CFTR involvement in nasal potential differences in mice and pigs studied using a thiazolidinone CFTR inhibitor
Danieli B. Salinas,1,2
Nicoletta Pedemonte,1
Chatchai Muanprasat,1
Walter F. Finkbeiner,3
Dennis W. Nielson,2 and
A. S. Verkman1
1Departments of Medicine and Physiology, Cardiovascular Research Institute, and Departments of 2Pediatrics and 3Pathology, University of California, San Francisco, California 94143
Submitted 6 October 2003
; accepted in final form 26 June 2004
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ABSTRACT
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Nasal potential difference (PD) measurements have been used to demonstrate defective CFTR function in cystic fibrosis (CF) and to evaluate potential CF therapies. We used the selective thiazolidinone CFTR inhibitor CFTRinh-172 to define the involvement of CFTR in nasal PD changes in mice and pigs. In normal mice infused intranasally with a physiological saline solution containing amiloride, nasal PD was 4.7 ± 0.7 mV, hyperpolarizing by 15 ± 1 mV after a low-Cl solution, and a further 3.9 ± 0.5 mV after forskolin. CFTRinh-172 produced 1.1 ± 0.9- and 4.3 ± 0.7-mV depolarizations when added after low Cl and forskolin, respectively. Systemically administered CFTRinh-172 reduced the forskolin-induced hyperpolarization from 4.7 ± 0.4 to 0.9 ± 0.1 mV but did not reduce the low Cl-induced hyperpolarization. Nasal PD was 12 ± 1 mV in CF mice after amiloride, changing by <0.5 mV after low Cl or forskolin. In pigs, nasal PD was 14 ± 3 mV after amiloride, hyperpolarizing by 13 ± 2 mV after low Cl and a further 9 ± 1 mV after forskolin. CFTRinh-172 and glibenclamide did not affect nasal PD in pigs. Our results suggest that cAMP-dependent nasal PDs in mice primarily involve CFTR-mediated Cl conductance, whereas cAMP-independent PDs are produced by a different, but CFTR-dependent, Cl channel. In pigs, CFTR may not be responsible for Cl channel-dependent nasal PDs. These results have important implications for interpreting nasal PDs in terms of CFTR function in animal models of CFTR activation and inhibition.
cystic fibrosis; cystic fibrosis transmembrane conductance regulator; nasal potential difference; chloride channels
NASAL POTENTIAL DIFFERENCE (PD) measurements have been used since the early 1980s as a relatively noninvasive functional assessment of cystic fibrosis transmembrane conductance regulator (CFTR) function as an epithelial Cl channel (1, 1315). As in the lower airways, the nose contains a secretory epithelium that generates a PD whose magnitude depends primarily on luminal membrane Na+ and Cl conductances and basolateral membrane K+ conductance and Na+/K+ pump activity. To interpret nasal PDs in terms of Cl channel function, the nasal mucosa is perfused topically with a physiological saline solution containing amiloride to reduce the influence of Na+ channels. The majority of Cl in the amiloride-containing solution is then replaced by a relatively impermeant anion such as gluconate to generate a diffusion potential (transepithelial hyperpolarization) that is generally interpreted as the cAMP-independent Cl conductance of the luminal membrane of the nasal epithelium. Addition of a cAMP agonist such as forskolin produces further hyperpolarization if cAMP-regulated Cl channels are activated. Typical hyperpolarizations of 12 and 8 mV are produced by low Cl and cAMP agonist addition, respectively, in normal humans, but <2 mV in cystic fibrosis (CF) subjects lacking functional CFTR (3, 15, 18, 31). However, the magnitude of the PD changes in CF humans correlates poorly with disease severity (6, 10, 26).
A similar protocol has been used to measure nasal PDs in mice. Baseline nasal PDs in mice in the range 7 to 27 mV have been reported, with hyperpolarizations of 317 mV and 111 mV produced by low Cl and cAMP agonist addition, respectively (4, 8, 9, 12). Various transport inhibitors have been used in an attempt to interpret mouse nasal PDs in terms of CFTR vs. non-CFTR Cl pathways. Brady and coworkers (4) suggested that the response to cAMP agonists is mediated mainly by CFTR, being sensitive to the Cl channel inhibitor diphenylamine-2-carboxylate (DPC) and the cAMP antagonist Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS), but not to DIDS, a Cl channel inhibitor with low CFTR specificity. In contrast, the response to low Cl was only partially reduced by Cl channel inhibitors (glibenclamide, DIDS, and DPC) and was not abolished by Rp-cAMPS (4). Nevertheless, the hyperpolarization caused by low Cl is absent or greatly reduced in CF (4, 14), suggesting that although the response to low Cl may not involve CFTR directly, it is CFTR dependent at some level. Nasal PDs have been used in mice to test various therapeutic strategies, including pharmacological modulation of
F508-CFTR biosynthesis and trafficking (7, 31), CFTR protein replacement (20), and CFTR gene transfer (11, 32).
Our lab established methodology for nasal PD measurements in mice and pigs to evaluate the utility of nasal PDs as minimally invasive indicators of CFTR function in CF animal models created using CFTR inhibitors. The pig was chosen as a large animal model because of its human-like airway/lung physiology. The thiazolidinone CFTR inhibitor CFTRinh-172 was used, having recently demonstrated its antidiarrheal efficacy in mice and rats (17, 24) and its antisecretory activity in submucosal glands of excised pig trachea (25). CFTRinh-172 reversibly inhibited CFTR Cl conductance in <2 min in a voltage-independent manner with Ki
300 nM (17, 23). In secretory diarrheas, CFTR is the common route for apical membrane Cl secretion. A single intraperitoneal injection of 20 µg of CFTRinh-172 in mice reduced by >90% cholera toxin-induced fluid secretion in the small intestine. In pig trachea, CFTRinh-172 reduced submucosal gland fluid secretions and increased secreted fluid viscosity and protein concentration, mimicking CF gland secretion properties (25). We reasoned that the use of a selective and potent CFTR inhibitor may permit clear-cut determination of the role of CFTR in nasal PDs, which has been problematic using the existing relatively nonselective inhibitors. The best CFTR inhibitor available until now for electrophysiological and other cell-based studies, glibenclamide, has been shown to inhibit other Cl transporters as well as K+ channels (5, 29). Here, the nasal PD method was optimized in mice and pigs by evaluation and selection of the details of the perfusion system, cannula positioning, and airway protection. We then investigated the ability of CFTRinh-172 to block CFTR-dependent potentials, comparing results to nasal PDs measured in CF mice and in normal mice/pigs using other CFTR activators such as genistein and inhibitors such as glibenclamide and DIDS. We obtained functional evidence for a substantial CFTR-independent Cl conductance that is not produced by CFTR itself but probably by a CFTR-regulated Cl channel. Our findings thus have implications for generation of CF animal models using inhibitors and for interpretation of nasal PDs in CF mutant mice treated with activators of mutant CFTRs.
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METHODS
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Animals.
Wild-type mice and CF mice (homozygous
F508 or G551D) were studied. All mice were backcrossed into a CD1 genetic background and bred at the University of California, San Francisco Animal Facility. Heterozygous mice were bred to generate homozygous mutant
F508 or G551D mutant mice. Wild-type mice were fed a standard diet, and the CF mice were fed Peptamen. Mice, equally distributed among males and females, were studied at age 1216 wk, with weights ranging from 20 to 25 g. A/J inbred mice were purchased from Jackson Laboratory and studied at age 12 wk (1820 g). Sixty-pound female Yucatan swine (Pork Power, Fresno, CA) were made available for nasal PD measurements after undergoing laparoscopic abdominal surgery for resident training. Animal protocols were approved by University of California, San Francisco Institutional Animal Care and Use Committee.
Compounds and solutions.
Baseline nasal PDs were measured during perfusion with PBS: (in mM) 136.5 NaCl, 2.68 KCl, 8 NaHPO4, 1.47 KH2PO4, 1 CaCl2, and 0.5 MgCl2 (pH 7.4). The majority of Cl was replaced by gluconate (final Cl 4.7 mM) in the "low-Cl" solution. Chemicals were purchased from Sigma (St. Louis, MO) if not stated otherwise. Amiloride (100 µM) was present in all perfusates after baseline measurement. The activators and inhibitors used in the various perfusates included 10 µM forskolin, 50 µM genistein (4',5,7-trihydroxy-isoflavone), 250 µM glibenclamide, 200 µM DIDS, 30 µM chromanol 293B (Tocris, Ellisville, MO), and 20 µM CFTRinh-172 (3-[(3-trifluoromethyl)phenyl]-5-[(3-carboxyphenyl) methylene]-2-thioxo-4-thiazolidinone), synthesized as described in Ref. 17.
Nasal PD measurements in mice.
PD was measured across murine nasal epithelia using a modification of protocols previously described (9). Mice were anesthetized using intraperitoneal ketamine (90120 mg/kg) and xylazine (510 mg/kg) and placed on a custom-made platform for orotracheal intubation. A 21-gauge angiocatheter was inserted into the trachea under direct visualization of the vocal cords by transillumination through the neck (Fig. 1A). Intubation generally took <10 s. After anesthesia and intubation, a PE-10 cannula, pulled to a tip diameter of 0.3 mm, was inserted using a micropositioner into one nostril 5 mm distal to the anterior nares. The tubing opening was cut at a 45° angle to maximize surface contact and positioned to make contact with the respiratory epithelium of the inferior turbinate. No further adjustments were made after cannula insertion. The cannula was connected though a 1 M KCl agar bridge to an Ag/AgCl electrode and a high-impedance digital voltmeter (input impedance 1012
; IsoMilivolt Meter, World Precision Instruments). The system electrical resistance was 1.1 x 106
. PD values were recorded at a rate of five samples per second using a 14-bit analog-to-digital converter. The nasal cannula was perfused with up to eight different solutions at 50 µl/min using dual microperfusion pumps (model 100 series; KD-Scientific) and a custom-built multireservoir perfusion apparatus. A winged 21-gauge needle filled with PBS was connected to a second Ag/AgCl electrode by a 1 M KCl agar bridge and inserted in the subcutaneous tissue in the abdomen as reference.

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Fig. 1. Potential difference (PD) recording methodology and characterization of cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor. A: mice were intubated using an orotracheal cannula. PE-10 tubing (attached to perfusion and electrical recording systems) was inserted into the right nostril. ET, endotracheal tube. (See text for details). B: schematic of PD recording showing PD changes after amiloride, followed by infusion of low-Cl solutions before and after addition of agonists (such as forskolin and genistein) and inhibitors (such as CFTRinh-172, glibenclamide, and DIDS). Amiloride was present in all solutions except for the initial and final "NaCl" solutions. PD changes ( PD) computed from individual curves for statistical comparisons are indicated. C: inhibition of CFTR-dependent short-circuit current (Isc) in primary cultures of tracheal epithelial cells from mouse (left) and pig (right). All solutions contained amiloride (10 µM). Where indicated, 8-(4-chlorophenylthio)-cAMP (CPT-cAMP; 0.1 mM) was added, followed by indicated concentrations of CFTRinh-172.
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Nasal PD protocols in mice.
For topical measurements, nasal PDs were measured continuously in response to infusion with PBS followed by various amiloride-containing solutions, including low-Cl solutions containing various agonists and/or inhibitors (Fig. 1B). See RESULTS and figure legends for individual protocols. For systemic administration, CFTRinh-172 was injected intraperitoneally (2 mg/kg) using a 1 mM solution in 12% DMSO and 88% ethanol. Vehicle controls were done as well.
Nasal PD measurements in pigs.
Pigs were anesthetized using intramuscular ketamine (20 mg/kg), xylazine (2 mg/kg), and atropine (0.04 mg/kg), followed by isoflurane (3.5% induction, 2.5% maintenance). An orotracheal tube was inserted as well as venous and arterial catheters. For nasal PD measurements, a 2-mm-inner-diameter silicon cannula (Tygon) was inserted into the nostril (at the canine level), 58 cm distal to the nares. The tubing opening was cut at a 45° angle and positioned to make contact with the nasal septum. Solutions were infused at a rate of 5 ml/min by gravity.
Data analysis.
Data are expressed as means ± SE. Statistical comparisons among groups were done using Student's t-test, with P < 0.05 considered as significant.
Primary tracheal cell cultures.
Mouse tracheas were obtained just after mice were killed by cervical dislocation, and tracheal epithelial cells were isolated and cultured as described (5). Tracheas from Yucatan swine were obtained from adult animals within 5 min postmortem. Swine tracheas were rinsed in ice-cold sterile PBS, and the surface epithelium was removed in strips and incubated overnight at 4°C in PBS containing protease (1 mg/ml) and antibiotics (penicillin, streptomycin, fungizone, and gentamicin). The following day, small sheets of cells were dislodged from the tissue strips, and cells were resuspended in PBS and separated by trypsin treatment. The trypsin was neutralized with the addition of medium containing 5% FCS, and cells were plated in Snapwells (0.4-µm pore size, Corning) coated previously with human placental collagen. Cells were grown in 2% Ultroser G serum substitute medium (BioSepra). The same procedure was used for mouse tracheal cells, except that the whole mouse trachea was immersed into the PBS-protease solution after rinsing.
Short-circuit current measurements.
Snapwell inserts containing mouse or pig tracheal cell monolayers with resistances >1,000
·cm2 were mounted in an Ussing chamber system (Easymount Chamber System; Physiologic Instruments, San Diego, CA). For studies in mouse cells, hemichambers were filled with Krebs solution as follows: (in mM) 120 NaCl, 25 NaHCO3, 3.3 KH2PO4, 0.8 K2HPO4, 1.2 MgCl2, 1.2 CaCl2, and 10 glucose (pH 7.3). For studies in pig cells, the basolateral membrane was permeabilized with amphotericin B (250 µg/ml), and in the apical solution 65 mM NaCl was replaced by sodium gluconate. Solutions were bubbled with 95% O2-5% CO2 and maintained at 37°C. Short-circuit current was recorded using a DVC-1000 voltage clamp (World Precision Instruments) with Ag/AgCl electrodes and 1 M KCl agar bridges.
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RESULTS
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The procedure for measurement of nasal PDs in mice was first optimized. We found that protection of the airway by orotracheal intubation eliminated the risk of fluid aspiration, substantially improved PD signal stability, and permitted full recovery from anesthesia after extubation (Fig. 1A). The intubation procedure, which involved transillumination of the vocal cords and catheter insertion, was completed in <10 s. Intranasal fluid infusion was done using a catheter inserted into the nares using a micropositioner, with the beveled tip positioned to make contact with the inferior turbinate. Constant flow at 50 µl/min, without interruption of flow during solution exchange, was also important for stable, reproducible recordings. Figure 1B shows the general experimental protocol. After recording baseline PD, there is initial amiloride-induced depolarization, followed by hyperpolarizations in response to the low-Cl solution and "agonist" (in the low-Cl solution), and depolarization in response to the "inhibitor" (also in the low-Cl solution). Amiloride was present in all low-Cl solutions. At the end of each experiment, PBS ("NaCl") was infused to verify minimal drift of baseline PD. In each experiment, the small (positive) junction potential resulting from the PBS vs. low-Cl solutions was measured (generally 23 mV) and used to correct PD values measured with low-Cl solutions.
To verify the efficacy of CFTRinh-172 for inhibition of mouse and pig CFTR, short-circuit current was measured on primary airway epithelial cell cultures from mouse and pig trachea. Representative data are provided in Fig. 1C, showing cAMP stimulation of short-circuit current and dose-dependent inhibition by CFTRinh-172. Measurements in pig cells were done after basolateral membrane permeabilization and in the presence of a Cl gradient because of poor stability in short-circuit current in nonpermeabilized cells. Ki values were in the range of 38 µM for the mouse and pig airway epithelial cell cultures. Subsequent nasal PD measurements were done using 20 µM CFTRinh-172.
The first set of nasal PD measurements was done to investigate inhibition of nasal PD by topical CFTRinh-172 in the absence of agonists. Representative original PD recordings from wild-type mice are shown in Fig. 2A. After amiloride-induced depolarization and low-Cl-induced hyperpolarization, inclusion of CFTRinh-172 or glibenclamide in the low-Cl solutions produced very small depolarizations, which were quite variable from mouse to mouse. DIDS produced a small but more consistent depolarization. Figure 2B summarizes averaged data from a series of mice, showing little inhibition of the large hyperpolarization produced by the low-Cl solution. To determine statistical significance, the changes in PD (
PD) were computed in individual mice, and averaged
PDs were compared with zero. Figure 2C summarizes
PDs for the low- Cl and inhibitor effects. There was no significant effect of CFTRinh-172 and glibenclamide, although there was a small but significant depolarization (inhibition) by DIDS. These results suggest that most or all of the hyperpolarization in nasal PD in response to infusion of a low-Cl solution does not involve Cl movement through CFTR.

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Fig. 2. Lack of CFTRinh-172 effect on agonist-independent nasal Cl conductance. A: original PD recordings showing responses to amiloride, low Cl, and the inhibitors CFTRinh-172 and DIDS (left) and glibenclamide (right). Inhibitor concentrations were 20 (CFTRinh-172), 200 (DIDS), and 250 (glibenclamide) µM. B: PD values (means ± SE, 4 mice) measured after indicated maneuvers deduced from experiments as in A. C: "paired" analysis of PD changes ( PD) measured after low Cl and inhibitor addition. *P < 0.01 for significant depolarization (inhibition).
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Similar studies were done with topical inhibitors given after CFTR activation by agonists. Figure 3A shows representative data where CFTR (and potentially other cAMP-dependent ion transporters) was activated by the cAMP agonist forskolin alone or forskolin followed by the flavone-type CFTR activator genistein. Forskolin and genistein produced hyperpolarizations in all wild-type mice. CFTRinh-172 application in the continued presence of agonist(s) resulted in consistent depolarization. Similar studies on CF mice did not show low Cl or agonist-induced hyperpolarizations or CFTRinh-172-induced depolarization. Figure 3B shows averaged PD values (after correction for junction potential). CFTRinh-172 reversed the agonist-induced hyperpolarization but had no effect on PD in CF mice. Figure 3C shows that by paired analysis, CFTRinh-172 produced significant depolarization after agonist additions (P < 0.001). The same protocol was used on A/J inbred mice reported to have a smaller response to low-Cl solution (4). A similar inhibitory effect was seen after forskolin as shown in Fig. 3C (P < 0.005). These results suggest that the forskolin- and genistein-induced hyperpolarizations in nasal PD involve Cl movement through CFTR.

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Fig. 3. CFTRinh-172 inhibition of CFTR agonist-dependent nasal Cl conductance. A: PD recordings showing response to amiloride, low Cl, agonists (forskolin, left; forskolin and genistein, right), and CFTRinh-172/DIDS. Concentrations were 10 (forskolin), 50 (genistein), 20 (CFTRinh-172), and 200 (DIDS) µM. Inset: PD measurement done on a cystic fibrosis (CF; G551D mutant) mouse. Data are representative of studies on 19 wild-type mice (forskolin protocol), 11 wild-type mice (forskolin/genistein protocol), and 9 CF mice (6 G551D, 3 F508). B: PD values (means ± SE) measured after indicated maneuvers deduced from experiments as in A. , Wild-type mice; , CF mice. C, left: paired analysis of PD for wild-type mice measured after agonist and inhibitor additions. *P < 0.001 for significant depolarization (inhibition). Right: PD (means ± SE) measured in 4 A/J mice (protocol as in A), with representative data shown as inset.
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The effect of systemically administered CFTRinh-172 on nasal PDs was studied, hypothesizing from the results above that the agonist-induced hyperpolarization may be blocked if the inhibitor penetrated sufficiently into the nasal epithelium. Previous studies showed that a single intraperitoneal injection of 2050 µg of CFTRinh-172 blocked cholera toxin-induced intestinal fluid secretion and that the inhibition persisted for several hours (24). On the basis of the results, nasal PD measurements were done at 23 h after CFTRinh-172 administration. Figure 4A shows representative nasal PD recordings using the same protocol as in Fig. 3 except that CFTRinh-172 was administered systemically rather than topically. The "vehicle" and "no vehicle" controls showed consistent hyperpolarization after low Cl and forskolin. However, the forskolin-induced hyperpolarization was remarkably reduced or absent after intraperitoneal CFTRinh-172 administration. Figure 4B summarizes
PD values for the low-Cl and forskolin-induced hyperpolarizations, showing no significant effect of CFTRinh-172 on the low-Cl response, but a significant reduction of the forskolin effect when CFTRinh-172 was administered at 23 h before nasal PD measurement. These results indicate that systemically administered CFTRinh-172 is able to penetrate into the nasal mucosa to inhibit CFTR and support the conclusion above that the forskolin-induced hyperpolarization in nasal PD involves CFTR Cl conductance.
Last, studies were done on pigs to investigate the utility of nasal PDs as a surrogate marker of CFTR activity in pig models of CF. The infusion protocols were first optimized for the pig nasal anatomy. Measurements were made in anesthetized pigs that were mechanically ventilated via an orotracheal tube. The infusion cannula and rates were similar to those used for human nasal PD measurements. Figure 5A shows excellent signal stability and signal-to-noise ratios for pig nasal PD recordings. Compared with nasal PDs in mice, there was remarkably greater hyperpolarization after forskolin, but little further hyperpolarization after genistein. Also, in contrast to mice, neither CFTRinh-172 nor glibenclamide produced depolarization after low Cl and agonists in any of the pigs studied, although DIDS produced a small depolarization in a few experiments, an example of which is shown. Figure 5B summarizes averaged PD values for the three different protocols used in the pig studies. The robust forskolin response is apparent as is the lack of effect of CFTRinh-172 and glibenclamide. The cAMP-dependent hyperpolarization could be due to the activation of an apical Cl channel or a basolateral cAMP-dependent K+ channel. However, the
PD analysis in Fig. 5C shows no effect on average of the Cl channel inhibitors CFTRinh-172, glibenclamide, or DIDS or (from separate experiments) an inhibitor (chromanol 239B) of cAMP-dependent K+ channels. These results provide functional evidence against CFTR-mediated Cl conductance in pig nasal mucosa.
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DISCUSSION
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The purpose of this study was to clarify the involvement of CFTR in nasal PD responses using the thiazolidinone CFTR inhibitor CFTRinh-172. CFTRinh-172 was identified by high-throughput screening of a collection of diverse small molecules followed by testing of thiazolidinone analogs (16). CFTRinh-172 inhibited CFTR Cl conductance reversibly with Kd of 0.20.3 µM by a mechanism that probably involves direct binding to a critical site on one of the CFTR cytoplasmic domains. At concentrations of CFTRinh-172 where CFTR is >95% inhibited (10 µM), there was no inhibition of other ATP-binding cassette transporters (MDR-1, SUR) or Cl channels (Ca2+ and volume-activated) tested (17). In mice, a single intraperitoneal injection of CFTRinh-172 reduced by >90% cholera toxin-induced fluid secretion in a closed-loop model of secretory diarrhea (24). Pharmacokinetic analysis in rodents indicated a large distribution volume with slow elimination by renal excretion with little metabolism (24). In addition, systemically administered CFTRinh-172 accumulates in liver and intestine by an enterohepatic recirculation mechanism but does not preferentially accumulate in lung or airways. CFTRinh-172 has been used in mice to investigate the involvement of CFTR in the depth and composition of airway surface liquid in distal airways (22) and in pigs to study the role of CFTR in airway submucosal gland fluid secretion (25). We reasoned that topical and systemic application of CFTRinh-172 could help resolve the uncertainty about the involvement of CFTR in the generation of nasal PD.
A central finding of this study was that CFTR inhibition blocked the cAMP-dependent but not the cAMP-independent hyperpolarization in nasal PD. To ensure effective CFTR inhibition, a relatively high concentration of CFTRinh-172 (20 µM) was used in the topical studies where CFTRinh-172 was added to the perfusate. We found previously that although the Kd for CFTR inhibition by CFTRinh-172 was 0.20.3 µM in patch-clamp studies and in short-circuit current measurements after basolateral membrane permeabilization in a variety of cell types, Kd in intact cells ranged from 0.2 µM to several micromolar (17, 24). The greater Kd in intact cells is probably a consequence of reduced intracellular concentrations of CFTRinh-172 because of the interior-negative membrane potential (CFTRinh-172 is negatively charged at physiological pH). The inability of CFTRinh-172 and glibenclamide to depolarize nasal PD after amiloride application and hyperpolarization by a low-Cl perfusate indicates that the low-Cl-induced hyperpolarization does not involve Cl movement through CFTR. However, this conclusion must be reconciled with the observation here and from several laboratories that the low-Cl induced hyperpolarization is absent in CF mice (CFTR null or
F508 or G551D) (4, 14). Two possible explanations, which cannot be resolved here, include regulation by CFTR of a non-CFTR Cl channel responsible for the low-Cl depolarization and downregulation of a non-CFTR Cl channel in CF mice. In any case, our results provide functional evidence for involvement of a non-CFTR Cl channel in the generation of nasal PD in mice.
The topical and systemic studies of CFTRinh-172 administration provide evidence that the hyperpolarization in nasal PD after CFTR agonists involves Cl movement through CFTR. Hyperpolarizations induced by forskolin and genistein were reversed by topical CFTRinh-172, and systemically administered CFTRinh-172 blocked the forskolin-induced hyperpolarization in nasal PD. Because of the less efficient accumulation of CFTRinh-172 in nasotracheal epithelium compared with liver, intestine, and kidney, we used a high dose of CFTRinh-172 for intraperitoneal administration (2 mg/kg
50 µg), as initial studies showed only small effects of a single intraperitoneal dose of 20 µg of CFTRinh-172 (an effective dose in inhibiting intestinal fluid secretion). These results thus support a direct role of CFTR in generating nasal PDs in mice, albeit only after CFTR is activated by agonists.
Several technical refinements substantially improved nasal PD signal stability and reproducibility compared with initial measurements and published recordings. Protection of the airways by orotracheal intubation prevented fluid aspiration events that often resulted in movement artifacts and mortality. The intubation method was fast and atraumatic and permitted full recovery of mice. Strict positioning of a 0.3-mm-diameter plastic microcatheter using a 4-axis micropositioner also improved PD signal stability and reproducibility from mouse to mouse, as did constant fluid infusion (50 µl/min) without interruption during fluid exchange. These technical improvements should be useful in evaluating various drug and gene therapies in CF mice where changes in nasal PDs may be quite subtle.
Nasal PDs in pigs were easily measured and quite stable and robust. Unexpectedly, neither CFTRinh-172 nor glibenclamide altered nasal PDs in pigs even after cAMP agonists and genistein. This observation provides functional evidence for the lack of involvement of CFTR in nasal PDs in pigs. The channel(s) responsible for the relatively large forskolin-dependent polarization in nasal PD after infusion of a low-Cl solution was not determined in the study here, since neither Cl channel inhibitors nor a cAMP-dependent K+ channel inhibitor caused significant depolarization. In any case, our findings indicate that measurements of nasal PDs will not provide a useful surrogate marker for CFTR activity in a CF pig model developed using CFTR inhibitors. Functional measurements in lower airways will be needed, as it has been demonstrated that CFTRinh-172 and glibenclamide inhibit Cl transport in pig trachea and airway submucosal glands (2, 25).
In summary, our results provide evidence that CFTR is the Cl-carrying channel responsible for nasal potential hyperpolarization after CFTR agonists. However, CFTR does not itself carry Cl during the hyperpolarization in response to a low-Cl solution in the absence of CFTR agonists. Whether the non-CFTR Cl channel is regulated by CFTR or downregulated in CF mice remains an unanswered question with important consequences for the interpretation of nasal PD measurements in CF mice after CFTR gene replacement or treatment with small-molecule activators of human disease-causing CFTR mutants.
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GRANTS
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This work was supported by Research Development Program and Drug Discovery grants from the Cystic Fibrosis Foundation and National Institutes of Health Grants HL-73856, HL-59198, EB-00415, EY-13574, and DK-35124. N. Pedemonte received partial support from the Fondazione Italiana per la Ricerca sulla Fibrosi Cistica.
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ACKNOWLEDGMENTS
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We thank Dr. Luis Galietta for critical reading of the manuscript and Drs. Ronald Baireuther, Lawrence Way, and Marshall Stoller for assistance with nasal potential difference measurements in pigs.
Present address of N. Pedemonte: Laboratory of Molecular Genetics, Giannina Gaslini Institute, Genova 16148, Italy.
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FOOTNOTES
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Address for reprint requests and other correspondence: A. S. Verkman, 1246 Health Sciences East Tower, Cardiovascular Research Institute, Univ. of California, San Francisco, CA 94143-0521 (E-mail: verkman{at}itsa.ucsf.edu)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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