1 Dalton Cardiovascular Research Center and Departments of Veterinary Biomedical Sciences, 2 Pharmacology, and 3 Biochemistry, University of Missouri-Columbia, Columbia, Missouri 65211
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ABSTRACT |
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The loss of cystic
fibrosis transmembrane conductance regulator (CFTR)-mediated
transepithelial HCO3 secretion contributes to the
pathogenesis of pancreatic and biliary disease in cystic fibrosis (CF)
patients. Recent studies have investigated P2Y2 nucleotide
receptor agonists, e.g., UTP, as a means to bypass the CFTR defect by
stimulating Ca2+-activated Cl
secretion.
However, the value of this treatment in facilitating transepithelial
HCO3
secretion is unknown. Gallbladder mucosae from
CFTR knockout mice were used to isolate the Ca2+-dependent
anion conductance during activation of luminal P2Y2 receptors. In Ussing chamber studies, UTP stimulated a transient peak
in short-circuit current (Isc) that declined to
a stable plateau phase lasting 30-60 min. The plateau
Isc after UTP was Cl
independent,
HCO3
dependent, insensitive to bumetanide, and
blocked by luminal DIDS. In pH stat studies, luminal UTP increased both
Isc and serosal-to-mucosal HCO3
flux (Js
m) during a
30-min period. Substitution of Cl
with gluconate in the
luminal bath to inhibit Cl
/HCO3
exchange did not prevent the increase in Js
m
and Isc during UTP. In contrast, luminal DIDS
completely inhibited UTP-stimulated increases in
Js
m and Isc. We
conclude that P2Y2 receptor activation results in a
sustained (30-60 min) increase in electrogenic HCO3
secretion that is mediated via an intracellular
Ca2+-dependent anion conductance in CF gallbladder.
cystic fibrosis; biliary system; P2Y2 receptor; nucleotide receptor; purinoceptor; chloride
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INTRODUCTION |
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CYSTIC FIBROSIS (CF)
disease is caused by mutations in the gene encoding the CF
transmembrane conductance regulator (CFTR) protein, a cyclic
nucleotide-activated anion channel (3, 5). CFTR mutations result in at least two abnormalities of transepithelial electrolyte and water transport that contribute to the pathogenesis of
CF. First, it is well documented that a deficiency in cAMP-dependent regulation of transepithelial salt and water secretion occurs in CF and
may lead to the dehydration of luminal mucus and debris (37). Second, it is also recognized that abnormal
transepithelial pH regulation occurs in CF epithelia (19,
32). Measurements of ionic currents across airway and
intestinal epithelia (16, 34), as well as
reports of deficient alkalinization of pancreatic juice, biliary
secretions, and the duodenal lumen, indicate a loss of
HCO3 secretion in CF (for review, see Refs. 11, 13,
19, 32). This deficiency likely relates to the role that CFTR plays in transepithelial HCO3
secretion and the possibility
that CFTR can function as a cAMP-activated HCO3
channel (9, 18, 20,
31).
In recent years, it has been proposed that extracellular nucleotide
(ATP, UTP) therapy may be useful in the symptomatic treatment of
CF. In a number of CF epithelial tissues (6,
10, 14, 23, 25),
topically applied UTP binds the P2Y2 nucleotide receptor, resulting in the activation of alternative Cl
conductances (e.g., CLCA) primarily via the phospholipase C/inositol 1,4,5-trisphosphate/intracellular Ca2+
(Cai2+) signaling pathway. Thus deficient
transepithelial salt and water secretion in CF may be partially
reversed through activation of Cl
secretion by a pathway
that bypasses the CFTR. Because the alternative conductances are anion
selective, we hypothesized that stimulation of the P2Y2
receptor may also result in transepithelial HCO3
secretion and therefore be useful in treating abnormal transepithelial pH regulation in CF.
Previous studies (6, 10, 33)
have linked the expression of the P2Y2 receptor with the
nucleotide-dependent stimulation of Cai2+-activated
anion secretion in murine, rat, and human biliary epithelia. However,
mRNA expression studies (29, 36) have also
shown that the CFTR is abundant in the gallbladder epithelia of both humans and mice. Functional studies verify the role of the CFTR in
cAMP-stimulated transepithelial anion secretion in these tissues, and,
recently, it was shown (24) that the CFTR primarily
mediates electrogenic HCO3 secretion rather than
Cl
secretion across the murine gallbladder. Therefore, to
isolate the effect of UTP on Cai2+-mediated
HCO3
secretion and to simulate the CF condition,
studies using the pH stat technique were performed with freshly excised
gallbladders from CFTR knockout mice.
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MATERIAL AND METHODS |
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Animals.
Weanling mice (2-4 mo of age) born to animals heterozygous for the
disrupted murine homologue of the cftr gene
(B6.129-Cftrtm/UNC;
C57BL/6J-Cftrtm/UNC) were used. The genotype of
each littermate was determined using a PCR technique employing primers
specific for murine cftr and the neomycin
resistance/cftr junction, as previously described (7). CFTR(/
) and CFTR(+/+) mice were homozygous for
the disrupted cftr gene and the wild-type cftr
gene, respectively. The mice were fed standard laboratory mouse chow
and water ad libitum until the evening before an experiment and then
only drinking water was provided. The drinking water for all mice
contained an osmotic laxative (polyethylene glycol) to prevent
intestinal impaction in the CFTR(
/
) mice (7). The
University of Missouri-Columbia Institutional Animal Care and Use
Committee approved all experiments involving the animals.
In vitro bioelectric and pH stat measurements. Mice were killed on the day of the experiment by brief exposure to an atmosphere of 100% CO2 to induce basal narcosis, followed by a surgically induced bilateral pneumothorax. The gallbladder and a portion of surrounding liver were excised en masse via an abdominal incision and immediately placed in ice-cold, oxygenated Ringer solution (containing 1 µM indomethacin to prevent prostanoid generation). Under a dissecting microscope, the gallbladder was dissected free of hepatic tissue, opened longitudinally, and placed mucosal-side up on coarse-gauge nylon mesh. The gallbladder on nylon mesh was mounted horizontally in a modified Ussing chamber (0.126 or 0.238 cm2 exposed surface area), and Parafilm "O" rings were used to minimize edge damage where the gallbladder was secured between chamber halves.
The bioelectric and pH stat studies were performed as recently described (9). The gallbladder preparations were bathed on the luminal surface with an unbuffered Ringer (NaCl) solution that was gassed with 100% O2 and contained (in mM) 143.8 NaCl, 5.2 KCl, 1.2 CaCl2, and 1.2 MgCl2. The serosal surface was bathed with a standard Krebs-Ringer-bicarbonate (KRB) solution gassed with 95% O2-5% CO2 and containing (in mM) 115 NaCl, 2.4 K2HPO4, 0.4 KH2PO4, 25 NaHCO3, 1.2 CaCl2, 1.2 MgCl2, and 10 glucose (pH 7.4). In some experiments, ClStatistics.
Student's paired or unpaired t-tests were used for
statistical comparisons. P 0.05 was considered
statistically significant. Unless otherwise indicated, data are
presented as means ± SE.
Materials. Immediately before each experiment, a stock solution of 10 mM UTP (Boehringer-Mannheim, Indianapolis, IN) was made in NaCl and titrated to pH 7.4. Indomethacin (Sigma Chemical, St. Louis, MO) was dissolved in DMSO at a stock concentration of 0.01 M. DIDS (Aldrich Chemical, Milwaukee, WI) was dissolved in NaCl solution at a stock concentration of 0.03 M. TTX (Sigma Chemical) was dissolved in 0.2% acetic acid at a stock concentration of 0.0001 M. All other reagents were obtained from either Sigma Chemical, Aldrich Chemical, or Fisher Scientific (Springfield, NJ).
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RESULTS |
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Ussing chamber studies.
Previous studies (10) of murine gallbladder epithelium
established the presence of a luminal membrane P2Y2
receptor that is stimulated by UTP and results in
Ca2+-activated, transepithelial anion secretion. In normal
murine gallbladder, CFTR is a basally active anion conductance that has been shown to mediate both electrogenic Cl and
HCO3
secretion (24). Because the
presence of CFTR complicates the analysis of Ca2+-activated
anion currents, studies evaluating the ionic basis of the
UTP-stimulated Isc were performed on freshly
excised gallbladder epithelium from CFTR knockout mice. As shown in
Fig. 1, cAMP-dependent stimulation of
CFTR(+/+) gallbladders by a forskolin treatment induced a large
Isc response, whereas treatment of the
CFTR(
/
) gallbladders was essentially without effect. In contrast,
UTP treatment of the luminal membrane stimulated a nearly equivalent Isc response in both CFTR(+/+) and CFTR(
/
)
gallbladders. Previous Ussing chamber studies of murine gallbladder
have established that 100 µM UTP induces a near-maximal
Isc response when added to the luminal bath but
is relatively ineffective when added to the serosal bath
(10).
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pH stat studies.
The preceding findings were consistent with the hypothesis that the
plateau phase of the Isc response after UTP
treatment represents electrogenic HCO3 secretion via
a Cai2+-activated anion conductance in murine
gallbladder. Therefore, the Isc and
Js
m were simultaneously measured using the pH stat technique (both parameters are expressed in µeq · cm
2 · h
1 for comparison). The flux
studies performed on intact CFTR(
/
) gallbladders consisted of two
periods, a 30-min basal flux period followed by addition of luminal UTP
and a 30-min treatment flux period (i.e., during the plateau phase of
the Isc response to luminal UTP). As shown in
Fig. 3, the baseline
Js
m across CFTR(
/
) gallbladder was
4.31 ± 0.64 µeq · cm
2 · h
1 and the Isc was 2.04 ± 0.28 µeq · cm
2 · h
1. During
the plateau phase beginning 5 min after luminal UTP addition, the
Js
m increased by 0.84 ± 0.21 µeq
· cm
2 · h
1, which was nearly
equivalent to the stable increase in Isc of 0.79 ± 0.32 µeq · cm
2 · h
1. Luminal UTP addition did not alter the
Gt between the two flux periods, indicating that
changes in the conductance of the paracellular pathway did not
contribute to the increased rate of HCO3
secretion
(basal Gt = 44.4 ± 5.3; UTP
Gt = 47.0 ± 6.1 mS/cm2).
To evaluate whether direct manipulation of Cai2+
mobilization would generate a response similar to that of UTP, additional experiments were performed using the Ca2+
ionophore ionomycin (1 µM) added to the luminal bath of CFTR(
/
) gallbladders. Ionomycin treatment initially caused a rapid
Js
m and Isc response
similar to UTP, but the plateau phase only lasted ~20 min. However,
both Js
m and Isc were
significantly increased during the 20-min period after ionomycin
treatment (
Js
m = 1.1 ± 0.3 µeq · cm
2 · h
1,
P < 0.05; peak
Isc after
ionomycin = 1.7 ± 0.6 µeq · cm
2
· h
1, P < 0.05;
Isc 20 min after ionomycin = 0.4 ± 0.2 µeq · cm
2 · h
1, not
significant; n = 4).
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DISCUSSION |
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Loss of functional CFTR activity in the epithelia of CF patients
results not only in a deficit of salt and water secretion, but also
severely limits regulated HCO3 permeation across
epithelia. The adverse effects of this deficit likely contribute to the
pathological changes in the pancreatic and biliary ducts of CF patients
(29, 33). Previously, it has been proposed
(21, 22) that topical UTP therapy directed at
luminal P2Y2 receptors in CF epithelia could restore salt
and water secretion via the activation of a
Cai2+-mediated anion conductance. On the basis of our
present results, we propose that UTP therapy will also restore a
portion of electrogenic HCO3
secretion in CF
epithelia expressing the Cai2+-mediated anion
conductance pathway. This effect may be useful in normalizing
transluminal pH across CF epithelia.
Murine CFTR knockout gallbladder provides a native epithelial model for
the measurement of P2Y2 receptor activation of the Cai2+-mediated anion conductance. Previously, we have
shown that murine gallbladder epithelial cells express P2Y2
receptor mRNA and that the receptor complement is functionally
localized to the luminal membrane (10). Furthermore,
activation of the P2Y2 receptor by UTP in murine
gallbladder epithelium induces dose-dependent increases in
inositol phosphate generation, intracellular Ca2+
mobilization, and Isc (10).
Characterization studies showed that the peak
Isc response to UTP is independent of luminal
Na+, DIDS sensitive, reduced by removal of either
Cl or HCO3
from the medium, and almost
completely abolished by removal of both anions. With the use of CFTR
knockout mice in the present study, Isc
measurements resulting from activation of the
Cai2+-mediated anion conductance were uncomplicated by
CFTR activity, a major conductive pathway for cAMP-mediated
electrogenic anion secretion in the murine gallbladder
(24, 29). In support of using the CF murine
gallbladder for isolation of the Cai2+-activated anion
conductance, an epithelial Cai2+-activated
Cl
channel (mCLCA) has recently been cloned and was shown
to be expressed at high levels in murine gallbladder (15,
17). Whether the mCLCA channel is solely responsible for
the Cai2+-activated anion channel activity demonstrated
in earlier studies (26) of biliary epithelium has yet to
be determined.
Two models have been proposed for electrogenic HCO3
secretion across epithelial tissues. In one model, an inward current is generated by HCO3
that permeates anion channels in
the luminal membrane (1, 12). In a second
model, the inward current is generated by Cl
permeation
of luminal membrane anion channels. Cl
secretion in the
latter model facilitates HCO3
secretion by
"recycling" Cl
entering via a luminal membrane
Cl
/HCO3
exchanger (28).
Several lines of evidence in the present study indicate that the
plateau phase of the UTP-induced Isc response represents a HCO3
secretory current carried by the
Cai2+-activated anion conductance (model 1).
First, the plateau Isc response to UTP was not
inhibited by bumetanide or complete Cl
substitution but
required HCO3
in the bathing medium. Second, the
rates of UTP-induced Js
m and
Isc were similar in magnitude both under control
conditions and during inhibition of
Cl
/HCO3
exchange by luminal
Cl
removal. Third, the UTP-stimulated increase in
Js
m and Isc during the
plateau phase of the response could be completely inhibited by
micromolar DIDS, a known blocker of the Cai2+-activated
anion conductance (4). Whether the recently cloned mCLCA
channel mediates this HCO3
conductance is not yet
known. To the best of our knowledge, neither the mCLCA channel nor the
Cai2+-activated anion channels described in biliary
epithelial cells (15, 26) have been evaluated
for HCO3
permeation.
Luminal Cl removal also revealed a significant amount of
Cl
/HCO3
exchange in the basal state of
the CF murine gallbladder [for comparison, electroneutral
HCO3
secretion in the murine duodenum is less than
one-third of this rate (9)]. If the residual
Js
m in the absence of luminal Cl
is taken as a measure of net paracellular flow of
HCO3
(~1 µeq · cm
2 · h
1), then the basal Js
m of the
murine gallbladder would be ~3 and 4 µeq · cm
2 · h
1 in CF and wild-type mice,
respectively. These values compare reasonably well with the basal
Js
m measured across the gallbladder of rabbit
and guinea pig (~3 and ~2 µeq · cm
2 · h
1, respectively) (30, 35).
Note, however, that the direction of net HCO3
movement (absorptive vs. secretory) across murine gallbladder in the
basal state could not be ascertained because mucosal-to-serosal flux of
HCO3
was not measured. Nonetheless,
Cl
/HCO3
exchange apparently dominates
the basal secretory flux of HCO3
in the mouse
gallbladder as it does in rabbit and guinea pig (30,
35). Although further studies will be necessary to
determine the molecular identity of luminal
Cl
/HCO3
exchange, it was apparent from
the studies shown in Fig. 5 that electroneutral HCO3
secretion was not highly sensitive to luminal DIDS (~12% decrease, not significant). This observation suggests that weakly DIDS-sensitive exchangers, such as the downregulated in adenoma protein (murine homologue) and the anion exchanger AE2 (2,
27), are candidate proteins for the luminal
Cl
/HCO3
exchange process in murine gallbladder.
In summary, it was shown that P2Y2 receptor activation of a
Cai2+-mediated anion conductance results in
electrogenic HCO3 secretion across CF murine
gallbladder epithelium. This finding suggests that nucleotide receptor
therapy may restore in CF patients the loss of regulated
HCO3
secretion that is necessary for transluminal pH
modulation in certain organs. In comparison to CFTR-mediated
HCO3
secretion in murine gallbladder, ~50% of this
activity can be induced by UTP treatment. However, the duration of the
response is relatively short lived (30-60 min). Efforts to prolong
activation of this pathway, perhaps by preventing P2Y2
receptor desensitization (10), may be beneficial. As
previously proposed for murine airway epithelia (8), the
dominance of the Cai2+-activated anion conductance in
other murine epithelial tissues may be responsible for the relative
lack of pancreatic and biliary disease in CF mouse models. Thus
increasing the activity and expression of components of the
Cai2+-activated anion conductance pathway in human
epithelia is a potential treatment for cystic fibrosis.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the gift of CFTR(+/) breeding animals
(B6.129-Cftrtm/UNC;
C57BL/6J-Cftrtm/UNC) from Dr. Beverly Koller
(Department of Medicine, University of North Carolina, Chapel Hill, NC).
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-48816 (L. L. Clarke), National Institute of Dental Research Grant DE-07389 (J. T. Turner), National Institute of General Medical Sciences Grant GM-36887 (G. A. Weisman) and grants from the University of Missouri-Columbia Food for the 21st Century Program (G. A. Weisman) and the Cystic Fibrosis Foundation (L. L. Clarke, J. T. Turner, G. A. Weisman).
Address for reprint requests and other correspondence: L. L. Clarke, 324D Dalton Cardiovascular Research Center, Research Park Dr., Univ. of Missouri-Columbia, Columbia, MO 65211 (E-mail: ClarkeL{at}missouri.edu).
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.
Received 9 July 1999; accepted in final form 31 January 2000.
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