Dual role of CFTR in cAMP-stimulated HCO<SUP>−</SUP><SUB>3</SUB> 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 65211

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

The role of the cystic fibrosis transmembrane conductance regulator (CFTR) in cAMP-stimulated HCO<SUP>−</SUP><SUB>3</SUB> secretion across the murine duodenum was investigated. Serosal-to-mucosal flux of HCO<SUP>−</SUP><SUB>3</SUB> (Jsright-arrow m, in µeq · cm-2 · h-1) and short-circuit current (Isc; in µeq · cm-2 · h-1) were measured by the pH stat method in duodenum from CFTR knockout [CFTR(-)] and normal [CFTR(+)] mice. Under control conditions, forskolin increased Jsright-arrow m and Isc (+1.7 and +3.5, respectively) across the CFTR(+) but not CFTR(-) duodenum. Both the forskolin-stimulated Delta Jsright-arrow m and Delta Isc were abolished by the CFTR channel blocker 5-nitro-2-(3-phenylpropylamino)benzoate, whereas inhibition of luminal Cl-/ HCO<SUP>−</SUP><SUB>3</SUB> exchange by luminal Cl- removal or DIDS reduced the Jsright-arrow m by ~18% without a consistent effect on the Delta Isc. Methazolamide also reduced the Jsright-arrow m by 39% but did not affect the Delta Isc. When carbonic anhydrase-dependent HCO<SUP>−</SUP><SUB>3</SUB> secretion was isolated by using a CO2-gassed, HCO<SUP>−</SUP><SUB>3</SUB>-free Ringer bath, forskolin stimulated the Jsright-arrow m and Isc (+0.7 and +2.0, respectively) across CFTR(+) but not CFTR(-) duodenum. Under these conditions, luminal Cl- substitution or DIDS abolished the Jsright-arrow m but not the Delta Isc. It was concluded that cAMP-stimulated HCO<SUP>−</SUP><SUB>3</SUB> secretion across the duodenum involves 1) electrogenic secretion via a CFTR HCO<SUP>−</SUP><SUB>3</SUB> conductance and 2) electroneutral secretion via a CFTR-dependent Cl-/ HCO<SUP>−</SUP><SUB>3</SUB> 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

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

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 1.5-2.0 and PCO2 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<SUP>−</SUP><SUB>3</SUB> across the epithelium (1, 16). The process of active HCO<SUP>−</SUP><SUB>3</SUB> secretion involves the concerted activities of an anion channel and Cl-/HCO<SUP>−</SUP><SUB>3</SUB> exchange in the luminal membrane and secretes HCO<SUP>−</SUP><SUB>3</SUB> 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<SUP>−</SUP><SUB>3</SUB> secretion is not well understood. In an excellent review of duodenal HCO<SUP>−</SUP><SUB>3</SUB> secretion, Allen et al. (1) have proposed that cAMP-stimulated HCO<SUP>−</SUP><SUB>3</SUB> secretion involves the activation of a luminal membrane HCO<SUP>−</SUP><SUB>3</SUB> channel. Alternatively, HCO<SUP>−</SUP><SUB>3</SUB> 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<SUP>−</SUP><SUB>3</SUB> exchanger (34).

Studies of cystic fibrosis transmembrane conductance regulator (CFTR), the dysfunctional cAMP-activated Cl- channel in cystic fibrosis (CF) disease (3, 5, 11), indicate that this channel may play a major role in cAMP-mediated HCO<SUP>−</SUP><SUB>3</SUB> 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<SUP>−</SUP><SUB>3</SUB> [HCO<SUP>−</SUP><SUB>3</SUB>-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<SUP>−</SUP><SUB>3</SUB> with a HCO<SUP>−</SUP><SUB>3</SUB>-to-Cl- permeability ratio of 1:2 (43). However, the hypothesis that CFTR functions as both a Cl- and a HCO<SUP>−</SUP><SUB>3</SUB> 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<SUP>−</SUP><SUB>3</SUB>) is greatly reduced in the presence of a more permeable anion (Cl-) (44).

Recently, direct measures of transepithelial HCO<SUP>−</SUP><SUB>3</SUB> flux have shown that CFTR is required for agonist-stimulated HCO<SUP>−</SUP><SUB>3</SUB> secretion across the mammalian duodenum. In pH stat studies of rat duodenum Guba et al. (21) demonstrated that HCO<SUP>−</SUP><SUB>3</SUB> 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<SUP>−</SUP><SUB>3</SUB> exchanger or ORCC, i.e., removal of Cl- from the luminal perfusate or treatment with DIDS. Although the mechanism of cAMP-stimulated HCO<SUP>−</SUP><SUB>3</SUB> 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<SUP>−</SUP><SUB>3</SUB> output is greatly reduced compared with the normal murine duodenum (25). However, the mechanistic role of CFTR in cAMP-stimulated HCO<SUP>−</SUP><SUB>3</SUB> 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<SUP>−</SUP><SUB>3</SUB> secretion. Direct measurements of HCO<SUP>−</SUP><SUB>3</SUB> secretory flux, using the pH stat method, were performed on duodena from the CFTR knockout mouse model, thereby allowing comparison of HCO<SUP>−</SUP><SUB>3</SUB> 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<SUP>−</SUP><SUB>3</SUB> 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<SUP>−</SUP><SUB>3</SUB> secretion involving luminal Cl-/HCO<SUP>−</SUP><SUB>3</SUB> exchange.

    MATERIAL AND METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

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-Cftrtm/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% CO2 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 cm2 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 152.6 mM NaCl solution gassed with 100% O2 and on the serosal surface with a standard Ringer solution gassed with 95% O2-5% CO2. 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 K2HPO4, 0.4 KH2PO4, 25 NaHCO3, 1.2 MgCl2, 1.2 CaCl2, 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 CaSO4 was added to overcome Ca2+ chelation). Before each experiment proximal duodenal tissues were equilibrated for 20-25 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 (Isc, in µeq · cm-2 · h-1) 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 Isc and automatic fluid resistance compensation current were applied through Ag-AgCl electrodes connected to the chamber baths via 4% agar-152.6 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 (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 HCO<SUP>−</SUP><SUB>3</SUB> (Jsright-arrow 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<SUP>−</SUP><SUB>3</SUB> (base) flux, taking into account the time and 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 immediately initiated, and then forskolin (10 µM) with or without various inhibitors was added to the bathing solutions. When the Jsright-arrow 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<SUP>−</SUP><SUB>3</SUB>, the luminal bath was gassed with 95% O2-5% CO2 and clamped at pH 5.1 (using 5 mM HCl). In the serosal bath HCO<SUP>−</SUP><SUB>3</SUB> was replaced equimolar with TES, and the solution was gassed with 100% O2 (pH 7.4). Initial studies using sodium gluconate (pKa 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 Na2SO4 (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 0.0001 M. In separate experiments DMSO and ethanol vehicles at concentrations equivalent to those used in each experiment (0.2 and 0.1%, respectively) produced no significant alterations of the basal Isc.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Control pH stat studies. Previous ion substitution studies of the CFTR(+) murine duodenum had shown that either Cl- or HCO<SUP>−</SUP><SUB>3</SUB> could carry an inward current across the epithelium and together account for >99% of the cAMP-stimulated Isc (23). Furthermore, the presence of HCO<SUP>−</SUP><SUB>3</SUB> in the bath medium resulted in a significant fraction of the cAMP-stimulated Isc that was insensitive to the Cl- transport inhibitor bumetanide. However, Isc measurements of epithelia treated with bumetanide or bathed in Cl--free medium may not reflect the actual rate of HCO<SUP>−</SUP><SUB>3</SUB> secretion (12, 40). Therefore, the rate of HCO<SUP>−</SUP><SUB>3</SUB> (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 Jsright-arrow m and Isc were measured under basal conditions (Fig. 1A). Addition of forskolin (cAMP) increased the Jsright-arrow m by 1.67 ± 0.15 µeq · cm-2 · h-1 and the Isc by 3.46 ± 0.38 µeq · cm-2 · h-1. The forskolin-stimulated Delta Jsright-arrow m was exceeded in magnitude by the Delta Isc, indicating that electrogenic Cl- secretion occurs simultaneously with HCO<SUP>−</SUP><SUB>3</SUB> secretion during cAMP stimulation of the duodenum. Subsequent addition of bumetanide to the serosal bath did not affect the mean Jsright-arrow m but significantly decreased the Isc. However, the postbumetanide Isc remained significantly elevated relative to the basal Isc. The transepithelial conductance (Gt) increased slightly over the course of the experiment with the main change occurring after forskolin treatment (basal Gt = 38.6 ± 2.8 mS/cm2; forskolin Gt = 43.7 ± 3.1 mS/cm2; bumetanide Gt = 48.2 ± 4.0 mS/cm2, P < 0.05). Together, these findings are consistent with the hypothesis that most of the Isc after bumetanide represents electrogenic HCO<SUP>−</SUP><SUB>3</SUB> secretion. To investigate whether cAMP-stimulated HCO<SUP>−</SUP><SUB>3</SUB> secretion occurs in the absence of CFTR, pH stat experiments were performed on CFTR(-) duodenum. As shown in Fig. 1B, a significant Jsright-arrow m was measured during the basal period and exceeded the mean Isc (which was slightly positive for the period). Sequential additions of forskolin and bumetanide had no significant effect on the Jsright-arrow m or Isc. The mean Gt of the CFTR(-) duodenum was unchanged through all three flux periods (baseline Gt = 32.3 ± 7.2 mS/cm2; forskolin Gt = 31.9 ± 6.8 mS/cm2; bumetanide Gt = 29.2 ± 7.7 mS/cm2).


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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<SUP>−</SUP><SUB>3</SUB> (Jsright-arrow m) and short-circuit current (Isc) 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: Jsright-arrow m and Isc responses of normal mice [CFTR(+)] duodenum, n = 7. B: Jsright-arrow m and Isc 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).

Effect of NPPB and DIDS on cAMP-stimulated Jsright-arrow m and Isc. To investigate whether the channel function per se of CFTR is required for cAMP-stimulated HCO<SUP>−</SUP><SUB>3</SUB> 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 Jsright-arrow m and reduced stimulation of the Isc by 84% (compare with Fig. 1A). The mean Gt in these preparations was unchanged by the NPPB-forskolin treatment (basal Gt = 44.1 ± 7.4 mS/cm2; NPPB-forskolin Gt = 45.4 ± 4.8 mS/cm2). To estimate the contribution of Cl-/HCO<SUP>−</SUP><SUB>3</SUB> exchange or a HCO<SUP>−</SUP><SUB>3</SUB> 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 Delta Jsright-arrow m (DIDS-forskolin Delta Jsright-arrow m = 1.38  ± 0.24 µeq · cm-2 · h-1) but had little effect on the Delta Isc (DIDS-forskolin Delta Isc = +3.33 ± 0.74 µeq · cm-2 · h-1) compared with control (Fig. 1A). The Gt measured in these experiments tended to increase with forskolin, but the changes were not statistically significant (basal Gt = 45.0 ± 5.4 mS/cm2; DIDS-forskolin Gt = 54.3 ± 8.6 mS/cm2).


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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 (3 × 10-4 M) or DIDS (3 × 10-4 M) in luminal bath for 10 min followed by forskolin (cAMP). The 2nd 30-min flux period began 15 min after forskolin. A: Jsright-arrow m and Isc responses of CFTR(+) duodenum pretreated with NPPB, n = 6. B: Jsright-arrow m and Isc responses of CFTR(+) duodenum pretreated with DIDS, n = 6. Bars, means ± SE. * Significantly different from basal (paired t-test).

Effect of luminal Cl- substitution on cAMP-stimulated Jsright-arrow m and Isc. The NPPB experiments indicated that cAMP-stimulated HCO<SUP>−</SUP><SUB>3</SUB> secretion is mediated via CFTR channel activity. This finding is consistent with the hypothesis that CFTR can function as a cAMP-stimulated HCO<SUP>−</SUP><SUB>3</SUB> channel. However, the results of the DIDS studies suggested that enhanced Cl-/HCO<SUP>−</SUP><SUB>3</SUB> activity, but not an electrogenic ORCC-mediated pathway, may also contribute to cAMP-stimulated HCO<SUP>−</SUP><SUB>3</SUB> 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 membrane to diminish or eliminate luminal Cl-/HCO<SUP>−</SUP><SUB>3</SUB> exchange activity. As shown in Fig. 3A, forskolin treatment in the absence of luminal Cl- stimulated the Jsright-arrow m to a mean value that was slightly less than in the control studies and comparable to the DIDS-forskolin treatment (1.39 ± 0.27 µeq · cm-2 · h-1). Forskolin treatment also significantly stimulated the Isc in the absence of luminal Cl-, but the mean Delta Isc (2.20 ± 0.6 µeq · cm-2 · h-1) was less than measured in the control experiments. However, interpretation of the Isc in this experiment was complicated by the fact that the Isc 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 Gt measured in these experiments was not different between the basal and forskolin-treated flux periods (basal Gt = 21.1 ± 1.2; cAMP Gt = 22.2 ± 1.3 mS/cm2). To test whether cAMP-stimulated HCO<SUP>−</SUP><SUB>3</SUB> secretion during inhibition of luminal Cl-/HCO<SUP>−</SUP><SUB>3</SUB> is mediated by CFTR, these experiments were performed in CFTR(-) duodenum. As shown in Fig. 3B, forskolin treatment had no significant effect on the Jsright-arrow m, Isc, or Gt (basal Gt = 22.3 ± 1.7; cAMP Gt = 25.7 ± 3.0 mS/cm2).


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Fig. 3.   Effect of luminal Cl- removal. CFTR(+) and CFTR(-) duodena were bathed with Cl--free Ringer in luminal bath (pH 7.4). Jsright-arrow m and Isc were recorded for two 30-min flux periods: before treatment (basal) and 15 min after forskolin (cAMP). A: Jsright-arrow m and Isc responses of CFTR(+) duodenum, n = 7. B: Jsright-arrow m and Isc responses of CFTR(-) duodenum, n = 4. Bars, means ± SE. * Significantly different from basal (paired t-test).

Effect of methazolamide on cAMP-stimulated Jsright-arrow m and Isc. Although the preceding experiments indicated that most cAMP-stimulated HCO<SUP>−</SUP><SUB>3</SUB> secretion is an electrogenic CFTR-dependent process, the evidence also suggested that a second mechanism involving luminal Cl-/HCO<SUP>−</SUP><SUB>3</SUB> exchange may contribute to cAMP-stimulated HCO<SUP>−</SUP><SUB>3</SUB> secretion. Previously, we had found that luminal Cl-/HCO<SUP>−</SUP><SUB>3</SUB> exchange was associated with carbonic anhydrase-dependent HCO<SUP>−</SUP><SUB>3</SUB> secretion across the duodenal epithelium (10). Therefore, we investigated the effect of carbonic anhydrase inhibition during cAMP stimulation of Jsright-arrow m and Isc. In these experiments CFTR(+) duodena were pretreated with a membrane-permeant inhibitor of carbonic anhydrase, methazolamide (Meth, 1 mM), before forskolin stimulation. As shown in Fig. 4, forskolin stimulated a significant increase in Jsright-arrow m in the methazolamide-pretreated duodenum, but the magnitude of the Delta Jsright-arrow m was significantly less than that found in the control studies shown in Fig. 1 (Meth-pretreated Delta Jsright-arrow m = +0.87 ± 0.22 vs. control Delta Jsright-arrow m = +1.67 ± 0.15 µeq · cm-2 · h-1). In contrast, forskolin stimulated the Isc in the methazolamide-pretreated duodenum to a level that was equivalent to that found in the control studies (Meth-pretreated Delta Isc = +3.78 ± 0.62 vs. control Delta Isc = +3.46 ± 0.38 µeq · cm-2 · h-1). In additional time control experiments, we found that methazolamide treatment per se caused a small reduction in the Jsright-arrow m and, paradoxically, increased the Isc in the second flux period compared with the first flux period (Meth Delta Jsright-arrow m = -0.14 ± 0.17 and Meth Delta Isc = +0.36 ± 0.46 µeq · cm-2 · h-1, 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 Delta Jsright-arrow m by 39%, compared with the control but did not affect the magnitude of the forskolin-stimulated Delta Isc (<3%). These observations indicated that the carbonic anhydrase-dependent fraction of cAMP-stimulated Jsright-arrow m does not involve an electrogenic process. Therefore, experiments were undertaken to isolate the mechanism responsible for duodenal secretion of endogenously generated HCO<SUP>−</SUP><SUB>3</SUB>.


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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).

Isolation of carbonic anhydrase-dependent HCO<SUP>−</SUP><SUB>3</SUB> secretion. To isolate duodenal secretion of carbonic anhydrase-generated HCO<SUP>−</SUP><SUB>3</SUB>, pH stat studies were performed on CFTR(+) duodena that were bathed with a NaCl solution gassed with 95% O2-5% CO2 in the luminal solution and an HCO<SUP>−</SUP><SUB>3</SUB>-free, TES-buffered Ringer gassed with 100% O2 in the basolateral solution. The purpose of this design was to provide a CO2 source for intracellular carbonic anhydrase generation of HCO<SUP>−</SUP><SUB>3</SUB> while preventing HCO<SUP>−</SUP><SUB>3</SUB> movement across the basolateral membrane via Na+-coupled uptake mechanisms, e.g., NaHCO3 cotransport (28). The luminal NaCl-5% CO2 solution equilibrated in the pH range of 4.9-5.3, and the PCO2 was 41 ± 1 mmHg (n = 3). Because clamping the NaCl-5% CO2 solution to pH 7.4 under this protocol would result in a significant HCO<SUP>−</SUP><SUB>3</SUB> 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 7.4 ± 0.01 (n = 22), and the PCO2 of the solution was found to be <0.4 mmHg (n = 4). In control studies using hemichambers without intestine, the 5% CO2 gassing resulted in a small spontaneous rate of HCO<SUP>−</SUP><SUB>3</SUB> 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 Jsright-arrow m measured in these studies was corrected for spontaneous HCO<SUP>−</SUP><SUB>3</SUB> production.

With the luminal NaCl-5% CO2 condition, the CFTR(+) duodenum yielded a basal Jsright-arrow m that was exceeded in magnitude by the Isc (Fig. 5A). Importantly, subsequent treatment of the duodenum with forskolin significantly increased the Jsright-arrow m by 0.69 ± 0.08 µeq · cm-2 · h-1 and the Isc by 2.04 ± 0.18 µeq · cm-2 · h-1 (n = 8). The mean Gt of the CFTR(+) duodenum in these experiments increased slightly during forskolin treatment (basal Gt = 41.1 ± 2.3; forskolin Gt = 48.6 ± 4.1 mS/cm2, P < 0.05). To investigate the requirement of carbonic anhydrase activity for the cAMP-stimulated Jsright-arrow 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 Jsright-arrow m to near the basal value (Meth Delta Jsright-arrow m = -0.66 ± 0.14; DMSO Delta Jsright-arrow m = -0.09 ± 0.19 µeq · cm-2 · h-1, n  = 4 each). The mean Isc significantly decreased during the third flux period for both the methazolamide-treated and DMSO-treated duodena, but the changes were not significantly different from each other (Meth Delta Isc = -0.91 ± 0.52; DMSO Delta Isc = -1.22 ± 0.37 µeq · cm-2 · h-1, n = 4 each). The mean Gt for both the methazolamide- and DMSO-treated duodenum increased significantly during the third flux period, but the differences between the two groups were not statistically significant (Meth Delta Gt = +6.7 ± 1.1; DMSO Delta Gt = +7.8 ± 3.0 mS/cm2, n = 4 each). To investigate the possibility that an acidic luminal solution per se was responsible for the cAMP-stimulated HCO<SUP>−</SUP><SUB>3</SUB> secretion, we lowered the luminal bath pH using an isotonic HCl solution and gassed the luminal bath with 100% O2 rather than 95% O2-5% CO2. Under these conditions, cAMP stimulation of CFTR(+) duodena did not increase the Jsright-arrow m but significantly stimulated the Isc (Delta Jsright-arrow m = -0.04 ± 0.12; Delta Isc = 1.73 ± 1.24 µeq · cm-2 ·h-1, n =  3). The above studies indicated that the duodenal epithelium was capable of increasing the secretion of endogenously generated HCO<SUP>−</SUP><SUB>3</SUB> via an electroneutral mechanism during intracellular cAMP stimulation of Cl- secretion.


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Fig. 5.   Control pH stat studies during isolation of endogenous HCO<SUP>−</SUP><SUB>3</SUB> production. CFTR(+) and CFTR(-) duodena were bathed with NaCl solution gassed with 95% O2-5% CO2 (pH 5.1) in luminal bath and HCO<SUP>−</SUP><SUB>3</SUB>-free Ringer solution in serosal bath (pH 7.4). Jsright-arrow m and Isc 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: Jsright-arrow m and Isc responses of CFTR(+) duodenum, n = 8. Insets: mean Delta Jsright-arrow m and Delta Isc after Meth or Veh, n = 4 each. B: Jsright-arrow m and Isc responses of CFTR(-) duodenum, n = 4. Bars, means ± SE. * Significantly different from basal (or Veh in 3rd flux period; paired t-test).

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 Jsright-arrow m was present under the basal conditions and exceeded the Isc. However, in contrast to the CFTR(+) duodenum, forskolin treatment did not significantly increase either Jsright-arrow m or Isc in the CFTR(-) duodenum. The mean Gt of the CFTR(-) duodenum increased after forskolin treatment but the change was not statistically significant (basal Gt = 38.1 ± 4.6; cAMP Gt = 45.1 ± 5.6 mS/cm2).

Effect of luminal Cl- removal or DIDS on carbonic anhydrase-dependent HCO<SUP>−</SUP><SUB>3</SUB> secretion. The involvement of luminal Cl-/HCO<SUP>−</SUP><SUB>3</SUB> exchange in cAMP-stimulated secretion of endogenously produced HCO<SUP>−</SUP><SUB>3</SUB> was investigated by inhibiting luminal Cl-/HCO<SUP>−</SUP><SUB>3</SUB> exchange via luminal Cl- substitution or DIDS treatment. As shown in Fig. 6A, removal of luminal Cl- prevented the forskolin-induced increase in Jsright-arrow m but did not diminish the Isc stimulation (Cl--free Delta Jsright-arrow m = +0.12 ± 0.14 and Delta Isc = +3.0 ± 0.6 µeq · cm-2 · h-1; compare with Fig. 5A). The mean Gt slightly increased after forskolin treatment in this series of experiments (basal Gt = 38.4 ± 3.9; cAMP Gt = 47.6 ± 5.0 mS/cm2, 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 Jsright-arrow m but had no effect on the forskolin-stimulated Isc compared with the control experiments (Fig. 5A). The mean Gt in these experiments also increased during the forskolin-treated flux period (basal Gt = 37.6 ± 2.2; cAMP Gt = 45.0 ± 1.6 mS/cm2, P < 0.05). These results demonstrate that during cAMP stimulation the murine duodenum is capable of increasing the secretion of carbonic anhydrase-generated HCO<SUP>−</SUP><SUB>3</SUB> via a mechanism involving CFTR-facilitated Cl-/HCO<SUP>−</SUP><SUB>3</SUB> exchange.