Chlorzoxazone or 1-EBIO increases Na+ absorption across cystic fibrosis airway epithelial cells

Lin Gao1, James R. Yankaskas2, Catherine M. Fuller3, Eric J. Sorscher3,4, Sadis Matalon3,5,6, Henry Jay Forman1, and Charles J. Venglarik1

Departments of 1 Environmental Health Sciences, 3 Physiology and Biophysics, 4 Medicine, 5 Anesthesiology, and 6 Comparative Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005; and 2 Department of Pulmonary and Critical Care Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7248


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previous studies demonstrated that chlorzoxazone or 1-ethyl-2-benzimidazolinone (1-EBIO) enhances transepithelial Cl- secretion by increasing basolateral K+ conductance (GK) (Singh AK, Devor DC, Gerlach AC, Gondor M, Pilewski JM, and Bridges RJ. J Pharmacol Exp Ther 292: 778-787, 2000). Hence these compounds may be useful to treat cystic fibrosis (CF) airway disease. The goal of the present study was to determine whether chlorzoxazone or 1-EBIO altered ion transport across Delta F508-CF transmembrane conductance regulator homozygous CFT1 airway cells. CFT1 monolayers exhibited a basal short-circuit current that was abolished by apical amiloride (inhibition constant 320 nM) as expected for Na+ absorption. The addition of chlorzoxazone (400 µM) or 1-EBIO (2 mM) increased the amiloride-sensitive Isc ~2.5-fold. This overlapping specificity may preclude use of these compounds as CF therapeutics. Assaying for changes in the basolateral GK with a K+ gradient plus the pore-forming antibiotic amphotericin B revealed that chlorzoxazone or 1-EBIO evoked an ~10-fold increase in clotrimazole-sensitive GK. In contrast, chlorzoxazone did not alter epithelial Na+ channel-mediated currents across basolateral-permeabilized monolayers or in Xenopus oocytes. These data further suggest that alterations in basolateral GK alone can modulate epithelial Na+ transport.

epithelial sodium channel; cystic fibrosis transmembrane conductance regulator; clotrimazole; amiloride; 1-ethyl-2-benzimidazolinone


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CYSTIC FIBROSIS (CF) is a common inherited disease caused by mutations in the gene coding for a cAMP-regulated epithelial anion channel called the CF transmembrane conductance regulator (CFTR) (28, 38). CF patients have impaired CFTR-mediated Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion and enhanced amiloride-sensitive Na+ absorption across the small airways (27). This causes an imbalance in the CF airway epithelial lining fluid composition that decreases mucociliary clearance and increases the susceptibility to bacterial infection (5). Hence one potential strategy to treat CF airway disease is to enhance anion secretion and/or reduce Na+ absorption pharmacologically.

Basolateral K+ conductance (GK) can play an important role in modulating vectorial Cl- secretion (see Ref. 7 for a review). K+ recycling via basolateral GK is essential to maintain the mass and charge balance across the Cl--secreting epithelial cell. Furthermore, increasing the GK will hyperpolarize the plasma membrane potential and thereby increase the driving force for Cl- efflux via CFTR or other apical anion channels. Hence basolateral K+ channels provide a target for therapeutics to overcome the CF Cl- secretory defect (32). In this regard, two potential lead compounds have been identified, namely, 1-ethyl-2-benzimidazolinone (1-EBIO) and chlorzoxazone (9, 10, 12, 31). Previous studies (9, 10, 12, 31, 34) showed that these compounds augment electrogenic Cl- secretion across CFTR-expressing epithelial cells by activating small- and intermediate-conductance Ca2+-activated K+ channels in the basolateral membranes. Because basolateral GK is also important in Na+ absorption (7, 35), it is possible that these compounds may increase amiloride-sensitive Na+ absorption. However, previous reports (6, 8-10, 12, 19, 25, 31, 32) have focused on the ability of 1-EBIO or chlorzoxazone to enhance Cl- secretion across cells expressing wild-type CFTR.

The goal of the present study was to quantify the possible effects of 1-EBIO or chlorzoxazone on ion transport across CFT1 airway cells that are homozygous for the Delta F508 mutation in the CFTR (39). We grew CFT1 cells as monolayers on filters and mounted them in Ussing chambers. We observed that the addition of either chlorzoxazone or 1-EBIO evoked an ~2.5- fold increase in amiloride-sensitive Na+ absorption. Next, we investigated the possibility that the ability of 1-EBIO and chlorzoxazone to increase Na+ absorption was mediated by an increase in basolateral GK. This was indeed the case. Finally, the addition of chlorzoxazone to either CFT1 monolayers treated with basolateral amphotericin B in the presence of an apical-to-basolateral Na+ gradient or Xenopus oocytes injected with cRNA for ENaC alpha -, beta -, and gamma -subunits elicited little enhancement of the amiloride-sensitive current. Together, these data indicate that basolateral K+ permeability can play an important role in modulating active Na+ absorption across airway epithelial cells. Furthermore, because enhanced Na+ absorption is thought to contribute to CF airway disease (27), caution should be observed before administration of these compounds to patients.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. The CFT1 cell line was derived from the trachea of a CF patient and is homozygous for Delta F508-CFTR (39). The clone 2 CFT1 cells used herein were cultured as previously described (14, 15, 39). Briefly, the cells were maintained in serum-free Ham's F-12 medium supplemented with seven hormones and growth factors: insulin (5 µg/ml), endothelial cell growth supplement (3.7 µg/ml), transferrin (5 µg/ml), epidermal growth factor (25 ng/ml), triiodothyronine (3 × 10-8 M), hydrocortisone (1 × 10-6 M), and cholera toxin (10 ng/ml) (15). Before being seeded on filters, CFT1 cells were passaged into serum-free F-12 medium formulated without cholera toxin. Confluent monolayers for Ussing chamber experiments were obtained by plating the cells on collagen-coated filter inserts (Falcon, Becton Dickinson, Franklin Lakes, NJ) at a density of 1.2 × 105 cells/cm2. On the day after seeding, the culture medium was replaced with the Ham's F-12 medium without cholera toxin diluted 1:1 with NIH/3T3 cell-conditioned DMEM containing 2% fetal bovine serum. Ussing chamber experiments were performed 7-10 days after seeding because a previous study (15) indicated that the transepithelial resistance was maximal during this period (15).

Epithelial voltage clamp. Filter inserts containing confluent monolayers were mounted in modified Ussing chambers (Jim's Instrument, Iowa City, IA). Unless otherwise specified, the cells were bathed on both sides with identical HEPES-buffered saline solutions containing (in mM) 128 NaCl, 5 sodium pyruvate, 4 KCl, 1 CaCl2, 1 MgCl2, 5 D-glucose, and 5 HEPES-NaOH (pH 7.4). The bath temperature was maintained at 37°C, and both solutions were stirred and gassed with room air. Short-circuit current (Isc) measurements were obtained with an epithelial voltage clamp (Physiologic Instruments VCC600, San Diego, CA) as previously described (1, 35, 36). A voltage pulse was imposed every 100 s to monitor the transepithelial resistance, which was calculated with Ohm's law. Amiloride (100 µM) was added to the apical solution to abolish the Isc component due to Na+ absorption (3). In one series of experiments, amiloride was added apically in increasing concentrations from 1 nM to 30 µM to determine the affinity of amiloride for the underlying Na+ channel.

In experiments designed to assay for changes in basolateral GK, transepithelial ion gradients were created by replacing the apical bath with a high-K+ solution that contained (in mM) 132 potassium gluconate, 1 CaCl2, 1 MgCl2, 5 D-glucose, and 5 HEPES-KOH (pH 7.4). Most of the apical bath Cl- was replaced by the impermeant anion gluconate to prevent cell swelling after amphotericin B treatment (17). The basolateral bathing solution was also formulated with low Cl- and contained (in mM) 128 sodium gluconate, 1 CaCl2, 1 MgCl2, D-glucose, and 5 HEPES-NaOH (pH 7.4). Amphotericin B (10 µM) was then added to the apical potassium gluconate solution in 0.3% DMSO. The pores formed by this polyene antibiotic functionally eliminate the apical membrane as a barrier to cation flow, and the resulting Isc provides a measure of the basolateral GK (17, 18, 21, 35). We added the K+ channel blocker clotrimazole to identify channel-mediated currents (8, 11, 16, 26, 29).

RNA synthesis. The pSPORT plasmid (GIBCO BRL, Life Technologies, Gaithersburg, MD) containing either rat alpha -, beta -, or gamma -ENaC (a generous gift from Dr. B. Rossier, University of Lausanne, Lausanne, Switzerland) was linearized by overnight incubation with NotI (Promega, Madison, WI), digested with 1% SDS and 200 µg/ml of proteinase K for 60 min at 50°C, extracted with phenol-chloroform, and precipitated. Full-length wild-type CFTR contained in pBluescript was treated similarly, with the exception that the plasmid was linearized with EcoRV. Sense RNA from each transcript was in vitro transcribed from precipitated plasmid DNA with a T7 mMessage Machine kit according to the manufacturer's instructions (Ambion, Austin, TX). The integrity and size of the resulting cRNAs were verified by electrophoresis through denaturing 1% agarose-formaldehyde gels.

Expression of ENaC or CFTR in oocytes and two-electrode voltage clamp. These methods have been previously described in detail (13). Briefly, partial ovariectomies were performed on anesthetized female Xenopus laevis toads (Xenopus Express, Beverly Hills, FL). Oocytes were manually defolliculated and incubated overnight in L15 medium diluted 1:1 with deionized water supplemented with penicillin (200 U/ml) and streptomycin (200 µg/ml). On the following day, the oocytes were injected with 8.3 ng of each rat ENaC subunit cRNA (25 ng of total cRNA) or 25 ng of CFTR cRNA in a 50-nl volume with a Nanoject microinjector (Drummond, Broomall, PA). Currents were evaluated 36-48 h after injection with a two-electrode voltage clamp (Geneclamp 500, Axon Instruments, Foster City, CA). The oocytes were bathed in a solution containing (in mM) 96 NaCl, 2 KCl, 1 MgCl2, 0.2 CaCl2, and 15 HEPES-NaOH (pH 7.6). Voltage was clamped to 0 mV, and inward and outward currents were recorded at 20-s intervals at holding potentials of ±100 mV. The amiloride-sensitive current was calculated by subtracting the inward current recorded after the addition of 10 µM amiloride from that before the addition.

Chemicals. Amphotericin B and 1-EBIO were the generous gifts from Drs. S. Lucania (Bristol-Myers Squibb, Princeton, NJ) and J. Tomich (Kansas State University, Manhattan, KS), respectively. Amiloride and clotrimazole were purchased from Sigma. Chlorzoxazone was obtained from Aldrich. All drugs were added to the Ussing chamber solutions in a small volume of concentrated stock. Stock solutions of 1-EBIO (0.1 M), chlorzoxazone (0.1 M), and amphotericin B (3 mM) were prepared in DMSO. Clotrimazole and amiloride stocks were made in ethanol (10 mM) and water (10 µM to 10 mM), respectively. Because of their lipophilic nature, 1-EBIO, chlorzoxazone, and clotrimazole were added to both sides of the monolayers (10, 11, 31).

Data analysis. The data are means ± SD; n is the number of different experiments. Curve fits were performed with the least squares routine in SigmaPlot (version 5.0 for Windows, SPSS Science). All dose-response relationships were initially fit to single-site Michaelis-Menten functions for activation {Isc = (Isc)max · [D]/([D] + Ks)} or inhibition {Isc = (Isc)max · Ki/([D] + Ki)}, where (Isc)max is the maximal Isc and the equilibrium constants Ks and Ki represent the concentration of drug ([D]) causing 50% stimulation or 50% inhibition, respectively. The Hill equation Isc = (Isc)max · [D<SUP><IT>n</IT><SUB>H</SUB></SUP>]/([D<SUP><IT>n</IT><SUB>H</SUB></SUP>}] K<UP><SUB>s</SUB><SUP><IT>n</IT><SUB>H</SUB></SUP></UP>) was used to fit the activation data having a slope >1 as defined by the Hill coefficient (nH) (30). Significance was determined by paired t-test analysis with P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chlorzoxazone or 1-EBIO increases amiloride-sensitive Isc across CFT1 monolayers. Filters containing CFT1 cells were mounted in Ussing chambers and bathed on both sides with standard HEPES-buffered saline solutions. CFT1 monolayers exhibited a basal or resting Isc of 4.0 ± 1.5 µA/cm2 and transepithelial resistance of 119 ± 22 Omega  · cm2 (n = 13). Figure 1 compares a representative current record after chlorzoxazone addition to a paired control. The addition of 600 µM chlorzoxazone caused the Isc across intact cells to increase ~2.5-fold (from 3.6 ± 1.5 to 9.4 ± 1.0 µA/cm2; n = 7). Subsequent addition of the Na+ channel blocker amiloride (100 µM) to both apical bath solutions abolished the basal and chlorzoxazone-stimulated currents. Active Na+ absorption mediated the Isc response to chlorzoxazone because it was eliminated by amiloride pretreatment (data not shown; n = 4; see Ref. 14). 1-EBIO was less potent than chlorzoxazone, requiring 2 mM to evoke a similar enhancement in amiloride-sensitive Isc (Fig. 2A). On average, the addition of 2 mM 1-EBIO caused Isc to increase from 4.4 ± 1.4 to 10.5 ± 2.7 µA/cm2 (n = 6). To quantify the dose-response relationships, we added 1-EBIO or chlorzoxazone in increasing concentrations as illustrated by the representative tracing shown in Fig. 2A. These data are summarized in Fig. 2B. Both data sets were poorly described by simple Michaelis-Menten activation functions, and the curves in Fig. 2B depict the least squares fits to the Hill equation (30). These data show that chlorzoxazone (Ks = 330 ± 21 µM) was a slightly more potent activator of Na+ absorption than 1-EBIO (Ks = 444 ± 48 µM). This Ks for 1-EBIO is similar to the value (490 µM) previously reported for the enhancement of Cl- secretion (10). The plots also show a twofold difference in the Hill coefficients for chlorzoxazone (nH = 5) vs. 1-EBIO (nH = 2.6). This analysis yields some mechanistic insights that are considered in DISCUSSION.


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 1.   Representative short-circuit current (Isc) records showing the response of a CFT1 monolayer after chlorzoxazone addition (dashed line) compared with a paired control (solid line). CFT1 cells were cultured on filters, mounted in Ussing chambers, bathed with symmetrical NaCl-containing solutions, and voltage clamped as described in MATERIALS AND METHODS. Chlorzoxazone (600 µM) was then added to both sides of 1 monolayer (left arrow) and was present continuously in the bathing solutions thereafter. Amiloride (100 µM) was subsequently added to both apical solutions (right 2 arrows) to abolish the Isc component due to active Na+ absorption (2, 3).



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 2.   Dose dependence of the 1-ethyl-2-benzimidazolinone (1-EBIO)- or chlorzoxazone-induced increase in amiloride-sensitive Isc (INa) across CFT1 monolayers. A: representative Isc tracing. Experiments were performed as described in Fig. 1 except for a stepwise increase in 1-EBIO concentration as indicated. B: dose-response relationships for 1-EBIO and chlorzoxazone. The curves depict the best fits to the Hill equation (30). Data are means ± SD; n = 4 different experiments/group.

We routinely added 100 µM amiloride to abolish the Isc due to active Na+ absorption as shown in Figs. 1 and 2A. The final series of experiments with intact CFT1 cells was designed to quantify the concentration-response relationship for amiloride blockade. These experiments were performed as described in MATERIALS AND METHODS, and the data are summarized in Fig. 3. The curves illustrate the fits to single-site Michaelis-Menten inhibition functions by nonlinear least squares regression. Amiloride blocked Isc with a high affinity for both the basal and chlorzoxazone-stimulated currents, with Ki values of 320 ± 27 and 245 ± 31 nM, respectively. This high affinity implicates the ENaC as mediating the Isc across cells (2). Furthermore, the Ki values were significantly different. Because amiloride blockade is voltage dependent (37), it is possible that the increased affinity results from membrane hyperpolarization after activation of basolateral K+ channels by chlorzoxazone.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 3.   Log dose-response relationships for inhibition of basal and chlorzoxazone-stimulated Isc by amiloride (n = 4 different experiments/group). Experiments were performed on CFT1 monolayers as described in Fig. 1 except for the stepwise addition of the Na+ channel blocker amiloride at the indicated concentrations ([amiloride]). One group of monolayers was pretreated with 400 µM chlorzoxazone. The curves show the best fits of the data to single-site Michaelis-Menten inhibition functions (30).

Chlorzoxazone or 1-EBIO augments the basolateral GK in CFT1 cells. Previous studies indicated that 1-EBIO and chlorzoxazone enhance transepithelial Cl- secretion by increasing the basolateral GK. Because active Na+ absorption also depends on the GK of the basolateral membranes (7, 35), it is likely that these compounds alter Na+ absorption by a similar mechanism. We tested this hypothesis using the technique described by Germann and colleagues (17, 18), Kirk and Dawson (21), and Venglarik and Dawson (35) where a K+ gradient is imposed across the monolayer and 10 µM amphotericin B is added to eliminate the apical membrane a barrier to the flow of monovalent cations. Figure 4 shows representative current records. An apical-to-basolateral K+ gradient was imposed across both monolayers before the start of the recordings as described in MATERIALS AND METHODS. Amphotericin B was then added to the apical bathing solution, which revealed a modest basal current. The subsequent addition of chlorzoxazone (500 µM) to both sides of one monolayer caused the Isc to increase approximately sevenfold. Addition of the K+ channel blocker clotrimazole (100 µM) to both bathing solutions eliminated most of the basal and chlorzoxazone-stimulated currents as illustrated by the representative traces in Fig. 4. 1-EBIO evoked similar increases in Isc as chlorzoxazone, although higher concentrations were required (Fig. 5). The concentration-response relationships for Isc activation were determined by exposing monolayers to increasing concentrations of 1-EBIO or chlorzoxazone as illustrated by the representative current trace shown in Fig. 5A. These data are summarized in Fig. 5B. Clotrimazole (100 µM) was used to block and thereby identify the channel-mediated K+ current (IK). The curves show the best fits to the Hill equation by nonlinear regression. Chlorzoxazone was a slightly more potent stimulator (Ks = 273 ± 5 µM) and had a twofold larger Hill coefficient (nH = 3.5) than 1-EBIO (Ks = 1,060 ± 282 µM; nH = 1.7). These results generally agree with the dose dependence of Isc activation in intact monolayers shown in Fig. 2. Oddly, both chlorzoxazone and 1-EBIO exerted a biphasic effect on IK, increasing it at low concentrations while inhibiting it at higher concentrations. This complicated dose dependency shows that it is important to compare the efficacy between K+ channel activators over a range of concentrations. Previously, 1-EBIO was reported to be more potent than chlorzoxazone based on the response to 1 mM chlorzoxazone (31). Although this conclusion is consistent with the results obtained at 1 mM chlorzoxazone as shown in Fig. 5B, our data show that chlorzoxazone was more potent than 1-EBIO throughout the lower concentration range.


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 4.   Representative Isc recordings illustrating the ability of chlorzoxazone to increase K+ current (IK) across an amphotericin B (ampho)-permeabilized CFT1 monolayer (dashed line) compared with a paired control (solid line). Filter-grown CFT1 cells were mounted in Ussing chambers in the presence of an apical-to-basolateral K+ gradient (135/4 mM) as described in MATERIALS AND METHODS. The pore-forming antibiotic amphotericin B was added to both apical bathing solutions (10 µM; left 2 arrows) to eliminate the apical membrane as a barrier to cation flow. Under these conditions, the Isc provides a measure of the basolateral K+ conductance (17, 18, 21, 35). Chlorzoxazone (500 µM) was then added to both sides of 1 monolayer at the time indicated (middle arrow). Clotrimazole (100 µM) was subsequently added to all solutions (right 2 arrows) because it was shown to block the 1-EBIO-activated basolateral K+ channels in Cl--secreting epithelial cells (11, 16, 26, 29).



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 5.   Dose dependence for 1-EBIO or chlorzoxazone activation of IK across CFT1 monolayers. A: representative Isc tracing. Experiments were performed as described in Fig. 4 except for the addition of 1-EBIO at increasing concentrations as indicated. B: dose-response relationships for 1-EBIO and chlorzoxazone. Clotrimazole (100 µM) was added to abolish the portion of IK mediated by basolateral small- or intermediate-conductance Ca2+-activated K+ channels (34). Curves show the best fits to the Hill equation. Values are means ± SD from 3 (chlorzoxazone) or 4 (1-EBIO) experiments. IK was reduced at the highest concentrations of 1-EBIO or chlorzoxazone tested. These data were not included in the dose-response analysis.

Finally, Fig. 6 shows the dose-response relationship for the inhibition of chlorzoxazone-stimulated IK by clotrimazole. The curve shows the best fit of the data to a simple Michaelis-Menten inhibition function, which yielded a Ki of 2.4 ± 0.4 µM. These data are consistent with the expected results for blockade of small- and intermediate-conductance Ca+2-activated K+ channels (11, 16, 26, 29, 34).


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 6.   Log dose response for inhibition of 1-EBIO-stimulated IK across CFT1 cells by clotrimazole. Experiments (n = 4) were performed as described in Fig. 4. 1-EBIO was added to increase IK (2 mM both sides) followed by addition of increasing concentrations of clotrimazole ([clotrimazole]; both sides; data not shown). The curve shows the best fit of the data to a simple Michaelis-Menten inhibition function, which yielded an inhibition constant of 2.4 µM.

Effect of chlorzoxazone on ENaC- or CFTR-mediated currents in oocytes. Previous studies (9, 31) showed that 1-EBIO and chlorzoxazone enhance transepithelial Cl- secretion by increasing both apical CFTR-mediated Cl- conductance and basolateral GK. Thus we considered the possibility that these compounds may also increase the apical ENaC channel activity in CFT1 cells. Initially, we tested for possible effects of chlorzoxazone on Isc across CFT1 monolayers exposed to an apical-to-basolateral Na+ gradient and treated with basolateral amphotericin B (200 µM). In these experiments, we observed a small Isc (3.1 µA/cm2) that was amiloride sensitive, and chlorzoxazone addition (400 µM) had no effect (n = 3). However, as a further test for possible activation of ENaC by chlorzoxazone, we expressed rat ENaC alpha -, beta -, and gamma -subunits in Xenopus oocytes and measured the amiloride-sensitive currents before and after chlorzoxazone. Oocytes expressing human wild-type CFTR were used as a control. The results are summarized in Fig. 7. Figure 7A compares amiloride-sensitive inward current (Iin) in ENaC-injected oocytes (n = 5) with the inward current from cAMP-stimulated CFTR-injected oocytes (n = 3) before and after the addition of chlorzoxazone (500 µM). Chlorzoxazone had only a small effect on the amiloride-sensitive currents while significantly increasing the current in CFTR-injected oocytes. The ability of chlorzoxazone to increase CFTR-mediated current is consistent with previous reports (9, 31). Figure 7B shows the dose response for CFTR activation as measured by the change in (Delta ) Iin. The solid line in Fig. 7B shows the best fit of the data to a Hill plot, which yielded a Ks of 377 ± 19 µM and a nH of 2.76 ± 0.4. 


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 7.   Effect of chlorzoxazone on epithelial Na+ channel (ENaC)- or cystic fibrosis transmembrane conductance regulator (CFTR)-mediated currents in Xenopus oocytes. A: amiloride-sensitive current in oocytes (n = 5 experiments) injected with rat alpha -, beta -, and gamma -ENaCs before (-) and after (+) chlorzoxazone (500 µM). We also tested for possible effects of chlorzoxazone on oocytes injected with CFTR (n = 3 experiments). CFTR-expressing oocytes were prestimulated with forskolin (10 µM) plus 8-(4-chlorophenylthio)-cAMP (200 µM) before chlorzoxazone addition. * Significant increase in inward current. B: dose dependence for activation of current in oocytes expressing CFTR. Solid line, best fit of these data to a Hill plot; , results from single experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The data presented here demonstrate that the addition of chlorzoxazone or 1-EBIO augments Na+ absorption across immortalized CF airway cells and that this response is mediated by an ~10-fold increase in basolateral GK. Although it is possible that chlorzoxazone or 1-EBIO may also increase apical Na+ channel activity, we consider it unlikely for two reasons. First, chlorzoxazone (500 µM) did not alter the amiloride-sensitive currents across CFT1 monolayers, with an apical-to-basolateral Na+ gradient in the presence of basolateral amphotericin B. Second, chlorzoxazone (500 µM) did not affect the amiloride-sensitive currents in Xenopus oocytes expressing alpha -, beta -, and gamma -subunits of the ENaC. In contrast, chlorzoxazone increased Cl- current across oocytes expressing CFTR. This enhancement of CFTR-mediated current is expected based on the known effects of these drugs on CFTR in permeabilized monolayers (9, 31) and also on the ability of these drugs to activate the K+ channels expressed in Xenopus oocytes (34). Thus we consider it likely that the ability of chlorzoxazone or 1-EBIO to augment Na+ absorption is mediated entirely by the increase in basolateral GK. This hypothesis is consistent with the reported effects of chlorzoxazone and 1-EBIO on basolateral K+ channels in Cl--secretory epithelial cells. It is also consistent with the obligatory coupling of apical Na+ and basolateral K+ currents imposed by the Koefoed-Johnson Ussing model for Na+ absorption (22; for a review, see Ref. 7).

Because the apical membrane resistance is 4- to 10-fold higher than that of the basolateral membrane, it is often assumed that changes in amiloride-sensitive Na+ channel activity alone account for agonist-induced alterations in epithelial Na+ absorption (2, 4, 33). This notion would be true if resistance were the only element determining the rate of ionic flow across the two plasma membranes in series. However, driving forces also play a crucial role. To maintain steady-state conditions, the higher resistance of the apical membrane must be precisely balanced by an increased electrochemical gradient for Na+ entry. This difference in driving force arises in part from the cell membrane potential, which favors Na+ entry and opposes K+ exit. Hence both membranes play an important role in determining the rate of transepithelial Na+ absorption (for a review, see Ref. 7). For example, Na+ absorption can be inhibited by K+ channel blockers such as Ba2+ (35) and clotrimazole (Venglarik, unpublished observations). Our data provide additional support for this notion by showing that known K+ channel activators promote transepithelial Na+ absorption across CFT1 airway cells. Chlorzoxazone and 1-EBIO should provide further insights regarding the role of basolateral GK in regulating Na+ absorption in future studies.

Furthermore, the observation that chlorzoxazone and 1-EBIO can increase Na+ absorption in addition to activating Cl- secretion also agrees with earlier studies (23, 24) showing that ENaC- and CFTR-mediated currents are present simultaneously in whole cell recordings of airway epithelial cells. If ENaC and CFTR are expressed in the same cell or in electrically coupled cells, then K+ channel openers such as chlorzoxazone or 1-EBIO are expected to increase both Na+ absorption and Cl- secretion (7). Although there is some evidence that 1-EBIO increases Na+ absorption across primary cultures of human bronchial epithelial cells (8), most previous reports (6, 8-10, 12, 19, 25, 31, 32) have focused on the ability of these compounds to enhance Cl- secretion via wild-type CFTR. In this regard, the CFT1 cell line (39) provides a unique model to study airway epithelial Na+ absorption without the confounding effects of CFTR-mediated anion secretion. Furthermore, data obtained with CFT1 cells are expected to be more relevant to CF patients. Because Na+ hyperabsorption may contribute to CF airway disease (27), our data suggest that caution should be observed before administering chlorzoxazone, 1-EBIO, or related compounds (32) to CF patients.

Finally, the mechanisms mediating K+ channel activation by benzimidazolinones or benzoxazolones remain unknown. However, our analysis of the dose-response relationships provides some insight. Specifically, all the activation data were described by Hill plots with coefficients > 1. This implies some form of signal amplification. In the case of hemoglobin, there are multiple binding sites that cooperatively increase affinity on increased occupancy (20). However, the Hill model per se cannot account for the approximately twofold difference in coefficients between 1-EBIO and chlorzoxazone. Together, our data indicate that K+ channel activation is mediated by a mechanism involving variable cooperativity or signal amplification that depends on the ligand. A recent patch-clamp study (34) further suggests that this activation mechanism depends on but is not mediated by Ca2+. Finally, the similarity in dose dependence for chlorzoxazone activation of Na+-absorptive current across CFT1 cells (Fig. 2B) and CFTR-mediated current in Xenopus oocytes (Fig. 7B) is suggestive of a common binding site and mechanism. This hypothesis needs to be tested in future studies.


    ACKNOWLEDGEMENTS

We are grateful to Dr. Lan Chen for assistance with oocyte injection and recording. We thank Drs. S. Lucania (Bristol-Myers Squibb), J. Tomich (Kansas State University), and B. Rossier (University of Lausanne, Lausanne, Switzerland) for their gifts of amphotericin B, 1-ethyl-2-benzimidazolinone, and epithelial sodium channel cDNAs, respectively. Finally, we thank Dr. Kevin Kirk for critiquing an earlier version of this article.


    FOOTNOTES

This work was supported by the Cystic Fibrosis Foundation (RDP464), the Webb Foundation, National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-54781, and National Heart, Lung, and Blood Institute Grants HL-46943, HL-37556, HL-31197, and HL51173.

Address for reprint requests and other correspondence: C. J. Venglarik, Dept. of Environmental Health Sciences, Univ. of Alabama at Birmingham, 760 McCallum, 1918 University Blvd., Birmingham, AL 35294 (E-mail: cjv{at}uab.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.

Received 13 February 2001; accepted in final form 21 June 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bebok, Z, Tousson A, Schwiebert LM, and Venglarik CJ. Improved oxygenation promotes CFTR maturation and trafficking in MDCK monolayers. Am J Physiol Cell Physiol 280: C135-C145, 2001[Abstract/Free Full Text].

2.   Benos, DJ, Cunningham S, Baker RR, Beason KB, Oh Y, and Smith PR. Molecular characteristics of amiloride-sensitive sodium channels. Rev Physiol Biochem Pharmacol 120: 31-113, 1992[ISI][Medline].

3.   Benos, DJ, Simon SA, Mandel LJ, and Cala PM. Effect of amiloride and some of its analogues of cation transport in isolated frog skin and thin lipid membranes. J Gen Physiol 68: 43-63, 1976[Abstract].

4.   Blazer-Yost, BL, Record RD, and Oberleithner H. Characterization of hormone-stimulated Na+ transport in a high-resistance clone of the MDCK cell line. Pflügers Arch 432: 685-691, 1996[ISI][Medline].

5.   Boat, TF, and Cheng PW. Epithelial cell dysfunction in cystic fibrosis: implications for airways disease. Acta Paediatr Scand Suppl 363: 25-29, 1989[Medline].

6.   Cuthbert, AW, Hickman ME, Thorn P, and MacVinish LJ. Activation of Ca2+- and cAMP-sensitive K+ channels in murine colonic epithelia by 1-ethyl-2-benzimidazolone. Am J Physiol Cell Physiol 277: C111-C220, 1999[Abstract/Free Full Text].

7.   Dawson, DC, and Richards NW. Basolateral K conductance: role in regulation of NaCl absorption and secretion. Am J Physiol Cell Physiol 259: C181-C195, 1990[Abstract/Free Full Text].

8.   Devor, DC, Bridges RJ, and Pilewski JM. Pharmacological modulation of ion transport across wild-type and Delta F508 CFTR-expressing human bronchial epithelia. Am J Physiol Cell Physiol 279: C461-C479, 2000[Abstract/Free Full Text].

9.   Devor, DC, Singh AK, Bridges RJ, and Frizzell RA. Modulation of Cl- secretion by benzimidazolones. II. Coordinate regulation of apical GCl and basolateral GK. Am J Physiol Lung Cell Mol Physiol 271: L785-L795, 1996[Abstract/Free Full Text].

10.   Devor, DC, Singh AK, Frizzell RA, and Bridges RJ. Modulation of Cl- secretion by benzimidazolones. I. Direct activation of a Ca2+-dependent K+ channel. Am J Physiol Lung Cell Mol Physiol 271: L775-L784, 1996[Abstract/Free Full Text].

11.   Devor, DC, Singh AK, Gerlach AC, Frizzell RA, and Bridges RJ. Inhibition of intestinal Cl- secretion by clotrimazole: direct effect on basolateral membrane K+ channels. Am J Physiol Cell Physiol 273: C531-C540, 1997[Abstract/Free Full Text].

12.   Devor, DC, Singh AK, Lambert LC, DeLuca A, Frizzell RA, and Bridges RJ. Bicarbonate and chloride secretion in Calu-3 human airway epithelial cells. J Gen Physiol 113: 743-760, 1999[Abstract/Free Full Text].

13.   DuVall, MD, Zhu S, Fuller CM, and Matalon S. Peroxynitrite inhibits amiloride-sensitive Na+ currents in Xenopus oocytes expressing alpha beta gamma -rENaC. Am J Physiol Cell Physiol 274: C1417-C1423, 1998[Abstract/Free Full Text].

14.   Gao, L, Broughman JR, Iwamoto T, Tomich JM, Venglarik CJ, and Forman HJ. Synthetic chloride channel restores glutathione secretion in cystic fibrosis airway epithelia. Am J Physiol Lung Cell Mol Physiol 281: L24-L30, 2001[Abstract/Free Full Text].

15.   Gao, L, Kim KJ, Yankaskas JR, and Forman HJ. Abnormal glutathione transport in cystic fibrosis airway epithelia. Am J Physiol Lung Cell Mol Physiol 277: L113-L118, 1999[Abstract/Free Full Text].

16.   Garcia, ML, Hanner M, Knaus HG, Koch R, Schmalhofer W, Slaughter RS, and Kaczorowski GJ. Pharmacology of potassium channels. Adv Pharmacol 39: 425-471, 1997[Medline].

17.   Germann, WJ, Ernst SA, and Dawson DC. Resting and osmotically induced basolateral K conductances in turtle colon. J Gen Physiol 88: 253-274, 1986[Abstract].

18.   Germann, WJ, Lowy ME, Ernst SA, and Dawson DC. Differentiation of two distinct K conductances in the basolateral membrane of turtle colon. J Gen Physiol 88: 237-251, 1986[Abstract].

19.   Hamilton, KL, Meads L, and Butt AG. 1-EBIO stimulates Cl- secretion by activating a basolateral K+ channel in the mouse jejunum. Pflügers Arch 439: 158-166, 1999[ISI][Medline].

20.   Hill, AV. The combinations of haemoglobin with oxygen and carbon monoxide. Biochem J 7: 471-480, 1913.

21.   Kirk, KL, and Dawson DC. Basolateral potassium channel in turtle colon. Evidence for single-file ion flow. J Gen Physiol 82: 297-329, 1983[Abstract].

22.   Koefoed-Johnsen, V, and Ussing HH. The nature of the frog skin potential. Acta Physiol Scand 42: 298-308, 1958[ISI].

23.   Kunzelmann, K, Kathofer S, and Greger R. Na+ and Cl- conductances in airway epithelial cells: increased response to amiloride and Na+ replacement in cystic fibrosis. Pflügers Arch 431: 1-9, 1995[ISI][Medline].

24.   Kunzelmann, K, Kathofer S, Hipper A, Gruenert DC, and Gregner R. Culture-dependent expression of Na+ conductances in airway epithelial cells. Pflügers Arch 431: 578-586, 1996[ISI][Medline].

25.   MacVinish, LJ, Hickman ME, Mufti DA, Durrington HJ, and Cuthbert AW. Importance of basolateral K+ conductance in maintaining Cl- secretion in murine nasal and colonic epithelia. J Physiol (Lond) 510: 237-247, 1998[Abstract/Free Full Text].

26.   Pedersen, KA, Schroder RL, Skaaning-Jensen B, Strobaek D, and Olesen SP. Activation of the human intermediate-conductance Ca2+-activated K+ channel by 1-ethyl-2-benzimidazolinone is strongly Ca2+-dependent. Biochim Biophys Acta 1420: 231-240, 1999[ISI][Medline].

27.   Pilewski, JM, and Frizzell RA. Role of CFTR in airway disease. Physiol Rev 79: S215-S255, 1999[Medline].

28.   Riordan, JR, Rommens JM, Kerem B, Alon N, Rozmahel R, Grzelczak Z, Zielenski J, Lok S, Plavsic N, Chou JL, Drumm MJ, Iannuzzi MC, Collins FS, and Tsui L. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245: 1066-1073, 1989[ISI][Medline].

29.   Rufo, PA, Jiang L, Moe SJ, Brugnara C, Alper SL, and Lencer WI. The antifungal antibiotic, clotrimazole, inhibits Cl- secretion by polarized monolayers of human colonic epithelial cells. J Clin Invest 98: 2066-2075, 1996[Abstract/Free Full Text].

30.   Segel, IH. Biochemical Calculations: How to Solve Mathematical Problems in General Biochemistry (2nd ed.). New York: Wiley, 1976.

31.   Singh, AK, Devor DC, Gerlach AC, Gondor M, Pilewski JM, and Bridges RJ. Stimulation of Cl- secretion by chlorzoxazone. J Pharmacol Exp Ther 292: 778-787, 2000[Abstract/Free Full Text].

32.   Singh, S, Syme CA, Singh AK, Devor DC, and Bridges RJ. Benzimidazolone activators of chloride secretion: potential therapeutics for cystic fibrosis and chronic obstructive pulmonary disease. J Pharmacol Exp Ther 296: 600-611, 2001[Abstract/Free Full Text].

33.   Stutts, MJ, Canessa CM, Olsen JC, Hamrick M, Cohn JA, Rossier BC, and Boucher RC. CFTR as a cAMP-dependent regulator of sodium channels. Science 269: 847-850, 1995[ISI][Medline].

34.   Syme, CA, Gerlach AC, Singh AK, and Devor DC. Pharmacological activation of cloned intermediate- and small-conductance Ca2+-activated K+ channels. Am J Physiol Cell Physiol 278: C570-C581, 2000[Abstract/Free Full Text].

35.   Venglarik, CJ, and Dawson DC. Cholinergic regulation of Na absorption by turtle colon: role of basolateral K conductance. Am J Physiol Cell Physiol 251: C563-C570, 1986[Abstract/Free Full Text].

36.   Venglarik, CJ, Schultz BD, DeRoos AD, Singh AK, and Bridges RJ. Tolbutamide causes open channel blockade of cystic fibrosis transmembrane conductance regulator Cl- channels. Biophys J 70: 2696-2703, 1996[Abstract].

37.   Warncke, J, and Lindemann B. Voltage dependence of the blocking rate constants of amiloride at apical Na channels. Pflügers Arch 405: S89-S94, 1985[ISI][Medline].

38.   Welsh, MJ, and Smith AE. Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis. Cell 73: 1251-1254, 1993[ISI][Medline].

39.   Yankaskas, JR, Haizlip JE, Conrad M, Koval D, Lazarowski E, Paradiso AM, Rinehart CA, Jr, Sarkadi B, Schlegel R, and Boucher RC. Papilloma virus immortalized tracheal epithelial cells retain a well-differentiated phenotype. Am J Physiol Cell Physiol 264: C1219-C1230, 1993[Abstract/Free Full Text].


Am J Physiol Lung Cell Mol Physiol 281(5):L1123-L1129
1040-0605/01 $5.00 Copyright © 2001 the American Physiological Society