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1 Physiologisches Institut, Cl
cystic fibrosis transmembrane conductance regulator; epithelial
transport; potassium channels; Ussing chamber; microelectrodes; transepithelial voltage; carbachol
SODIUM CHLORIDE AND WATER secretion across the human
colon is generated mainly by epithelial cells lining the crypts but
also and to a lesser degree by the surface epithelium (9, 19). Secretion is under the control of a variety of hormones and
neurotransmitters and is affected in common diseases like secretory
diarrhea and cystic fibrosis (CF). Therefore, detailed knowledge about
the ion conductances involved is essential for the understanding of electrolyte secretion in human colon. Activators of electrolyte transport can be subdivided into those that act via the intracellular cAMP-, cGMP-dependent pathway and others that require an increase in
intracellular Ca2+. During
stimulation of electrolyte transport by either pathway, ion channels
are activated in apical or basolateral membranes of colonic epithelial
cells. Previous reports demonstrated that an apical
Cl Whereas CFTR and KvLQT1 are regulated by intracellular cAMP, other
classes of ion channels are activated by intracellular Ca2+. Accordingly, basolateral
K+ channels with a larger
single-channel conductance (~16 pS) are activated by agonists that
increase intracellular Ca2+ such
as carbachol (CCH) (5, 28). These channels are inhibited by the common
K+ channel blockers
Ba2+ and tetraethylammonium
(TEA+) but not by chromanols. It
has been shown for the rat colonic epithelium that
Ca2+-dependent
K+ channels are shut off when the
small-conductance K+ channels are
turned on during an increase of intracellular cAMP (34). It is,
however, not clear whether or not an increase of intracellular
Ca2+ also leads to the activation
of apically localized
Ca2+-regulated
Cl The aim of the present study was to gain a more detailed knowledge
about the process of electrolyte secretion in the human colonic
epithelium. To this end, it was essential to make use of freshly
isolated human colonic epithelium rather than cultured cells. Using a
novel type of miniature Ussing chamber allowing for the continuous
exchange of luminal and basolateral bath solutions, we demonstrate the
presence of a recently cloned new type of
K+ channel in the basolateral
membrane of human colonic epithelial cells. This channel could be an
important pharmaceutical target for the treatment of secretory diarrhea
(21). Moreover, the results uncover the relationship between
Ca2+- and cAMP-activated
electrolyte secretion in the human colon. Former studies demonstrated a
relationship between cholinergic and cAMP-dependent colonic ion
secretion and described defective function for both in CF (11, 15,
31-33). The conclusions from this study are essential for the
understanding of previous results showing altered
Ca2+ (i.e., CCH)-induced
electrolyte secretion in CF (32, 33).
Patients. Colonic tissue preparations
were obtained from 29 patients with a mean age of 55.9 ± 4.5 yr
(ranging from 1 mo to 88 yr) who underwent routine surgical procedures
at the University Hospital Freiburg. Two- to three-millimeter forceps
biopsies were taken either from surgical resections or directly from
the patients. There was no muscle layer left after superficial forceps
biopsies. Intestinal segments examined in the present study comprised
distal descendent colon, sigmoidal colon, and rectum. The responses in the presence of indomethacin were similar in the preparations derived
from the various segments. The tissues used for Ussing chamber
experiments were not affected by the primary disease that was the cause
for surgical intervention. The study was approved by the ethics
committee and the patients had given their written informed consent.
Ussing chamber experiments. Small
pieces of the removed colon that were not affected by the primary
disease 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 10 g/l
albumin. Small samples (2-4 mm in diameter) were taken from the
tissue and mounted into a modified Ussing chamber. To obtain stable
measurements even with small pieces of tissue, we constructed a
sandwich chamber with a circular aperture of 0.95 mm2. The luminal and basolateral
sides of the epithelium were perfused continuously at a rate of
10-20 ml/min (chamber volume 1 ml), allowing for the paired
examination of the effects of CCH in the presence or absence of cAMP.
The bath solution, which was replaced continuously, had the following
composition (mmol/l): 145 NaCl, 0.4 KH2PO4,
1.6 K2HPO4,
5 D-glucose, 1 MgCl2, and 1.3 calcium gluconate.
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
with values for transepithelial voltage (Vt) referring
to the serosal side of the epithelium. We found open-circuit
measurements more adequate because
1) they more accurately reflect the
in vivo situation, 2) we were able
to keep the tissue preparations functional and responding for a longer
time period (up to 7 h), and 3) the
resulting calculated short-circuit current (Isc) was larger
and tissues responded better to stimulation with the agonists used in
this study. Transepithelial resistance
(Rt) was
determined by applying short (1 s) current pulses [change ( Compounds and analysis. Amiloride,
indomethacin, TEA+,
Ba2+, and IBMX were all obtained
from Sigma and Merck (Deisenhofen and Darmstadt, Germany). Forskolin
and
trans-6-cyano-4-(N-ethylsulfonyl-N-methylamino)-3-hydroxy-2,2-dimethyl&2-chromane (293B) were obtained from Hoechst (Frankfurt, Germany). All used chemicals were of highest grade of purity available. Data are shown as
individual recordings or as mean ± SE
(n = number of observations). Paired
Student's t-test was used for
analysis of paired data (P < 0.05).
Basal properties of human rectal
mucosa. Under control conditions,
Isc of the tissue
biopsies was Cooperativity of
Ca2+- and
cAMP-dependent Cl
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
secretion in the colon can be activated by an increase of either
intracellular Ca2+ or cAMP. In
this study we examined a possible interdependence of the two
second-messenger pathways in human colonic epithelium. When measured in
a modified Ussing chamber, carbachol (CCH; 100 µmol/l, basolateral),
via an increase in cytosolic Ca2+
concentration
([Ca2+]i),
activated a transient lumen-negative equivalent short-circuit current
(Isc)
[change (
) in
Isc = 
79.4 ± 7.5 µA/cm2].
Previous studies indicated that intracellular
Ca2+ directly acts on basolateral
K+ channels, thus enhancing
driving force for luminal
Cl exit. Increased
intracellular cAMP (by basolateral addition of 100 µmol/l IBMX and 1 µmol/l forskolin) activated a sustained lumen-negative current
(Isc = 42.4 ± 7.2 µA/cm2)
that was inhibited by basolateral
trans-6-cyano-4-(N-ethylsulfonyl-N-methylamino)-3-hydroxy-2,2-dimethyl&2-chromane (10 µmol/l), a blocker of KvLQT1 channels. In the presence of elevated cAMP, the CCH-activated currents were augmented
(Isc = 167.7 ± 32.7 µA/cm2), suggesting
cooperativity of the Ca2+- and
cAMP-mediated responses. Inhibition of endogenous cAMP production by
indomethacin (10 µmol/l) significantly reduced CCH-activated currents
and even reversed the polarity in 70% of the experiments. The
transient lumen-positive
Isc was probably
due to activation of apical K+
channels because it was blocked by luminal
Ba2+ (5 mmol/l) and
tetraethylammonium (10 mmol/l). In the presence of indomethacin (10 µmol/l, basolateral), an increase of cAMP activated a sustained
negative Isc.
Under these conditions, CCH induced a large further increase in
lumen-negative
Isc
(Isc = 100.0 ± 21.0 µA/cm2). We conclude that CCH
acting via
[Ca2+]i
can induce Cl secretion
only in the presence of cAMP, i.e., when luminal
Cl channels are already
activated. The activation of a luminal and basolateral
K+ conductance by CCH may be
essential for transepithelial KCl secretion in human colon.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
conductance is activated
when intracellular cAMP is enhanced to upregulate
Cl secretion (13). This
apical Cl conductance is
formed by the CF transmembrane conductance regulator (CFTR), the
protein that was demonstrated to be defective in CF (26). In addition
to the opening of apical Cl
conductances, basolateral K+
channels are activated (34) in rat colonic epithelium. The basolateral
K+ conductance is formed by very
small-conductance K+ channels,
corresponding to the recently cloned KvLQT1 channel. This novel type of
K+ conductance can be inhibited
specifically by a new class of chromanol compounds (4).
channels in colonic
epithelial cells. Furthermore, CCH might further upregulate
cAMP-dependent Cl channels.
Alternatively, CCH-induced
Cl secretion could be
solely due to an activation of basolateral K+ channels, which hyperpolarizes
these cells and thus enhances the driving force for luminal
Cl exit (6). These
hypotheses have thus far not been examined in human colonic epithelium.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
)
in I = 0.5 µA]. Voltage
deflections obtained under conditions without the mucosa present in the
chamber were subtracted from those obtained in the presence of the
tissues. Rt was
calculated according to Ohm's law
(Rt = Vt /I).
Tissue preparations were only accepted if Rt
exceeded that obtained for an empty chamber at least by a factor of 2. From each of patients
1-6, in most cases three biopsies were examined and recordings were usually stable
for 3-4 h. Typically, after stabilization of basal
Vt and Rt, amiloride (10 µmol/l) was added to the luminal side of the colonic mucosa. Under
these conditions the effect of basolaterally added CCH (100 µmol/l)
was examined. Subsequently, the effect of CCH was examined in the
presence of activators of the intracellular cAMP pathway (IBMX, 100 µmol/l, and forskolin, 1 µmol/l, basolateral solution). In another
series of experiments, recordings were performed in the presence of
indomethacin (10 µmol/l, basolateral solution) to suppress synthesis
of endogenous prostaglandins and intracellular cAMP.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
52.3 ± 5.3 µA/cm2
(n = 41). Basal
Vt was 1.6 ± 0.3 mV and
Rt was 27.8 ± 3.1 
· cm2.
Addition of amiloride (10 µmol/l) to the mucosal side of the epithelium reduced
Vt and
Isc slightly but
significantly to 1.5 ± 0.2 mV and 48.1 ± 5.1 µA/cm2, respectively
(n = 41).
Rt under these
conditions was 29.1 ± 3.0 · cm2.
secretion. In the presence of amiloride,
Ca2+-dependent
Cl secretion was stimulated
by adding CCH (100 µmol/l) to the basolateral side of the epithelium.
CCH invariably increased the lumen-negative Isc from
41.3 ± 5.7 to 122.4 ± 22.7 µA/cm2
(n = 10).
Vt was increased
from 1.0 ± 0.4 to 2.4 ± 0.5 mV and Rt was slightly
reduced from 24.6 ± 5.8 to 23.2 ± 5.2 · cm2
(n = 10) (Fig.
1, A and
B). The effect of CCH was only
transient, and
Isc returned to
control values within 2-4 min and was due to increase of
intracellular Ca2+ without any
change of intracellular cAMP (unpublished data from our laboratory).
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Fig. 1.
Effect of basolateral carbachol (CCH, 100 µmol/l) on transepithelial
voltage (Vt)
and resistance
(Rt) of human
colon epithelium.
Rt was determined
from the Vt
downward deflections obtained by pulsed current injection. Impact of
parallel activation of cAMP pathway is shown.
A: CCH transiently enhanced
lumen-negative Vt
under control (Con) conditions. Activation of the cAMP pathway by
stimulation with IBMX (100 µmol/l) and forskolin (Fors, 1 µmol/l)
persistently enhanced the lumen-negative
Vt and thus
increased equivalent short-circuit current
(Isc = Vt /Rt).
In the presence of IBMX and forskolin, effect of CCH on
Vt was more
pronounced and thus activation of
Isc by CCH was
augmented by costimulation via the cAMP-dependent pathway. Time gap
between both records was 5 min. B:
summary of Isc
obtained from experiments as shown in
A. IBMX and forskolin induced a slight
and nontransient increase in
Isc. No. in
parentheses indicates no. of experiments. § Effect of CCH was
significantly enhanced in presence of parallel activation of the cAMP
pathway. All experiments were performed in presence of 10 µmol/l
amiloride. * Significantly different from control
(P < 0.05).
Intracellular cAMP was enhanced by the inhibitor of phosphodiesterase
IBMX (100 µmol/l) and the stimulator of the adenylate cyclase
forskolin (1 µmol/l), both applied to the basolateral side of the
epithelium. Intracellular Ca2+ was
not affected by these agonists (unpublished data from our laboratory).
This enhanced lumen-negative
Isc from
40.5 ± 7.1 to 71.8 ± 9.8 µA/cm2
(Vt increased
from 1.0 ± 0.4 to 2.0 ± 0.5 mV;
Rt decreased from
34.2 ± 3.1 to 33.6 ± 2.8 · cm2,
n = 28). After activation of the
cAMP-dependent pathway, the effects of CCH on lumen-negative
Isc were
significantly enhanced: Isc was enhanced
from 71.8 ± 9.8 to 248.3 ± 38.2 µA/cm2
(Vt was increased
from 1.0 ± 0.4 to 5.2 ± 0.8 mV;
Rt was decreased from 25.3 ± 3.8 to 22.7 ± 3.8 · cm2,
n = 10) (Fig. 1,
A and
B). These paired experiments
indicate that Ca2+ and cAMP
increase Cl secretion cooperatively.
Role of basolateral
K+ channels for
Cl secretion.
We further examined the impact of basolateral
K+ channels, activated by either
cAMP or Ca2+, on
Cl secretion in human colon
epithelium. After stimulation of the tissues by IBMX and forskolin, the
effects of BaCl2 (5 mmol/l) and a
specific blocker of the cAMP-activated KvLQT1
K+ channel [chromanol 293B
(21)] were added to the basolateral side. In this series, IBMX
and forskolin enhanced
Isc from
43.4 ± 3.5 to 72.9 ± 5.0 µA/cm2
(Vt was increased
from 1.5 ± 0.2 to 2.4 ± 0.3 mV and
Rt was decreased
from 33.4 ± 3 to 32.1 ± 2.4 cm2,
n = 36). Addition of
BaCl2 (5 mmol/l) to the
basolateral side completely inhibited
Isc activated by
increase of intracellular cAMP and reduced total
Isc to
17.3 ± 3.1 µA/cm2
(n = 6) (Fig.
2, A and
C). This effect was mimicked by the
chromanol 293B (10 µmol/l), which also led to complete inhibition of
Isc activated by
the increase of intracellular cAMP and reduced total Isc to
29.8 ± 3.0 µA/cm2
(n = 36) (Fig. 2,
B and
C). Figure
2D depicts the concentration-response curve for 293B. The approximate
IC50 value was 5 µmol/l.
Therefore, the activation of a basolateral
K+ conductance is essential for
cAMP-dependent stimulation of electrolyte secretion in the human colon,
and the K+ channel involved is
most likely the KvLQT1 channel (4).
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secretion, the colonic
tissue was first stimulated by IBMX and forskolin, and subsequently the
effects of CCH were examined in the presence or absence of
Ba2+ or 293B, respectively. CCH
(100 µmol/l) enhanced
Isc from
47.1 ± 6.6 to 151.4 ± 27.5 µA/cm2
(n = 27). The effect of CCH was
completely abolished in the presence of
Ba2+
(Isc = 1.0 ± 3.3 µA/cm2,
Rt = 1.4 ± 0.2 · cm2,
n = 6) (Fig.
3, B and
C). In contrast, 293B inhibited
sustained lumen-negative
Isc activated by
cAMP from 72.3 ± 5.2 µA/cm2 to 32.5 ± 3.5 µA/cm2
(Rt = 0.7 ± 0.1 · cm2),
but the CCH-induced transient changes of
Isc were not
attenuated by 293B
(Isc = 102.2 ± 14.4 µA/cm2,
Rt = 4.1 ± 0.31, n = 26) (Fig. 3,
A and
C). We conclude from these data that
different types of K+ channels are
activated during stimulation of electrolyte secretion in the human
colon by the two second messengers cAMP and
Ca2+. One type of
K+ conductance must be activated
to maintain electrolyte transport. In this respect, the properties of
the human colon as found in the present study are very similar to those
found in rat colon (34).
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Ca2+-dependent
Cl secretion requires activation of
the cAMP-dependent pathway.
To further investigate a possible cooperativity of
Ca2+- and cAMP-activated
Cl secretion, we examined
in paired experiments the effects of CCH under three different
conditions: 1) under control
conditions, 2) in the presence of
indomethacin (10 µmol/l) to suppress endogenous production of
prostaglandins and thus intracellular cAMP, and 3) after maximal activation of the
cAMP-dependent pathway by IBMX and forskolin. Before treatment with
indomethacin, basal
Isc was 32.1 ± 3.7 µA/cm2
(Vt = 1.1 ± 0.3 mV, Rt = 33.2 ± 4.5 · cm2) and
was further increased by CCH to 99.6 ± 13.7 µA/cm2
(n = 26). Subsequently, indomethacin
was added to the basolateral side of the mucosa and the effect of CCH
was examined repetitively in intervals of 10-20 min. After only
~1 h of perfusion with indomethacin, the basal
Isc was inhibited
almost completely to 8.8 ± 1.8 µA/cm2
(Vt = 0.4 ± 0.1 mV, Rt = 38.3 ± 3.6 · cm2,
n = 26). In 18 of 26 experiments
(70%), we observed positive deflections of
Vt after the
application of CCH (Fig.
4A)
resulting in a transient increase of
Isc to 14.3 ± 4.7 µA/cm2
(Rt = 3.6 ± 0.5 · cm2)
In 6 of these 18 experiments the response was monophasic and consisted
only of a lumen-positive
Isc. In 20 experiments, residual negative deflections of
Vt were observed
with Isc
increasing to 14.3 ± 2.1 µA/cm2
(Rt = 0.1 ± 0.2 · cm2). In
eight experiments the response was monophasic negative. This
variability was observed for all colonic segments and rectal tissues,
respectively, and is most likely due to variable inhibition of
endogenous cAMP synthesis in different tissue preparation because of
variable incubation with indomethacin.
|
0.4 ± 0.1 vs. 1.6 ± 0.2 mV). Rt fell (36.7 ± 3.0 vs. 32.2 ± 2.6 · cm2) and
lumen-negative
Isc was increased
from 7.6 ± 1.7 µA/cm2
to 55.7 ± 8.0 µA/cm2
(n = 26). Now, the effect of CCH was
examined in the presence of both indomethacin and IBMX and forskolin. A
further increase of the lumen-negative
Isc to
151.4 ± 27.5 µA/cm2 was observed
(Vt = 3.5 ± 0.4 mV,
Rt = 30.4 ± 2.7 · cm2,
n = 26) (Fig. 4,
A and
B). As shown above for the absence
of indomethacin, the CCH response was significantly enhanced under these conditions. These results suggest that
Ca2+-dependent
Cl secretion in human
colonic epithelium requires coactivation of the cAMP-dependent pathway
and is only demonstrable when the endogenous cAMP pathway is activated,
e.g., due to stimulation by the major autacoid prostaglandin. These
results also suggest that the only relevant apical
Cl conductance in human
colon epithelial cells is that by cAMP-dependent Cl channels, corresponding
to CFTR. Because, in CF, CFTR is mutated and cannot function as a
Cl channel, colonic
Cl secretion is defective,
thus leading to the well-described intestinal manifestations of CF
(12).
CCH activates luminal
K+ secretion.
The reversed lumen-positive response induced by CCH after inhibition of
the cAMP pathway could be either due to activation of a basolateral
Cl conductance or, more
likely, due to an unmasked parallel activation of a luminal
K+ conductance. We addressed this
question by comparing the effects of CCH on lumen-positive
Vt in the
presence or absence of luminal BaCl2 and
TEA+. In nine paired experiments
with indomethacin in the bath, CCH (100 µmol/l) induced an increase
of lumen-positive
Isc from
6.3 ± 3.1 to 20.9 ± 6.7 µA/cm2
(Rt = 4.8 ± 0.5 · cm2)
(n = 9). After addition of
BaCl2 (5 mmol/l) and
TEA+ (10 mmol/l) to the luminal
side, the lumen-positive CCH-induced Isc was
completely abolished and only a very small lumen-negative Isc of
4.6 ± 4.2 µA/cm2 remained. Furthermore,
inhibition of the lumen-positive
Isc by BaCl2 and
TEA+ was completely reversible on
removal (Isc = 29.5 ± 5.7 µA/cm2,
n = 9; Fig.
5, A and
B). These experiments clearly
indicate activation of a K+
conductance in the luminal membrane of human colonic epithelial cells
by CCH, which is unmasked when cAMP-dependent apical
Cl channels are blocked by
indomethacin.
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DISCUSSION |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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cAMP and Ca2+ activate different types of K+ channels. The results of the present study indicate that at least two different types of K+ channels exist in the basolateral membrane of human colonic epithelial cells: one activated by Ca2+ and the other by cAMP. Intracellular Ca2+ was increased by CCH, which binds to M3- type receptors on the basolateral side of colonic epithelial cells (25). Although cholinergic stimulation may increase intracellular inositol trisphosphate and Ca2+ as well as diacylglycerol and thus may activate protein kinase C (PKC), Ca2+ probably is the primary mediator because basolateral K+ channels in the colon are directly activated by an increase in intracellular Ca2+ (5). Both types of K+ channels can be distinguished on the basis of their sensitivity toward the recently designed K+ channel blocker 293B (21); the cAMP-activated K+ channel is inhibited by 293B, whereas the Ca2+-activated K+ channel is not (5, 34). Thus electrophysiological properties of the human colon resemble those of the rat. Although the molecular nature of the Ca2+-activated K+ channel is not definitively clarified at this stage (5, 28), the present data strongly suggest that the cAMP-activated K+ channels in the basolateral membranes of human colonic epithelial cells are most likely identical to the KvLQT1 channels that were recently cloned from human heart (2, 29). In addition, overexpression of KvLQT1 in COS-7 cells and Xenopus oocytes identified KvLQT1 as the target for 293B (4, 7, 23). Activation of KvLQT1 channels by cAMP in the basolateral membrane of human colon epithelial cells is essential for cAMP-dependent electrolyte secretion. In this respect, the compound 293B may add a new therapeutic tool for the treatment of secretory diarrhea (21).
Activation of luminal K+ conductance by CCH. Inhibition of the endogenous production of prostaglandins and hence a fall in cytosolic cAMP unmasked activation of apical K+ conductance by an increase in intracellular Ca2+. This occurs in parallel to the activation of basolateral K+ channels. Apical K+ channels were identified in the rat colon in previous reports (8, 30). Normally, the positive Isc due to activation of apical K+ conductance is masked by the parallel activation of luminal Cl channels. As a net
result, CCH enhances lumen-negative
Isc. Because of
difficulties in performing patch-clamp recordings from luminal membrane
of colonic epithelial cells (unpublished observations from our
laboratory), it is not currently clear which class of K+ channels accounts for the
apical K+ conductance. According
to a previous report, expression of luminal K+ channels is modified by dietary
K+ and by aldosterone (10, 22,
27). Thus colonic KCl secretion apparently is activated by an increase
in intracellular Ca 2+ and depends
on dietary K+ uptake and aldosterone.
Cooperativity of
Ca2+- and
cAMP-activated membrane conductances.
Previous patch-clamp studies identified
Ca2+-activated
Cl channels in nonpolarized
cultured colonic epithelial cells, whereas other studies failed to
demonstrate Ca2+-activated
Cl channels in colonic
epithelial cells (6, 20). The data of the present study on native human
colonic tissue demonstrate that Ca2+-induced
Cl secretion requires
activation of apical CFTR
Cl channels and therefore
confirm results of previous studies (3, 11, 14, 15, 24, 31). Moreover,
inhibitors of Ca2+-activated
Cl channels such as DIDS
failed to show inhibitory effects on CCH-induced Cl secretion when applied
to the luminal side of the epithelium (data not shown). Hence, a
separate Ca2+-regulated
Cl conductance could not be
demonstrated in the luminal membrane of human colonic crypts. The
absence of a Ca2+-activated
Cl conductance in the
present experiments could also be caused if this specific conductance
would require the coactivation of CFTR by cAMP. In fact, in previous
experiments with HT-29 colonic carcinoma cells, the amplitude and time
course of the Ca2+-activated
Cl currents depended on the
expression of CFTR and its prestimulation with cAMP (1). At any rate,
although contribution of other cAMP-dependent
Cl conductances cannot be
ruled out completely by this and previous studies (17, 18), CFTR seems
to be the predominant Cl
conductance in the luminal membrane of colonic crypt cells.
channels through the
activated PKC pathway (16). When CFTR is mutated and thus the apical
Cl conductance is impaired
as in CF and in CFTR (/) knockout mice, colonic
Cl secretion is abolished
(3, 11, 14, 15, 31, 33). From these reports and the present study it
becomes obvious that CFTR is essential not only for cAMP-dependent but
also for Ca2+-dependent
Cl secretion in the colon.
CCH responses in CF colon and implications for the
measurement of CFTR activity. As demonstrated in the
present report, characteristic changes as they occur in CF can be
mimicked by treatment of the tissue with inhibitors of the
prostaglandin synthesis. In this respect the results of another report
(12) have to be reconsidered. For that report, residual activation of
Isc on
CCH-dependent stimulation of colonic mucosa biopsies derived from CF
patients were taken as a measure for the severity of the disease. To
that end, the tissues were treated with indomethacin for only 10 min,
which is, according to our data, too short for complete inhibition of prostaglandin synthesis. Therefore, data obtained under these conditions are difficult to interpret. We suggest that quantification of residual CFTR function by measuring CCH-induced
Isc in colonic biopsies of CF patients with different CF phenotype should be obtained
only in paired experiments. To that end, endogenous prostaglandin synthesis should be completely inhibited by indomethacin and the effect
of CCH should be examined in both the absence or presence of cAMP in
paired fashion.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the expert technical assistance of H. Schauer and G. Kummer.
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FOOTNOTES |
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K. Kunzelmann is supported by a Heisenberg fellowship. This work was supported by Deutsche Forschungsgemeinshaft Grant Ku756/2-2, AFLM, Zentrum Klinische Forschung 1, and Fritz Thyssen Stiftung.
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: K. Kunzelmann, Physiologisches Institut,
Albert-Ludwigs-Universität Freiburg, Hermann-Herder-Stra
e 7, 79104 Freiburg, Germany.
Received 15 May 1998; accepted in final form 24 August 1998.
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REFERENCES |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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