Extracellular UTP stimulates electrogenic bicarbonate secretion across CFTR knockout gallbladder epithelium

Lane L. Clarke1, Matthew C. Harline1, Lara R. Gawenis1, Nancy M. Walker1, John T. Turner2, and Gary A. Weisman3

1 Dalton Cardiovascular Research Center and Departments of Veterinary Biomedical Sciences, 2 Pharmacology, and 3 Biochemistry, University of Missouri-Columbia, Columbia, Missouri 65211


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 (Jsright-arrow 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 Jsright-arrow m and Isc during UTP. In contrast, luminal DIDS completely inhibited UTP-stimulated increases in Jsright-arrow 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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
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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.


    MATERIAL AND METHODS
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ABSTRACT
INTRODUCTION
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, Cl- was replaced with an equimolar concentration of gluconate- (3 mM CaSO4 was added to overcome Ca2+ chelation). The solutions were circulated throughout the experiment by gas lift and warmed to 37°C by water-jacketed reservoirs. Before each experimental protocol, gallbladders were equilibrated for 20 min under short-circuited conditions with TTX (0.1 µM) in the serosal bath to minimize variation due to intrinsic neural tone.

Transepithelial short-circuit current (Isc, in µeq · cm-2 · h-1) was measured using an automatic voltage clamp (VCC-600, Physiologic Instruments, San Diego, CA) and calomel electrodes that were connected to the chambers by 4% agar-3 M KCl bridges, as previously described (9). Isc and automatic fluid resistance compensation current were applied through Ag-AgCl electrodes connected to the chamber baths via 4% agar-NaCl bridges. Every 5 min during an experiment, a 5-mV pulse was passed across the gallbladder tissue to determine the total tissue conductance (Gt, mS/cm2 tissue surface area) by measuring the magnitude of the resulting current deflections and applying Ohm's law. The serosal bath served as ground in all experiments.

The serosal-to-mucosal flux of HCO3- (Jsright-arrow m, in µeq · cm-2 · h-1) was measured by pH stat titration of the luminal bath (4 ml) to pH 7.4 using 5 mM HCl delivered by either a computer-aided titrimeter (Fisher, model 455/465) or manual addition of titrant. The volume of added acid was used to calculate the HCO3- (base) flux, taking into account the time period and the surface area of the tissue. Typically, Jsright-arrow m stabilized within 30 min after the tissue was mounted, and the luminal solution was replaced to refresh transepithelial ion gradients and remove secreted mucus. A 30-min basal flux period was initiated, and then UTP (100 µM) was added to the luminal bath. Isc assumed a plateau phase after 5 min, and a second 30-min flux period was initiated.

Statistics. 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).


    RESULTS
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ABSTRACT
INTRODUCTION
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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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Fig. 1.   Comparison of the maximal change in short-circuit current (Isc) after treatment with either 10 µM forskolin (Forsk) or 100 µM UTP in gallbladder epithelium from cystic fibrosis transmembrane conductance regulator (CFTR)(+/+) and CFTR(-/-) mice (n = 5 each). Delta Isc has been normalized to 1 cm2 of mucosal surface area. * Significantly different from CFTR(+/+).

An Isc recording trace of the UTP response in a CFTR(-/-) gallbladder epithelium with NaCl in the luminal bath and KRB in the serosal bath is shown in Fig. 2A. Typical of Cai2+-activated transepithelial anion secretion, the Isc after UTP treatment immediately increased to a maximum value and then decreased within minutes to a stable plateau where it remained elevated over baseline. The plateau Isc in the UTP-treated CFTR(-/-) gallbladder typically was elevated for over 30 min. Subsequent treatment of the gallbladder with bumetanide, an inhibitor of the Na+-K+-2 Cl- cotransporter, did not affect the Isc, suggesting that the plateau Isc was not due to Cl- secretion. To evaluate the Cl- dependence of the UTP-induced Isc response, CFTR(-/-) gallbladders were bathed in identical solutions except that Cl- was replaced with gluconate-. As shown in Fig. 2B, the time course of the immediate Isc response to UTP was reduced in the Cl--free Ringer solution, whereas the plateau Isc phase was not affected. These findings are consistent with the hypothesis that the plateau phase of the UTP-induced Isc response in CFTR(-/-) gallbladder epithelium represents electrogenic HCO3- secretion. To evaluate the HCO3- dependence of the sustained Isc response, CFTR(-/-) gallbladders were bathed in HCO3--free Ringer solution (TES buffered, gassed with 100% O2) and treated with 100 µM methazolamide (luminal + serosal) to inhibit endogenous HCO3- production (Fig. 2C). This condition markedly attenuated the initial Isc response and abolished the Isc plateau after UTP treatment. Next, we asked whether the plateau Isc after UTP could be inhibited at the luminal membrane with DIDS (300 µM), a blocker of the Cai2+-activated anion conductance (4, 8). As shown in Fig. 2D, DIDS treatment completely abolished the plateau Isc response after UTP addition. DIDS at 100 µM concentration also completely inhibited the plateau phase of the UTP-induced Isc response (data not shown). As a control, 300 µM DIDS was added to the serosal bath after UTP and did not decrease but, unexpectedly, caused an increase in the Isc (+2.3 and +1.9 µA, n = 2).


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Fig. 2.   Recordings showing the effect of different conditions on Isc in UTP-stimulated (100 µM) gallbladder epithelium from CFTR(-/-) mice. A: Isc response in gallbladder epithelium bathed with NaCl Ringer solution on the luminal surface and Krebs-Ringer-bicarbonate (KRB) solution on the serosal surface. Bumetanide (Bumet; 100 µM) was added to the serosal bath (arrow). Basal Isc = 7.7 µA (mean = 6.4 ± 1.7 µA or 1.67 ± 0.44 µeq · cm-2 · h-1, n = 3). B: Isc response in gallbladder epithelium bathed bilaterally with Cl--free solutions (gluconate substitution in luminal and serosal baths). Basal Isc = 6.0 µA (mean = 5.3 ± 0.4 µA or 1.38 ± 0.10 µeq · cm-2 · h-1, n = 3). C: Isc response in gallbladder epithelium bathed bilaterally with HCO3--free solutions (TES substitution in serosal bath + 100 µM methazolamide). Basal Isc = 4.8 µA (mean = 2.1 ± 1.4 µA or 0.54 ± 0.36 µeq · cm-2 · h-1, n = 3). D: Isc response in gallbladder epithelium bathed with NaCl Ringer solution on the luminal surface and KRB solution on the serosal surface. DIDS (300 µM) was added to the luminal bath (arrow). Basal Isc = 5.5 µA (mean = 5.9 ± 2.1 µA or 1.56 ± 0.55 µeq · cm-2 · h-1, n = 3). Dotted line indicates the steady-state basal Isc before UTP treatment. Isc traces have not been normalized to mucosal surface area.

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 Jsright-arrow 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 Jsright-arrow 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 Jsright-arrow 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 Jsright-arrow m and Isc response similar to UTP, but the plateau phase only lasted ~20 min. However, both Jsright-arrow m and Isc were significantly increased during the 20-min period after ionomycin treatment (Delta Jsright-arrow m = 1.1 ± 0.3 µeq · cm-2 · h-1, P < 0.05; peak Delta Isc after ionomycin = 1.7 ± 0.6 µeq · cm-2 · h-1, P < 0.05; Delta Isc 20 min after ionomycin = 0.4 ± 0.2 µeq · cm-2 · h-1, not significant; n = 4).


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Fig. 3.   Effect of luminal UTP (100 µM) on the serosal-to-mucosal flux of HCO3- (Jsright-arrow m), Isc and tissue conductance (Gt) of gallbladder epithelia from CFTR(-/-) mice (n = 5). Gallbladder epithelia were bathed with NaCl Ringer solution on the luminal surface and KRB solution on the serosal surface. Jsright-arrow m, Isc, and Gt have been normalized to 1 cm2 of mucosal surface area. * Significantly different from basal.

Previous studies have provided evidence that cAMP-dependent stimulation of normal murine gallbladder induces electrogenic HCO3- secretion via a CFTR-dependent process (24). For purposes of comparison with the UTP response, we measured the effect of cAMP-dependent stimulation on Jsright-arrow m and Isc in intact CFTR(+/+) gallbladders. Using forskolin (10 µM) treatment to increase intracellular cAMP, the Delta Jsright-arrow m and Delta Isc were 1.76 ± 0.42 and 1.98 ± 0.47 µeq · cm-2 · h-1, respectively, over basal values (basal Jsright-arrow m and Isc were 5.18 ± 0.66 and 1.41 ± 0.27 µeq · cm-2 · h-1, respectively; n = 6). Thus the plateau phase of the response to UTP treatment in a CFTR(-/-) gallbladder yields a Delta Jsright-arrow m that is 48% and a Delta Isc that is 40% of the responses to forskolin treatment in CFTR(+/+) gallbladder.

The nearly equivalent increases in Delta Jsright-arrow m and Isc after UTP treatment suggest a process of electrogenic HCO3- secretion mediated by a Cai2+-activated HCO3- conductance. However, it is also possible that UTP stimulates a Cai2+-activated Cl- conductance that increases the activity of a luminal membrane Cl-/HCO3- antiporter system (28). To investigate this possibility, CFTR(-/-) gallbladders were bathed with a Cl--free solution in the luminal bath to inhibit activity of luminal membrane Cl-/HCO3- exchange. As shown in Fig. 4, substitution of luminal Cl- with gluconate- greatly reduced the basal Jsright-arrow m, indicating that Cl-/HCO3- exchange contributes significantly to basal HCO3- secretion. The basal Isc in the absence of luminal Cl- was reversed in polarity, largely as a result of uncompensated junction potentials. Nevertheless, UTP treatment in the absence of luminal Cl- stimulated increases in Jsright-arrow m and Isc that were similar to the UTP responses in luminal NaCl solution (Delta Jsright-arrow m = 1.13 ± 0.23 µeq · cm-2 · h-1; Delta Isc = 1.34 ± 0.35 µeq · cm-2 · h-1). UTP treatment under these conditions had no effect on Gt (basal Gt = 19.8 ± 3.1 mS/cm2; UTP Gt = 18.7 ± 2.7 mS/cm2).


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Fig. 4.   Effect of luminal Cl- removal (gluconate substitution) on Jsright-arrow m, Isc, and Gt before and after luminal treatment with 100 µM UTP in CFTR(-/-) gallbladder epithelia (n = 5). Jsright-arrow m, Isc, and Gt have been normalized to 1 cm2 of mucosal surface area. * Significantly different from basal.

We observed that treatment with luminal DIDS abolishes the plateau phase of the Isc response (Fig. 2), consistent with inhibition of the Cai2+-activated anion conductance (4). Therefore, we asked whether luminal DIDS treatment would inhibit UTP-dependent stimulation of both Jsright-arrow m and Isc during the plateau phase of the response. As shown in Fig. 5, luminal treatment with DIDS (300 µM) during the plateau phase of the UTP response eliminated both the increases in Jsright-arrow m and Isc in the CFTR(-/-) gallbladder. The Gt was not different between the basal and UTP treatment periods under these conditions (basal Gt = 30.1 ± 5.7 mS/cm2; UTP Gt = 29.0 ± 3.4 mS/cm2). The ability of luminal DIDS to block both the Isc and Jsright-arrow m is consistent with the conclusion that Cai2+-activated anion channels mediate electrogenic HCO3- secretion across the murine CF gallbladder epithelium.


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Fig. 5.   Effect of luminal DIDS (300 µM) on Jsright-arrow m, Isc, and Gt after luminal treatment with 100 µM UTP in gallbladder epithelia from CFTR(-/-) mice (n = 5). DIDS was added to the luminal bath 5 min after UTP. Jsright-arrow m, Isc, and Gt have been normalized to 1 cm2 of mucosal surface area.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 Jsright-arrow 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 Jsright-arrow 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 Jsright-arrow 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 Jsright-arrow 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 Jsright-arrow 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.


    ACKNOWLEDGEMENTS

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).


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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