Cystic Fibrosis Transmembrane Conductance Regulator Currents in Guinea Pig Pancreatic Duct Cells: Inhibition by Bicarbonate Ions

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1 GASTROENTEROLOGY 2000;118: Cystic Fibrosis Transmembrane Conductance Regulator Currents in Guinea Pig Pancreatic Duct Cells: Inhibition by Bicarbonate Ions CATHERINE M. O REILLY, JOHN P. WINPENNY, BARRY E. ARGENT, and MICHAEL A. GRAY Department of Physiological Sciences, University Medical School, Newcastle upon Tyne, England See editorial on page Background & Aims: Cystic fibrosis transmembrane conductance regulator (CFTR) Cl channels play an important role in HCO 3 secretion by pancreatic duct cells (PDCs). Our aims were to characterize the CFTR conductance of guinea pig PDCs and to establish whether CFTR is regulated by HCO 3. Methods: PDCs were isolated from small intralobular and interlobular ducts, and their Cl conductance was studied using the whole-cell patch clamp technique. Results: Activation of a typical CFTR conductance by adenosine 3,5 cyclic monophosphate (camp) was observed in 114 of 204 cells (56%). A larger (10-fold), time- and voltagedependent Cl conductance was activated in 39 of 204 cells (19%). Secretin had a similar effect. Coexpression of both conductances in the same cell was observed, and both conductances had similar anion selectivity and pharmacology. Extracellular HCO caused a dose-dependent inhibition of both currents (K i, D7 mmol/l), which was independent of intracellular and extracellular ph, and the PCO 2 and CO 2 content of the bathing solutions. Conclusions: Two kinetically distinct Cl conductances are activated by camp in guinea pig PDCs. Because these conductances are coexpressed and exhibit similar characteristics (anion selectivity, pharmacology, and HCO 3 inhibition), we conclude that CFTR underlies them both. The inhibition of CFTR by HCO 3 has implications for the current model of pancreatic ductal HCO 3 secretion. Pancreatic duct cells (PDCs) secrete the HCO found in pancreatic juice. This alkaline ductal secretion has 2 functions. First, it flushes digestive enzymes secreted by the acinar portion of the gland down the ductal tree toward the gut. Second, it helps neutralize acid chyme that enters the duodenum from the stomach. 1 3 Evidence exists that accumulation of HCO 3 across the basolateral membrane of the PDC occurs (1) via backward transport of protons, either on an Na /H exchanger 4 or a proton pump, 5 and (2) by forward transport of HCO 3 on an Na(HCO 3 ) n cotransporter. 6 It has been proposed that HCO 3 secretion across the apical membrane occurs by parallel operation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl channels and Cl /HCO exchangers. 7,8 In this scheme the CFTR channel can be viewed as having 2 functions. First, it provides luminal Cl for operation of the anion exchangers. Second, it acts as a leak pathway to dissipate intracellular Cl accumulated as the exchangers cycle. This CFTR anion exchanger model for HCO 3 secretion has been largely derived from studies on rat PDCs that secrete a relatively low concentration of HCO ( 70 mmol/l) in their pancreatic juice. 1 3 Whether the same scheme can explain the secretion of a pancreatic juice containing a high concentration of HCO 3 ( 150 mmol/l) that occurs in cats, dogs, guinea pigs, and humans is doubtful. 9 The principal problem is that with a low Cl concentration in the lumen of the duct, intracellular Cl concentration would have to be very low indeed to drive HCO 3 secretion on an anion exchanger. Moreover, at high luminal HCO 3 concentrations, the exchanger would reverse, favoring HCO 3 absorption rather than secretion. Although we do not have a clear picture of how HCO 3 -rich pancreatic juice is secreted, CFTR must play an important role in that process as evidenced by the reduced HCO 3 secretion and pancreatic pathology that occurs in cystic fibrosis. 10 Previously, we have studied the whole-cell CFTR conductance in rat and mouse PDCs. 11,12 Now our aims were (1) to characterize the CFTR conductance in a species (guinea pig) that, like humans, Abbreviations used in this paper: CFTR, cystic fibrosis transmembrane conductance regulator; DIDS, 4,4 -diisothiocyanostilbene-2,2 disulfonic acid; DMSO, dimethyl sulfoxide; EGTA, ethylene glycolbis( -aminoethyl ether)-n,n,n,n -tetraacetic acid; E rev, reversal potential; I, current flow; I/V, current/voltage; ORCC, outwardly rectifying Cl conductance; p, partial pressure; P, permeability; PDC, pancreatic duct cell; pf, input capacitance; R s, series resistance; V m, membrane potential; V p, pipette potential by the American Gastroenterological Association /00/$10.00 doi: /gast

2 1188 O REILLY ET AL. GASTROENTEROLOGY Vol. 118, No. 6 secretes near isotonic NaHCO 3, 13 and (2) to investigate whether CFTR channel function is regulated by the high concentrations of HCO 3 found in the pancreatic juice of most species. Materials and Methods Isolation of PDCs Duncan Hartley and Newcastle-bred pigmented guinea pigs ( g) of either sex were used. Small intralobular and interlobular ducts were isolated from the pancreas, cultured, and dissociated into single cells as described previously. 12,14 Electrophysiology An EPC-7 amplifier (List Electronic, Darmstadt, Germany) was used to record whole-cell currents from single duct cells at 21 C 23 C by holding the membrane potential (V m )at 0 mv and then alternately clamping to 60 mv for 1 second. Between each pulse, V m was clamped to 0 mv for 1 second. Current/voltage (I/V) relationships were obtained by holding V m at 0 mv and clamping to 100 mv in 20-mV steps. Data were filtered at 1 khz, sampled at 2 khz with a CED 1401 interface (Cambridge Electronic Design, Cambridge, England), and stored on computer hard disc and a digital tape recorder. I/V plots were constructed using the average current measured over a 200-millisecond period starting 180 milliseconds into the voltage pulse. The currents were not leak subtracted. Series resistance (R s ) compensation was routinely used (50% 60%). V m was corrected for current flow (I) across the uncompensated fraction of R s using the relationship V m V p IR s, where V p is the pipette potential. V m was also corrected for junction potentials. Reversal potentials (E rev ) were calculated from I/V plots by interpolation after fitting a third-order polynomial using least squares regression analysis. Input capacitance (pf) was measured using the EPC-7 amplifier. Solutions The pipette solution contained 110 mmol/l CsCl, 2 mmol/l MgCl 2, 5 mmol/l ethylene glycol-bis( -aminoethyl ether)-n,n,n,n -tetraacetic acid (EGTA), 10 mmol/l HEPES, and 1 mmol/l Na 2 ATP (ph 7.2) with CsOH. The osmolarity of the pipette solution was 240 mosm/l. The standard bath solution contained 145 mmol/l NaCl, 4.5 mmol/l KCl, 2 mmol/l CaCl 2, 1 mmol/l MgCl 2, 10 mmol/l HEPES, and 5 mmol/l glucose (ph 7.4) with NaOH (300 mosm/l). To make the anion replacement solutions, Cl in the standard bath solution was replaced with equimolar concentrations of either HCO 3, ClO 4, I, Br, or aspartate. The HCO 3 - containing solutions were not routinely gassed with CO 2,so their ph was higher than that of the normal bath solution (ph 7.4). In some experiments the HEPES buffer was omitted from the HCO 3 solutions. In other experiments the HEPES-free HCO 3 -containing solutions were gassed with 5% CO 2 in air. The HCO 3 concentration, PCO 2, and ph of these solutions were measured using a blood gas analyzer (StatProfile 5; Nova Biochemical, Waltham, MA). When we did not measure HCO 3 concentration, the nominal value is given in the text. Duct cells were stimulated with either an adenosine 3,5 cyclic monophosphate (camp) cocktail consisting of 5 µmol/l forskolin and 100 µmol/l dibutyryl camp, with forskolin alone (50 nmol/l to 10 µmol/l) or with secretin (1 pmol/l to 10 nmol/l). Chemicals Stock solutions of forskolin (50 mmol/l) were made up in ethanol, whereas the glybenclamide stock (50 mmol/l) was made up in dimethyl sulfoxide (DMSO). Secretin stocks were made up in bath solution containing 1% (wt/vol) bovine serum albumin. Unless otherwise stated, all chemicals were from Sigma Chemical Co. (Poole, Dorset, UK). Statistics Data are presented as mean SEM. Significance of differences between mean values of 2 groups was tested by the Student paired or unpaired t test as appropriate. Alternatively, for multiple comparisons between control and treatment groups, data were analyzed using 1-way analysis of variance (ANOVA), followed up by the Tukey Kramer multiple comparisons test. A probability of P 0.05 was considered statistically significant. Results camp-stimulated Conductances Exposure of PDCs to the camp cocktail while whole-cell recording was in progress resulted in stimulation of Cl selective conductances in 153 of 204 cells tested (75%). The remaining 51 cells (25%) did not respond to stimulation. These findings contrast to our previous results obtained with rat and murine PDCs in which preexposure of the cells to the cocktail (i.e., before patch clamping) was required to detect camp-activated whole-cell Cl currents. 11,12 Two kinetically distinct conductances were activated in guinea pig PDCs by the camp cocktail (Figure 1). Essentially time-and voltage-independent currents were observed in 114 of 153 (75%) of the responding cells (Figure 1A C). Whole-cell current density increased from to pa/pf at E rev 60 mv and from to pa/pf at E rev 60 mv (P 0.001). The mean input capacitance of this group of cells was pf. The I/V plots exhibited slight outward rectification with a reversal potential of mv (Figure 1C), which is close to the predicted equilibrium potential for Cl ( 7.9 mv). A positive shift in reversal potential was observed when 100 mmol/l extracellular Cl was replaced by aspartate ( E rev, mv; n 26), indicating that the currents were predominantly Cl selective (P Asp /P Cl, ). The

3 June 2000 CFTR AND HCO Figure 1. Biophysical characteristics of whole-cell Cl currents recorded from guinea pig pancreatic duct cells. (A C) CFTR. (D F)Cl tv-dep.(a and D) Basal currents. (B and E) currents recorded from the same cells after exposure to the camp cocktail. Note the different vertical scales for CFTR and Cl tv-dep currents. (B) CFTR currents have time- and voltage-independent kinetics. (E) In contrast, Cl tv-dep currents have time- and voltage-dependent kinetics and exhibit clear tail currents. (C and F) I/V plots for the current traces shown. Note that the CFTR plot (C) is near linear and the Cl tv-dep plot (F) is outwardly rectified. biophysical characteristics of these camp-activated wholecell currents (time- and voltage-independent kinetics and near linear I/V plot) suggest that they were carried by CFTR Cl channels. Large, outwardly rectifying, time- and voltagedependent conductances with marked tail currents were stimulated by the camp cocktail in the remaining 39 of 153 (25%) cells that responded (Figure 1D F). Wholecell current density increased from to pa/pf at E rev 60 mv and from to pa/pf at E rev 60 mv (P 0.001) (Figure 1D F). The mean capacitance in this group of cells was pf. The time- and voltage-dependent currents were about 10-fold larger than the CFTR-like currents observed in the majority of duct cells. These large currents were Cl selective as judged by a positive shift in E rev after replacement of 100 mmol/l Cl by aspartate ( E rev, ; P Asp /P Cl, ; n 7). Because of its time- and voltage-dependent kinetics, this Cl current has been designated Cl tv-dep. Note that the shifts in E rev after replacement of extracellular Cl with aspartate for both CFTR and Cl tv-dep were lower than predicted by the Nernst equation for a perfectly Cl -selective conductance (26.3 mv). This may either reflect the fact that Cl channels such as CFTR have a finite permeability to large anions 15 or that a small cation permeability exists in the duct cells. Secretin-Stimulated Whole-Cell Currents The peptide hormone secretin increases camp levels in PDCs and is an important physiological regulator of ductal HCO 3 secretion in the pancreas. 1 3 Of 24 cells tested, 19 (79%) responded to 10 nmol/l secretin, showing that most of the isolated PDCs retain secretin receptors. CFTR-like currents were observed in 8 of 19 cells (42%) and whole-cell current density increased from to pa/pf at E rev 60 mv and from to pa/pf at E rev 60 mv (P 0.05). The mean capacitance of this group of cells was pf. The I/V plots (not shown) exhibited slight outward rectification and reversed at mv. Larger Cl tv-dep currents were observed in 11 of 19 cells (58%) with the whole-cell current density increasing from to pa/pf at E rev 60 mv and from to pa/pf at E rev 60 mv. The currents reversed at mv, and the mean capacitance of this group of cells was pf. In this series of experiments with secretin, a smaller proportion of cells developed CFTR currents compared with when camp was the stimulant (42% vs. 75%, respectively) and vice versa for the Cl tv-dep

4 1190 O REILLY ET AL. GASTROENTEROLOGY Vol. 118, No. 6 currents (58% vs. 25%, respectively). However, this difference may simply reflect the fact that fewer cells were exposed to secretin (24 cells) compared with the camp cocktail (204 cells). The important point is that secretin, a physiological regulator of pancreatic ductal HCO 3 secretion, can activate both currents in PDCs. Evidence for Coexpression of CFTR and Cl tv-dep Conductances To test whether CFTR and Cl tv-dep conductances were coexpressed in the same cell and whether their appearance was related to either the degree or type of stimulation, we performed dose-response experiments on individual PDCs. Figure 2 shows cumulative doseresponse curves for the effect of secretin and forskolin on CFTR and Cl tv-dep current densities in a total of 14 PDCs. As expected, PDCs were much more sensitive to stimulation by secretin compared with forskolin (Figure 2A and D). There was also considerable cell-to-cell variation in the dose-response relationships for the 2 stimulants. In particular, there was a marked reduction in the size of the currents at higher doses of stimulant in some cells, giving a bell-shaped dose-response curve (Figure 2A and D). Seven of the 14 cells tested (50% overall; 4 of 8 cells with secretin and 3 of 6 cells with forskolin) exhibited typical CFTR currents (time- and voltage-independent kinetics and near linear I/V plot) at all doses of stimulant used. The dose-response relationships for these CFTR cells are shown in Figure 2A, and examples of their CFTR currents in Figure 2B1, B2, C1, and C2. In contrast, 2 of the 14 cells tested (14% overall; 1 of 8 cells with secretin and 1 of 6 cells with forskolin) exhibited typical Cl tv-dep currents (time- and voltage-dependent kinetics, outwardly rectifying I/V plot and clear tail currents) at all doses of stimulant. The dose-response relationships for these 2 Cl tv-dep cells are shown in Figure 2D (secretin, closed triangles; forskolin, open circles). The remaining 5 cells shown in Figure 2D (36% overall; 3 of 8 cells with secretin and 2 of 6 cells with forskolin) exhibited CFTR currents at low doses of stimulant, but Cl tv-dep currents at higher doses. Examples of the currents recorded from 2 of these cells are shown in Figure 2E1, E2, F1, and F2. The recorded currents were easily classified as either CFTR or Cl tv-dep and we never observed intermediates states. These data show that the majority of the PDCs tested exhibited either only the CFTR conductance (50%) or only the Cl tv-dep conductance (14%), regardless of the dose of stimulant used. In contrast, a smaller proportion of the cells (36%) exhibited CFTR at low doses of stimulant and Cl tv-dep at higher doses. Thus, about one third of the cells in the guinea pig pancreatic ductal epithelium coexpress the CFTR and Cl tv-dep conductances. Anion Selectivity of CFTR and Cl tv-dep The anion selectivity sequence of the 2 conductances was assessed by replacing 100 mmol/l extracellular Cl with a range of anions. The selectivity sequence was determined from the E rev values using the Goldman Hodgkin Katz equation (Table 1). Similar selectivity sequences were obtained for both currents. The sequence for CFTR was: Br (1.5) I (1.0) Cl (1.0) HCO 3 (0.5) ClO 4 (0.3) aspartate (0.2), whereas that for Cl tv-dep was: Br (1.3) I (1.2) Cl (1.0) ClO 4 (0.5) HCO 3 (0.4) aspartate (0.2). Pharmacological Inhibition of CFTR and Cl tv-dep Glybenclamide has been reported to inhibit CFTR channels, 16 although its effects are not specific and it is known to inhibit other anion channels. 4,4 -Diisothiocyanostilbene-2,2 -disulfonic acid (DIDS) does not affect CFTR, 17 inhibits Ca 2 -activated chloride channels in some but not all pancreatic duct cell preparations, 21 and blocks outwardly rectifying Cl channels. 22 Glibenclamide (100 µmol/l) inhibited both CFTR and Cl tv-dep currents in guinea pig PDCs (CFTR, 8 cells: 37% and 55% inhibition at E rev 60 mv, respectively; Cl tv-dep,4 cells: 37% and 55% inhibition at the same potentials). Note that glibenclamide inhibited both CFTR and Cl tv-dep currents to the same degree. Current inhibition was observed in all 12 cells that were exposed to glibenclamide, and the effects of the inhibitor were not reversed after 2 5 minutes of bath washout. In contrast, 500 µmol/l DIDS had no effect on either current (CFTR, 9 cells; Cl tv-dep, 4 cells). Effect of HCO 3 on CFTR and Cl tv-dep Figure 3 shows that replacement of 100 mmol/l extracellular Cl with HCO 3 caused a marked inhibition of both inward and outward currents carried by CFTR and Cl tv-dep. Inhibition began within 20 seconds of the start of bath Cl replacement with HCO 3 and reversed slowly after the extracellular Cl was restored (Figure 3). Inhibition of the outward currents (Cl influx) might be caused by the reduced Cl concentration in the extracellular solution. However, extracellular HCO 3 also inhibited inward current (Cl efflux) through CFTR and Cl tv-dep (Figure 3). The reduction in inward current indicates that external HCO 3 is causing transinhibition of Cl efflux. In one series of experiments, nominally 100 mmol/l HCO 3 inhibited outward and inward currents, measured at E rev 60 mv, through CFTR by 63.5% 6.0% and 64.8% 6.8%, respectively (n 12 cells; P 0.05). For Cl tv-dep, the degree of

5 June 2000 CFTR AND HCO Figure 2. Dose-response relationships for the effect of secretin and forskolin on whole-cell Cl current density. (A C) Data for CFTR cells; (D F) data for Cl tv-dep cells. (A) Cumulative dose-response curves for the effect of secretin (closed symbols) and forskolin (open symbols) on CFTR cells. These cells exhibited only CFTR currents. The inward CFTR current densities recorded at 60 mv are plotted, after subtraction of basal currents, for individual cells (4 cells with secretin and 3 cells with forskolin) as a percentage of the response to either 10 8 mol/l secretin or 10 5 mol/l forskolin. Basal current densities were not significantly different (secretin group, pa/pf [n 4 cells]; forskolin group, pa/pf [n cells]). (B1 and B2) Currents from the secretin-stimulated cell indicated by the closed triangles and arrow in A obtained with 10 9 mol/l (B1) and 10 8 mol/l secretin (B2). (C1 and C2) Currents from the forskolin-stimulated cell indicated by the open triangles and arrow in A obtained with 10 6 mol/l (B1) and 10 5 mol/l forskolin (B2). (D) Cumulative dose-response curves for the effect of secretin (closed symbols) and forskolin (open symbols)oncl tv-dep cells. The inward Cl tv-dep current densities recorded at 60 mv are plotted for individual cells (4 cells with secretin and 3 cells with forskolin). Basal currents were not significantly different for secretin group ( pa/pf; n 4 cells) and forskolin group (5.9.7 pa/pf; n cells). Most of these cells (3 of 4 with secretin and 2 of 3 with forskolin) exhibited CFTR currents at low doses of the stimulants and Cl tv-dep currents at higher doses. (E and F) Currents recorded from cells that converted between the 2 current types. (E1 and E2) Currents from the secretin-stimulated cell indicated by the closed circles and arrow in D obtained with mol/l (E1) and 10 9 mol/l secretin (E2). (F1 and F2) Currents from the forskolin-stimulated cell indicated by the open squares and arrow in D obtained with mol/l (F1) and (E2, F2) 10 5 mol/l forskolin (F2). E1 and F1 are CFTR currents, whereas E2 and F2 are Cl tv-dep currents. Note the difference in magnitudes of the Cl tv-dep and the CFTR currents (B1, B2, C1, C2, E1, F1). When performing the cumulative dose-response curves (A and D), the total time of exposure to either secretin or forskolin was about 30 minutes for all cells. To obtain the current traces in sections B, C, E, and F, cells were voltage clamped over the range 100 mv to 100 mv in 20-mV steps.

6 1192 O REILLY ET AL. GASTROENTEROLOGY Vol. 118, No. 6 Table 1. Anion Selectivity of Whole-Cell Currents Anion E rev CFTR P X /P Cl n E rev Cl tv-dep P X /P Cl n P Br NS I NS HCO NS ClO NS Aspartate NS NOTE. Change in reversal potentials of camp-activated whole-cell currents in pancreatic duct cells after replacement of 100 mmol/l extracellular Cl with 100 mmol/l of the anions shown. Anion selectivity sequences were calculated from the E rev values using the Goldman Hodgkin Katz equation: P X /P Cl ([Cl o ] b exp F/RT Erev ) [Cl o ] a /[X o ]; where [Cl o ] b is [Cl ] in the bath solution before anion substitution; [Cl o ] a is [Cl ] in the bath solution after anion substitution; [X o ] is bath concentration of anion that was substituted for Cl ; and E rev is change in reversal potential after anion substitution. inhibition of outward and inward currents at the same potentials was 64.5% 9.6% and 57.8% 11.2%, respectively (n 5 cells, P 0.05). These data show that HCO 3 inhibits CFTR and Cl tv-dep conductances to a similar degree, and that the inhibitory effect of the anion is not voltage dependent. Other anions that we tested also had an inhibitory effect on the CFTR and Cl tv-dep currents (Table 2). However, only ClO 4 and I gave inhibitions comparable with those obtained with HCO 3 (Table 2), and the effect of I was not reversible. Note that the inhibitory effects of Br and aspartate were much smaller than that obtained with HCO 3 (Table 2). Figure 4 shows that the inhibitory effect of HCO 3 on inward currents exhibited a clear dose dependency that was particularly steep around the normal plasma HCO 3 concentration of 25 mmol/l. There was no significant difference between the percentage inhibitions of the 2 currents at any of the HCO 3 concentrations tested. The K i values were mmol/l and mmol/l for the CFTR and Cl tv-dep conductances, respectively. Maximal inhibition values were 71.1%.9% for CFTR and 70.2% 6.7% for Cl tv-dep. Thus, with the exception of current magnitude and kinetics, the properties of the CFTR and Cl tv-dep conductances seem very similar. Both conductances are activated by an increase in intracellular camp and by secretin, are coexpressed in some cells, have similar anion selectivities, are inhibited by glybenclamide but not by DIDS, and are both blocked to a similar degree by HCO 3. Given these marked similarities, we think it is likely that the same channel (i.e., CFTR) underlies both conductances. There- Figure 3. Inhibitory effect of HCO 3 on CFTR and Cl tv-dep currents. The duct cells were stimulated with camp cocktail throughout the experiments, and the currents were sampled at V m of 60 mv. Upward deflections of the traces are outward currents (Cl influx) flowing at 60 mv, and downward deflections are inward currents (Cl efflux) flowing at 60 mv. The normal bath solution was switched to one containing nominally 100 mmol/l HCO 3 (replacement of 100 mmol/l Cl in the normal bath solution) at the arrow marked HCO 3. The normal bath solution was restored at the arrow marked wash. The pulse protocol was interrupted at various points to collect I/V data. Note that the fast onset of HCO 3 inhibition and its slow reversal may reflect the anion s relatively low K i value ( 7 mmol/l) and the kinetics of solution exchange in the tissue bath. To establish a half-inhibitory concentration of HCO 3 in the bath ( 7 mmol/l) would require only 7% exchange of the bath Cl solution with the 100 mmol/l HCO 3 solution. In contrast, for half-recovery, 93% of the 100 mmol/l HCO 3 in the bath would have to be exchanged for Cl.

7 June 2000 CFTR AND HCO Table 2. Inhibition of Inward Currents by Extracellular Anions Anion CFTR n Cl tv-dep n HCO ClO I Br Aspartate NOTE. Percent inhibition (mean SE) of camp-activated whole-cell inward currents in pancreatic duct cells after replacement of 100 mmol/l extracellular Cl with 100 mmol/l of the anions shown. Because E rev shifts occur with the different anions, inward currents were measured at E rev 60 mv to maintain the same electrochemical driving force on each anion. fore, in the following sections, in which we have investigated the mechanism of HCO 3 inhibition, data for both conductances have been combined. The Mechanism of HCO 3 Inhibition The HCO 3 buffer system consists of HCO 3, H, and CO 2 ; thus either of these components could be responsible for the observed current inhibition. Another possible candidate is the carbonate ion (CO 2 ), although the concentration of CO 2 will be low in HCO solutions buffered at near physiological ph values. 23 Figure 4. Dose-response curves for the inhibitory effect of external HCO 3 on CFTR and Cl tv-dep currents., CFTR;, Cl tv-dep. The actual HCO 3 concentration in the bath solutions was measured using a blood gas analyzer. Inward currents were measured at E rev 60 mv so that the electrochemical driving force on the HCO 3 remained constant. Data points (mean SE) contain observations from between 8 and 14 cells. The curves have been fit to a Michaelis Menten equation: I I maximum [HCO 3 ]/K i [HCO 3 ], where I is percentage inhibition, I maximum is the maximum percentage inhibition, and K i is the bicarbonate concentration giving half-maximal inhibition. Effect of varying ph o and ph i. Exposing stimulated cells to a HCO 3 -free solution with a ph o of 7.9 (in the range of ph values measured for solutions containing 100 mmol/l HCO 3 ) had no effect on the currents (n 8 cells). Shifting ph o in the acid direction, to ph 6.8, was also without effect (n cells). We also considered 2 other possibilities to explain the inhibition caused by HCO 3 : (1) CO 2 generated spontaneously in the HCO 3 containing extracellular solution might diffuse into the cell and acidify ph i, and (2) HCO 3 itself might diffuse into the cell and alkalinize ph i. We tested the first possibility by adding 20 mmol/l acetate to the bath solution (a procedure that should acidify ph i ), 4,7 but this had no effect on the size of the currents (n 8 cells). Furthermore, lowering the ph of the pipette solution (and, therefore, ph i ) from the standard value of 7.2 to 6.2 also had no effect (n 13 cells). Finally, increasing the ph of the pipette solution to 7.9 did not change the magnitude of the currents (n 8 cells). From these data we conclude that the inhibitory effect of extracellular HCO 3 on the camp-activated whole-cell currents cannot be explained by changes in either ph o or ph i. Effect of CO 2. Our approach was to expose duct cells to solutions containing markedly different partial pressures (p) of CO 2. Solution 1 was the standard, nominally 100 mmol/l HCO 3 solution, which had an pco 2 of kpa (ph ; [HCO 3 ], mmol/l; n 5). Solution 2 was produced by simply leaving the HEPES buffer out of the standard HCO 3 solution, which caused the pco 2 to decrease to kpa (ph ; [HCO 3 ], mmol/l; n 12). The lower pco 2 in this solution presumably resulted from its higher ph value. To obtain solution 3 we gassed the HEPES-free HCO 3 solution with 5% CO 2 in air, which increased the pco 2 to the expected value of kpa (ph ; [HCO 3 ], mmol/l; n 9). Thus, the pco 2 levels in these 3 solutions varied over a 4-fold range from 3 to 12 kpa. Of course, the ph and HCO 3 concentration in these solutions also varied. However, based on the nil effect of changes in ph o and ph i on the currents, and the relatively shallow slope of the dose-response relationship for HCO 3 inhibition around 100 mmol/l (Figure 4), we anticipated that these variations could be ignored. The percentage inhibitions of the camp-activated currents observed after exposing duct cells to the 3 solutions were as follows: solution 1, 54.3% 7.5% (n 8 cells); solution 2, 38.6% 5.4% (n 14 cells); and solution 3, 40.8% 6.1% (n 14 cells) (ANOVA, P 0.05). These values are not statistically different, suggesting that pco 2 itself is unlikely to be the inhibitory agent.

8 1194 O REILLY ET AL. GASTROENTEROLOGY Vol. 118, No. 6 Effect of CO 2. The calculated CO 2 concentrations in the 3 solutions described earlier were 0.6, 1.5, and 1.1 mmol/l for solutions 1, 2, and 3, respectively. 23 Because the 2 3-fold difference in CO 2 concentration of these solutions was not correlated with a difference in their inhibitory effect on the Cl currents, we conclude that CO 2 is unlikely to be the inhibitory factor. Having ruled out an effect of pco 2 and CO 2, and also changes in ph i and ph o, we are left with the possibility that inhibition results from a direct interaction of HCO with the camp-activated Cl conductance pathway, i.e., with CFTR. Discussion Identity of the Conductances Activated by camp and Secretin Stimulation of the guinea pig duct cells with a camp cocktail resulted in the activation of 2 kinetically distinct Cl conductances. The small conductance was observed in 75% of responding cells and can be identified as CFTR on the basis of its biophysical properties (near linear I/V plot together with time and voltage independence) and inhibition by glibenclamide but not DIDS. The large conductance was termed Cl tv-dep because of its time- and voltage-dependent kinetics. This conductance was observed in 25% of the cells responding to the camp cocktail and its current density was about 10-fold larger than CFTR. The 2 conductances were also observed in cells stimulated with the peptide hormone secretin, which is an important physiological regulator of pancreatic HCO 3 secretion. 1 3 The biophysical characteristics of Cl tv-dep are similar to those of Ca 2 -activated chloride channels present in many epithelial cell types including mouse 12,24 and human PDC. 21 However, the presence of 5 mmol/l EGTA in the pipette solution, which should buffer intracellular Ca 2 concentration at a low level, makes it unlikely that Cl tv-dep isaca 2 -activated conductance. Moreover, in a separate series of experiments we have identified the Ca 2 -activated chloride conductance in guinea pig duct cells; its current density is low compared with Cl tv-dep ( 56 pa/pf at 60 mv). CFTR has been shown to regulate an outwardly rectifying Cl conductance (ORCC) in airway epithelial cells. 25,26 However, unlike Cl tv-dep, ORCC is blocked by DIDS. Also, both ORCC and the Ca 2 -activated Cl conductance have an I to Cl permeability ratio of about 2:1, 21,25 whereas the ratio for Cl tv-dep is 1.2:1. Besides the different kinetics and current densities, in all other respects Cl tv-dep seems similar to CFTR. Both conductances are activated by an increase in intracellular camp concentration, have similar anion selectivities, and are inhibited to a similar degree by glibenclamide but not DIDS. Both conductances are also blocked to a similar degree by HCO 3. The fact that we could sometimes record typical CFTR and Cl tv-dep currents from the same cell (depending on the dose of stimulant used) suggests that the 2 conductances are coexpressed. Given these marked similarities, we think it likely that the same channel, i.e., CFTR, underlies both conductances. We speculate that some cells in the guinea pig ductal epithelium express CFTR at very high levels and that the time- and voltage-dependent kinetics of the large Cl tv-dep currents might be caused by a high density of CFTR channels in the apical membrane. Note that the time- and voltage-dependent kinetics of Cl tv-dep are most marked on the inward currents (Figure 1). Such kinetic phenomena could be explained by Cl depletion at the internal face of the CFTR channels as the large inward currents flow. Thus, our overall conclusion from this part of the study is that guinea pig PDCs, like those of the rat and the mouse, express only 1 camp-activated conductance, CFTR. However, unlike rat and mouse pancreas, some cells in the guinea pig ductal epithelium express CFTR at very high levels. Further work will be required to establish whether this difference is related to the guinea pig s ability to secrete HCO 3 at high concentration in its pancreatic juice. Interactions Between CFTR and HCO We calculated HCO 3 /Cl permeability ratios for CFTR and Cl tv-dep in guinea pig duct cells of (n 12) and (n 5), respectively (P 0.05) (Table 1). These values are at the higher end of the range reported previously for CFTR in other species, 11,15,17,27,28 but are probably overestimates of the true HCO 3 /Cl permeability because of inaccuracies in determining changes in reversal potential after inhibition of the whole-cell currents by HCO 3. The inhibitory effect of HCO 3 was rapid and reversible, and occurred over the physiological range of extracellular HCO concentrations (K i values of 6.2 mmol/l and 8.2 mmol/l for CFTR and Cl tv-dep conductances, respectively). Our data show that HCO 3 inhibited both inward and outward currents flowing through CFTR. The inhibition of outward current (Cl influx) could be explained by the lower Cl concentration in the HCO 3 -containing solutions. However, the reduced inward current suggests that extracellular HCO 3 is also blocking Cl efflux through CFTR. Extracellular perchlorate and iodide also inhibited inward currents and the magnitude of the inhibition was similar to that observed with HCO 3. Single channel CFTR currents are also blocked by iodide. 29,30 Finally, we

9 June 2000 CFTR AND HCO found that bromide and aspartate were much less effective inhibitors than the other anions. The small inhibitory effect of aspartate (a relatively impermeant anion) rules out the possibility that a reduction in extracellular Cl concentration is the inhibitory factor because aspartate would then be expected to be as effective a blocker as HCO 3. The bicarbonate buffer system contains 4 components: HCO 3, H, CO 2, and CO 2, and we sought to determine which of these components was responsible for causing the inhibition. Neither an increase nor a decrease in ph o (to 7.9 and 6.8) inhibited the CFTR currents in guinea pig PDCs. Similar results have been reported for the CFTR conductance in the human sweat gland duct. 31 Increasing ph o to 8.4 has also been reported to have no effect on the conductance of human CFTR expressed in Chinese hamster ovary cells. 15 We also found that reducing the ph of the pipette solution (ph i ) from 7.2 to 6.2 did not reproduce the inhibitory effect of HCO 3.In contrast, Reddy et al. 31 reported that a reduction in ph i over the same range caused about a 50% inhibition of the CFTR conductance in human sweat ducts. At this point in time we have no explanation as to why guinea pig and human CFTR should respond differently to a reduction in ph i. A different method was used to acidify the ph i of PDC (exposure to 20 mmol/l sodium acetate) and was also without effect on the currents, confirming that cytoplasmic ph is not a regulator of guinea pig CFTR. Having ruled out changes in ph o and ph i, we next checked whether CO 2 might be responsible for the inhibition by exposing cells to 3 HCO 3 -containing solutions with different pco 2 levels ( 3, 7, and 12 kpa). Our data suggest that pco 2 itself is unlikely to be the inhibitory agent. The same applies to CO 2. Therefore, we are left with the possibility that HCO 3 interacts directly with the CFTR channel to cause its inhibition. The shape of the dose-inhibition plot (Figure 4) would imply that a single binding site is involved in HCO inhibition. How Does HCO 3 Inhibit CFTR? Initially, we were very surprised by our finding that HCO 3 inhibits the whole-cell CFTR conductance in guinea pig duct cells because we had not detected a marked inhibitory effect of HCO 3 in our earlier single channel work on rat CFTR. 17 A 35% reduction in conductance did occur with the rat channel, and we noted that some block of the rat CFTR was occurring with HCO 3 because its conductance ratio was lower than that measured for gluconate (a supposedly impermeant anion). 17 However, neither our own single channel work on rat CFTR, 17 nor the more recent studies of Linsdell et al. 15 on human CFTR, found any evidence of marked transinhibition of Cl efflux by extracellular HCO 3 (which is clearly evident from our whole-cell studies; see Figure 3). These earlier observations, together with the fact that the inhibitory effect of HCO 3 is not voltage dependent, suggest to us that the anion is exerting its inhibitory effect by an action on either CFTR gating (open state probability) or on the number of active channels in the plasma membrane, rather than by reducing the size of single channel currents. Because the inhibitory effect of HCO 3 is not voltage dependent, we anticipate that the ion will interact with positively charged residues (arginine, lysine, histidine) that do not sense the transmembrane voltage, i.e., that are probably located outside the pore. The full sequence for guinea pig CFTR is not available; however, there are many positively charged residues in the extracellular loops of human CFTR, 32 e.g., K114, R104, and R117 in extracellular loop 1; K329 in extracellular loop 3; H897, K892, and R899 in extracellular loop 4; and R1128 in extracellular loop 6, that are candidates for an HCO 3 binding site. Physiological Implications of HCO Inhibition of CFTR At first sight, an inhibitory effect of HCO 3 on CFTR seems paradoxical in that it would inhibit HCO secretion. At the maximum concentration of HCO found in guinea pig pancreatic juice ( 150 mmol/l), 13 the CFTR conductance would be 70% blocked (Figure 4). In guinea pig ducts, basal HCO 3 secretion is Cl dependent and blocked by DIDS, suggesting that it occurs via a Cl /HCO 3 exchanger. 33,34 In contrast, camp-stimulated HCO 3 secretion is unaffected by removal of extracellular Cl and must, therefore, occur via some other Cl -independent pathway. 33,34 That pathway may be CFTR because (1) our data show that CFTR has a finite permeability to HCO 3, and (2) no other candidate HCO 3 transporters have been identified on the apical plasma membrane of the duct cell. We speculate that inhibiting CFTR as the luminal HCO concentration increases might be advantageous in this respect. Such a negative feedback mechanism from the lumen would limit apical membrane depolarization and maintain the electrical driving force for HCO 3 secretion via the uninhibited fraction of CFTR. References 1. Case RM, Argent BE. Pancreatic duct cell secretion. Control and mechanisms of transport. In: Go VLM, DiMagno EP, Gardner JD, Lebenthal E, Reber HA, Scheele G, eds. The pancreas: biology, pathobiology, and diseases. Volume 2. New York: Raven, 1993: Argent BE, Case RM. Pancreatic ducts. Cellular mechanism and

10 1196 O REILLY ET AL. GASTROENTEROLOGY Vol. 118, No. 6 control of bicarbonate secretion. In: Johnson LR, ed. Physiology of the gastrointestinal tract. Volume 2. 3rd ed. New York: Raven, 1994: Argent BE, Gray MA. Regulation and formation of fluid and electrolyte secretions by pancreatic ductal epithelium. In: Sirica AE, Longnecker DS, eds. Biliary and pancreatic ductal epithelia. Pathobiology and pathophysiology. New York: Marcel Dekker, 1997: Novak I, Greger R. Electrophysiological study of transport systems in isolated perfused pancreatic ducts: properties of the basolateral membrane. Pflugers Arch 1988;411: Ræder MG. The origin of and subcellular mechanisms causing pancreatic bicarbonate secretion. Gastroenterology 1992;103: Ishiguro H, Steward MC, Lindsay ARG, Case RM. Accumulation of intracellular HCO 3 by Na -HCO 3 cotransport in interlobular ducts from guinea-pig pancreas. J Physiol 1996;495.1: Novak I, Greger R. Properties of the luminal membrane of isolated perfused rat pancreatic ducts. Effect of cyclic AMP and blockers of chloride transport. Pflugers Arch 1988;411: Gray MA, Greenwell JR, Argent BE. Secretin-regulated chloride channel on the apical plasma membrane of pancreatic duct cells. J Membr Biol 1988;105: Sohma Y, Gray MA, Imai Y, Argent BE. A mathematical model of the pancreatic ductal epithelium. J Membr Biol 1996;154: Durie PR, Forstner GG. Pathophysiology of the exocrine pancreas in cystic fibrosis. J R Soc Med 1989;82(suppl 16): Gray MA, Plant S, Argent BE. camp-regulated whole cell chloride currents in pancreatic duct cells. Am J Physiol 1993;264:C591 C Gray MA, Winpenny JP, Porteous DJ, Dorin JR, Argent BE. CFTR and calcium-activated chloride currents in pancreatic ducts of a transgenic CF mouse. Am J Physiol 1994;266:C213 C Padfield PJ, Garner A, Case RM. Patterns of pancreatic secretion in the anaesthetised guinea-pig following stimulation with secretin, cholecystokinin octapeptide, or bombesin. Pancreas 1989;4: Argent BE, Arkle S, Cullen MJ Green R. Morphological, biochemical and secretory studies on rat pancreatic ducts maintained in tissue culture. Q J Exp Physiol 1986;71: Linsdell P, Tabcharani JA, Rommens JM, Hou Y-X, Chang X-B, Tsui L-C, Riordan JR, Hanrahan JW. Permeability of wild type and mutant cystic fibrosis transmembrane conductance regulator chloride channels to polyatomic anions. J Gen Physiol 1997;110: Sheppard DN, Welsh MJ. Effect of ATP sensitive K channel regulators on cystic fibrosis transmembrane conductance regulator chloride currents. J Gen Physiol 1992;100: Gray MA, Pollard CE, Harris A, Coleman L, Greenwell JR, Argent BE. Anion selectivity and block of the small conductance chloride channel on pancreatic duct cells. Am J Physiol 1990;259:C752 C Chan HC, Cheung WT, Leung PY, Wu LJ, Chew SBC, Ko WH, Wong PYD. Purinergic regulation of anion secretion by cystic fibrosis pancreatic duct cells. Am J Physiol 1996;271:C469 C Nguyen TD, Koh DS, Moody MW, Fox NR, Savard CE, Kuver R, Hille B, Lee SP. Characterization of two distinct chloride channels in cultured dog pancreatic duct epithelial cells. Am J Physiol 1997; 272:G172 G Al-Nakkash L, Cotton CU. Bovine pancreatic duct cells express camp- and Ca 2 -activated apical membrane Cl conductances. Am J Physiol 1997;273:G204 G Winpenny JP, Harris A, Hollingsworth MA, Argent BE, Gray MA. Calcium-activated chloride conductance in pancreatic adenocarcinoma cell line of ductal origin (HPAF) and in freshly isolated human pancreatic duct cells. Pflugers Arch 1998;413: Bridges RJ, Worrell RT, Frizzell RA, Benos DJ. Stilbene disulphonate blockade of colonic secretory Cl channels in planar lipid bilayers. Am J Physiol 1989;256:C902 C Butler JN. Carbon dioxide equilibria and their applications. Chelsea, Michigan: Lewis, Winpenny JP, Verdon B, McAlroy HL, Colledge WH, Ratcliff R, Evans MJ, Gray MA, Argent BE. Calcium-activated chloride conductance is not increased in pancreatic duct cells of CF mice. Pflugers Arch 1994;430: Schwiebert EM, Flotte T, Cutting GR, Guggino WB. Both CFTR and outwardly rectifying chloride channels contribute to campstimulated whole cell chloride currents. Am J Physiol 1994;266: C1464 C Schwiebert EM, Egan ME, Hwang T-H, Fulmer SB, Allen SS, Cutting GR, Guggino WB. CFTR regulates outwardly rectifying chloride channels through an autocrine mechanism involving ATP. Cell 1995;81: Hanrahan JW, Tabcharani JA, Grygorczyk R. Patch clamp studies of apical membrane chloride channels. In: Dodge JA, Brock DJH, Widdicombe JH, eds. Cystic fibrosis current topics. Volume 1. London: Wiley, 1993: Poulsen JH, Fischer H, Illek B, Machen TE. Bicarbonate conductance and ph regulatory capability of cystic fibrosis transmembrane conductance regulator. Proc Natl Acad Sci U S A 1994;91: Tabcharani JA, Chang XB, Riordan JR, Hanrahan JW. The cystic fibrosis transmembrane conductance regulator chloride channel: iodide block and permeation. Biophys J 1992;62: Tabcharani JA, Linsdell P, Hanrahan JW. Halide permeation in wild-type and mutant cystic fibrosis transmembrane conductance regulator chloride channels. J Gen Physiol 1997;110: Reddy MM, Kopito RR, Quinton PM. Cytosolic ph regulates G cl through control of phosphorylation states of CFTR. Am J Physiol 1998;275:C1040 C Riordan JR, Rommens JM, Kerem B-S, Alon N, Rozmahel R, Grzelczak Z, Zielenski J, Lok S, Plavsic N, Chou J-L, Drumm ML, Iannuzzi MC, Collins FS, Tsui L-C. Identification of the cystic fibrosis gene: cloning and characterization of complimentary DNA. Science 1989;245: Ishiguro H, Steward MC, Wilson RW, Case RM. Bicarbonate secretion in interlobular ducts from guinea-pig pancreas. J Physiol 1996;495.1: Ishiguro H, Naruse S, Steward MC, Kitagawa M, Ko SBH, Hayakawa T, Case RM. Fluid secretion in interlobular ducts isolated from guinea-pig pancreas. J Physiol 1998;511: Received June 22, Accepted March 2, Address requests for reprints to: Barry E. Argent, Ph.D., University Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, England. b.e.argent@ncl.ac.uk; fax: (44) Supported by the Cystic Fibrosis Trust (United Kingdom) and a Wellcome Trust Travelling Fellowship (grant no ; to J.P.W.). Dr. O Reilly s present address is: Biomedical Imaging Group & Department of Physiology, University of Massachusetts Medical School, 373 Plantation Street, Worcester, Massachusetts Dr. Winpenny s present address is: School of Health Sciences, Pasteur Building, University of Sunderland, Sunderland SR1 3SD, United Kingdom. The authors thank David Stephenson for skilled technical assistance.