Introduction

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Cystic fibrosis (CF) is the most common life-shortening inherited disease among the Caucasian population, and in North America occurs in ~1 in 2500 live births (Boat and Cheng, 1989; Strong et al., 1992). The genetic basis of this autosomal recessive disease has been traced to a defect in the gene on chromosome 7 that encodes for a cAMP-regulated chloride channel, the cystic fibrosis transmembrane conductance regulator (CFTR). Defective cAMP-mediated chloride secretion and increased apical membrane sodium absorption results in abnormal airway surface liquid, defective mucocilliary clearance, and bacterial infection in patients with CF (Pilewski and Frizzell, 1999). At the molecular level, there are several mechanisms whereby mutations in CFTR produce a loss or impaired cAMP-dependent Cl conductance (Welsh and Smith, 1993). One potential strategy to treat CF patients is to identify pharmacological agents that will restore normal function to the mutant forms of CFTR and/or activate alternative ion conductances (e.g., Ca²⁺-dependent Cl or K⁺ channels) to stimulate net Cl secretion. Devor et al. (1996b) demonstrated that the benzimidazolone 1-ethyl-2-benzimidazolinone (1-EBIO) stimulates Cl secretion across several epithelial cell types via the direct activation of K_Ca, which provides the necessary driving force for chloride secretion.

In our pursuit to identify other novel and specific high-affinity modulators of K_Ca that might be useful for CF, we searched several databases, including the list of all Food and Drug Administration (FDA)-approved drugs. The aim was to identify FDA-approved drugs for their potential off-label use for treatment of CF. This search for structures similar to 1-EBIO uncovered chlorzoxazone (Parafon Forte DSC). Chlorzoxazone is a centrally acting agent used clinically as a muscle relaxant for painful musculoskeletal conditions (Physicians Desk Reference, 1996). In this study we determined whether chlorzoxazone and its structural analog zoxazolamine (2-amino-5-chlorzoxazole) were capable of modulating Cl secretion via the direct activation of K_Ca in various epithelial cell lines and in human nasal epithelium in vivo. Our results demonstrate that chlorzoxazone and zoxazolamine have the same response profile as the benzimidazolone 1-EBIO, namely, it stimulates Cl secretion in the same in vitro assays and via the same mechanisms of action. Furthermore, in in vivo studies on healthy human volunteers, chlorzoxazone induced a chloride secretory response similar to that seen in vitro. We hypothesize that administration of drugs within this class of compounds may restore a significant Cl secretory response in the airway epithelium of CF patients with mutations that result in some residual CFTR activity.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

T84 Cell Culture. T84 cells were grown in Dulbecco's modified Eagle's medium and Ham's F-12 (1:1) supplemented with 15 mM HEPES, 14 mM NaHCO₃, and 10% fetal bovine serum (FBS). The cells were incubated in a humidified atmosphere containing 5% CO₂ at 37°C. For measurements of short-circuit current (I_sc), T84 cells were seeded onto Costar Transwell cell culture inserts (0.33 cm²) and the culture media changed every 48 h. I_sc measurements were performed on filters after 14 to 21 days in culture. Patch-clamp experiments were performed on single cells placed onto glass coverslips 18 to 48 h before use.

Primary Cultures of Human Bronchial Epithelium (HBE). HBE was obtained from excess pathologic tissue remaining after lung transplantation under a protocol approved by the University of Pittsburgh Investigational Review Board. Tissue expressing wild-type CFTR was obtained following lung transplantation for a variety of pathologic conditions, including emphysema, primary pulmonary hypertension, pulmonary fibrosis, and 1-antitrypsin deficiency. All CF tissue used in this study was homozygous for the F508 CFTR mutation by allele-specific hybridization (performed at Genzyme Genetics, Framingham, MA). Second through sixth generation bronchi were dissected, rinsed thoroughly, and incubated overnight at 4°C in minimal essential medium (MEM) containing 0.1% protease (type XIV; Sigma Chemical Co., St. Louis, MO). The epithelial cells were isolated by centrifugation and washed in MEM containing 5% FBS. Following centrifugation, the cells were resuspended in serum-free bronchial epithelial growth media (Clonetics, San Diego, CA) and plated into type VI human placental collagen (Sigma Chemical Co.)-coated t-25 tissue culture flasks. On reaching 80 to 90% confluence, the cells were trypsinized, resuspended in MEM plus 5% FBS, and seeded onto human placental collagen-coated Costar Transwell filters (0.33 cm²) at a density of ~2 × 10⁶/cm². After 24 h, the media was changed to Dulbecco's modified Eagle's medium:F-12 (1:1) plus 2% Ultroser G (BioSepra, Inc.; Cedex, France) and an air interface at the apical membrane established. The media bathing the basolateral surface was changed every 48 h. Measurements of I_sc were performed after ~10 to 20 additional days in culture.

Solutions. For measurements of I_sc, the bath solution contained 120 mM NaCl, 25 mM NaHCO₃, 3.3 mM KH₂PO₄, 0.8 mM K₂HPO₄, 1.2 mM MgCl₂, 1.2 mM CaCl₂, and 10 mM glucose. The pH of this solution was 7.4 when gassed with a mixture of 95% O₂/5% CO₂ at 37°C. The effects of chlorzoxazone and zoxazolamine on apical membrane Cl currents (I_Cl) were assessed after permeabilization of the serosal membrane with nystatin (360 µg/ml) and the establishment of a mucosa to serosa Cl concentration gradient. Serosal NaCl was replaced by equimolar sodium gluconate, and CaCl₂ was increased to 4 mM to compensate for the Ca²⁺-buffering capacity of the gluconate. Nystatin was added to the serosal membrane 10 to 25 min before the addition of drugs. Successful permeabilization of the basolateral membrane was based on the recording of a negative I_Cl that was not sensitive to inhibition by bumetanide (20 µM; see below).

I_sc Measurements. Costar Transwell cell culture inserts were mounted in an Ussing chamber (Jim's Instruments, Iowa City, IA) and the monolayers continuously short-circuited (University of Iowa, Department of Bioengineering). Transepithelial resistance was measured by periodically applying a 5-mV pulse, and the resistance calculated with Ohm's Law. Forskolin, chlorzoxazone, 6-hydroxychlorzoxazone, and zoxazolamine were added to both sides of the monolayers at the indicated concentrations. Bumetanide was added only to the serosal-bathing solution, whereas amiloride was added only to the mucosal-bathing solution. Changes in I_sc are calculated as a difference current between the sustained phase of the response and their respective baseline values.

Single-Channel Recording. Single-channel currents were recorded in the inside-out patch-clamp recording configuration with a List EPC-7 amplifier (Medical Systems, Greenvale, NY) and were recorded on videotape for later analysis, as described previously (Devor and Frizzell, 1993). Pipettes were fabricated from KG-12 glass (Wilmad, Buena, NY). All recordings were done at a holding voltage of 100 mV. The voltage is referenced to the extracellular compartment as the standard method for membrane potentials. Inward currents are defined as the movement of positive charge from the extracellular compartment to the intracellular compartment and are presented as downward deflections from baseline in all recording configurations.

⁸⁶Rb⁺ Uptake Studies. ⁸⁶Rb⁺ uptake was measured with the method of Gasko et al. (1976) as modified by Garty et al., (1983) and our laboratories (Bridges et al., 1988; Devor et al., 1997b). In this method, tracer uptake is driven by a large electrochemical potential gradient by passing K₂SO₄-loaded vesicles down a cation exchange column. The removal of the extravesicular K⁺ creates a chemical gradient for K⁺ loss from the vesicles, and because the intravesicular counterion SO₄², is less permeant than K⁺, an inside-negative diffusion potential is generated by the outward K⁺ gradient. We estimate the membrane potential to be nearly 200 mV, vesicle interior negative.

In Vivo Human Nasal Potential Difference (PD) Measurements. Human nasal PD measurements were made with techniques previously described by Knowles et al. (1991). PD measurements were made along the floor of the nose, under the inferior turbinate, with a probing electrode made of pliable polypropylene tubing attached to a syringe to allow perfusion of different solutions with a Harvard pump. Reference and probing electrodes were constructed from agar-filled i.v. tubing placed in calomel half-cells containing 3 M KCl solution. The half-cells are connected to a voltmeter, which interfaces with a Fisher data recorder for continuous voltage monitoring.

5

Chemicals. Nystatin was a generous gift from Dr. S. Lucania (Bristol-Meyers Squibb, New York, NY). Chlorzoxazone, 6-hydroxychlorzoxazone, and forskolin were obtained from Calbiochem (La Jolla, CA). Zoxazolamine and 1-EBIO were obtained from Aldrich Chemical (Milwaukee, WI). Amiloride and bumetanide was obtained from Sigma Chemical Co. Charybdotoxin (CTX) was obtained from Accurate Chemicals and Scientific (Westbury, NY) and was made as a 10 µM stock solution in standard bath solution. 1-EBIO, chlorzoxazone, 6-hydroxychlorzoxazone, and zoxazolamine were made as 1000-fold stock solutions in dimethyl sulfoxide. Nystatin was made as a 360-mg/ml stock solution in dimethyl sulfoxide and was sonicated for 30 s just before use. Forskolin and bumetanide were made as 1000-fold stock solutions in ethanol. Cell culture medium was obtained from Life Technologies (Grand Island, NY).

Data Analysis. All data are presented as means ± S.E., where n indicates the number of experiments. Statistical analysis was performed with Student's t test. A value of P < .05 was considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Effect of Chlorzoxazone on I_sc and Blockade by CTX. Coordinated regulation of both apical Cl and basolateral K⁺ channels is required to stimulate transepithelial Cl secretion. We have previously shown that 1-EBIO stimulates a basolateral membrane K⁺ channel (K_Ca), and apical membrane Cl conductance (G_Cl) and thereby stimulates transepithelial Cl secretion in several epithelia (Devor et al., 1996b). Therefore, we determined whether a structurally related benzoxazolone, chlorzoxazone (Fig. 1), would stimulate a transepithelial Cl secretory response in the model Cl secretory epithelium T84. In this experiment, chlorzoxazone was only tested for its effect at 300 µM, a higher concentration than reported for the plasma level concentration (100-200 µM) in patients taking it for skeletal muscle relaxation (Elenbaas, 1980; Stewart and Janaki, 1987). The results of one representative experiment are shown in Fig. 2. Chlorzoxazone (300 µM) induced a sustained Cl secretory current that was sensitive to block by CTX (50 nM), a known blocker of the maxi-K⁺ channel (McKay et al., 1994; Olessen et al., 1994; Sellers and Ashford, 1994), as well as K_Ca (Devor and Frizzell, 1993; Devor et al., 1996b). The baseline I_sc in these tissues averaged 1.5 ± 0.4 µA/cm² (n = 10). Subsequent addition of chlorzoxazone (300 µM) induced a sustained I_sc of 175 ± 10 µA/cm². We previously demonstrated that 1-EBIO induced a CTX-sensitive I_sc due to the direct activation of K_Ca (Devor and Frizzell, 1993; Devor et al., 1996b). Therefore, the effect of CTX on the chlorzoxazone-induced I_sc was determined. As shown in Fig. 2, CTX (50 nM) reduced by 130 µA/cm² (>70%) the chlorzoxazone-induced I_sc (n = 10). Because chlorzoxazone and 1-EBIO are structurally related, we assumed that chlorzoxazone may be regulating K_Ca. The T84 cells are not known to express maxi-K⁺ channels (Devor et al., 1996b), hence this inhibition by CTX suggests that chlorzoxazone, like 1-EBIO, activates the basolateral membrane K_Ca channel. In addition, the chlorzoxazone induced I_sc was not inhibited by the selective maxi-K⁺ channel inhibitor iberiotoxin (Galvez et al., 1990; data not shown) or by paxilline (Knaus et al., 1994; data not shown). This further suggests that a maxi-K⁺ channel is not involved in the secretory response to chlorzoxazone. However, the magnitude of the I_sc response generated by chlorzoxazone does suggest that like 1-EBIO, it also activates an apical membrane Cl conductances (see below in Results). Due to solubility restrictions in the Ussing chamber buffer, chlorzoxazone could not be tested for its effect on transepithelial Cl secretion at concentrations >500 µM. Hence, the half-maximal concentration of chlorzoxazone could not be evaluated.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Chemical structures of 1-EBIO, chlorzoxazone, zoxazolamine, and 6-hydroxychlorzoxazone.

View larger version (9K): [in this window] [in a new window]   Fig. 2.   Effect of chlorzoxazone on short-circuit current (Isc) in T84 monolayers. The chlorzoxazone-induced (300 µM; mucosal and serosal addition) Isc response was >70% inhibited by CTX (serosal).

Transepithelial I_K and I_Cl Measurements. To further determine whether chlorzoxazone was activating a basolateral K⁺ conductance (G_K) and an apical Cl conductance (G_Cl) in the intact T84 monolayer, the pore-forming antibiotic nystatin was used to permeabilize either the apical or basolateral membrane, and the appropriate transepithelial ion gradients established to measure the I_K or I_Cl (see Materials and Methods). The effect of chlorzoxazone on I_K is shown in Fig. 3A. Nystatin increased the transepithelial current by only 30 ± 5 µA/cm² (n = 12). After nystatin permeabilization, subsequent addition of chlorzoxazone (200 µM) increased I_K by an average of 80 ± 10 µA/cm² (n = 6). This chlorzoxazone-induced current response was inhibited an average of 84 ± 3% by CTX (50 nM). This result suggests that chlorzoxazone activates a CTX-sensitive G_K, in T84 cells.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Effect of chlorzoxazone (200 µM) (A) and zoxazolamine (200 µM) (B) on basolateral membrane K+ currents after establishment of a mucosa-to-serosa K+ gradient and permeabilization of the mucosal membrane with nystatin (see Materials and Methods). Monolayer illustration indicates direction of the ion gradient and the membrane, which was permeabilized with nystatin (dashed line in monolayer illustration). CTX (50 nM; serosal) inhibited the current response of both agonists.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   A, effect of chlorzoxazone (200 µM) on Cl currents after establishment of mucosa-to-serosa Cl gradient and permeabilization of the serosal membrane with nystatin (see Materials and Methods). The inward current is consistent with an absorptive Cl flow. B, effect of forskolin (10 µM; mucosal and serosal) after nystatin permeabilization and addition of chlorzoxazone (200 µM; mucosal and serosal). Bumetanide did not block this current, consistent with permeabilization of the serosal membrane. Glibenclamide (Gliben, 300 µM; serosal and mucosal addition) blocked this absorptive Cl current response (A and B).

⁸⁶Rb⁺ Uptake in Membrane Vesicles. The above-mentioned data strongly suggest that chlorzoxazone and zoxazolamine activate a CTX-sensitive basolateral K⁺ channel. To further test this proposal by an independent method, we measured ⁸⁶Rb⁺ uptake into membrane vesicles prepared from T84 cells (Devor et al., 1996b). Both chlorzoxazone and zoxazolamine stimulated ⁸⁶Rb⁺ uptake in a concentration-dependent manner; however, both compounds precipitated out of solution at concentrations below those required for maximal stimulation. Therefore, these compounds were tested at 1 mM, the maximal soluble concentration in this assay. In 14 experiments, chlorzoxazone and zoxazolamine were tested and compared with the ability of 1-EBIO to stimulate CTX-sensitive ⁸⁶Rb⁺ uptake. On average, 1-EBIO produced a 9-fold increase in CTX-sensitive uptake compared with control (control, 7.62 ± 2.18; 1-EBIO 69.66 ± 12.14 pmol ⁸⁶Rb⁺/100 µg protein; P < .01). In seven experiments, chlorzoxazone and zoxazolamine stimulated 81.83 ± 4.0 and 69.37 ± 6.48%, respectively, of the CTX-sensitive 1-EBIO-stimulated uptake (P < .01; Fig. 5A). Therefore, the rank order of potency for stimulation of CTX-sensitive ⁸⁶Rb⁺ uptake at 1 mM was 1-EBIO > chlorzoxazone = zoxazolamine (P < .01). The effect of 6-hydroxychlorzoxazone on ⁸⁶Rb⁺ uptake also was assessed. In five experiments, this metabolite neither stimulated CTX-sensitive uptake alone nor antagonized the stimulatory effects of chlorzoxazone (Fig. 5B: control, 10.02 ± 4.06; 6-hydroxychlorzoxazone, 15.24 ± 6.45; chlorzoxazone, 64.45 ± 17.68; and chlorzoxazone in the presence of 6-hydroxychlorzoxazone, 65.45 ± 24.1 pmol ⁸⁶Rb⁺/100 µg protein).

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Effect of chlorzoxazone, zoxazolamine, and 6-hydroxychlorzoxazone on CTX-sensitive 86Rb+ uptake into T84 membrane-derived vesicles. A, both chlorzoxazone and zoxazolamine stimulate CTX-sensitive 86Rb+ uptake into T84 membrane-derived vesicles. Vesicles were incubated with the appropriate agonist in either the presence or absence of CTX (100 nM). Both chlorzoxazone and zoxazolamine (1 mM) produced a 3- to 4-fold stimulation over control activity. The data are normalized to uptake produced by the agonist 1-EBIO (1 mM). Control uptake was only 11.98 ± 2.39% of 1-EBIO-stimulated uptake; whereas chlorzoxazone and zoxazolamine stimulated 81.83 ± 3.05 and 69.37 ± 6.48% (n = 7; P < .01 of the 1-EBIO-stimulated uptake. B, the primary chlorzoxazone metabolite 6-hydroxychlorzoxazone neither stimulates nor inhibits uptake. The data are normalized to the CTX-sensitive uptake produced by chlorzoxazone (1 mM). 6-Hydroxychlorzoxazone (1 mM) failed to stimulate uptake (control, 12.98 ± 7.88 and 6-hydroxychlorzoxazone, 28.22 ± 9.33% of CTX-sensitive chlorzoxazone-stimulated uptake). In addition, the metabolite failed to compete with chlorzoxazone, resulting in 90.84 ± 11.58% of the CTX-sensitive chlorzoxazone-stimulated uptake (n = 5).

Excised Patch Single-Channel Records. Previous studies have shown that 1-EBIO activates a CTX-sensitive, Ca²⁺-activated K⁺ channel (K_Ca) (Devor and Frizzell, 1993; Devor et al., 1996b). To further characterize the mechanism of action of chlorzoxazone, the effects of chlorzoxazone on K_Ca were assessed with excised inside-out patch-clamp recordings. The result of one representative experiment performed with chlorzoxazone is shown in Fig. 6. Under control conditions with 400 nM Ca²⁺ in the bath, very little K_Ca channel activity (NP_o) was observed (0.06 ± 0.02; n = 6), whereas subsequent addition of chlorzoxazone (100 µM) produced a spontaneous increase in NP_o to 1.69 ± 0.49 (n = 6; P < .02) (Fig. 6). No lag in activation or recovery of channel activity was observed. In the same patch, the Ca²⁺ dependence of this activation was assessed. After chlorzoxazone washout (0.04 ± 0.02; n = 6) and removal of bath Ca²⁺ (0.00; n = 3), re-addition of chlorzoxazone did not activate the channels (0.01 ± 0.01; n = 3), whereas subsequent addition of 400 nM Ca²⁺ in the continued presence of chlorzoxazone (100 µM) in the bath resulted in the reactivation of this channel (0.34 ± 0.11).

View larger version (30K): [in this window] [in a new window]   Fig. 6.   Activation of Ca2+-dependent K+ channel by chlorzoxazone (100 µM) in excised inside-out patch (left) in the presence of 400 nM free Ca2+. In nominally Ca2+-free solutions, chlorzoxazone failed to activate the channel in the same patch, whereas subsequent addition of 400 nM Ca2+ reactivated the channel (right). Bath and pipette contained symmetric potassium gluconate. Membrane was voltage clamped to 100 mV, with voltage references to the extracellular compartment. Arrows indicate closed state of the channel.

View larger version (39K): [in this window] [in a new window]   Fig. 7.   Effect of 6-hydroxychlorzoxazone, the primary metabolite of chlorzoxazone, in excised inside-out patch clamp recordings. 6-Hydroxychlorzoxazone (100 µM) had no effect on channel activity, whereas subsequent addition of chlorzoxazone (100 µM) to the same patch produced a large increase in the Ca2+-dependent K+ channel activity. Arrows indicate closed state of the channel.

View larger version (33K): [in this window] [in a new window]   Fig. 8.   Reversible activation of Ca2+-dependent K+ channel by zoxazolamine (100 µM) in excised inside-out patch. No lag in the activation and recovery was observed. Arrows indicate closed state of the channel.

Effect of Chlorzoxazone and Zoxazolamine on HBE. The effect of chlorzoxazone on transepithelial Cl current in primary cultures of human bronchial epithelium was determined (see Materials and Methods). The results of one representative experiment are shown in Fig. 9A. The monolayer developed a spontaneous I_sc that is due to electrogenic sodium absorption. After amiloride (10 µM) inhibition of this Na⁺ transport, chlorzoxazone induced a concentration-dependent Cl secretory current that was partially sensitive to block by CTX (50 nM). The I_sc was further inhibited by serosal application of Na⁺-K⁺-2 Cl contransport inhibitor bumetanide (20 µM). In five monolayers, the spontaneous current averaged 20 ± 10 µA/cm². Amiloride (10 µM) reduced this current to 8 ± 4 µA/cm². Addition of chlorzoxazone (500 µM) increased I_sc to 12 ± 4 µA/cm², which was blocked by CTX to an average of 10 ± 5 µA/cm² (P < .05). Subsequent addition of bumetanide (20 µM) inhibited this I_sc to 6 ± 3 µA/cm². Effects of zoxazolamine on transepithelial Cl current in primary cultures of human bronchial epithelium also were determined. Zoxazolamine had a similar response profile as chlorzoxazone, as shown in Fig. 9B. In three monolayers, the spontaneous current averaged 54 ± 4 µA/cm². Amiloride (10 µM) reduced this current to 24 ± 3 µA/cm². Addition of zoxazolamine (300 µM) increased I_sc to 37 ± 7 µA/cm², which was blocked by CTX to an average of 31 ± 5 µA/cm² (P < .05) Subsequent addition of bumetanide (20 µM) inhibited this I_sc to 10 ± 1 µA/cm².

View larger version (14K): [in this window] [in a new window]   Fig. 9.   A, dose-dependent stimulation of Isc across primary cultures of HBE by chlorzoxazone (300 and 500 µM; serosal and mucosal) after inhibition of Na+ absorption by amiloride (10 µM; mucosal addition). Chlorzoxazone-induced Isc was inhibited by CTX (50 nM; serosal) and bumetanide (20 µM; serosal). B, effect of zoxazolamine (300 µM; serosal and mucosal) on Isc in HBEs after amiloride (10 µM; mucosal) inhibition of spontaneous Na+-absorptive current. Isc was partially inhibited by CTX (50 nM; serosal) and >75% inhibited by bumetanide (20 µM; serosal). C, lack of any effect of forskolin (10 µM) followed by chlorzoxazone (300 µM) on primary cultures of HBE derived from CF patients with the F508 mutation of CFTR.

Nasal PD Measurements. The effects of chlorzoxazone on nasal epithelial ion transport were determined in eight healthy volunteers (non-CF) and five CF patients. Representative PD tracings are shown in Fig. 10 and the mean changes for the low Cl, chlorzoxazone, and isoproterenol conditions are given in Fig. 11. As anticipated, the CF patients had higher basal PD values [32.2 ± 3.6 mV (mean ± S.E.) in CF versus 14.6 ± 0.9 mV in non-CF] and a greater depolarization of PD after perfusion with amiloride (13.3 ± 1.7 mV in CF versus 5.2 ± 0.7 mV in non-CF). Moreover, the total chloride secretory response (sum of the response to perfusion with low chloride solution and low chloride solution containing isoproterenol) in the control nostril was a hyperpolarizing 10.7 ± 1.9 mV in normal volunteers. In the CF cohort, there was a further depolarization of 8.6 ± 2.5 mV. Perfusion with 500 µM chlorzoxazone induced a mean hyperpolarization of 7.5 ± 2.4 mV in non-CF, a response that was not statistically different (P = .5 by t test) from the 5.2 ± 1.2-mV change observed with perfusion of isoproterenol. In contrast, in CF patients, there was a small depolarization with both chlorzoxazone and isoproterenol perfusion (mean depolarizations of 3.4 ± 1.4 and 3.0 ± 1.9 mV, respectively; not statistically different). Thus, as demonstrated by the in vitro studies, chlorzoxazone can stimulate a Cl secretory response in non-CF epithelia in vivo. However, the response to chlorzoxazone appears to require a functional CFTR in the apical membrane.

View larger version (12K): [in this window] [in a new window]   Fig. 10.   Chlorzoxazone induces chloride secretion in human respiratory epithelium. Changes in transepithelial PD were used to assess the acute effects of chlorzoxazone on chloride secretion in airway epithelium in vivo. Shown are representative PD tracings from one CF patient (A) and two normal volunteers (B and C). Each perfusion was for 3 min, or until a stable baseline was established. The arrows indicate the time at which perfusion solutions were changed. Arrow 1 indicates the addition of amiloride to Ringer's solution, arrow 2 the change to low chloride solution containing amiloride, arrow 3 the change to low chloride solution containing chlorzoxazone, arrow 4 the change from chlorzoxazone to isoproterenol in low chloride solution, and arrow 5 the change to Ringer's solution. Note hyperpolarization of PD in normal volunteers (B and C) with perfusion of low chloride (arrow 2) and chlorzoxazone (arrow 3), but small depolarization in CF patient (A).

View larger version (17K): [in this window] [in a new window]   Fig. 11.   Summary of PD responses. Shown are the means ± S.E. change in PD in response to perfusion of nasal epithelium with low chloride solution, chlorzoxazone, and isoproterenol, in eight normal volunteers (NI) and five CF patients.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The stimulation of epithelial Cl secretion requires the activation of apical membrane Cl channels and basolateral membrane K⁺ channels. Naturally occurring secretory agonists achieve the activation of both Cl and K⁺ channels via the binding to plasma membrane receptors and the stimulation of various intracellular signal transduction cascades that regulate the activities of these channels. Pharmacological agents also may stimulate Cl secretion by acting via the same cascades and can in addition act by the direct binding to the ion channels to modulate their activity. 1-EBIO, chlorzoxazone, and zoxazolamine appear to stimulate the secretion of Cl via the direct binding and activation of K_Ca and CFTR. The evidence for the direct activation of K_Ca by these agents is more substantial than is their direct action on CFTR.

Chlorzoxazone and Zoxazolamine Directly Activate K_Ca. Several lines of evidence in different epithelia demonstrate that chlorzoxazone, like 1-EBIO, activates the same K_Ca that has been previously characterized in these epithelial cells. First, as true of the response to 1-EBIO, the Cl secretory response was blocked by CTX, a known blocker of K_Ca (Fig. 2). Following permeabilization of the apical membrane of T84 cells with nystatin and establishment of a mucosa-to-serosa K⁺ gradient, chlorzoxazone stimulated a basolateral membrane G_K, which was inhibited by CTX (Fig. 3). In membrane vesicles prepared from T84 cells, chlorzoxazone stimulated ⁸⁶Rb⁺ uptake in a CTX-sensitive manner (Fig. 5). In excised, inside-out patches from T84 (Devor and Frizzell, 1993; Devor et al., 1996b, 1997b), Calu-3 (Devor et al., 1999), and HBE (D.C.D. and R.J.B., unpublished observations) cell lines mentioned above, chlorzoxazone activated an inwardly rectifying K⁺ channel, K_Ca, that was inhibited by CTX. Like 1-EBIO, this activation of the K⁺ channel was dependent on the presence of resting levels of Ca²⁺ (Fig. 6). 6-Hydroxychlorzoxazone, the major metabolite of chlorzoxazone, did not activate the basolateral membrane K_Ca (Fig. 7), whereas another structurally similar compound, zoxazolamine, which has a stronger electron donating group at position 2, was as potent as chlorzoxazone in activating this channel in excised membrane patches (Fig. 8). These results suggest that these compounds activate K_Ca by directly binding to the channel protein or to a closely associated regulatory protein that is maintained in the membrane vesicles and in an excised membrane patch. Heterologous expression of K_Ca (hIK1) and its activation by these compounds supports the notion of direct binding to the channel protein as the mechanism of activation (A.K.S., D.D., C. Syme, and R.J.B., unpublished data).

Chlorzoxazone and Zoxazolamine Activate CFTR. The identity of the Cl channel activated by 1-EBIO, chlorzoxazone, and zoxazolamine is less certain. Our results demonstrate that these compounds activate an apical membrane Cl conductance that can be inhibited by glibenclamide. Stimulation of a Cl secretory current by chlorzoxazone was observed in HBE cells derived from patients expressing wild-type CFTR but not with cells from CF patients with the F508 mutation of CFTR. Although consistent with the notion, these results alone are not adequate to conclude CFTR is the activated Cl channel. However, 1-EBIO is a benzimidazolone and other benzimidazolones {e.g., 1,3-dihydro-1-(5-chloro-2-hydroxyphenyl)-5-trifluoromethyl-2H-benzimidazol-2-one (NS004) and 1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)- phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS1619)} have been reported to activate CFTR in cell-attached patches of cells that express the protein heterologously (Gribkoff et al., 1994; Champigny et al., 1995). These studies do not preclude the possibility that the benzimidazolones act indirectly via a signal transduction cascade to activate CFTR. Further studies will be necessary to confirm that CFTR is the activated Cl channel as well as to elucidate the mechanism of activation by these compounds.

Modulation of Nasal PD by Chlorzoxazone. Results obtained from the T84, Calu-3 (data not shown) as well as primary cultures of HBE cell lines were extrapolated to clinical nasal PD measurements in non-CF human subjects. With the methods pioneered by Knowles et al. (1991), we have demonstrated that chlorzoxazone induced a hyperpolarization of the transepithelial nasal PD in normal volunteers after inhibition of the basal Na⁺ absorption with amiloride and establishment of a blood-to-lumen Cl concentration gradient (Fig. 10). This hyperpolarization indicates that chlorzoxazone induced a Cl secretory response in non-CF airway epithelium. No change was seen after a 3-min perfusion in CF (F508 homozygous) subjects. Northern blot analysis and patch-clamp studies on primary cultures of HBE cells have demonstrated the functional expression of K_Ca (data not shown). Hence, the lack of response in CF patients homozygous for F508 suggests the prerequisite for a chlorzoxazone Cl secretory response is the expression of functional CFTR in the plasma membrane, in vivo results that confirm the in vitro studies indicating that CFTR is the Cl channel activated by chlorzoxazone. Although the G551D mutation does reach the plasma membrane, its channel function is significantly impaired and this may explain why we did not observe a Cl secretory response in the G551D/F508-CFTR CF patient. Further work is in progress to evaluate chlorzoxazone in CF patients with mutations in which some functional CFTR is present in the plasma membrane.