Bicarbonate Secretion in the Murine Gallbladder

Alan W Cuthbert
Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Hill's Road. Cambridge,
United Kingdom
Summary
The epithelium lining the gallbladder of
mammalian species has absorptive and
secretory functions. An important function is
the secretion of a bicarbonate rich fluid that
helps neutralise stomach acid and provides an
appropriate environment for intestinal enzymes.
In cystic fibrosis (CF) this secretory function is
lost. This study concerns the bicarbonate
secreting activity of murine gallbladders in
vitro using wild type and CF mice and four
main questions are considered as follows: a)
Does the murine gallbladder secrete
bicarbonate electrogenically and is this
prevented in CF? b) Can the secretory activity
in CF gallbladders be restored by gene therapy
or pharmacologically? c) How is the cystic
fibrosis transmembrane conductance regulator
(CFTR) involved in bicarbonate secretion? d)
Does the data offer prospects for the treatment
of CF?. Work from both the author's laboratory
and the literature will be reviewed.
Consideration of the currently available data
indicates that the wild type murine gallbladder
does secrete bicarbonate electrogenically and
that this is absent in CF mice. Further it has
been demonstrated that bicarbonate secretory
activity can be restored by both gene therapy
and by the use of drugs. The role of CFTR in
bicarbonate secretion remains equivocal. Much
evidence suggests that CFTR can act as a
channel for HCO3
ions as well as Clions,
while others propose a parallel arrangement of
CFTR with a Cl-/HCO3
exchanger is necessary.
The matter is further complicated by the
regulatory role of CFTR on other transporting
activities. Opportunities for possible application
to man are discussed.
Secretion of Bicarbonate by the Mouse
Gallbladder
Mouse bile contains around 40 mEq/L of
bicarbonate that is within the normal range of
most mammals. Agents that increase cAMP
either by activating adenylate cyclase, such as
forskolin and vasoactive intestinal polypeptide
(VIP), or in other ways, such as
isobutylmethylxanthine (IBMX) and di-butyryl
cAMP, cause an increase in short circuit current
(SCC) when applied to isolated gallbladders
mounted in Ussing chambers and voltage
clamped at zero potential. The basal SCC in
gallbladders is 7.2±4.3 µA/cmand increased
by 48.2±6.1µA/cm(n=21) following treatment
with forskolin (Figure 1). The direction of the
current and its insensitivity to amiloride
suggests it is due to the secretion of anions [1].
Several pieces of evidence indicate that the
current is generated by the transport of
bicarbonate in the serosal to apical direction.
First the secretory current is significantly

Page 2
JOP. J Pancreas (Online) 2001; 2(4 Suppl):257-262.
JOP. Journal of the Pancreas – http://www.joplink.net – Vol.2, No.4 Suppl. – July 2001
258
reduced when HCO3
and COare removed
from the bathing fluid (Figure 2). This
procedure reduced the current by 84% (P<0.05,
n=5). Removal of bicarbonate alone caused a
reduction in the response to forskolin of 78%
(P<0.05, n=5), suggesting the bicarbonate ion is
more important than the hydration of CO2.
Removal of HCO3
-/COfrom the basolateral
side only causes a significant reduction in the
response to forskolin (58% compared to
controls, P<0.003, n=9), while HCO3
-/CO2
removal from the apical side had no effect
(103% compared to controls, NS, n=4). Thus
removal of bicarbonate from the side from
which it is transported reduces the response to
the secretagogue, while no effect is apparent
when it is absent from the receiving side [1].
Furosemide, an inhibitor of electrogenic
chloride secretion has no significant effect on
the forskolin induced current, while
acetazolamide, a carbonic anhydrase inhibitor,
significantly reduced the current (from
48.2±6.1µA/cmby 18.2±2.1µA/cm2, P<0.05,
n=21). Even though significant effects of
furosemide are not demonstrable the distinct
reductions in current sometimes seen following
its addition, plus other data argue for a minor
component of chloride secretion.
While the experimental set up did not allow the
measurement of serosal to mucosal bicarbonate
flux, others using small bath volumes, were
able to determine Js-m for bicarbonate by
titration [2]. The increased flux in response to
forskolin in wild type gallbladders was
1.76±0.42 µEq/cm2/h. This corresponds to a
steady state increase in current of 47.2±11.3
µA/cm2, virtually identical to the value given
above.
The presence of CFTR is essential for the
response of gallbladders to forskolin (Figure 3)
[3]. Neither CF null (Cftrtm1Cam) or CF∆F508
(Cftrtm2Cam) gallbladders showed significant
responses to forskolin. Thus CFTR is essential
Figure 1. SCC recordings from a mouse gallbladder over
8 hours. Sequential additions of forskolin 10 µM (F),
furosemide 1 mM (Fu), and acetazolamide 100 µM (A)
were made as indicated. Redrawn from [1].
Figure 2. Effect of HCO3
-/COremoval on gallbladder
responses. Sequential additions of forskolin 10 µM (F),
furosemide 1 mM (Fu), and acetazolamide 100µM (A)
were made as indicated. Redrawn from [1].

Page 3
JOP. J Pancreas (Online) 2001; 2(4 Suppl):257-262.
JOP. Journal of the Pancreas – http://www.joplink.net – Vol.2, No.4 Suppl. – July 2001
259
for HCO3
secretion in the murine gallbladder
when stimulated by agonists which increase
cAMP, which includes endogenous ones.
Restoration of Bicarbonate Secreting
Activity in Murine CF Gallbladder
In vivo gene transfer in CF mice by instilling a
plasmid containing the cDNA for CFTR
complexed with liposomes either into the
trachea or the nose restored the forskolin
bicarbonate secretory activity to the
gallbladder, measured in vitro. Briefly the
plasmid pTrial10-CFTR2 was mixed with DC-
Chol/dioleoyl
phosphatidylethanolamine
(DOPE) liposomes as detailed in Curtis et al.
[3], and the mice sacrificed two days after
transfection. It is important to realise that
hCFTR was reintroduced rather than the murine
version. Figure 3 shows the profile of responses
in transfected mice gallbladders, which is
similar to that of wild type and unlike those in
CF gallbladders. The procedure used also
restored the responses of the mouse tracheal
epithelium [4] and that in the nose [5] to
forskolin, but had no effect on the epithelia of
the intestine.
The route taken by the genetic material from
the airways to the gallbladder is still unclear.
We were able to detect mRNA for human
CFTR in the gallbladders of transfected mice.
Furthermore using either pGL-3 luc or pCMV-
luc (containing the cDNA coding for luciferase)
lipoplex we were able to detect luciferase
activity in gallbladders when given by the nasal
or intratracheal route, but not when given
orally, or by intramuscular, intraperitoneal or
subcutaneous routes. When these lipoplex were
given intravenously some luciferase activity
was found in gallbladders, but only 3% of that
found with the airway route [3]. When the
plasmid pTrial10-CFTR2 was given alone into
the airways no transfer of genetic material to
the gallbladder occurred, yet it was possible to
transfect the gallbladder in vitro by exposure to
plasmid to restore bicarbonate secretion. Viral
vectors with or without cationic amphiphiles
can be used to transfect gallbladders in vitro or
in vivo (retrograde perfusion of the bile duct)
[6] and provide a further way to restore CFTR
function to the gallbladder.
Bicarbonate secretion in CF gallbladders does
not necessarily require the restoration of CFTR
function. Agents that increase Ca2+
i
also
increase bicarbonate secretion by activating
Ca2+ sensitive Clchannels. For example,
ionomycin increased SCC by 14.8±3.9µA/cm2
(n=10) in wild type gallbladders and in CF null
and CF ∆F508 gallbladders by 25.4±13.4
µA/cm(n=9) and 12.3±3.5 µA/cm(n=11)
respectively, none of these values being
significantly different from each other. Uridine
triphosphate (UTP), via activation of P2Y2
receptors also stimulates bicarbonate secretion
in CF mice (Cftrtm/UNC) measured both as SCC
and as Js-m for bicarbonate [2]. In conclusion,
gene transfer to gallbladders correlates with the
restoration of cAMP-dependent HCO3
-
secretion, but alternative pathways support
bicarbonate secretion without CFTR.
Figure 3. Responses to mouse gallbladders to forskolin
(10 µM), furosemide (1 mM) and acetazolamide (100
µM). In (a) are the data for wild type while in (c) and (d)
the responses are for CF null and CF ∆F508 gallbladders
respectively. In (b) are the data for CF null gallbladders
where the mice had been lipofected through the airways
two days before the measurements were made.

Page 4
JOP. J Pancreas (Online) 2001; 2(4 Suppl):257-262.
JOP. Journal of the Pancreas – http://www.joplink.net – Vol.2, No.4 Suppl. – July 2001
260
CFTR Involvement in Bicarbonate Secretion
Various mechanisms have been proposed for
bicarbonate secretion in epithelia as follows: a)
HCO3
leaves the cell via CFTR channels; b) a
parallel arrangement of CFTR with a chloride
bicarbonate exchanger; c) complex models
dependent on CFTR regulation of other
transporting activities.
Certainly CFTR has a bicarbonate conductance
although it is lower than for chloride [7].
However it has been argued that the driving
force for HCO3
exit must be small [8].
Experimental evidence for the electrogenic
secretion of bicarbonate via CFTR centres on
the failure of chloride removal from the apical
face to stop secretion. This opposes the
classical view that Cl-
lowering, by exit
through CFTR, triggers the Cl-/HCO3
-
exchanger to affect net HCO3
transport [9].
Figure 4 shows that chloride removal from the
apical surface of mouse gallbladder has only a
modest effect on HCO3
secretion caused by
forskolin. Statistically the reduction is
significant, the response to forskolin being
reduced from 89.0±14.4 µA/cmto 59.3±14.2
µA/cm(P<0.02) in three experiments like the
one illustrated. Thus 67% of the current
remains when the Cl-/HCO3
exchanger is
blocked, suggesting the exit route for HCO3
is
via CFTR. The 30% reduction in current may
mean that this fraction normally uses the
exchanger mechanism. Others have made
similar arguments for HCO3
secretion in the
airways [10], intestine [11] and colon [12]. In
other situations chloride removal blocks HCO3
-
secretion, and in these tissues the activity of the
exchanger and CFTR are tightly linked, as for
example in PANC-1 cells derived from the
pancreatic duct [13].
Returning to the mouse gallbladder, it is known
the UTP can stimulate bicarbonate secretion in
CF tissue via activation of P2Yreceptors.
Removal of apical chloride fails to inhibit
secretion, as in the normal gallbladder
stimulated with forskolin. The conclusion,
therefore, is that the Ca2+-activated chloride
channel can also conduct bicarbonate ions in
the bladder [2].
It is always a mistake to assume that absence of
CFTR has no other consequences apart from
the removal of a chloride conductance. For
example, the presence of CFTR in the
membrane, but not its conductance function,
has been shown to be necessary for the
activation of the Cl-/HCO3
exchanger by
cAMP [14, 15]. Consequently in tissues such as
submandibular gland ducts and pancreatic ducts
from CF mice the exchanger was not activated
by forskolin as it is in wild type tissues
Recently, Wheat et al. have shown that CFTR
is necessary for the expression of down
regulated in adenoma (DRA) which in turn is
responsible for the upregulation of the Cl-
/HCO3
exchanger. It is suggested that the
failure of bicarbonate secretion in CF airways
may be due in part to the down regulation of
the exchanger caused by the absence of DRA
[16]. An intriguing finding has been reported
by Cremaschi and colleagues [17]. Using patch
Figure 4. Effect of Clremoval from the apical bathing
solution. Forskolin (F, 10µM), furosemide (Fu, 1 mM)
and acetazolamide (A, 100 µM) were added sequentially
as indicated.

Page 5
JOP. J Pancreas (Online) 2001; 2(4 Suppl):257-262.
JOP. Journal of the Pancreas – http://www.joplink.net – Vol.2, No.4 Suppl. – July 2001
261
clamp analysis, with rabbit gallbladder cells, it
was found that inhibition of the Cl-/HCO3
-
exchanger from the outer surface caused the
appearance of a anion selective conductance
with a Pgluconate/Pchloride value of 0.18. As
chloride removal from the apical surface also
inhibits the exchanger it may well be important
to look for this phenomena elsewhere. It may
provide an alternative pathway for bicarbonate
exit in the absence of external chloride without
HCO3
ions leaving via CFTR. In conclusion,
evidence suggests that bicarbonate ions exit
through CFTR in the gallbladder and in other
epithelia, but direct proof for this exit route
remains an urgent problem.
Lessons for the Treatment of Cystic Fibrosis
It would appear that retrograde perfusion offers
a way to specifically target the gallbladder by
gene therapy. However the procedure is
invasive and unlikely to be useful until long
lived benefit without significant risk is
available. Consideration might also be given to
recruiting the gallbladder as a vehicle for other
functions, for example the secretion of
pancreatic enzymes. Clinically useful
restoration of lung function in CF by gene
therapy is the goal of many groups. When this
is achieved it would be worthwhile to look at
indicators of gallbladder function.The unusual
route for gallbladder transfection detailed here
might have its counterpart in patients.
key words Cystic Fibrosis Transmembrane
Conductance Regulator; Gene therapy;
Liposomes; Luciferase; Mice, Inbred CFTR
Abbreviations CF: cystic fibrosis; CFTR:
cystic fibrosis transmembrane conductance
regulator;
DOPE:
dioleoyl
phosphatidylethanolamine; DRA: down
regulated
in
adenoma;
IBMX:
isobutylmethylxanthine; SCC: short circuit
current; UTP: uridine triphosphate; VIP:
vasoactive intestinal polypeptide
Acknowledgements The author is grateful for
support from the Medical Research Council and
a Showcase Award from the Wellcome Trust.
Correspondence
Alan W Cuthbert
Department of Medicine
University of Cambridge
Addenbrooke's Hospital (Box 157)
Hill's Road
Cambridge CB2 2QQ
United Kingdom
Phone: +44-1223-336.853
Fax: +44-1223-336.846
E-mail address: awc1000@cam.ac.uk
References
1. Martin LC, Hickman ME, Curtis CM, MacVinish
LJ, Cuthbert AW. Electrogenic bicarbonate secretion in
the mouse gallbladder. Am J Physiol 1998: 274:G1045-
52. [98359064]
2. Clarke LL, Harline MC, Gawenis LR, Walker NM,
Turner JT, Weisman GA. Extracellular UTP stimulates
electrogenic bicarbonate secretion across CFTR
knockout gallbladder epithelium. Am J Physiol 2000;
279:G132-8. [20357578]
3. Curtis CM, Martin LC, Higgins CF, Colledge WH,
Hickman ME, Evans MJ, et al. Restoration by
intratracheal gene transfer of bicarbonate secretion in
cystic fibrosis mouse gallbladder. Am J Physiol 1998:
274:G1053-60. [98359065]
4. MacVinish LJ, Gill DR, Hyde SC, Mofford KA,
Evans MJ, Higgins CF, et al. Chloride secretion in the
trachea of null cystic fibrosis mice: the effects of
transfection with pTrial10-CFTR2. J Physiol 1997;
499:677-87.
5. MacVinish LJ, Goddard C, Colledge WH, Higgins
CF, Evans MJ, Cuthbert AW. Normalisation of ion
transport in murine cystic fibrosis nasal epithelium using
gene transfer. Am J Physiol 1997; 273:C734-40.
[97423429]
6. McKay TR, MacVinish LJ, Carpenter B, Themis M,
Jezzard S, Goldin R, et al. Selective in vivo transfection

Page 6
JOP. J Pancreas (Online) 2001; 2(4 Suppl):257-262.
JOP. Journal of the Pancreas – http://www.joplink.net – Vol.2, No.4 Suppl. – July 2001
262
of murine biliary epithelia using selctive polycation-
enhanced adenovirus. Gene Therapy 2000; 7:644-52.
7. Gray MA, Pollard CE, Harris A, Coleman L,
Greenwell JR, Argent BE. Anion selectivity and the
block of the small conductance chloride channel on
pancreatic duct cells. Am J Physiol 1990: 259:C752-61.
[91051856]
8. Kunzelmann K, Gerlach L, Frobe U, Greger R.
Bicarbonate permeability of epithelial chloride channels.
Pflugers Arch 1991; 417:616-21.
9. Novak I, Greger R. Properties of the luminal
membrane of isolated perfused rat pancreatic ducts.
Pflugers Arch 1988: 411:546-53. [88262412]
10. Poulsen JH, Machen TE. HCO3
--dependent pHI
regulation in tracheal epithelial cells. Pflugers Arch
1996; 432:546-54. [96281574]
11. Hogan DL, Crombie DL, Isenberg JI, Svendsen P,
Schaffalitzky-de-Muckadell OB, Ainsworth MA. CFTR
mediates cAMP- and Ca2+-actiavted duodenal epithelial
HCO3
-
secretion. Am J Physiol 1997; 272:G872-8.
[97287792]
12. Geibel JP, Singh S, Rajendran VM, Binder HJ.
HCO3
secretion in rat colonic crypts is closely linked to
Cl-
secretion. Gastroenterology 2000; 118:101-7.
[20079124]
13. Zsembery A, Strazzabosco M, Graf J. Ca2+-activated
Clchannels can substitute for CFTR in stimulation of
pancreatic duct bicarbonate secretion. FASEB J 2000;
14:2345-56. [20507653]
14. Lee MG, Wigley WC, Zeng W, Noel LE, Marino
CR, Thomas PJ, Muallem S. Regulation of Cl-/HCO3
-
exchange by cystic fibrosis conductance regulator
expressed in NIH 3T3 and HEK 293 cells. J Biol Chem
1999; 274:3414-21. [99121077]
15. Lee MG, Choi JY, Luo X, Strickland E, Thomas PJ,
Muallem S. Cystic fibrosis transmembrane conductance
regulator regulates luminal Cl-/HCO3
exchange in mouse
submandibular and pancreatic ducts. J Biol Chem 1999;
274:14670-7. [99262614]
16. Wheat VJ, Shumaker H, Burnham C, Shull G,
Yankaskas JR, Soleimani M. CFTR induces the
expression of DRA along with Cl-/HCO3
exchange
activity in tracheal epithelial cells. Am J Physiol 2000;
279:C62-71. [20357597]
17. Meyer G, Porta C, Garavaglia M, Cremaschi D.
Inhibitors of Cl-/HCO3
exchanger activate an anion
channel with similar features in the epithelial cells of
rabbit gallbladder: patch-clamp analysis. Pflugers Arch
2001; 441:467-73. [21079733

There are no products listed under this category.