Intestinal Bicarbonate Secretion in Cystic

Lane L Clarke, Xavier Stien, Nancy M Walker
Dalton Cardiovascular Research Center and Department of Biomedical Sciences, University of
Missouri. Columbia, MO, USA
Summary
Gene-targeted disruption of the cystic fibrosis
transmembrane conductance regulator (CFTR)
in mice results in an intestinal disease
phenotype that is remarkably similar to bowel
disease in cystic fibrosis patients. In the
intestinal segment downstream from the
stomach (i.e., the duodenum), CFTR plays an
important role in bicarbonate secretion that
protects the epithelium from acidic gastric
effluent. In this report, we examine the role of
CFTR in cAMP-stimulated bicarbonate
secretion in the murine duodenum and the
mechanisms of acid-base transport that are
revealed in CFTR knockout (CF) mice. Ion
substitution, channel blocker and pH stat
studies comparing duodena from wild-type and
CF mice indicate that CFTR mediates a HCO3
-
conductance across the apical membrane of the
epithelium. In the presence of a favorable cell-
to-lumen HCO3
-
gradient, the CFTR-mediated
HCO3
-
current accounts for about 80% of
stimulated HCO3
-
secretion. Exposure of the
duodenal mucosa to acidic pH reveals another
role of CFTR in facilitating HCO3
-
secretion via
an electroneutral, 4,4’-diisothiocyanato-
stilbene-2,2’ disulfonic acid (DIDS) sensitive-
Cl
-
/HCO3
-
exchange process. In CF duodenum,
other apical membrane acid-base transporters
retain function, thereby affording limited
control of transepithelial pH. Activity of a Cl
-
-
dependent anion exchanger provides near-
constant HCO3
-
secretion in CF intestine, but
under basal conditions the magnitude of
secretion is lessened by simultaneous activity of
a Na
+
/H
+
exchanger (NHE). During cAMP
stimulation of CF duodenum, a small increase
in net base secretion is measured but the change
results from cAMP inhibition of NHE activity
rather than increased HCO3
-
secretion.
Interestingly, a small inward current that is
sensitive to the anion channel blocker, 5-nitro-
2(3-phenylpropyl amino)-benzoate (NPPB), is
also activated during cAMP stimulation of the
CFTR-null intestine but the identity of the
current is yet to be resolved. Studies to identify
the proteins involved in non-CFTR mediated
HCO3
-
secretion are on-going and potentially
will provide targets to correct deficient HCO3
-
secretion in the CF intestine.
In cystic fibrosis patients and CFTR knockout
mice, duodenal bicarbonate transport is greatly
diminished, resulting in abnormal pH regulation
at the mucosal surface. Two transport pathways
at the apical cell membrane are involved in
bicarbonate secretion - an anion conductance(s)
and Cl
-
/HCO3
-
(OH
-
) exchanger(s). Stimulation
of intracellular cAMP yields electrogenic
bicarbonate secretion that requires the activity
of CFTR to either provide a bicarbonate
conductance and/or a chloride conductance that
recycles Cl
-
entering the cell via a Cl
-
/HCO3
-
exchanger. Although patch clamp and apical
membrane preparation studies have shown that
CFTR is moderately permeable to HCO3
-

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264
(PCl:PHCO= about 0.25) [1, 2, 3] it has been
difficult to determine whether CFTR mediates a
bicarbonate conductance under physiological
conditions in native duodenal epithelium. Two
lines of evidence have emerged. First, in vivo
measurements and pH stat studies have shown
that CFTR is required for electrogenic
bicarbonate secretion under conditions that
inhibit apical membrane Cl
-
/HCO3
-
exchange
activity [4, 5, 6, 7, 8]. Second, anion
substitution studies of intact duodenal mucosa,
such as the study of murine duodenum shown
in Figure 1, indicate that cAMP-stimulated
CFTR can carry a bumetanide-insensitive
HCO3
-
current when other anions that have
significant permeability in CFTR are removed
from the bathing medium.
Interestingly, careful examination of CFTR
knockout duodenum reveals a finite increase in
bicarbonate (base) secretion in response to
cAMP stimulation. As shown in Figure 2a,
bicarbonate secretion (Jsm
HCO
3) is minimal
under basal conditions. Following treatment
with forskolin (cAMP), Jsm
HCO
increases by 1
µEq/cm²·h and this change is accompanied by a
similar increase in short-circuit current (Isc).
Note, however, that the cAMP-induced changes
Permeant anions in medium
sc
(µA/cm²)
0
20
40
60
80
100
Basal
cAMP
+ Bumet
Cl+
HCO3
-
Cl-
HCO3
-
a
b
c
a
a
b
a
b
b
Figure 1. Effect of anion substitution in the bathing
medium on basal and cAMP-stimulated short-circuit
current across wild-type murine duodenum.
Transepithelial short-circuit current (Isc, an index of
anion secretion) was measured in Ringers media
containing both chloride and bicarbonate (Cl-+ HCO3-),
only chloride (Cl-) or only bicarbonate (HCO3
-) as
CFTR-permeant anions. Measurements were made
during sequential periods: Basal, during cAMP
stimulation by bilateral addition of 10 µM forskolin +
100 µM isobutyl methylxanthine (cAMP), and following
addition of 100 µM bumetanide (an inhibitor of Cl-
secretion) to the serosal bath (Bumet). The data provide
evidence that CFTR carries bicarbonate current during
cAMP stimulation. First, a significant bumetanide-
insensitive Isc is present only when HCO3
is present in
the bathing medium. Second, a significant cAMP current
is stimulated when HCO3
is the only permeant anion
available for transport by CFTR. In contrast, these
responses are minimal in CFTR knockout duodenum
(data not shown). Letters indicate differences between
means within each group, < 0.05.
30
35
40
45
50
55
60
J
sm
(µeq/cm²·h)
-1
0
1
2
r2=0.00009
y0=0.2
*
*
HCO3
I
sc
0
1
2
Basal
cAMP
J
s-m
µeq/cm²·h
0
1
2
Figure 2. Effect of intracellular cAMP stimulation on
bicarbonate secretion, Isc and transepithelial conductance
in CF muri ne duodenum. In pH stat experiments (2a)
performed under voltage-clamped conditions, stimulation
of intracellular cAMP with a forskolin/IBMX cocktail
induces simultaneous increases in the serosal-to-mucosal
flux of bicarbonate (Js m
HCO
3) and the Isc. However, the
transepithelial conductance, Gt, was significantly
increased by stimulation of cAMP (Basal G= 45.2 ±0.7;
cAMP G= 553 ± 0.7, < 0.05, n = 10). * Significantly
different from Basal, p < 0.05). To estimate the effect of
transepithelial conductance (2b) on the Jsm
HCO
3, the Gt
from the CF mice experiments were regressed against the
Jsm
HCO
3. However, a significant correlation was not
apparent.
2a
∆∆ G(mS/cm²)
4
5
6
7
8
9
10
11
12
∆∆
sm
(µeq/cm²·h)
0
1
2
3
r² = 0.02335
y= 1.18
HCO
3-
2b

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265
in the bioelectric properties include a
significant increase in total tissue conductance
(Gt), a measure of the paracellular pathway in
the intestine [9]. To evaluate the possibility that
the cAMP-induced ∆ Jsm
HCO
is a consequence
of both the imposed transepithelial bicarbonate
gradient (see Methods) and an increase in
paracellular permeability, the cAMP-induced
Jsm
HCO
was correlated with the Gt. As
shown in Figure 2b, no relationship existed
between the Jsm
HCO
and Gunder either
condition. This finding confirms earlier studies
of murine duodenum showing that Jsm
HCO
does
not correlate with Gduring cAMP stimulation
[10].
The above findings indicated an active
bicarbonate secretory process, therefore, the
involvement of luminal Cl
-
/HCO3
-
exchange
activity during cAMP stimulation of the CF
duodenum was investigated by replacing Cl
-
in
the luminal bath with the poorly permeable
solute, isethionate. Interestingly, this maneuver
resulted in net acid secretion during the basal
period that was then abolished by cAMP
treatment (Figure 3a). The basal Isc of the
duodenum was greatly accentuated under these
conditions but did not increase with cAMP
treatment. Acid secretion during inhibition of
luminal Cl
-
/HCO3
-
exchange is consistent with
activity of luminal Na
+
/H
+
exchange activity as
recently suggested by pH measurements of the
luminal content in wild-type and NHE3
knockout mice [11]. We tested this hypothesis
by exposing the luminal membrane to the NHE
exchange inhibitor, 5-(N-Ethyl-N-isopropyl)
amiloride (EIPA), at a concentration (100 µM)
that inhibits the major intestinal isoforms,
NHE2 and NHE3, in media containing
physiological concentrations of Na
+
[12]. As
shown in Figure 3b, EIPA treatment increased
the basal Jsm
HCO
to about 1 µEq/cm²·h and
prevented the increase in Jsm
HCO
during cAMP
stimulation. Thus, the increase in Jsm
HCO
3
measured during cAMP stimulation of the CF
duodenum is likely due to cAMP inhibition of
NHE activity, which reveals activity of a Cl
-
-
dependent anion exchanger(s). This latter
conclusion was confirmed by the lack of an
EIPA effect on the CF duodenum during Cl
-
substitution in the luminal bath (data not
shown).
Although the cAMP change in Jsm
HCO
was
abolished by the amiloride analog EIPA,
forskolin stimulated a small but significant
increase in the Isc of the CF duodenum. Thus,
the cAMP-induced Isc in the CFTR knockout
duodenum was dissociated from the Jsm
HCO
in
both the luminal Cl
-
substitution and EIPA
experiments, indicating that the current is not
µeq/cm²·h
0
1
2
Isc
0
1
2
*
*
HCO3
Js-m
0
1
4
Basal
cAMP
Isc
µeq/cm²·h
-1
0
1
ClFree (L)
ClFree (L)
EIPA 100 µM
EIPA 100 µM
Js-m
HCO3
Figure 3. Effects of luminal Clremoval and EIPA on
bicarbonate secretion and Isc
across CF murine
duodenum. Replacing luminal Clcontent (3a) with the
poorly permeable anion, isethionate, resulted in net acid
secretion by CF duodenum that was abolished by
subsequent cAMP stimulation using a forskolin/IBMX
cocktail. The imposed cell-to-lumen and mucosal-to-
serosal chloride gradient resulted in high basal Isc in the
CF duodenum. However, cAMP stimulation did not
increase Isc under this condition (n = 13). To determine
whether a change in proton secretion via a luminal
membrane Na+/Hexchanger was responsible for the
change in Jsm
HCO
3, the CF duodenum was treated with
EIPA (3b), an inhibitor of intestinal Na+/Hexchangers
(NHE2 and NHE3). EIPA treatment resulted in a stable
increase in the basal bicarbonate secretion and prevented
cAMP stimulation of Jsm
HCO
3. However, a significant
increase in Isc
was still apparent following
forskolin/IBMX in the presence of EIPA (n = 8).
* Significantly different from Basal.
3a
3b

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JOP. Journal of the Pancreas – http://www.joplink.net – Vol.2, No.4 Suppl. – July 2001
266
carried by HCO3
-
. Evaluation of the individual
cAMP-induced Isc indicates the presence of a
subpopulation of CF mice that have robust
responses (see Figure 4a), suggesting that a
cohort of CF mice surviving to adulthood may
be selected for the expression of an alternate
conductance. Inhibitor studies of the cAMP
stimulated Isc in CF duodenum indicate partial
blockade by the anion conductance inhibitors,
DIDS and NPPB (Figure 4b). Although the
identity of the conductive pathway has not been
resolved, these findings indicate the presence of
an alternate cAMP-sensitive anion channel that
may modify the physiological consequences of
gene-targeted deletion of CFTR in murine
intestine.
Methods
Animals. Wild-type (WT) and CFTR knockout
(CF) mice 2-4 months of age were used. The
mice were fasted overnight before
experimentation (water was provided ad-
libitum).
Ussing chamber studies. Freshly-excised
duodenum was stripped of the underlying
muscle layers and mounted on standard Ussing
chambers with 0.25 cm² exposed surface area.
All sections were treated with 1 µM
indomethacin and 0.1 µM tetrodotoxin (serosal)
prior to experimentation. The duodenal sections
were voltage-clamped using an automatic
voltage clamp (Physiologic Instruments, San
Diego, CA, USA).
pH stat. The duodenal studies consisted of two
sequential 30 min flux periods: a basal period
and a treatment period using either 10 µM
forskolin (cAMP) or 100 µM EIPA (EIPA).
All drugs were obtained from Sigma Chemicals
(St. Louis, USA). The luminal surface of
duodenum was bathed with 4 mL of an
unbuffered NaCl solution containing (in mM):
Na
+
, 144.0; Cl
-
, 154.0; K
+
, 5.2; Ca
2+
, 1.2; Mg
2+
,
1.2. The mucosal bath pH was clamped at 7.4
by neutralizing the appearance of base with 5
mM HCl or acid with 5 mM NaOH using an
automatic titrator (Radiometer, Radiometer
Analytical, Lyon, France). The mucosal
solution was gassed with 100% O2. The serosal
surface was bathed with Krebs bicarbonate
Ringers solution (KBR) containing (in mM):
Na
+
, 140.0; Cl
-
, 120.0; HCO3
-
, 25.0; H2PO4
-
,
0.4; HPO4
2-
, 2.4; K
+
, 5.2; Ca
2+
, 1.2; Mg
2+
, 1.2;
glucose, 10; pH 7.4 (gassed with 95% O2: 5%
CO2). Both solutions were warmed to 37 °C by
water-jacketed reservoirs. For Cl
-
free lumen
experiments, chloride was replaced with 91
mM gluconate and either 25 mM SO4
2-
plus 25
mM mannitol or 50 mM isethionate.
Statistics. Paired t-test was used to compare two
sequential treatment periods and an unpaired t-
% Inhibition of cAMP
∆∆
sc
-30
-15
0
15
30
45
60
Glybc. DIDS
NPPB Bumet
*
sc
(µeq/cm²·h)
-1
0
1
2
3
Basal
cAMP
Basal
cAMP
Figure 4. Inhibitor studies of the cAMP -induced Isc in
CF murine duodenum. Analysis of the cAMP-induced Isc
in the duodena from individual CF mice (4a) indicated a
subpopulation of mice with robust responses to
forskolin/IBMX (right). The effects of anion transport
inhibitors on the cAMP-induced Isc
response (4b)
indicated that NPPB significantly reduced the Isc by 39%
(n = 4 - 6).
*Significantly different from Isc before treatment.
4a
4b

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267
test was used to compare two treatment groups.
Repeated measures ANOVA was used to
compare three sequential treatment periods and
a one-way ANOVA was used to compare
multiple treatments groups. A value less than
0.05 was considered statistically significant. All
data are given as means±SEM.
Key words Antiporters; Chloride Channels;
Cyclic AMP; Cystic Fibrosis Transmembrane
Conductance Regulator; Duodenum; Sodium-
Hydrogen Antiporter
Abbreviations CF: CFTR knockout mice;
CFTR: cystic fibrosis transmembrane
conductance
regulator;
DIDS: 4,4’-
diisothiocyanato-stilbene-2,2’ disulfonic acid;
EIPA: 5-(N-Ethyl-N-isopropyl) amiloride; Gt:
total tissue conductance; Isc: short-circuit
current; Jsm
HCO
3: serosal-to-mucosal bicarbonate
flux; KBR: Krebs bicarbonate Ringers solution;
NHE: Na
+
/H
+
exchanger: NPPB: 5-nitro-2(3-
phenylpropyl amino)-benzoate; WT: wild-type
Acknowledgements The study was funded by
grants from the Cystic Fibrosis Foundation and
National Institutes of Health (DK48816).
Correspondence
Lane L Clarke
University of Missouri-Columbia
Dalton Cardiovascular Research Center
Research Park Drive
Columbia, MO 65211-3300
USA
Phone: +1-573-882.7049
Fax: +1-573-884-4232
E-mail address: clarkel@missouri.edu
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