Dalton Cardiovascular Research Center and Department of Veterinary Biomedical Sciences, University of Missouri-Columbia, Columbia, Missouri 65211
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ABSTRACT |
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The role of the cystic
fibrosis transmembrane conductance regulator (CFTR) in
cAMP-stimulated
secretion across the murine duodenum was investigated.
Serosal-to-mucosal flux of
(Js
m, in
µeq · cm
2 · h
1)
and short-circuit current (Isc; in
µeq · cm
2 · h
1)
were measured by the pH stat method in duodenum from CFTR knockout [CFTR(
)] and normal [CFTR(+)] mice. Under control conditions, forskolin increased Js
m and
Isc (+1.7 and +3.5, respectively) across the
CFTR(+) but not CFTR(
) duodenum. Both the forskolin-stimulated
Js
m and
Isc were
abolished by the CFTR channel blocker 5-nitro-2-(3-phenylpropylamino)benzoate, whereas inhibition of luminal
Cl
/
exchange by
luminal Cl
removal or DIDS reduced the
Js
m by ~18% without a consistent effect on
the
Isc. Methazolamide also reduced the
Js
m by 39% but did not affect the
Isc. When carbonic anhydrase-dependent
secretion was isolated by using a
CO2-gassed,
-free Ringer
bath, forskolin stimulated the Js
m and
Isc (+0.7 and +2.0, respectively) across CFTR(+) but not CFTR(
) duodenum. Under these conditions, luminal
Cl
substitution or DIDS abolished the
Js
m but not the
Isc. It was concluded that cAMP-stimulated
secretion across the duodenum involves 1) electrogenic
secretion via a CFTR
conductance and
2) electroneutral secretion via a CFTR-dependent
Cl
/
exchange process
that is closely associated with the carbonic anhydrase activity of the
epithelium.
cystic fibrosis; cystic fibrosis transmembrane conductance regulator; chloride secretion; chloride/bicarbonate exchanger; anion exchanger; pH stat
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INTRODUCTION |
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THE DUODENAL EPITHELIUM produces an
alkaline mucus secretion as protection against the acidic
effluent from the stomach (16). After a meal the gastric effluent may
have a pH of 1.5-2.0 and PCO2 values
exceeding 400 mmHg (24, 36, 38). A major component of the alkaline
secretion is regulated by intracellular cAMP, resulting in both passive
(i.e., paracellular) and active transport of
across the epithelium (1, 16). The
process of active
secretion involves the concerted activities of an anion channel and
Cl
/
exchange in the
luminal membrane and secretes
taken
up across the basolateral membrane or generated by intracellular
carbonic anhydrase activity (1). However, the interaction of these
transport processes during cAMP stimulation of
secretion is not well understood. In
an excellent review of duodenal
secretion, Allen et al. (1) have proposed that cAMP-stimulated
secretion involves the activation of
a luminal membrane
channel.
Alternatively,
secretion may follow the model proposed for pancreatic duct epithelium, which predicts a
cAMP-regulated Cl
channel that "recycles"
Cl
entering across the luminal membrane by means of a
Cl
/
exchanger (34).
Studies of cystic fibrosis transmembrane conductance regulator (CFTR),
the dysfunctional cAMP-activated Cl channel in cystic
fibrosis (CF) disease (3, 5, 11), indicate that this channel may play a
major role in cAMP-mediated
secretion. Fueled by observations of deficient transluminal pH regulation and loss of transepithelial anion current activity across CF
epithelial tissues (15, 20, 23, 32, 37, 40), bioelectric studies of
recombinant and wild-type CFTR have shown that CFTR is permeable to
[
-to-Cl
permeability
ratios range from 1:8 to 1:4 (19, 27, 35)]. Likewise, the
outward-rectifying Cl
channel (ORCC), a channel
reportedly regulated by CFTR (14, 18), also conducts
with a
-to-Cl
permeability
ratio of 1:2 (43). However, the hypothesis that CFTR functions as both
a Cl
and a
channel
under physiological conditions is complicated by the fact that CFTR
also displays anomalous mole fraction behavior, whereby the channel
conductance of a less permeable anion
(
) is greatly reduced in the
presence of a more permeable anion (Cl
) (44).
Recently, direct measures of transepithelial
flux have shown that CFTR is required
for agonist-stimulated
secretion
across the mammalian duodenum. In pH stat studies of rat duodenum Guba
et al. (21) demonstrated that
secretion stimulated by cGMP agonists could be specifically inhibited
by 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB), a blocker of the
CFTR channel (4), but not by maneuvers that inhibit the activity of the
Cl
/
exchanger or ORCC,
i.e., removal of Cl
from the luminal perfusate or
treatment with DIDS. Although the mechanism of cAMP-stimulated
secretion was also evaluated in the
study by Guba et al. (21), interpretation of the findings was
confounded by the fact that cAMP agonists were applied after cGMP
stimulation with guanylin. More recently, in vivo perfusion studies of
the duodenum from CFTR knockout mice have demonstrated that
cAMP-stimulated duodenal
output is
greatly reduced compared with the normal murine duodenum (25). However,
the mechanistic role of CFTR in cAMP-stimulated
secretion was not evaluated, probably
owing to the difficulty of controlling transepithelial electrochemical gradients in that preparation.
In the present study we investigate the role that CFTR plays in the
process of cAMP-stimulated duodenal secretion. Direct measurements of
secretory flux, using the pH stat method, were performed on duodena
from the CFTR knockout mouse model, thereby allowing comparison of
secretion in the presence and absence
of CFTR. We hypothesized that CFTR functions as an anion channel that
is responsible for both electrogenic Cl
and
secretion during cAMP stimulation of
the duodenum. The evidence supports this hypothesis but also indicates
that CFTR is required in an electroneutral mechanism of cAMP-stimulated
secretion involving luminal
Cl
/
exchange.
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MATERIAL AND METHODS |
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Animals.
All studies were performed on weanling mice (2-4 mo of age) born
to breeding animals heterozygous for the disrupted murine homologue of the cftr gene
(B6.129-Cftrtm/UNC;
C57BL/6J-Cftr tm/UNC). The mice were
either purchased from Jackson Laboratories (Bar Harbor, ME) or received
as a generous gift from Dr. Beverly Koller (Dept. of Medicine, Univ. of
North Carolina, Chapel Hill, NC). Each littermate was genotyped using a
PCR technique employing primers specific for murine cftr and
the neomycin resistance-cftr junction (neo was used for
gene disruption) (8). Littermate mice, which were either homozygous or
heterozygous for the wild-type cftr gene, were used as controls
[designated as CFTR(+) mice]. Only one to two heterozygous
cftr(+/) mice were used per treatment group. The CFTR
knockout mice were homozygous for the disrupted cftr gene
[designated as CFTR(
) mice]. The mice were maintained on standard
laboratory mouse chow and water ad libitum. The drinking water provided
to all mice contained an osmotic laxative (polyethylene glycol; PEG) to
prevent intestinal impaction in the CFTR(
) mice (8). Before each
experiment the mice were fasted >2 h but were provided the
PEG-containing drinking water ad libitum. All experiments involving
animals were approved by the University of Missouri-Columbia Institutional Animal Care and Use Committee.
In vitro bioelectric and pH stat measurements.
The mice were killed on the day of the experiment by brief exposure to
an atmosphere of 100% CO2 to induce basal narcosis, which
was followed by a surgically produced pneumothorax. The proximal
duodenum (from ~2 mm distal to the pylorus to the common bile duct
ampulla) was removed via an abdominal incision, immediately placed in
ice-cold, oxygenated Ringer solution, and opened along the mesenteric
border. Indomethacin (106 M) was present in the rinse
and experimental Ringer solutions to prevent prostanoid generation
during tissue manipulation (6). The proximal duodenum was stripped of
the outer muscle layers and then mounted on a standard Ussing chamber
(0.25 cm2 exposed surface area). Parafilm "O" rings
were used to minimize edge damage to the intestine where it was secured
between the chamber halves.
Statistics.
Student's t-test was used for comparisons of the mean
responses between CFTR(+) and CFTR() genotypes, or between basal and treatment periods. When two sequential treatment periods were compared
with a basal period, a one-way repeated measures ANOVA followed by a
post hoc Bonferroni's test was used. P
0.05 was considered
statistically significant (41). Unless otherwise indicated data are
presented as means ± SE.
Materials.
Unless otherwise stated reagents were obtained from either Sigma
Chemical (St. Louis, MO), Aldrich Chemical (Milwaukee, WI), or Fisher
Scientific (Springfield, NJ). Indomethacin, methazolamide [to inhibit
intracellular carbonic anhydrase activity (7)], forskolin, and NPPB
were dissolved in DMSO at stock concentrations of 0.01, 1.0, 0.01, or
0.3 M, respectively. Bumetanide [to inhibit Na+-K+-2Cl cotransport (22)]
was dissolved in ethanol at a stock concentration of 0.1 M. DIDS was
dissolved in the appropriate Ringer solution at a concentration of 0.03 M. TTX was dissolved in 0.2% acetic acid at a stock concentration of
0.0001 M. In separate experiments DMSO and ethanol vehicles at
concentrations equivalent to those used in each experiment (0.2 and
0.1%, respectively) produced no significant alterations of the basal
Isc.
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RESULTS |
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Control pH stat studies.
Previous ion substitution studies of the CFTR(+) murine duodenum had
shown that either Cl or
could carry an inward current across the epithelium and together account for >99% of the cAMP-stimulated Isc (23). Furthermore, the presence of
in the bath medium resulted in a
significant fraction of the cAMP-stimulated Isc
that was insensitive to the Cl
transport inhibitor
bumetanide. However, Isc measurements of epithelia
treated with bumetanide or bathed in Cl
-free medium may
not reflect the actual rate of
secretion (12, 40). Therefore, the rate of
(base) secretion before and during
cAMP stimulation was directly measured using pH stat titration of
voltage-clamped murine duodenum.
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Effect of NPPB and DIDS on cAMP-stimulated Jsm and
Isc.
To investigate whether the channel function per se of CFTR is required
for cAMP-stimulated
secretion, CFTR(+) duodenal tissues were treated before forskolin stimulation with
NPPB, an extracellular blocker of CFTR (4, 21). As shown in Fig.
2A, NPPB completely prevented
forskolin stimulation of Js
m and reduced
stimulation of the Isc by 84% (compare with Fig.
1A). The mean Gt in these preparations was
unchanged by the NPPB-forskolin treatment (basal
Gt = 44.1 ± 7.4 mS/cm2;
NPPB-forskolin Gt = 45.4 ± 4.8
mS/cm2). To estimate the contribution of
Cl
/
exchange or a
conductance through the ORCC, CFTR(+)
duodena were treated before forskolin stimulation with the distilbene
derivative DIDS, which has inhibitory actions on both transport
processes (4, 13, 28). As shown in Fig. 2B, DIDS pretreatment
slightly reduced the mean forskolin-stimulated
Js
m (DIDS-forskolin
Js
m = 1.38 ± 0.24
µeq · cm
2 · h
1)
but had little effect on the
Isc (DIDS-forskolin
Isc = +3.33 ± 0.74 µeq · cm
2 · h
1)
compared with control (Fig. 1A). The Gt
measured in these experiments tended to increase with forskolin, but
the changes were not statistically significant (basal
Gt = 45.0 ± 5.4 mS/cm2;
DIDS-forskolin Gt = 54.3 ± 8.6
mS/cm2).
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Effect of luminal Cl substitution on cAMP-stimulated
Js
m and Isc.
The NPPB experiments indicated that cAMP-stimulated
secretion is mediated via CFTR
channel activity. This finding is consistent with the hypothesis that
CFTR can function as a cAMP-stimulated
channel. However, the results of the
DIDS studies suggested that enhanced
Cl
/
activity, but not
an electrogenic ORCC-mediated pathway, may also contribute to
cAMP-stimulated
secretion. Therefore,
pH stat studies were performed on duodenal sections bathed with an
unbuffered Cl
-free solution (Na+ gluconate,
pH 7.4) on the luminal membrane to diminish or eliminate luminal
Cl
/
exchange activity.
As shown in Fig. 3A, forskolin
treatment in the absence of luminal Cl
stimulated the
Js
m to a mean value that was slightly less than in the control studies and comparable to the DIDS-forskolin treatment (1.39 ± 0.27
µeq · cm
2 · h
1).
Forskolin treatment also significantly stimulated the
Isc in the absence of luminal Cl
,
but the mean
Isc (2.20 ± 0.6
µeq · cm
2 · h
1)
was less than measured in the control experiments. However, interpretation of the Isc in this experiment was
complicated by the fact that the Isc before
forskolin was greatly increased, probably as a result of establishing a
large concentration gradient for Cl
secretion across
both the apical membrane and via the paracellular pathway. The
Gt measured in these experiments was not different between the basal and forskolin-treated flux periods (basal
Gt = 21.1 ± 1.2; cAMP
Gt = 22.2 ± 1.3 mS/cm2). To test
whether cAMP-stimulated
secretion during inhibition of luminal
Cl
/
is mediated by
CFTR, these experiments were performed in CFTR(
) duodenum. As shown
in Fig. 3B, forskolin treatment had no significant effect on
the Js
m, Isc, or Gt (basal
Gt = 22.3 ± 1.7; cAMP
Gt = 25.7 ± 3.0 mS/cm2).
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Effect of methazolamide on cAMP-stimulated Jsm and
Isc.
Although the preceding experiments indicated that most cAMP-stimulated
secretion is an electrogenic CFTR-dependent process, the evidence also suggested that a second mechanism involving luminal
Cl
/
exchange may
contribute to cAMP-stimulated
secretion. Previously, we had found that luminal
Cl
/
exchange was
associated with carbonic anhydrase-dependent
secretion across the duodenal
epithelium (10). Therefore, we investigated the effect of carbonic
anhydrase inhibition during cAMP stimulation of
Js
m and Isc. In these
experiments CFTR(+) duodena were pretreated with a membrane-permeant
inhibitor of carbonic anhydrase, methazolamide (Meth, 1 mM), before
forskolin stimulation. As shown in Fig. 4, forskolin stimulated a significant increase in
Js
m in the methazolamide-pretreated duodenum,
but the magnitude of the
Js
m was
significantly less than that found in the control studies shown in Fig.
1 (Meth-pretreated
Js
m = +0.87 ± 0.22 vs. control
Js
m = +1.67 ± 0.15 µeq · cm
2 · h
1).
In contrast, forskolin stimulated the Isc in the
methazolamide-pretreated duodenum to a level that was equivalent to
that found in the control studies (Meth-pretreated
Isc = +3.78 ± 0.62 vs. control
Isc = +3.46 ± 0.38 µeq · cm
2 · h
1).
In additional time control experiments, we found that methazolamide treatment per se caused a small reduction in the
Js
m and, paradoxically, increased the
Isc in the second flux period compared with the
first flux period (Meth
Js
m =
0.14 ± 0.17 and Meth
Isc = +0.36 ± 0.46
µeq · cm
2 · h
1,
n = 3). Using these values to adjust the
forskolin-stimulated response in methazolamide-pretreated duodenum, we
still found that methazolamide pretreatment significantly reduced the
forskolin-stimulated
Js
m by 39%, compared
with the control but did not affect the magnitude of the
forskolin-stimulated
Isc (<3%). These
observations indicated that the carbonic anhydrase-dependent fraction
of cAMP-stimulated Js
m does not involve an
electrogenic process. Therefore, experiments were undertaken to isolate
the mechanism responsible for duodenal secretion of endogenously
generated
.
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Isolation of carbonic anhydrase-dependent
secretion.
To isolate duodenal secretion of carbonic anhydrase-generated
, pH stat studies were performed on
CFTR(+) duodena that were bathed with a NaCl solution gassed with 95% O2-5% CO2 in the luminal solution and an
-free, TES-buffered Ringer gassed with
100% O2 in the basolateral solution. The purpose of this
design was to provide a CO2 source for intracellular carbonic anhydrase generation of
while preventing
movement across the
basolateral membrane via Na+-coupled uptake mechanisms,
e.g., NaHCO3 cotransport (28). The luminal NaCl-5%
CO2 solution equilibrated in the pH range of 4.9-5.3, and the PCO2 was 41 ± 1 mmHg
(n = 3). Because clamping the NaCl-5% CO2
solution to pH 7.4 under this protocol would result in a significant
concentration in the luminal bath,
the luminal solution was clamped to pH 5.1. In contrast to the luminal bath, the TES-buffered solution in the basolateral bath remained constant at pH 7.4 ± 0.01 (n = 22), and the
PCO2 of the solution was found to be <0.4
mmHg (n = 4). In control studies using hemichambers without
intestine, the 5% CO2 gassing resulted in a small
spontaneous rate of
production in
both the NaCl solution (+0.049 µeq/h, n = 3) and in the
Cl
-free solution (+0.029 µeq/h, n = 3).
Therefore, the Js
m measured in these studies
was corrected for spontaneous
production.
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Effect of luminal Cl removal or DIDS on carbonic
anhydrase-dependent
secretion.
The involvement of luminal
Cl
/
exchange in
cAMP-stimulated secretion of endogenously produced
was investigated by inhibiting
luminal Cl
/
exchange
via luminal Cl
substitution or DIDS treatment. As shown
in Fig. 6A, removal of luminal
Cl
prevented the forskolin-induced increase in
Js
m but did not diminish the
Isc stimulation (Cl
-free
Js
m = +0.12 ± 0.14 and
Isc = +3.0 ± 0.6
µeq · cm
2 · h
1;
compare with Fig. 5A). The mean Gt slightly
increased after forskolin treatment in this series of experiments
(basal Gt = 38.4 ± 3.9; cAMP
Gt = 47.6 ± 5.0 mS/cm2,
P < 0.05). CFTR(+) duodena were also treated with DIDS
before cAMP stimulation. As shown in Fig. 6B DIDS completely
inhibited the forskolin-induced increase in
Js
m but had no effect on the
forskolin-stimulated Isc compared with the control
experiments (Fig. 5A). The mean Gt in these
experiments also increased during the forskolin-treated flux period
(basal Gt = 37.6 ± 2.2; cAMP Gt = 45.0 ± 1.6 mS/cm2,
P < 0.05). These results demonstrate that during cAMP
stimulation the murine duodenum is capable of increasing the secretion
of carbonic anhydrase-generated
via a
mechanism involving CFTR-facilitated
Cl
/
exchange.