomethacin and further increased by stimulation with IBMX and forskolin. We examined whether this inverse cAMP response in CF tissues is caused by activation of luminal K+ channels and applied Ba2+ (5 mmol/l, luminal side) in the absence and presence of IBMX and forskolin. In this series of experiments, which were performed in the presence of amiloride and indomethacin, activation with IBMX and forskolin significantly increased Isc from -3.1 ± 1.6 µA/cm2 to 8.3 ± 1.5 µA/cm2. When Ba2+ was added to the luminal side, Isc was significantly and reversibly inhibited to -2.3 ± 1.4 µA/cm2 (n = 5). No significant effect was observed when Ba2+ was applied in the absence of IBMX and forskolin (Fig. 6), suggesting cAMP-dependent activation of a luminal K+ conductance in CF subjects.


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Fig. 6.   Effect of Ba2+ on lumen-positive Vte and Isc induced by activation of cAMP pathway in CF subjects. In presence of amiloride and indomethacin, a lumen-positive Vte (A) and Isc (B) was activated by IBMX/forskolin (100 µmol/l and 1 µmol/l). Lumen-positive response was reversibly inhibited by luminal Ba2+ (5 mmol/l). Values are means ± SE. * Indicates statistical significance for effects of IBMX/forskolin and Ba2+. Number in parentheses indicates number of subjects.


    DISCUSSION
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ABSTRACT
INTRODUCTION
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DISCUSSION
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Previous studies reported defective cAMP- and Ca2+-mediated Cl- secretion in distal colon from CF patients (2, 12, 17). An inverse lumen-positive Isc response was observed upon cholinergic stimulation (34, 35). Similar observations were reported for surgical resections from non-CF colonic tissues after deactivation of CFTR by blockage of prostaglandin production (27). In the present study, we investigated the ion conductances underlying this reversed lumen-positive response. Theoretically, lumen-positive Vte and Isc could be caused by activation of either luminal K+ channels or basolateral Cl- channels, which have both been described previously in the colonic crypt (18, 31).

We have shown time-dependent inhibition of Cl- secretion by the cyclooxygenase inhibitor indomethacin in non-CF tissues that mimics a CF phenotype (6, 7). Using the K+-channel blockers Ba2+ and TEA+, we demonstrated that the lumen-positive CCh response is caused by activation of a luminal K+ conductance. CCh-, cAMP-, and aldosterone-induced K+ secretion have been localized in rat distal colon (13, 18, 19). Here, we have shown macroscopic K+ secretion in rectal biopsies from CF and non-CF individuals. To characterize this luminal Ca2+-activated K+ conductance further, the effect of more specific K+ channel blockers was examined. The recently cloned small-conductance K+ channel is expressed in human intestine and was therefore considered a possible candidate (20). However, clotrimazole, an inhibitor of the small-conductance K+ channel, had no effect on cholinergic K+ secretion. Moreover, the chromanol 293B, a specific inhibitor of KvLQT1 K+ channels expressed in colonic epithelia, was also without effect on CCh-induced lumen-positive Isc (3, 24). Thus the molecular nature of the luminal K+ channel remains to be determined.

Interestingly, cholinergic K+ secretion was significantly enhanced in CF subjects. The data suggest that CFTR might be somehow involved in the regulation of apical K+ conductance by inhibiting luminal K+ channels. In non-CF subjects, inhibition of Cl- secretion by blocking the Na+-K+-2Cl- cotransporter with bumetanide (25) unmasked cholinergic K+ secretion that was significantly attenuated in the presence of IBMX and forskolin (Fig. 5B). Attenuation of luminal K+ channels by CFTR would decrease the luminal membrane voltage, reduce the driving force for Na+ reabsorption, and assist colonic crypt cells in switching from Na+ absorption to Cl- secretion. In contrast, in CF tissues, hyperpolarization of the apical cell membrane by an increased K+ conductance would contribute to increased Na+ absorption observed in CF subjects (14, 26, 28, 33). Along these lines, the data also present some evidence for a cAMP-activated luminal K+ conductance in CF distal colon (Fig. 6). In non-CF tissues, cAMP-dependent K+ secretion is probably masked by Cl- secretion. However, under experimental conditions in which Cl- transport was inhibited by both indomethacin and bumetanide, no cAMP-activated K+ conductance could be detected in non-CF tissues (data not shown).

The perfused micro-Ussing chamber allows us to examine cholinergic Cl- secretion in both the absence and presence of cAMP stimulation. We show that in non-CF tissues, the CCh response depends on stimulation of CFTR. Typically, a Cl- secretory response (negative Isc) of variable magnitude is observed under baseline conditions. However, an inverse, i.e., lumen-positive, CCh response was detected in 28% of non-CF subjects. Inhibition of CFTR by indomethacin abolished Cl- secretion and mimicked a CF phenotype. A clear separation of CF and non-CF tissues was only possible after cAMP-dependent stimulation. Therefore, discrimination of CF and non-CF tissues on the basis of Isc measurements, as suggested previously (34, 35), requires a more extensive analysis of conductance properties to avoid false-positive results.

Assessment of increased amiloride-sensitive epithelial Na+ conductance, defective Ca2+- and cAMP-dependent Cl- conductance, and enhanced K+ secretion on cholinergic stimulation clearly identifies the CF phenotype. In future, Isc measurements could be applied as a functional test when the clinical diagnosis of CF is unclear (30). Because cholinergic Cl- secretion depends on CFTR activity, the technique is of particular interest for detection of residual CFTR function in the case of class III, IV, and V mutations (36). Interestingly, even in some patients carrying severe class I and II mutations (Delta F508 and R553X), a small residual Cl- secretion was observed that was cAMP-dependent and amounted to ~3% compared with non-CF patients (data not shown). These observations from native CF tissue fit well to biochemical and patch-clamp analysis of Delta F508 (8, 23). The role of residual Cl- secretion in different CF genotypes will be subject to further studies and, in future, might become important for the prognosis and pharmacotherapy of CF.


    ACKNOWLEDGEMENTS

We gratefully thank Dr. P. Greiner at the Children's Hospital, University of Freiburg, for performing rectoscopy procedures. We further acknowledge the expert technical assistance of S. Hirtz and C. Hodler.


    FOOTNOTES

This work was supported by Deutsche Forschungsgemeinschaft (DFG) KU1228/1-1, DFG Ku 756/4-1, and Zentrum Klinische Forschung 1, Albert-Ludwigs-Universität Freiburg.

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, Universitäts-Kinderklinik, Albert-Ludwigs-Universität Freiburg, Mathildenstrasse 1, 79106 Frieburg, Germany (E-mail: mall{at}ruf.uni-freiburg.de)

Received 13 July 1999; accepted in final form 2 December 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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