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 |
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
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INTRODUCTION |
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
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.
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EXPERIMENTAL PROCEDURES |
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 (
I = 0.5 µA) and by recording the corresponding changes in transepithelial
voltage
(
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
· cm2, and
the voltage deflections obtained under conditions without the mucosa
present (
V'te)
were subtracted from those obtained in the presence of the tissues.
Rte was
calculated according to Ohms law
(Rte =
Vte
V'te/
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 |
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
· 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
· 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).
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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
(
Vte = 0.4 ± 0.1 mV) was observed and
Rte was
significantly increased by 4.5 ± 0.7
· 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
· 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.
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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
· cm2. Thus
Isc-eq was
significantly decreased from
13.3 ± 1.2 to
3.0 ± 0.6 µA/cm2
(
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
· cm2, and
Isc-eq was
significantly inhibited from
17.4 ± 3.8 to
0.4 ± 0.9 µA/cm2
(
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.
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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
(
Vte =
1.1 ± 0.1 mV,
Rte =
3.1 ± 0.7
· cm2,
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
· 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).
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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
· cm2.
Accordingly,
Isc-eq
was significantly inhibited from
32.3 ± 2.3 to
25.8 ± 2.1 µA/cm2, corresponding
to
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
(
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
(
Isc-eq = 3.0 ± 0.7 µA/cm2;
n = 70;
P < 0.0001), with only minimal
changes of Vte
and Rte (
Vte = 0.1 ± 0.0 mV,
Rte = 0.1 ± 0.1
· 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.
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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
· cm2.
Isc-eq was
inhibited from
14.2 ± 3.4 to 4.9 ± 1.4 µA/cm2
(
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
(
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
· cm2.
Isc-eq was
significantly inhibited from
14.8 ± 3.0 to 5.0 ± 1.4 µA/cm2. However, the
amiloride-sensitive
Isc-eq
(
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.
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 |
DISCUSSION |
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.
 |
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