Dual role of CFTR in camp-stimulated HCO 3 secretion across murine duodenum

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1 Dual role of CFTR in camp-stimulated HCO 3 secretion across murine duodenum LANE L. CLARKE AND MATTHEW C. HARLINE Dalton Cardiovascular Research Center and Department of Veterinary Biomedical Sciences, University of Missouri-Columbia, Columbia, Missouri Clarke, Lane L., and Matthew C. Harline. Dual role of CFTR in camp-stimulated HCO 3 secretion across murine duodenum. Am. J. Physiol. 274 (Gastrointest. Liver Physiol. 37): G718 G726, The role of the cystic fibrosis transmembrane conductance regulator (CFTR) in camp-stimulated HCO 3 secretion across the murine duodenum was investigated. Serosal-to-mucosal flux of HCO 3 (J s=m,inµeq cm 2 h1 ) and short-circuit current (I sc ;inµeq cm 2 h1 ) were measured by the ph stat method in duodenum from CFTR knockout [CFTR()] and normal [CFTR()] mice. Under control conditions, forskolin increased J s=m and I sc (1.7 and 3.5, respectively) across the CFTR() but not CFTR() duodenum. Both the forskolin-stimulated J s=m and I sc were abolished by the CFTR channel blocker 5-nitro-2-(3-phenylpropylamino)benzoate, whereas inhibition of luminal Cl / HCO 3 exchange by luminal Cl removal or DIDS reduced the J s=m by 18% without a consistent effect on the I sc. Methazolamide also reduced the J s=m by 39% but did not affect the I sc. When carbonic anhydrase-dependent HCO 3 secretion was isolated by using a CO 2 -gassed, HCO 3 -free Ringer bath, forskolin stimulated the J s=m and I sc (0.7 and 2.0, respectively) across CFTR() but not CFTR() duodenum. Under these conditions, luminal Cl substitution or DIDS abolished the J s=m but not the I sc. It was concluded that campstimulated HCO 3 secretion across the duodenum involves 1) electrogenic secretion via a CFTR HCO 3 conductance and 2) electroneutral secretion via a CFTR-dependent Cl / HCO 3 exchange process that is closely associated with the carbonic anhydrase activity of the epithelium. cystic fibrosis; cystic fibrosis transmembrane conductance regulator; chloride secretion; chloride/bicarbonate exchanger; anion exchanger; ph stat THE DUODENAL EPITHELIUM produces an alkaline mucus secretion as protection against the acidic effluent from the stomach (16). After a meal the gastric effluent may have a ph of and PCO 2 values exceeding 400 mmhg (24, 36, 38). A major component of the alkaline secretion is regulated by intracellular camp, resulting in both passive (i.e., paracellular) and active transport of HCO 3 across the epithelium (1, 16). The process of active HCO 3 secretion involves the concerted activities of an anion channel and Cl /HCO 3 exchange in the luminal membrane and secretes HCO 3 taken up across the basolateral membrane or generated by intracellular carbonic anhydrase activity (1). However, the interaction of these transport processes during camp stimulation of HCO 3 secretion is not well understood. In an excellent review of duodenal HCO 3 secretion, Allen et al. (1) have proposed that camp-stimulated HCO 3 secretion involves the activation of a luminal membrane HCO 3 channel. Alternatively, HCO 3 secretion may follow the model proposed for pancreatic duct epithelium, which predicts a camp-regulated Cl channel that recycles Cl entering across the luminal membrane by means of a Cl /HCO 3 exchanger (34). Studies of cystic fibrosis transmembrane conductance regulator (CFTR), the dysfunctional campactivated Cl channel in cystic fibrosis (CF) disease (3, 5, 11), indicate that this channel may play a major role in camp-mediated HCO 3 secretion. Fueled by observations of deficient transluminal ph regulation and loss of transepithelial anion current activity across CF epithelial tissues (15, 20, 23, 32, 37, 40), bioelectric studies of recombinant and wild-type CFTR have shown that CFTR is permeable to HCO 3 [HCO 3 -to-cl permeability ratios range from 1:8 to 1:4 (19, 27, 35)]. Likewise, the outward-rectifying Cl channel (ORCC), a channel reportedly regulated by CFTR (14, 18), also conducts HCO 3 with a HCO 3 -to-cl permeability ratio of 1:2 (43). However, the hypothesis that CFTR functions as both a Cl and a HCO 3 channel under physiological conditions is complicated by the fact that CFTR also displays anomalous mole fraction behavior, whereby the channel conductance of a less permeable anion (HCO 3 ) is greatly reduced in the presence of a more permeable anion (Cl ) (44). Recently, direct measures of transepithelial HCO 3 flux have shown that CFTR is required for agoniststimulated HCO 3 secretion across the mammalian duodenum. In ph stat studies of rat duodenum Guba et al. (21) demonstrated that HCO 3 secretion stimulated by cgmp agonists could be specifically inhibited by 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB), a blocker of the CFTR channel (4), but not by maneuvers that inhibit the activity of the Cl /HCO 3 exchanger or ORCC, i.e., removal of Cl from the luminal perfusate or treatment with DIDS. Although the mechanism of camp-stimulated HCO 3 secretion was also evaluated in the study by Guba et al. (21), interpretation of the findings was confounded by the fact that camp agonists were applied after cgmp stimulation with guanylin. More recently, in vivo perfusion studies of the duodenum from CFTR knockout mice have demonstrated that camp-stimulated duodenal HCO 3 output is greatly reduced compared with the normal murine duodenum (25). However, the mechanistic role of CFTR in camp-stimulated HCO 3 secretion was not evaluated, probably owing to the difficulty of controlling transepithelial electrochemical gradients in that preparation. In the present study we investigate the role that CFTR plays in the process of camp-stimulated duodenal HCO 3 secretion. Direct measurements of HCO 3 secretory flux, using the ph stat method, were per- G /98 $5.00 Copyright 1998 the American Physiological Society

2 CFTR AND DUODENAL HCO 3 SECRETION IN CF MICE G719 formed on duodena from the CFTR knockout mouse model, thereby allowing comparison of HCO 3 secretion in the presence and absence of CFTR. We hypothesized that CFTR functions as an anion channel that is responsible for both electrogenic Cl and HCO 3 secretion during camp stimulation of the duodenum. The evidence supports this hypothesis but also indicates that CFTR is required in an electroneutral mechanism of camp-stimulated HCO 3 secretion involving luminal Cl /HCO 3 exchange. MATERIAL AND METHODS Animals. All studies were performed on weanling mice (2 4 mo of age) born to breeding animals heterozygous for the disrupted murine homologue of the cftr gene (B6.129-Cftr tm/unc ; C57BL/6J-Cftr tm/unc ). The mice were either purchased from Jackson Laboratories (Bar Harbor, ME) or received as a generous gift from Dr. Beverly Koller (Dept. of Medicine, Univ. of North Carolina, Chapel Hill, NC). Each littermate was genotyped using a PCR technique employing primers specific for murine cftr and the neomycin resistance-cftr junction (neo was used for gene disruption) (8). Littermate mice, which were either homozygous or heterozygous for the wild-type cftr gene, were used as controls [designated as CFTR() mice]. Only one to two heterozygous cftr(/) mice were used per treatment group. The CFTR knockout mice were homozygous for the disrupted cftr gene [designated as CFTR() mice]. The mice were maintained on standard laboratory mouse chow and water ad libitum. The drinking water provided to all mice contained an osmotic laxative (polyethylene glycol; PEG) to prevent intestinal impaction in the CFTR() mice (8). Before each experiment the mice were fasted 2 h but were provided the PEG-containing drinking water ad libitum. All experiments involving animals were approved by the University of Missouri-Columbia Institutional Animal Care and Use Committee. In vitro bioelectric and ph stat measurements. The mice were killed on the day of the experiment by brief exposure to an atmosphere of 100% CO 2 to induce basal narcosis, which was followed by a surgically produced pneumothorax. The proximal duodenum (from 2 mm distal to the pylorus to the common bile duct ampulla) was removed via an abdominal incision, immediately placed in ice-cold, oxygenated Ringer solution, and opened along the mesenteric border. Indomethacin (10 6 M) was present in the rinse and experimental Ringer solutions to prevent prostanoid generation during tissue manipulation (6). The proximal duodenum was stripped of the outer muscle layers and then mounted on a standard Ussing chamber (0.25 cm 2 exposed surface area). Parafilm O rings were used to minimize edge damage to the intestine where it was secured between the chamber halves. The bioelectric and ph stat studies were performed using a variation of the method recently described by Guba et al. (21). The duodenal preparations were bathed independently on the luminal surface with mm NaCl solution gassed with 100% O 2 and on the serosal surface with a standard Ringer solution gassed with 95% O 2-5% CO 2. The solutions were circulated throughout the experiment by gas-lift and were warmed to 37 C by water-jacketed reservoirs. The Ringer solution contained (in mm) 115 NaCl, 4 K 2 HPO 4, 0.4 KH 2 PO 4, 25 NaHCO 3, 1.2 MgCl 2, 1.2 CaCl 2, and 10 glucose with ph 7.4. In some experiments Cl was replaced in the luminal solution with an equimolar concentration of gluconate (3 mm CaSO 4 was added to overcome Ca 2 chelation). Before each experiment proximal duodenal tissues were equilibrated for min under short-circuited conditions with TTX (0.1 µm) in the serosal bath to minimize variation in the intrinsic neural tone of the intestine, as previously described (39). Transepithelial short-circuit current (I sc,inµeq cm 2 h1 ) was measured using an automatic voltage clamp (VCC-600; Physiologic Instruments, San Diego, CA) and calomel electrodes connected to the chamber halves with 4% agar-3 M KCl bridges, as previously described (9). The I sc and automatic fluid resistance compensation current were applied through Ag-AgCl electrodes connected to the chamber baths via 4% agar mm NaCl bridges. In experiments requiring replacement of Cl in the luminal bath with gluconate, the spontaneous transepithelial voltage was corrected for the asymmetric junction potential difference using the method of Frizzell and Schultz (17). Every 5 min during an experiment, a 5-mV pulse was passed across the duodenal tissue to determine the total tissue conductance (G t, ms/cm 2 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 HCO 3 (J s=m in µeq cm 2 h 1 ) was measured by continuously titrating the luminal bath solution (4 ml) to ph 7.4 with 5 mm HCl, using either a computer-aided titrimeter (Fisher, model 455/465) or by manual addition of titrant. The volume of added acid was used to calculate the HCO 3 (base) flux, taking into account the time and surface area of the tissue. Typically, J s=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 immediately initiated, and then forskolin (10 µm) with or without various inhibitors was added to the bathing solutions. When the J s=m stabilized (15 min), a second 30-min flux period was initiated. In some studies, the tissue preparations were given a second treatment and a third 30-min flux period was performed. In ph stat experiments designed to measure secretion of endogenously generated HCO 3, the luminal bath was gassed with 95% O 2-5% CO 2 and clamped at ph 5.1 (using 5 mm HCl). In the serosal bath HCO 3 was replaced equimolar with TES, and the solution was gassed with 100% O 2 (ph 7.4). Initial studies using sodium gluconate (pk a 3.6) to replace NaCl in the luminal bath revealed that the solution had a significant buffering capacity at ph 5.1. Therefore, a luminal solution containing Na 2 SO 4 (78.1 mm) with sufficient mannitol (78.1 mm) to balance the transepithelial osmolarity was used for these experiments (33). Statistics. Student s t-test was used for comparisons of the mean responses between CFTR() and CFTR() genotypes, or between basal and treatment periods. When two sequential treatment periods were compared with a basal period, a one-way repeated measures ANOVA followed by a post hoc Bonferroni s test was used. P 0.05 was considered statistically significant (41). Unless otherwise indicated data are presented as means SE. Materials. Unless otherwise stated reagents were obtained from either Sigma Chemical (St. Louis, MO), Aldrich Chemical (Milwaukee, WI), or Fisher Scientific (Springfield, NJ). Indomethacin, methazolamide [to inhibit intracellular carbonic anhydrase activity (7)], forskolin, and NPPB were dissolved in DMSO at stock concentrations of 0.01, 1.0, 0.01, or 0.3 M, respectively. Bumetanide [to inhibit Na -K -2Cl cotransport (22)] was dissolved in ethanol at a stock concentration of 0.1 M. DIDS was dissolved in the appropriate Ringer solution at a concentration of 0.03 M. TTX was dissolved in 0.2% acetic acid at a stock concentration of M. In separate experiments DMSO and ethanol vehicles at concentrations equivalent to those used in each experiment (0.2 and

3 G720 CFTR AND DUODENAL HCO 3 SECRETION IN CF MICE 0.1%, respectively) produced no significant alterations of the basal I sc. RESULTS Control ph stat studies. Previous ion substitution studies of the CFTR() murine duodenum had shown that either Cl or HCO 3 could carry an inward current across the epithelium and together account for 99% of the camp-stimulated I sc (23). Furthermore, the presence of HCO 3 in the bath medium resulted in a significant fraction of the camp-stimulated I sc that was insensitive to the Cl transport inhibitor bumetanide. However, I sc measurements of epithelia treated with bumetanide or bathed in Cl -free medium may not reflect the actual rate of HCO 3 secretion (12, 40). Therefore, the rate of HCO 3 (base) secretion before and during camp stimulation was directly measured using ph stat titration of voltage-clamped murine duodenum. In CFTR() duodenum bathed with the control solution on the luminal membrane (i.e., unbuffered NaCl, ph 7.4), a significant J s=m and I sc were measured under basal conditions (Fig. 1A). Addition of forskolin (camp) increased the J s=m by µeq cm 2 h1 and the I sc by µeq cm 2 h1. The forskolinstimulated J s=m was exceeded in magnitude by the I sc, indicating that electrogenic Cl secretion occurs simultaneously with HCO 3 secretion during camp Fig. 1. Control ph stat studies. Duodena were bathed with a NaCl solution maintained at ph 7.4 in luminal bath. Serosal-to-mucosal flux of HCO 3 (J s=m ) and short-circuit current (I sc ) were recorded for 3 30-min flux periods: before treatment (basal), 15 min after forskolin (camp), and 10 min after bumetanide (Bumet). CFTR, cystic fibrosis transmembrane conductance regulator. A: J s=m and I sc responses of normal mice [CFTR()] duodenum, n 7. B: J s=m and I sc responses of CFTR knockout mice [CFTR()] duodenum, n 4. Bars, means SE. a,b,c Within group, means with different letters are significantly different (1-way repeated measure ANOVA with post hoc Bonferroni s test). stimulation of the duodenum. Subsequent addition of bumetanide to the serosal bath did not affect the mean J s=m but significantly decreased the I sc. However, the postbumetanide I sc remained significantly elevated relative to the basal I sc. The transepithelial conductance (G t ) increased slightly over the course of the experiment with the main change occurring after forskolin treatment (basal G t ms/cm 2 ; forskolin G t ms/cm 2 ; bumetanide G t ms/cm 2, P 0.05). Together, these findings are consistent with the hypothesis that most of the I sc after bumetanide represents electrogenic HCO 3 secretion. To investigate whether camp-stimulated HCO 3 secretion occurs in the absence of CFTR, ph stat experiments were performed on CFTR() duodenum. As shown in Fig. 1B, a significant J s=m was measured during the basal period and exceeded the mean I sc (which was slightly positive for the period). Sequential additions of forskolin and bumetanide had no significant effect on the J s=m or I sc. The mean G t of the CFTR() duodenum was unchanged through all three flux periods (baseline G t ms/cm 2 ; forskolin G t ms/cm 2 ; bumetanide G t ms/cm 2 ). Effect of NPPB and DIDS on camp-stimulated J s=m and I sc. To investigate whether the channel function per se of CFTR is required for camp-stimulated HCO 3 secretion, CFTR() duodenal tissues were treated before forskolin stimulation with NPPB, an extracellular blocker of CFTR (4, 21). As shown in Fig. 2A, NPPB completely prevented forskolin stimulation of J s=m and reduced stimulation of the I sc by 84% (compare with Fig. 1A). The mean G t in these preparations was unchanged by the NPPB-forskolin treatment (basal G t ms/cm 2 ; NPPB-forskolin G t ms/cm 2 ). To estimate the contribution of Cl /HCO 3 exchange or a HCO 3 conductance through the ORCC, CFTR() duodena were treated before forskolin stimulation with the distilbene derivative DIDS, which has inhibitory actions on both transport processes (4, 13, 28). As shown in Fig. 2B, DIDS pretreatment slightly reduced the mean forskolin-stimulated J s=m (DIDS-forskolin J s=m µeq cm 2 h1 ) but had little effect on the I sc (DIDS-forskolin I sc µeq cm 2 h1 ) compared with control (Fig. 1A). The G t measured in these experiments tended to increase with forskolin, but the changes were not statistically significant (basal G t ms/cm 2 ; DIDS-forskolin G t ms/cm 2 ). Effect of luminal Cl substitution on camp-stimulated J s=m and I sc. The NPPB experiments indicated that camp-stimulated HCO 3 secretion is mediated via CFTR channel activity. This finding is consistent with the hypothesis that CFTR can function as a campstimulated HCO 3 channel. However, the results of the DIDS studies suggested that enhanced Cl /HCO 3 activity, but not an electrogenic ORCC-mediated pathway, may also contribute to camp-stimulated HCO 3 secretion. Therefore, ph stat studies were performed on duodenal sections bathed with an unbuffered Cl - free solution (Na gluconate, ph 7.4) on the luminal

4 CFTR AND DUODENAL HCO 3 SECRETION IN CF MICE G721 Fig. 2. Effect of 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB) or DIDS. Duodena were bathed with a NaCl solution maintained at ph 7.4 in luminal bath. After 30-min flux period (basal), duodena were pretreated with either NPPB ( M) or DIDS ( M) in luminal bath for 10 min followed by forskolin (camp). The 2nd 30-min flux period began 15 min after forskolin. A: J s=m and I sc responses of CFTR() duodenum pretreated with NPPB, n 6. B: J s=m and I sc responses of CFTR() duodenum pretreated with DIDS, n 6. Bars, means SE. * Significantly different from basal (paired t-test). membrane to diminish or eliminate luminal Cl /HCO 3 exchange activity. As shown in Fig. 3A, forskolin treatment in the absence of luminal Cl stimulated the J s=m to a mean value that was slightly less than in the control studies and comparable to the DIDS-forskolin treatment ( µeq cm 2 h1 ). Forskolin treatment also significantly stimulated the I sc in the absence of luminal Cl, but the mean I sc ( µeq cm 2 h1 ) was less than measured in the control experiments. However, interpretation of the I sc in this experiment was complicated by the fact that the I sc before forskolin was greatly increased, probably as a result of establishing a large concentration gradient for Cl secretion across both the apical membrane and via the paracellular pathway. The G t measured in these experiments was not different between the basal and forskolin-treated flux periods (basal G t ; camp G t ms/cm 2 ). To test whether campstimulated HCO 3 secretion during inhibition of luminal Cl /HCO 3 is mediated by CFTR, these experiments were performed in CFTR() duodenum. As shown in Fig. 3B, forskolin treatment had no significant effect on the J s=m, I sc, or G t (basal G t ; camp G t ms/cm 2 ). Effect of methazolamide on camp-stimulated J s=m and I sc. Although the preceding experiments indicated that most camp-stimulated HCO 3 secretion is an electrogenic CFTR-dependent process, the evidence also suggested that a second mechanism involving Fig. 3. Effect of luminal Cl removal. CFTR() and CFTR() duodena were bathed with Cl -free Ringer in luminal bath (ph 7.4). J s=m and I sc were recorded for two 30-min flux periods: before treatment (basal) and 15 min after forskolin (camp). A: J s=m and I sc responses of CFTR() duodenum, n 7. B: J s=m and I sc responses of CFTR() duodenum, n 4. Bars, means SE. * Significantly different from basal (paired t-test). luminal Cl /HCO 3 exchange may contribute to campstimulated HCO 3 secretion. Previously, we had found that luminal Cl /HCO 3 exchange was associated with carbonic anhydrase-dependent HCO 3 secretion across the duodenal epithelium (10). Therefore, we investigated the effect of carbonic anhydrase inhibition during camp stimulation of J s=m and I sc. In these experiments CFTR() duodena were pretreated with a membranepermeant inhibitor of carbonic anhydrase, methazolamide (Meth, 1 mm), before forskolin stimulation. As shown in Fig. 4, forskolin stimulated a significant increase in J s=m in the methazolamide-pretreated duo- Fig. 4. Effect of methazolamide. Duodena were bathed with a NaCl solution maintained at ph 7.4 in luminal bath. After 30-min flux period (basal), duodena were pretreated with either vehicle (0.1% DMSO) or methazolamide (1 mm, Meth) in serosal bath for 10 min and then forskolin (camp). The 2nd flux period began 15 min after forskolin. Bars, means SE. * Significantly different from basal (paired t-test).

5 G722 CFTR AND DUODENAL HCO 3 SECRETION IN CF MICE denum, but the magnitude of the J s=m was significantly less than that found in the control studies shown in Fig. 1 (Meth-pretreated J s=m vs. control J s=m µeq cm 2 h1 ). In contrast, forskolin stimulated the I sc in the methazolamidepretreated duodenum to a level that was equivalent to that found in the control studies (Meth-pretreated I sc vs. control I sc µeq cm 2 h1 ). In additional time control experiments, we found that methazolamide treatment per se caused a small reduction in the J s=m and, paradoxically, increased the I sc in the second flux period compared with the first flux period (Meth J s=m and Meth I sc µeq cm 2 h1, n 3). Using these values to adjust the forskolin-stimulated response in methazolamide-pretreated duodenum, we still found that methazolamide pretreatment significantly reduced the forskolin-stimulated J s=m by 39%, compared with the control but did not affect the magnitude of the forskolin-stimulated I sc (3%). These observations indicated that the carbonic anhydrasedependent fraction of camp-stimulated J s=m does not involve an electrogenic process. Therefore, experiments were undertaken to isolate the mechanism responsible for duodenal secretion of endogenously generated HCO 3. Isolation of carbonic anhydrase-dependent HCO 3 secretion. To isolate duodenal secretion of carbonic anhydrase-generated HCO 3, ph stat studies were performed on CFTR() duodena that were bathed with a NaCl solution gassed with 95% O 2-5% CO 2 in the luminal solution and an HCO 3 -free, TES-buffered Ringer gassed with 100% O 2 in the basolateral solution. The purpose of this design was to provide a CO 2 source for intracellular carbonic anhydrase generation of HCO 3 while preventing HCO 3 movement across the basolateral membrane via Na -coupled uptake mechanisms, e.g., NaHCO 3 cotransport (28). The luminal NaCl-5% CO 2 solution equilibrated in the ph range of , and the PCO 2 was 41 1 mmhg (n 3). Because clamping the NaCl-5% CO 2 solution to ph 7.4 under this protocol would result in a significant HCO 3 concentration in the luminal bath, the luminal solution was clamped to ph 5.1. In contrast to the luminal bath, the TES-buffered solution in the basolateral bath remained constant at ph (n 22), and the PCO 2 of the solution was found to be 0.4 mmhg (n 4). In control studies using hemichambers without intestine, the 5% CO 2 gassing resulted in a small spontaneous rate of HCO 3 production in both the NaCl solution (0.049 µeq/h, n 3) and in the Cl -free solution (0.029 µeq/h, n 3). Therefore, the J s=m measured in these studies was corrected for spontaneous HCO 3 production. With the luminal NaCl-5% CO 2 condition, the CFTR() duodenum yielded a basal J s=m that was exceeded in magnitude by the I sc (Fig. 5A). Importantly, subsequent treatment of the duodenum with forskolin significantly increased the J s=m by µeq cm 2 h1 and the I sc by µeq cm2 h1 (n 8). The mean G t of the CFTR() duodenum in Fig. 5. Control ph stat studies during isolation of endogenous HCO 3 production. CFTR() and CFTR() duodena were bathed with NaCl solution gassed with 95% O 2-5% CO 2 (ph 5.1) in luminal bath and HCO 3 -free Ringer solution in serosal bath (ph 7.4). J s=m and I sc were recorded for two 30-min flux periods: before treatment (basal) and 15 min after forskolin (camp). CFTR() duodena were then divided into 2 groups (4 each) and treated with either methazolamide (Meth) or its vehicle (0.1% DMSO, Veh) for 10 min. This was followed by a 3rd 30-min flux period. A: J s=m and I sc responses of CFTR() duodenum, n 8. Insets: mean J s=m and I sc after Meth or Veh, n 4 each. B: J s=m and I sc responses of CFTR() duodenum, n 4. Bars, means SE. * Significantly different from basal (or Veh in 3rd flux period; paired t-test). these experiments increased slightly during forskolin treatment (basal G t ; forskolin G t ms/cm 2, P 0.05). To investigate the requirement of carbonic anhydrase activity for the camp-stimulated J s=m the duodenal tissues were treated with either methazolamide or its vehicle DMSO in a third flux period (Fig. 5A, insets). Compared with the DMSO-treated duodena, methazolamide significantly decreased the forskolin-stimulated J s=m to near the basal value (Meth J s=m ; DMSO J s=m µeq cm 2 h 1, n 4 each). The mean I sc significantly decreased during the third flux period for both the methazolamide-treated and DMSOtreated duodena, but the changes were not significantly different from each other (Meth I sc ; DMSO I sc µeq cm 2 h 1, n 4 each). The mean G t for both the methazolamide- and DMSO-treated duodenum increased significantly during the third flux period,

6 CFTR AND DUODENAL HCO 3 SECRETION IN CF MICE G723 Fig. 6. Effect of luminal Cl removal or DIDS treatment during isolation of endogenous HCO 3 production. CFTR() duodena were bathed with either a Cl -free Ringer (A) or NaCl solution (B) gassed with 95% O 2-5% CO 2 maintained at ph 5.1 in luminal bath and HCO 3 -free Ringer solution in serosal bath (ph 7.4). J s=m and I sc were recorded for two 30-min flux periods: before treatment (basal) and 15 min after forskolin (camp). A: J s=m and I sc responses of CFTR() duodenum bathed with luminal Cl -free solution, n 5. B: J s=m and I sc responses of CFTR() duodenum pretreated with DIDS ( M) in luminal bath. Bars, means SE. * Significantly different from basal (paired t-test). but the differences between the two groups were not statistically significant (Meth G t ; DMSO G t ms/cm 2, n 4 each). To investigate the possibility that an acidic luminal solution per se was responsible for the camp-stimulated HCO 3 secretion, we lowered the luminal bath ph using an isotonic HCl solution and gassed the luminal bath with 100% O 2 rather than 95% O 2-5% CO 2. Under these conditions, camp stimulation of CFTR() duodena did not increase the J s=m but significantly stimulated the I sc (J s=m ; I sc µeq cm 2 h1, n 3). The above studies indicated that the duodenal epithelium was capable of increasing the secretion of endogenously generated HCO 3 via an electroneutral mechanism during intracellular camp stimulation of Cl secretion. Next, to test whether CFTR is required for the carbonic anhydrase-dependent secretory process, we performed ph stat experiments on CFTR() duodenum. As shown in Fig. 5B, a significant J s=m was present under the basal conditions and exceeded the I sc. However, in contrast to the CFTR() duodenum, forskolin treatment did not significantly increase either J s=m or I sc in the CFTR() duodenum. The mean G t of the CFTR() duodenum increased after forskolin treatment but the change was not statistically significant (basal G t ; camp G t ms/cm 2 ). Effect of luminal Cl removal or DIDS on carbonic anhydrase-dependent HCO 3 secretion. The involvement of luminal Cl /HCO 3 exchange in camp-stimulated secretion of endogenously produced HCO 3 was investigated by inhibiting luminal Cl /HCO 3 exchange via luminal Cl substitution or DIDS treatment. As shown in Fig. 6A, removal of luminal Cl prevented the forskolin-induced increase in J s=m but did not diminish the I sc stimulation (Cl -free J s=m and I sc µeq cm 2 h1 ; compare with Fig. 5A). The mean G t slightly increased after forskolin treatment in this series of experiments (basal G t ; camp G t ms/cm 2, P 0.05). CFTR() duodena were also treated with DIDS before camp stimulation. As shown in Fig. 6B DIDS completely inhibited the forskolin-induced increase in J s=m but had no effect on the forskolinstimulated I sc compared with the control experiments (Fig. 5A). The mean G t in these experiments also increased during the forskolin-treated flux period (basal G t ; camp G t ms/cm 2, P 0.05). These results demonstrate that during camp stimulation the murine duodenum is capable of increasing the secretion of carbonic anhydrase-generated HCO 3 via a mechanism involving CFTR-facilitated Cl /HCO 3 exchange. DISCUSSION In the present study it was demonstrated that CFTR participates in two mechanisms of camp-stimulated HCO 3 secretion across the murine duodenum. The requirement for a functional CFTR in camp-stimulated HCO 3 secretion was shown by 1) the inability to generate a camp-stimulated transepithelial J s=m and I sc across the CFTR() duodenum and 2) the nearly complete blockade of a camp-stimulated J s=m flux and I sc across the CFTR() duodenum by the CFTR channel blocker NPPB. A major role for CFTR in campstimulated electrogenic HCO 3 secretion was first demonstrated by finding that forskolin stimulated a simultaneous increase in J s=m and I sc under conditions that inhibit other pathways for HCO 3 efflux, i.e., luminal membrane Cl /HCO 3 exchange or the ORCC. These data are consistent with the function of CFTR in the duodenal epithelium as a camp-stimulated HCO 3 conductance (1, 21). A second role for CFTR in HCO 3 secretion was demonstrated by its requirement for camp stimulation of luminal Cl /HCO 3 exchange activity. Here the HCO 3 secretion was electroneutral and largely dependent on a carbonic anhydrase-generated source of HCO 3. These findings are consistent with a mechanism for HCO 3 secretion whereby CFTR, possibly functioning as a Cl conductance, facilitates a luminal membrane Cl /HCO 3 exchanger (34). The ph stat method, which to the best of our knowledge has not previously been applied to murine intestine, measured rates of J s=m across the mouse duodenum that compare favorably with duodenal J s=m measured in other mammalian species (range µeq cm 2 h1 for rat and rabbit) (21, 26, 46).

7 G724 CFTR AND DUODENAL HCO 3 SECRETION IN CF MICE Furthermore, our observations of the relative differences in camp-stimulated HCO 3 secretion between the CFTR() and CFTR() duodenum also agree with the studies of Hogan et al. (25) in which duodenal HCO 3 output was measured by an in vivo perfusion technique (25). Unlike the perfusion studies, we did not find that the basal rates of J s=m in the CFTR() duodenum were less than in the CFTR() duodenum. This difference is probably methodological, reflecting the fact that our duodenal preparations were routinely pretreated with indomethacin and TTX blockade. Both prostaglandins and intrinsic neural activity likely maintain a basal level of intracellular camp and CFTR activity in the CFTR() duodenum in vivo (26, 46). Similar to other studies of intestinal HCO 3 secretion (33), we also found it difficult to discern the contribution of luminal Cl / HCO 3 exchange to basal HCO 3 secretion. Although the basal J s=m during perfusion of the luminal membrane with Cl -free Ringer was reduced relative to perfusion with Cl -replete solutions, the difference only attained statistical significance when all the experiments on CFTR() duodena in the study were compared (NaCl J s=m µeq cm 2 h1, n 17; Cl -free J s=m µeq cm 2 h1,n 7, P 0.05). It is increasingly recognized that CFTR may function as a camp-stimulated HCO 3 channel in the luminal membrane of epithelial cells. Ion channel blocker studies of guanylin-stimulated intestine by Guba et al. (21) provided the first evidence that CFTR is a cyclic nucleotide-stimulated HCO 3 channel in duodenal epithelium. In the present study we focused on campstimulated HCO 3 secretion and also found that the forskolin-induced J s=m across the CFTR() murine duodenum was only modestly affected by inhibition of luminal membrane Cl /HCO 3 exchange or ORCC, but could be completely inhibited by NPPB and was not elicited in the CFTR() duodenum. These findings are consistent with the role of CFTR as a camp-stimulated HCO 3 channel. By comparing the J s=m and I sc measured during inhibition of luminal Cl /HCO 3 exchanger activity (1.4 µeq cm 2 h1 ) to that measured under our control conditions (1.7 µeq cm 2 h1 ), it can be estimated that as much as 80% of the campstimulated J s=m is conducted through CFTR and accounts for 50% of the camp-stimulated I sc. These findings suggest that the permeability limitations of HCO 3 conductance through CFTR are overcome by favorable electrochemical driving forces for HCO 3 efflux across the apical membrane. Thus the bulk of evidence supports the model proposed by Allen et al. (1) that a HCO 3 channel, i.e., CFTR, provides the dominant means of camp-stimulated HCO 3 secretion across the mammalian duodenum. A novel result was that CFTR also participated in a fraction of camp-stimulated HCO 3 secretion that was dependent on the activity of luminal Cl /HCO 3 exchange and endogenous carbonic anhydrase. Although the possibility of this second CFTR-dependent mechanism was first suggested by the small decrease in the camp-stimulated HCO 3 secretion during inhibition of luminal Cl /HCO 3 exchange, the fact that methazolamide significantly inhibited camp-stimulated HCO 3 secretion without affecting the increase in I sc strongly indicated stimulation of electroneutral HCO 3 secretion. Experimental conditions were then designed to isolate camp-stimulated secretion of endogenously generated HCO 3 by providing luminal CO 2 for carbonic anhydrase activity but preventing the uptake of extracellular HCO 3 across the basolateral membrane (using an HCO 3 -free, Cl -replete basolateral solution). Under these conditions forskolin stimulated a significant increase in both J s=m and I sc. However, in contrast to the studies with HCO 3 -replete medium, the J s=m was abolished by luminal Cl substitution or DIDS treatment without affecting the I sc (which presumably represented only Cl secretion). The lack of a response in the CFTR() duodenum, under these conditions, demonstrated a requirement for CFTR and indicated that increased intracellular camp alone does not stimulate luminal membrane Cl /HCO 3 exchange (13). Together, these results show camp stimulation of CFTRdependent Cl /HCO 3 exchange activity, a process most consistent with the model of HCO 3 secretion proposed by Novak and Greger (34) for the pancreatic duct. In that model a camp-stimulated Cl channel (CFTR) functions to recycle Cl to a luminal membrane Cl / HCO 3 exchanger, resulting in camp-stimulated HCO 3 secretion. The fact that NPPB in the control studies completely inhibited camp-stimulated J s=m suggests that CFTR Cl channel function is necessary for the stimulatory effect. However, the camp-stimulated I sc was not significantly reduced during inhibition of either Cl /HCO 3 exchange (Fig. 6) or carbonic anhydrase (Fig. 5A, insets). Thus the Cl channel activity of CFTR in this mechanism may not generate a transepithelial current but, instead, prevents the accumulation of exchanger-transported Cl on the cytosolic side of the luminal membrane. Alternatively, CFTR may serve to directly regulate the activity of the luminal Cl /HCO 3 exchanger during camp stimulation of the murine duodenum. The studies designed to isolate the endogenous source of HCO 3 demonstrated a close association between stimulated activity of luminal membrane Cl /HCO 3 exchange and the carbonic anhydrase activity of the epithelium. This observation is consistent with recent studies in red blood cells that demonstrate both a functional and an intermolecular association between intracellular carbonic anhydrase activity and band 3, a Cl /HCO 3 exchanger (29, 45). Given that carbonic anhydrase II activity in both the rat and human duodenum is specifically localized to the villus epithelium (30, 31), it is possible that a functional complex between carbonic anhydrase and the luminal membrane Cl /HCO 3 exchanger may serve the alkaline mucus barrier of murine villi. Another pathway for camp-mediated transmural HCO 3 secretion across the duodenum results from increased paracellular movement of HCO 3 (1, 21). In general, we found that camp stimulation of CFTR() duodenum caused an increased G t, which primarily

8 CFTR AND DUODENAL HCO 3 SECRETION IN CF MICE G725 reflects a change in the paracellular conductance of the small intestine (17). The camp-induced G t depended on a functional CFTR because G t changes were not found in the CFTR() duodenum or during NPPB treatment of the CFTR() duodenum. However, the percent changes of G t in the CFTR() experiments were small and did not always correlate with the J s=m [see the experiments on CFTR() duodenum bathed with a Cl -free luminal solution (Fig. 3A)]. Furthermore, previous anion substitution studies using whole thickness (outer muscle layers intact) duodenum (23) and jejunum (9) have shown that the G t significantly decreases during camp stimulation of murine intestine. Thus the physiological significance of campstimulated changes in paracellular HCO 3 movement is presently unclear and will require further evaluation. In conclusion, CFTR appears to function both as a HCO 3 conductance and a facilitator of luminal membrane Cl /HCO 3 exchange during camp stimulation of the murine duodenum. Both mechanisms may operate simultaneously during stimulated secretion; however, the relative contribution of each mechanism to total camp-activated HCO 3 secretion may differ depending on the luminal environment and the activity of the basolateral mechanisms of HCO 3 uptake [e.g., NaHCO 3 cotransport (28)]. Whereas 80% of camp-stimulated HCO 3 secretion may result from CFTR HCO 3 channel activity in the absence of luminal CO 2, the studies of carbonic anhydrase-dependent secretion suggested that up to one-half of this amount may result from enhanced Cl /HCO 3 exchange when luminal CO 2 is present. In addition, the activities of the two CFTR-dependent mechanisms of HCO 3 secretion are potentially segregated along the crypt-villus axis. Electrogenic HCO 3 secretion may primarily occur at the crypt epithelium where CFTR is expressed at the greatest levels (2, 42). In contrast, CFTR is expressed at lower levels and carbonic anhydrase activity is expressed at higher levels in the villus epithelium (2, 30, 31, 42). Thus the mechanism coupling the CFTR with a carbonic anhydrase-dependent Cl /HCO 3 exchanger may be primarily localized to the villus. Because villi are readily exposed to the luminal environment, this mechanism would be appropriate to regulate the rate of duodenal HCO 3 secretion during postprandial changes in the CO 2 tension of the gastric effluent (1, 24, 36, 38). The authors thank Lara Gawenis, Nancy Walker, Julie Schultz, and Sue Spalding for expert technical assistance. We also gratefully acknowledge Dr. Mark Milanick (Dept. of Medical Physiology, Univ. of Missouri-Columbia) for helpful advice in the preparation of the manuscript. Financial support for this work was provided by the National Institute of Diabetes and Digestive and Kidney Diseases Grant DK (L. Clarke) and a Cystic Fibrosis Foundation Grant P973. Address for reprint requests: L. L. Clarke, 324D Dalton Cardiovascular Research Center, Research Park, Univ. of Missouri-Columbia, Columbia, MO Received 19 September 1997; accepted in final form 8 December REFERENCES 1. Allen, A., G. Flemstrom, A. Garner, and E. Kivilaakso. Gastroduodenal mucosal protection. Physiol. Rev. 73: , Ameen, N. A., T. Ardito, M. Kashgarian, and C. R. Marino. A unique subset of rat and human intestinal villus cells express the cystic fibrosis transmembrane conductance regulator. Gastroenterology 108: , Anderson, M. P., R. J. Gregory, S. Thompson, D. W. Souza, S. Paul, R. C. Mulligan, A. E. Smith, and M. J. Welsh. Demonstration that CFTR is a chloride channel by alteration of its anion selectivity. Science 253: , Anderson, M. P., D. N. Sheppard, H. A. Berger, and M. J. Welsh. Chloride channels in the apical membrane of normal and cystic fibrosis airway and intestinal epithelia. Am. J. Physiol. 263 (Lung Cell. Mol. Physiol. 7): L1 L14, Bear, C. E., C. Li, N. Kartner, R. J. Bridges, T. J. Jensen, M. Ramjeesingh, and J. R. Riordan. Purification and functional reconstitution of the cystic fibrosis transmembrane conductance regulator (CFTR). Cell 68: , Bukhave, K., and J. Rask-Madsen. Saturation kinetics applied to in vitro effects of low prostaglandin E 2 and F 2a concentrations on ion transport across human jejunal mucosa. Gastroenterology 78: 32 42, Candia, O. A. Forskolin-induced HCO 3 current across apical membrane of the frog corneal epithelium. Am. J. Physiol. 259 (Cell Physiol. 28): C215 C233, Clarke, L. L., L. R. Gawenis, C. L. Franklin, and M. C. Harline. Increased survival of CFTR knockout mice using an oral osmotic laxative. Lab. Anim. Sci. 46: , Clarke, L. L., and M. C. Harline. CFTR is required for camp inhibition of intestinal Na absorption in a cystic fibrosis mouse model. Am. J. Physiol. 270 (Gastrointest. Liver Physiol. 33): G259 G267, Clarke, L. L. and M. C. Harline. Bicarbonate-dependent fraction of the camp-stimulated chloride current across murine duodenum (Abstract). Physiologist 39: 146, Collins, F. S. Cystic fibrosis: molecular biology and therapeutic implications. Science 256: , Dagher, P. C., L. Balsam, J. T. Weber, R. W. Egnor, and A. N. Charney. Modulation of chloride secretion in the rat colon by intracellular bicarbonate. Gastroenterology 103: , Dunk, C. R., C. D. A. Brown, and L. A. Turnberg. Stimulation of Cl/HCO 3 exchange in rat duodenal brush border membrane vesicles by camp. Pflügers Arch. 414: , Egan, M., T. Flotte, S. Afione, R. Solow, P. L. Zeitlin, B. J. Carter, and W. B. Guggino. Defective regulation of outwardly rectifying Cl channels by protein kinase A corrected by insertion of CFTR. Nature 358: , Eggermont, E., and K. De Boeck. Small-intestinal abnormalities in cystic fibrosis patients. Eur. J. Pediatr. 150: , Flemstrom, G. Gastric and duodenal mucosal bicarbonate secretion. In: Physiology of the Gastrointestinal Tract, edited by L. R. Johnson. New York: Raven, 1987, p Frizzell, R. A., and S. G. Schultz. Ionic conductances of extracellular shunt pathway in rabbit ileum: influence of shunt on transmural sodium transport and electrical potential differences. J. Gen. Physiol. 59: , Gabriel, S. E., L. L. Clarke, R. C. Boucher, and M. J. Stutts. CFTR and outward rectifying chloride channels are distinct proteins with a regulatory relationship. Nature 363: , Gray, M. A., S. Plant, and B. E. Argent. camp-regulated whole cell chloride currents in pancreatic duct cells. Am. J. Physiol. 264 (Cell Physiol. 33): C591 C602, Grubb, B. R. Ion transport across the jejunum in normal and cystic fibrosis mice. Am. J. Physiol. 268 (Gastrointest. Liver Physiol. 31): G505 G513, Guba, M., M. Kuhn, W.-G. Forssmann, M. Classen, M. Gregor, and U. Seidler. Guanylin strongly stimulates rat duodenal HCO 3 secretion: proposed mechanism and comparison with other secretagogues. Gastroenterology 111: , 1996.

9 G726 CFTR AND DUODENAL HCO 3 SECRETION IN CF MICE 22. Haas, M. Properties and diversity of (Na-K-Cl) cotransporters. Annu. Rev. Physiol. 51: , Harline, M. C., and L. L. Clarke. Duodenal mucosal anion transport in the cystic fibrosis mouse model (Abstract). Pediatr. Pulmonol. Suppl. 10: 197, Harmon, J. W., M. Woods, and N. J. Gurll. Different mechanisms of hydrogen ion removal in stomach and duodenum. Am. J. Physiol. 235 (Endocrinol. Metab. Gastrointest. Physiol. 4): E692 E698, Hogan, D. L., D. L. Crombie, J. I. Isenberg, P. Svendsen, O. B. Schaffalitzky de Muckadell, and M. A. Ainsworth. CFTR mediates camp- and Ca 2 -activated duodenal epithelial HCO 3 secretion. Am. J. Physiol. 272 (Gastrointest. Liver Physiol. 35): G872 G878, Hogan, D. L., B. Yao, J. H. Steinbach, and J. I. Isenberg. The enteric nervous system modulates mammalian duodenal mucosal bicarbonate secretion. Gastroenterology 105: , Illek, B., J. R. Yankaskas, and T. E. Machen. camp and genistein stimulate HCO 3 conductance through CFTR in human airway epithelia. Am. J. Physiol. 272 (Lung Cell. Mol. Physiol. 16): L752 L761, Isenberg, J. I., M. Ljungstrom, B. Safsten, and G. Flemstrom. Proximal duodenal enterocyte transport: evidence for Na -H and Cl -HCO 3 exchange and NaHCO 3 cotransport. Am. J. Physiol. 265 (Gastrointest. Liver Physiol. 28): G677 G685, Kifor, G., M. R. Toon, A. Janoshazi, and A. K. Solomon. Interaction between red cell membrane band 3 and cytosolic carbonic anhydrase. J. Membr. Biol. 134: , Lonnerholm, G., L. Knutson, P. J. Wistrand, and G. Flemstrom. Carbonic anhydrase in the normal rat stomach and duodenum and after treatment with omeprazole and ranitidine. Acta Physiol. Scand. 136: , Lonnerholm, G., O. Selking, and P. J. Wistrand. Amount and distribution of carbonic anhydrase I and II in the gastrointestinal tract. Gastroenterology 88: , Lopez, M. J., and R. J. Grand. Hereditary and childhood disorders of the pancreas. In: Gastrointestinal Disease, edited by M. H. Sleisenger and J. S. Fordtran. Philadelphia, PA: Saunders, 1993, p Minhas, B. S., S. K. Sullivan, and M. Field. Bicarbonate secretion in rabbit ileum: electrogenicity, ion dependence, and effects of cyclic nucleotides. Gastroenterology 105: , Novak, I., and R. Greger. Properties of the luminal membrane of isolated perfused rat pancreatic ducts. Pflügers Arch. 411: , Poulsen, J. H., H. Fischer, B. Illek, and T. E. Machen. Bicarbonate conductance and ph regulatory capability of cystic fibrosis transmembrane conductance regulator. Proc. Natl. Acad. Sci. USA 91: , Rhodes, J., H. T. Apsimon, and J. H. Lawrie. ph of the contents of the duodenal bulb in relation to duodenal ulcer. Gut 7: , Robinson, P. J., A. L. Smith, and P. D. Sly. Duodenal ph in cystic fibrosis and its relationship to fat malabsorption. Dig. Dis. Sci. 35: , Rune, S. J., and F. W. Henriksen. Carbon dioxide tensions in the proximal part of the canine gastrointestinal tract. Gastroenterology 56: , Sheldon, R. J., M. E. Malarchik, D. A. Fox, T. F. Burks, and F. Porreca. Pharmacological characterization of neural mechanisms regulating mucosal ion transport in mouse jejunum. J. Pharmacol. Exp. Ther. 249: , Smith, J. J., and M. J. Welsh. camp stimulates bicarbonate secretion across normal, but not cystic fibrosis airway epithelia. J. Clin. Invest. 89: , Steel, R. G. D., and J. H. Torrie. Principles and Procedures of Statistics. New York: McGraw-Hill, 1980, p Strong, T. V., K. Boehm, and F. S. Collins. Localization of cystic fibrosis transmembrane conductance regulator mrna in the human gastrointestinal tract by in situ hybridization. J. Clin. Invest. 93: , Tabcharani, J. A., T. J. Jensen, J. R. Riordan, and J. W. Hanrahan. Bicarbonate permeability of the outwardly-rectifying anion channel. J. Membr. Biol. 12: , Tabcharani, J. A., J. M. Rommens, Y. X. Hou, X. B. Chang, L. C. Tsui, J. R. Riordan, and J. W. Hanrahan. Multi-ion pore behavior in the CFTR chloride channel. Nature 366: 79 82, Vince, J. W., J. M. Grushcow, and A. F. Reithmeier. Interaction of the carboxyl-terminus of human band 3 with red cell proteins (Abstract). Biophys. J. 72: A197, Yao, B., D. L. Hogan, K. Bukhave, M. A. Koss, and J. I. Isenberg. Bicarbonate transport by rabbit duodenum in vitro: effect of vasoactive intestinal polypeptide, prostaglandin E 2, and cyclic adenosine monophosphate. Gastroenterology 104: , 1993.