Electrogenic bicarbonate secretion in mouse
gallbladder
L. C.
Martin,
M. E.
Hickman,
C. M.
Curtis,
L. J.
MacVinish, and
A. W.
Cuthbert
Department of Pharmacology, University of Cambridge, Cambridge CB2
1QJ, United Kingdom
 |
ABSTRACT |
Mouse gallbladders (4 mm2) were investigated using the
short-circuit current
(Isc)
technique. Responses of 50 µA/cm2 were obtained in response
to forskolin and agents that stimulated the adenylate cyclase system
(IBMX and dibutyryl-cAMP). The calcium ionophore
ionomycin increased
Isc to 30% of
the forskolin-stimulated increase. The forskolin-dependent current was
inhibited 40% by acetazolamide but was insensitive to furosemide.
Forskolin responses were dependent on the presence of bicarbonate ions;
removal from both sides of the membrane or the basolateral side alone
caused a significant reduction in responses. Removal of chloride ions from the basolateral side had no effect, while removal from the apical
side caused a significant reduction in the forskolin responses, but
only by 30%. It is argued that the remaining current (70%) cannot
result from a parallel arrangement of a chloride channel and a
chloride-bicarbonate exchanger and that bicarbonate is secreted through
the apical membrane by a predominantly conductive mechanism. Apparently, forskolin converts a near electrically silent epithelium to
an electrogenically secreting tissue.
electrogenic transport; forskolin; acetazolamide
 |
INTRODUCTION |
THERE HAVE BEEN FEW electrophysiological studies of the
mouse gallbladder epithelium (18, 22). The organ in the mouse is a
small, ovoid sac, with a long axis of 2-3 mm, making the conventional short-circuit current
(Isc) approach
technically demanding. However, the advent of transgenic mice, in which
genes of interest are "knocked out" or "knocked in," has
led to important understandings of function. Consequently, the
physiology of this species has become an important area of study. Here
we have made observations of the electrogenic transport of ions across
the gallbladder epithelium to gain insight into the mechanisms of bile
secretion in this organ.
The literature on bile secretion is derived from species other than the
mouse. It is known that bile is concentrated in the gallbladder by
fluid absorption driven by active salt transport. Bile formation occurs
in the bile canaliculi by bile salt-dependent and -independent
secretion of salts and water. Bile salt-independent secretion extends
into the bile duct and the gallbladder itself, both the secretory and
absorptive functions being the responsibility of a simple columnar
epithelium (7). However, the apparently identical columnar epithelial
cells have heterogeneous functions (1). Human gallbladder epithelial
cells have both Ca2+- and
cAMP-dependent Cl
efflux
pathways, and natural secretion may be under the control of
vasoactive intestinal polypeptide (VIP) (7). A variety of agents can
modify the transporting activities of gallbladder epithelial cells
in a species-dependent manner. In the dog an increase in intracellular
Ca2+ converts absorption to
secretion by a prostaglandin-dependent mechanism (16); in humans
somatostatin analogs decrease the secretion of bile acids,
HCO
3, and lipase (17). The effects in
both tissues are due to interactions with the adenylate cyclase system.
Here we show that agents that increase intracellular cAMP in mouse
gallbladder epithelium cause electrogenic anion secretion, with
HCO
3 carrying the major fraction of
the current.
 |
MATERIALS AND METHODS |
Animals.
All experiments were carried out in 2- to 3-mo-old mice killed by
exposure to an increasing concentration of
CO2. The care and use of the
animals conformed to the requirements set by the Home
Office. The gallbladder and as much of the common bile
duct as possible were removed by fine dissection under a microscope. Tissues were placed in cold Krebs-Henseleit solution (KHS) immediately and mounted, after opening, as soon as possible.
Isc recording.
Gallbladders were mounted in a specially constructed chamber, cushioned
with silicone washers and with a window area of 4 mm2, and bathed on both sides with
KHS (20 ml warmed to 37°C), continually circulated by bubbling with
95% O2-5%
CO2. The chambers were initially assembled without tissues for the purpose of balancing the electrodes. In experiments of long duration, periodic checks were made to ensure
the electrodes remained in balance. However, if asymmetry was
introduced by having solutions of different composition on either side
of the tissue, the asymmetric electrode potentials introduced an
apparent change in basal
Isc. The tissues
were short circuited with a World Precision Instruments dual-voltage
clamp with series resistance compensation, and the
Isc was recorded continuously with a MacLab and associated AppleMac computer. KHS had
the following composition (in mM): 117 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4,
25 NaHCO3, and 11.1 glucose (pH 7.4 at 37°C). In
Cl
-free solution, sodium
isethionate, potassium sulfate, and calcium sulfate replaced the
corresponding Cl
salts. To
make HCO
3-free solutions,
NaHCO3 and
KH2PO4
were replaced either with sucrose or NaCl and KCl and the solutions
were buffered with either 5 mM Tris or 10 mM HEPES. In general,
HCO
3-free solutions were bubbled with
100% O2 and had a pH of 7.3 at
37°C. On occasion HCO
3-free solution was bubbled with 95%
O2-5%
CO2 when the pH dropped to 6. Several agents were used frequently throughout this study as follows:
10 µM amiloride applied apically, 10 µM forskolin applied to both
sides, 1 mM furosemide applied basolaterally, and 100 µM
acetazolamide applied to both sides.
Base content of mouse bile.
A Radiometer VIT90 videotitrator with an ABU91 autoburette fitted with
a 1-ml syringe used in the pH-stat mode was employed to construct a
linear calibration curve for the volume of 0.01 N HCl added to maintain
pH at 3.5 when aliquots of NaHCO3
solution (25 mM) of 5-25 µl were added to a starting volume of 2 ml. Small volumes of bile were withdrawn directly from the
gallbladders of freshly killed mice, using a Hamilton syringe. Volumes
of bile from 2 to 8.5 µl were obtained. The concentration of base in
the bile was calculated as NaHCO3,
using the calibration curve.
Statistical analysis.
Student's t-test (paired or unpaired)
was used as appropriate to test for significance.
P < 0.05 was considered significant.
 |
RESULTS |
Electrogenic ion transport in wild-type mouse gallbladder
epithelium.
In an initial series of experiments, forskolin, an adenylate cyclase
activator, was applied to short-circuited mouse gallbladders. The basal
Isc was 7.2 ± 4.3 µA/cm2
(n = 21), and addition of 10 µM
forskolin to the solution bathing both sides of the tissue caused a
rapid increase in current of 48.2 ± 6.1 µA/cm2
(n = 21). The large standard errors
underlie the considerable variation in both basal and stimulated
Isc, with some
tissues having virtually no
Isc before
addition of forskolin. The transepithelial potential (TEP) in
unstimulated bladders was close to zero but increased after forskolin.
In a series of four measurements, the TEP was 0 mV with an
Isc of
1.25 ± 5.75 µA/cm2.
After forskolin, the mean TEP increased to 1.02 ± 0.37 mV and the
mean Isc to 39.0 ± 5.5 µA/cm2, indicating a
low transepithelial resistance (TER) of 26
· cm2.
However, the low surface area-to-circumference ratio (i.e., 0.5×
radius) in these small preparations adversely affects edge damage (6)
and lowers TEP. As the TEP was close to zero in untreated bladders, the
TER was measured by recording the current required to impose a
potential of 1.5 mV across the epithelium. For seven untreated
gallbladders with a basal
Isc of
3.2 ± 4.4 µA/cm2, the resistance
obtained by imposing a potential was 16.6 ± 2.6
· cm2.
Although we have used forskolin throughout to activate adenylate
cyclase, some further preliminary experiments were made to substantiate
the hypothesis that the responses were due to cAMP generation in the
epithelial cells. A high concentration (100 µM) of the
phosphodiesterase inhibitor IBMX also increased
Isc, as did
forskolin, whereas a low IBMX concentration (1 µM) had no
effect alone but was able to potentiate the response to dibutyryl-cAMP (DBcAMP). Alone DBcAMP only produced a modest effect, even at a high concentration (Fig. 1). Finally, the
peptide hormone VIP, which acts through receptors coupled to adenylate
cyclase, also produced responses comparable to those of forskolin at a
concentration of 100 nM, but only when added basolaterally (data not
shown). Secretin, even in the presence of IBMX, was unable to increase the Isc in mouse
gallbladders.

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Fig. 1.
Effects of cAMP on short-circuit current
(Isc) in mouse
gallbladder. A: all records are from
the same gallbladder epithelium (area 4 mm2). Agents were applied as
indicated to bathing solutions on both sides of the membrane, with
extensive washing between top,
middle, and
bottom traces. Forskolin and
dibutyryl-cAMP (DBcAMP) were used at concentrations of 10 µM and 1 mM, respectively, unless indicated otherwise.
B: record from a second gallbladder
epithelium with DBcAMP alone. Basal
Isc is shown at
the beginning of each trace.
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To establish whether Ca2+
ionophores could also stimulate electrogenic anion secretion, we
applied ionomycin (5 µM) to the bathing solution on either side of
the tissue. Some tissues showed no response, but their viability was
unquestioned, as they subsequently responded to other secretagogues
(see Fig. 6A). The mean response in
10 gallbladders, of which seven responded, was 14.8 ± 3.9 µA/cm2, only 30% of the
response obtained with forskolin. Piroxicam (5 µM), a cyclooxygenase
inhibitor, had no effect on the response to ionomycin when applied
during the plateau phase.
Effects of transport inhibitors.
Individual gallbladder preparations were able to withstand multiple
solution changes, as shown in Fig.
2. Furosemide and then acetazolamide were added after
Isc was increased
with forskolin. The whole sequence was repeated after extensive washing
at 90-min intervals. The low value of the basal
Isc, shown at the
beginning of each trace, indicates the protocol of washing and
reequilibration is adequate to reverse the effects of previous drug
exposure. Initially, furosemide had no effect on current, whereas
acetazolamide caused inhibition. Later, furosemide did cause a small
inhibition of
Isc, and the
responses to acetazolamide remained constant. In other instances (see
Figs. 4 and 6), furosemide caused a minor stimulation in
Isc when first
added. Results from 12 experiments were pooled in which the sequence of
agents (forskolin, furosemide, and acetazolamide) was added more than
once to gallbladders. The data are given in Fig.
3, which shows that acetazolamide caused a
significantly greater effect than furosemide
(P < 0.0002 in applications 1 and 2, n = 12) and that furosemide caused no changes that were
significantly different from zero. The mean inhibition by acetazolamide
of the forskolin response on first application was 38% (
18.2 ± 2.1 µA/cm2 for forskolin
responses of 48.2 ± 6.1 µA/cm2,
n = 21).

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Fig. 2.
Effects of forskolin, furosemide, and acetazolamide on
Isc in mouse
gallbladder. The sequence of forskolin (10 µM, applied to both
sides), furosemide (1 mM, applied basolaterally), and acetazolamide
(100 µM, applied to both sides), with 10 min between each addition,
was repeated four times with extensive washing between each addition
over a period of 8 h. Basal
Isc at the
beginning of each trace is indicated at
left.
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Fig. 3.
Changes in the responses to furosemide and acetazolamide with time.
Using the protocol depicted in Fig. 2, we measured the inhibition of
the Isc responses
to forskolin by furosemide (1 mM) and acetazolamide (100 µM). In each
experiment, the whole protocol was repeated a second, and in some
instances a third, time at 90-min intervals after thorough washing.
Means ± SE are shown. The responses to acetazolamide remain
constant over time, unlike those to furosemide. The
x-axis refers to the 3 repetitions of
the protocol.
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Apparently, no electrogenic Na+
absorption occurs in the murine gallbladder epithelium, because
addition to the apical face of 10 µM amiloride, a concentration that
completely blocks Na+ channels
(5a), had no effect on
Isc either before
or after forskolin.
Effects of ion substitutions on responses to forskolin in mouse
gallbladder epithelium.
The responses to acetazolamide reported above strongly suggest that
HCO
3 is involved in the anion
transport processes of the gallbladder. When
HCO
3 and CO2 were removed and replaced by
sucrose and O2, respectively, the
responses to forskolin were severely attenuated by 84% (Table 1), as depicted in Fig.
4. Removal of
HCO
3 alone, leaving
CO2 present, caused 78%
inhibition (Table 1), but in this situation the pH of the bathing
solution dropped to 6. In both sets of experiments, tissues were used
to measure the effect of forskolin both in KHS and in the modified
solution, with each tissue acting as its own control. Care was taken to ensure that the first exposure was alternately to KHS or the modified solution in a series of experiments or that the responses in the modified solution were bracketed between responses in KHS. This precaution was to avoid bias from any adverse effects of the modified solutions. Further evidence for the role of
HCO
3 was sought by examining the
effects of unilateral removal of HCO
3 and CO2, replacing
HCO
3 with NaCl and substituting O2 for 95%
O2-5%
CO2 on the appropriate side.
Again, care was taken to alternate the solution to which the tissue was
first exposed. No effect was seen when
HCO
3 was removed from the apical side
(Fig. 5), i.e., the compartment into which anions were being transported. However, when
HCO
3 was removed from the basolateral
side, presumably the source of the transported ions, the response was
reduced in eight of the nine experiments by a mean of 58% compared
with controls. Not only was there a significant reduction in the
responses in the absence of basolateral
HCO
3
(P < 0.003), but the responses were
less well maintained after the initial peak (Fig. 5).

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Fig. 4.
Effects of HCO 3 and
CO2 removal on the responses to
forskolin. Again using the protocol depicted in Fig. 2, we obtained
sequences of Isc
responses to forskolin (F; 10 µM), furosemide (Fr; 1 mM), and
acetazolamide (A; 100 µM) at 90-min intervals. The protocol was
repeated 5 times, but the solutions bathing the tissue were alternated
between Krebs-Henseleit solution (KHS) and
HCO 3 and
CO2-free solution. The extent of
the forskolin responses is shown in
µA/cm2 at
bottom. The
x-axis refers to the 5 repetitions of
this protocol.
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Fig. 5.
Effects of unilateral removal of HCO 3
and CO2 on responses to forskolin.
Pairs of traces shown in A and
B are
Isc records from
single bladders that were exposed to forskolin, both in KHS and in the
modified solution in which HCO 3 and
CO2 were absent. Modified solution
was applied basolaterally in A and
apically in B.
C,
left: data from 9 experiments
identical to that shown in A.
C,
right: data from 4 experiments
identical to that shown in B. NS, not
significant.
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Similar sets of experiments were undertaken to study the effects of
Cl
removal, from either one
or both sides of the epithelium. Figure 6B shows
the effects of complete removal of
Cl
. A similar result was
obtained in a further experiment, with responses to forskolin reduced
from 69.1 to 6.8 µA/cm2 (means
of 2 experiments) when Cl
was removed. More interesting data were obtained when
Cl
was removed
unilaterally. In the absence of
Cl
on the apical side, the
responses to forskolin were well maintained; on average 70% of the
response was sustained, although the reduction was significant
(P < 0.02; Fig.
7 and Table 2).
Figure 6A shows how removing all
Cl
from the basolateral
side, creating an unfavorable gradient for Cl
secretion, actually
increased the response to forskolin compared with the control. This
experiment was repeated nine more times, alternating as usual the
solution to which the tissues were first exposed and without prior
exposure to ionomycin, as in Fig. 6A. In four experiments, the responses were again larger in the absence of
basolateral Cl
and smaller
in the other three experiments. Overall, the responses were one-third
smaller in the absence of basolateral
Cl
, but no significant
difference was apparent (Table 2).

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Fig. 6.
Effects of Cl removal from
both sides of the epithelium and from the basolateral side only on the
responses to forskolin. Two bladders were subjected to solution changes
as indicated, and the effects of forskolin (F; 10 µM), furosemide
(Fr; 1 mM), acetazolamide (A; 100 µM), and ionomycin (I; 5 µM) were
recorded. A:
Cl was absent from the
basolateral side only on the first exposure to the drugs and
subsequently responses were obtained in KHS.
B: responses in the complete
absence of Cl are shown
between responses obtained in KHS. F, forskolin; Fr, furosemide; A,
acetazolamide; I, ionomycin.
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Fig. 7.
Effects of Cl removal from
the apical bathing solution only on the responses to forskolin. Shown
are Isc records
of a gallbladder subjected to the sequential drug regimen of forskolin
(F; 10 µM), furosemide (Fr; 1 mM), and acetazolamide (A: 100 µM),
followed by extensive washing and repeat application of the same drugs
1 h later. Tissue was exposed to the protocol 5 times with
Cl -free apical solution
replacing KHS in alternate sequences. The extent of the forskolin
responses in µA/cm2 is shown at
bottom.
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Base content of mouse bile.
A simple pH-stat method was used to estimate the total base content of
mouse bile, expressing the result as
HCO
3. In 10 measurements, the base
equivalent corresponded to 38.9 ± 3.8 meq/l
(range 21.4-58.0 meq/l).
 |
DISCUSSION |
It is shown that the murine gallbladder epithelium is capable of
electrogenic ion transport. The basal current, which has not been
investigated, is small and often close to zero, but after treatment
with forskolin to activate adenylate cyclase, an increase in both TEP
and Isc occurs.
The epithelium is of the leaky type, with a low TER of 15-30
· cm2,
comparable to those of other leaky epithelia, such as
Necturus gallbladder (13), goldfish
intestine, frog choroid plexus, and the rectal gland of
Squalus
acanthias (see Ref. 9). However, we
were not able to allow for edge damage, which is accentuated in tissues
of small dimensions (6), although we took possible precautions by using
soft silicone washers to cushion the tissue.
The direction of the current flow and the lack of effect of low
concentrations of amiloride indicate the current is due to the
electrogenic secretion of anions. This behavior is reminiscent of
studies with the guinea pig gallbladder, in which cAMP converts electroneutral transport to electrogenic secretion (19, 21), which in
the mouse can be activated by VIP. No response to secretin was seen,
although this peptide is effective in the human gallbladder (7). The
effects of other agents that interact with the cAMP cascade, such as an
inhibitor of phosphodiesterase (IBMX) and the lipid-soluble cAMP analog
DBcAMP, justify the assumption that the secretory effect is cAMP
dependent. Although the Ca2+
ionophore ionomycin did stimulate
Isc in the
gallbladder, the responses were relatively small compared with those
with forskolin. They did not appear to be due to prostaglandin
formation, as in the dog (16). We did not further investigate how
increased intracellular Ca2+
causes secretion.
To discover the nature of the major transported species, changes in the
composition of the bathing fluid plus the use of inhibitors were
informative. Acetazolamide inhibited the
forskolin-sensitive Isc by 40%.
Although not complete, this degree of inhibition is greater than that
found in other HCO
3-secreting epithelia (15) and similar to that found in the human duodenum (12).
Inhibition at this level may mean that although carbonic anhydrase-dependent hydration of
CO2 is important in generating intracellular HCO
3, it is not the only
or even the predominant way that HCO
3
enters the cell. Uncatalyzed formation of
HCO
3 may play a part, but retention of
CO2 in the absence of
HCO
3 was not more efficient in
maintaining secretion than was removing both
HCO
3 and
CO2 (Table 1). It seems likely that HCO
3 enters the basolateral face
of the epithelial cells directly, for example by using an
Na+-HCO
3
cotransporter (23) or
Cl
/HCO
3
exchanger.
Complete removal of HCO
3 and
CO2 from the bathing solution
reduced the forskolin response to 16% of normal (Table 1). Because the
concentration of the major permanent anion, Cl
, was 120 mM, it is
likely that conventional Cl
secretion involving a basolateral
Na+-K+-2Cl
cotransporter and apical Cl
channels would have been maintained in this situation. This suggests that only a small fraction of the secretory response could be due to
Cl
secretion. When
HCO
3 and
CO2 were removed only from the
basolateral side, the response to forskolin was significantly reduced
and not well maintained (Fig. 5), a situation that would have
attenuated the activity of any basolateral
HCO
3 cotransporters. However, with
unilateral solution changes it is possible that
HCO
3 crosses the epithelium from the
apical side by a nonelectrogenic process, especially as the epithelium
is of the low-resistance type, preventing a true
HCO
3-free situation remaining in close
proximity to the basolateral face and accounting for the residual
response. In contrast, HCO
3 and
CO2 removal from the apical
solution made no difference in the forskolin responses, which were as
well maintained as in the normal situation.
Complete removal of Cl
from
the bathing solutions reduced the forskolin response to a low level,
10% of normal. However, the total amount of permeant anion was reduced
to only 25 mM and was accounted for entirely by
HCO
3. Two explanations can be offered
for the failure of secretion in
Cl
-free conditions. First,
HCO
3 might cross the basolateral face
of the epithelial cells by a
Cl
/HCO
3
exchange mechanism, which would fail in the complete absence of
Cl
. However, removal of
Cl
from the basolateral
side alone fails to cause a significant reduction in secretion,
suggesting that basolateral
Cl
is not essential for the
forskolin response, although leakage of
Cl
from the apical side
cannot be assumed to have no effect. Second, the absence of
sufficient permeant anion, when
Cl
is totally removed, may
lead to cell shrinkage such that the secretory processes are disrupted.
A more significant result is the effect of
Cl
removal from the apical
side alone. Anion secretion in response to forskolin is significantly
reduced, but only by 33%. It has been suggested, for pancreatic duct
cells, that HCO
3 secretion occurs via
a Cl
channel in parallel
with a
Cl
/HCO
3
exchanger (10). When Cl
is
absent from the apical solution, any efflux of
Cl
via
Cl
channels would be
infinitely diluted, given the large volume of the rapidly stirred
bathing solution, so that
Cl
/HCO
3
exchange could not occur. Yet, in this situation, 70% of the response
to forskolin remains. It is concluded that the major fraction of the
secretory response is due to the direct secretion of
HCO
3 via anion channels. The minor
fraction of the secretory response could be due to the operation of the
parallel channel-exchanger mechanism or simply to
Cl
secretion. Although no
significant effects of furosemide were measured, it is difficult not to
accept that in some instances furosemide had a real, but minor,
inhibitory effect (Fig. 2), perhaps representing a minor contribution
of conventional Cl
secretion.
Anion channels with a finite permeability to both
Cl
and
HCO
3 have been described previously
(11, 14), so direct efflux of HCO
3
through the apical face is possible if appropriate electrochemical
gradients are present after forskolin stimulation. In summary, the
major fraction of the electrogenic anion secretory response in the
murine gallbladder is sensitive to acetazolamide, insensitive to
furosemide, prevented by HCO
3 removal,
does not depend on basolateral or apical
Cl
, and is attenuated by
HCO
3 removal from the basolateral
side. Many investigations have been made with the guinea pig
gallbladder with results closely similar to those found here for the
mouse. For example, the cAMP-dependent secretion was insensitive to
piretanide but sensitive to acetazolamide (2) and dependent on
basolateral HCO
3 (21). It was
concluded, for the guinea pig, that
HCO
3 exits the apical face of the
epithelium mainly by a conductance pathway and partly by a parallel
channel-exchanger mechanism. Furthermore, similar conclusions were
reached for transport in the mammalian epididymis (4), frog gastric
mucosa (3), and turtle bladder (20). In the only other studies of
electrogenic transport in the mouse gallbladder (18, 22), it was
assumed that forskolin caused a
Cl
secretory response
powered by the basolateral
Na+-K+-2Cl
cotransporter in series with apical
Cl
channels. The simple
expedient of using appropriate inhibitors would have shown this was
incorrect. Here, the retention of a major part of the secretory
response in the absence of apical Cl
and with retained
sensitivity to acetazolamide (Fig. 7) indicates that electrogenic
secretion of HCO
3 proceeds directly
through apical anion channels. Although we have not measured HCO
3 flux, because of the small window
area, we could find no value in the literature for the
HCO
3 content of mouse bile. The values
for the HCO
3 content in human, dog,
rabbit, guinea pig, and turkey bile range from 20 to 60 µeq/l, and
values range from 10 to 30 µeq/l in rats and sheep (8). The mean
value and range found here for the mouse, expressed as base, are
comparable to those found in the majority of other species. The low
value for rat bile could be related to the absence of a gallbladder in
this species, so that concentration by electroneutral absorption from
the gallbladder cannot occur.
As mentioned earlier, the main reason for investigating anion secretion
in the mouse gallbladder epithelium was the advent of transgenic
animals. As shown by Curtis et al. (5), using cystic fibrosis animals,
the apically located anion channels responsible for
HCO
3 secretion are the cystic fibrosis
transmembrane conductance regulator channels.
 |
ACKNOWLEDGEMENTS |
This work was supported by a Sir Henry Wellcome Commemorative Award
for Innovative Research to A. W. Cuthbert.
 |
FOOTNOTES |
Address for reprint requests: A. W. Cuthbert, Dept. of Pharmacology,
Univ. of Cambridge, Tennis Court Rd., Cambridge CB2 1QJ, UK.
Received 29 October 1997; accepted in final form 3 February 1998.
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