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
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ABSTRACT |
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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
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INTRODUCTION |
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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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EXPERIMENTAL PROCEDURES |
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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 F508, and three were compound
heterozygous for
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 (
F508; R117H and S108F in exon 4; R347P, R347H, I336K, and
T338I in exon 7; S549N, G551D, R553X, G542X, Q552X, and 1717-1
G
A in exon 11; W1282X and 3905insT in exon 20; N1303K in exon 21;
and 3849 + 10kB C
T 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 (I = 0.5 µA), and the corresponding changes in transepithelial voltage (
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 (
Vte') were subtracted from
those obtained in the presence of the tissues. Rte
was calculated according to Ohm's law [Rte = (
Vte
Vte')/
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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RESULTS |
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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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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
(
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 (
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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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
(
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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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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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 (
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
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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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