INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

THE HALLMARK ION TRANSPORT DEFECTS in cystic fibrosis (CF) are a diminished or absent Cl secretory response to cAMP-mediated agonists and Na⁺ hyperabsorption. The most common mutation in the CF transmembrane conductance regulator (CFTR) gene product, a deletion of phenylalanine at amino acid 508 (F508), is particularly insidious, in that it leads to both a defect in the trafficking of the mutant protein to the apical membrane (55) as well as a channel that exhibits defective gating (1, 44). Pharmacologically, there are theoretically several means to ameliorate this primary ion transport defect. First, one can correct the trafficking defect associated with F508 CFTR (5). This approach was highlighted by Denning et al. (9) who demonstrated that decreasing the temperature at which F508 CFTR-expressing cells are grown increases the trafficking of the mutant protein to the plasma membrane.

A second strategy is to develop agents capable of directly interacting with any F508 CFTR expressed in the apical membrane. Highlighting this possibility, Kalin et al. (31) recently demonstrated that F508 CFTR can be expressed at the apical membrane of CF airway. The benzimidazolone, 5-trifluoromethyl-1-(5-chloro-2-hydroxyphenyl)-1,3-dihydro-2H-benzimidazole-2-one (NS004) was the first compound shown to activate both wild-type and F508 CFTR in excised patch-clamp recordings (23). However, we demonstrated that neither NS004 nor its structurally related analog, NS1619, stimulated Cl secretion in either the T84 cell line or in primary cultures of murine tracheal epithelium (MTE), despite the fact that these compounds increased apical membrane Cl conductance (13). Nguyen et al. (41) first demonstrated that the flavones quercetin and kaempferol stimulated Cl secretion across the T84 model secretory epithelium in a cAMP-independent manner. Subsequently, it has been shown that the related compound, genistein, also stimulates Cl secretion (28). More recent evidence indicates that genistein directly interacts with CFTR to increase the open probability of the channel (25, 54).

Finally, one can bypass the CFTR defect altogether by modulating the activity of alternative ion conductive pathways. For example, increasing intracellular Ca²⁺ has been shown to stimulate Cl secretion across CF airway (57). Indeed, Mason et al. (37) demonstrated that the Ca²⁺-dependent agonist, UTP, acting at P_2y2 receptors, stimulates Cl secretion across CF tracheal epithelium. We previously characterized the basolateral membrane K⁺ channel activated by Ca²⁺-mediated agonists (K_Ca) in colonic and airway epithelia (10, 16). We demonstrated that direct pharmacological activation of K_Ca by the benzimidazolone, 1-ethyl-2-benzimidazolinone (1-EBIO), resulted in the stimulation of Cl secretion across T84 and Calu-3 cells as well as primary cultures of MTE (13, 15). These results suggest that basolateral K⁺ channels may represent unique pharmacological targets for CF therapy (13, 15, 16).

Although 1-EBIO, genistein, 8-methoxypsoralen (Methoxsalen; 8-MOP), and NS004 have been shown to stimulate Cl secretion across T84 cells and MTE, the effects of these compounds on Cl secretion across human airway have not been evaluated. Therefore, we determined the effects of these agonists on primary cultures of human bronchial epithelia (HBE) expressing wild-type (wt HBE) or F508 (F-HBE) CFTR. We demonstrate that 1-EBIO, NS004, 8-MOP, and genistein stimulate a sustained Cl secretory response in wt HBE. In F-HBE both NS004 and genistein stimulate a small Cl secretory response, whereas 1-EBIO and 8-MOP do not. Additionally, following incubation of the cells at 26°C, the responses to 1-EBIO, NS004, and genistein are all potentiated. These results indicate that an apical membrane Cl conductance, perhaps F508 CFTR, is expressed and can be pharmacologically modulated in F-HBE. Further, these results demonstrate that, despite the low levels of expression of CFTR in native tissue, pharmacological agents directed at either apical Cl or basolateral K⁺ channels are capable of modulating Cl secretion, supporting the notion that they may be therapeutically useful for CF.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Primary Cultures of HBE

I_sc Measurements

Chemicals

Data Analysis

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In total, we evaluated 350 wt CFTR-expressing HBE monolayers from 40 patients under short-circuit conditions in symmetric standard bath solution. The baseline I_sc averaged 27.0 ± 0.6 µA/cm² with a transepithelial potential difference (PD_te) and resistance (R_te) of -15.6 ± 0.5 mV and 646 ± 18  · cm², respectively. Addition of amiloride (10 µM) to the mucosal chamber reduced I_sc an average of 13.5 ± 0.5 µA/cm² to a new plateau value of 13.5 ± 0.4 µA/cm² (n = 317). In contrast, in 200 monolayers, from 16 patients homozygous for the F508 CFTR mutation (F-HBE), the baseline I_sc averaged 33.8 ± 1.2 µA/cm² with a PD_te and R_te of 17.5 ± 0.6 mV and 568 ± 24  · cm², respectively. Addition of amiloride to these F-HBE reduced I_sc by 29.6 ± 1.5 µA/cm² to a new plateau value of 5.1 ± 0.2 µA/cm² (n = 116). Thus amiloride reduces I_sc an average of 50% in wt HBE, whereas it reduces I_sc by 87% in F-HBE, demonstrating a significant Na⁺ hyperabsorption in our F-HBE cultures (P < 0.0001). Our data on wt CFTR-expressing HBE are presented first, and data on F-HBE are presented in later sections.

Effect of Forskolin on Ion Transport Across HBE

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Effect of forskolin on ion transport across wild-type (wt) cystic fibrosis transmembrane conductance regulator (CFTR)-expressing human bronchial epithelia (wt HBE). A: in the presence of mucosal and serosal NaCl-containing solutions, forskolin (Forsk; 10 µM) stimulated a sustained increase in short-circuit current (Isc) subsequent to amiloride (Amil; 10 µM). Bumetanide (Bumet; 20 µM) reduced Isc to below the amiloride-dependent baseline, and this Isc was further reduced by the addition of the carbonic anhydrase inhibitor acetazolamide (Acetazol; 100 µM) and the Na+/H+ exchange inhibitor 5-(N-ethyl-N-isopropyl)-amiloride (EIPA; 5 µM, serosal). B: in the absence of mucosal and serosal Cl, forskolin induced a sustained, bumetanide-insensitive Isc that was inhibited by the subsequent addition of acetazolamide. C: in the absence of mucosal and serosal Cl, serosal 4,4'-dinitrostilben-2,2'-disulfonic acid (DNDS; 3 mM) inhibited the forskolin-dependent increase in Isc. Dashed lines, zero current level.

Our results suggest that a portion of the amiloride-insensitive I_sc is due to Cl secretion. However, the I_sc does not approach zero in the presence of the combination of amiloride plus bumetanide, suggesting an additional ongoing active transport process. Smith and Welsh (47) previously demonstrated that canine trachea was capable of secreting HCO₃ in response to cAMP-mediated agonists and also demonstrated that primary cultures of human airway respond to forskolin in Cl-free solutions, suggesting a HCO₃ secretory process. Also, we recently demonstrated that Calu-3 cells secrete HCO₃ in response to elevated cAMP (16). Thus, in an initial attempt to determine whether a portion of the amiloride-insensitive I_sc observed may be due to HCO₃ secretion, we utilized a combination of the carbonic anhydrase inhibitor acetazolamide (100 µM) and the Na⁺/H⁺ exchange inhibitor EIPA (5 µM). As shown in Fig. 1A, acetazolamide plus EIPA reduced I_sc an additional 3.6 ± 0.4 µA/cm² (n = 4), suggesting this basal I_sc may be due to HCO₃ secretion. The magnitude of this inhibition is similar to what was reported by Smith and Welsh (47) using acetazolamide (1 mM) and serosal amiloride (1 mM) in canine trachea.

To further evaluate the possibility that forskolin is stimulating HCO₃ secretion across wt HBE, we performed ion substitution experiments in which Cl or both Cl and HCO₃ were removed from both the mucosal and serosal solutions (see METHODS). As shown in Fig. 1B, in the absence of Cl, forskolin stimulated a bumetanide-insensitive increase in I_sc that was partially inhibited by acetazolamide. In 11 experiments forskolin increased I_sc an average of 6.3 ± 1.3 µA/cm² in the absence of Cl. By comparison, in the absence of both Cl and HCO₃, forskolin increased I_sc by only 0.9 ± 0.4 µA/cm² (n = 6). These experiments demonstrate that forskolin stimulates a transepithelial current response that is dependent on HCO₃ in the bathing solution.

We recently demonstrated that the human airway cell line Calu-3 secretes HCO₃ by a Na⁺-dependent mechanism in response to forskolin and that this could be inhibited by serosal DNDS (16). High concentrations of DNDS (1-3 mM) have been shown to inhibit the Na⁺-HCO₃ cotransporter (4, 56), suggesting that this cotransporter was responsible for HCO₃ entry across the serosal membrane of Calu-3 cells [ribonuclease protection assays confirm expression of a Na⁺- HCO₃ cotransporter (NBC) in Calu-3 cells as well as HBE (Gangopadhyay NN and Bridges RJ, unpublished observations)]. We therefore determined whether DNDS would similarly inhibit forskolin-mediated anion transport across wt HBE. As shown in Fig. 1C, in the absence of mucosal and serosal Cl, serosal DNDS (3 mM) partially inhibited the forskolin-induced I_sc, and this was further reduced by the addition of acetazolamide. In five monolayers, forskolin increased I_sc from 3.4 ± 0.3 to 7.4 ± 1.0 µA/cm², and this was reduced to 5.4 ± 0.8 and 3.7 ± 0.5 µA/cm² by DNDS and acetazolamide, respectively. These results suggest that a DNDS-sensitive Na⁺- HCO₃ cotransporter is partially responsible for serosal HCO₃ entry in HBE, a mechanism similar to that which we described in Calu-3 cells (16).

Although it is likely that CFTR represents the apical membrane Cl channel activated by forskolin, the basolateral membrane K⁺ channels involved in maintaining the driving force for Cl secretion have received little attention. Therefore, we evaluated the effect of several known K⁺ channel blockers on the forskolin-stimulated I_sc. We previously demonstrated that CTX inhibits Ca²⁺- but not cAMP-dependent Cl secretion across T84 cells (15). Similarly, in wt HBE cells, CTX (50 nM) had no effect on forskolin-stimulated Cl secretion (Fig. 2). Similar results were obtained in six additional experiments. Lohrmann et al. (34) first described the chromanol, 293B, as a highly selective inhibitor of the basolateral membrane cAMP-dependent K⁺ channel. We determined the effect of 293B (100 µM) on the forskolin-stimulated I_sc in wt HBE (Fig. 2). In 12 monolayers, forskolin increased I_sc from 17.1 ± 2.8 µA/cm² to a sustained value of 23.4 ± 3.4 µA/cm², and this was reduced by 67% to 19.2 ± 2.7 µA/cm² by 293B (P < 0.001). These results demonstrate that, similar to colonic epithelia (15, 34), human airway expresses a 293B-sensitive K⁺ conductance.

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Effect of K+ channel blockers on the forskolin-dependent increase in Isc across wt CFTR-expressing HBE. Subsequent to amiloride (10 µM), forskolin induced a sustained increase in Isc that was insensitive to block by charybdotoxin (CTX; 50 nM, serosal). However, the subsequent addition of both 293B (100 µM, serosal) and Ba2+ (5 mM, serosal) inhibited this Isc. Bumetanide (20 µM) further reduced Isc. Dashed line, zero current level.

Although it is clear that 293B inhibits a significant portion (67%) of the cAMP-mediated I_sc, a large 293B-independent I_sc remains (Fig. 2). Our results above (Fig. 1) suggest that a portion of this current is due to Cl secretion based on its bumetanide sensitivity and requirement for Cl. Thus we determined whether other nonselective K⁺ channel blockers would further inhibit this basal Cl secretion. Subsequent to 293B, addition of Ba²⁺ (5 mM) reduced I_sc to a sustained value of 8.5 ± 1.5 µA/cm² (n = 10; Fig. 2), with bumetanide further reducing I_sc to 3.7 ± 0.5 µA/cm² (n = 10). Subsequent to 293B, quinine further reduced I_sc from 9.8 ± 0.6 to 4.3 ± 0.8 µA/cm² (n = 4). Finally, clofilium (100 µM) inhibited both the forskolin-dependent and -independent current, reducing I_sc from 42.0 ± 12.5 to 4.7 ± 1.2 µA/cm² (n = 4). These results suggest that there is a Ba²⁺-, clofilium-, and quinine-sensitive basolateral K⁺ conductance that underlies the bumetanide-sensitive Cl secretion induced by amiloride and forskolin.

Effect of the CFTR Openers NS004, 8-MOP, and Genistein on Cl Secretion Across wt HBE

NS004. Gribkoff et al. (23) characterized NS004 as the first known opener of both wt and F508 CFTR in the Xenopus oocyte heterologous expression system. Subsequently, we demonstrated that, although NS004 increased apical membrane Cl conductance in the T84 cell line, it failed to induce a Cl secretory response (13). Also, NS004 failed to stimulate Cl secretion in primary cultures of MTE (13). In contrast to these results, NS004 (10 µM) stimulated a sustained, bumetanide-sensitive increase in I_sc across wt HBE, subsequent to amiloride (Fig. 3A). This concentration of NS004 was chosen based on both the K_s (see below) for NS004 as well as our previous studies, demonstrating that higher concentrations of NS004 disrupted epithelial integrity in T84 cells (13). In 18 experiments, NS004 increased I_sc by 10.8 ± 1.7 µA/cm², from 13.4 ± 0.9 to 24.2 ± 2.1 µA/cm². In eight of these experiments, the subsequent addition of bumetanide reduced I_sc to 10.4 ± 0.6 µA/cm². In nine additional monolayers, a complete concentration-response curve for NS004 was generated. These data were fitted to a Michaelis-Menten function having an apparent K_s of 1.2 ± 0.3 µM (Fig. 3B).

View larger version (11K): [in this window] [in a new window]   Fig. 3.   Effect of 5-trifluoromethyl-1-(5-chloro-2-hydroxyphenyl)-1,3-dihydro-2H-benzimidazole-2-one (NS004; 10 µM) on Isc across wt CFTR-expressing HBE. A: subsequent to amiloride, NS004 induced a sustained, bumetanide-sensitive increase in Isc. B: concentration-response curve for the NS004-induced Isc response in wt HBE, subsequent to amiloride. Data were fitted to a Michaelis-Menten function with an apparent stimulatory constant (Ks) of 1.2 ± 0.3 µM. C: after inhibition of Na+ absorption with amiloride, Ba2+ (5 mM, serosal) further reduced Isc to near zero (dashed line). Subsequent addition of NS004 induced a small increase in Isc, and this was further increased following addition of 1-ethyl-2-benzimidazolinone (1-EBIO; 300 µM). Addition of CTX (50 nM, serosal) inhibited the 1-EBIO-induced current, with the subsequent addition of bumetanide inducing a further decrease in Isc.

Psoralens. We previously demonstrated that the psoralens increase apical membrane Cl conductance and stimulate Cl secretion across both T84 cells and MTE (14). Similar to our results with NS004, stimulation of transepithelial Cl secretion required the addition of a K⁺ channel agonist such as 1-EBIO or carbachol (14). Based on blocker pharmacology, we proposed that the psoralens stimulated Cl secretion via an activation of CFTR (14). We evaluated the effect of 8-MOP in wt HBE following inhibition of basal Na⁺ absorption with amiloride. The maximal effective concentration of 8-MOP for increasing apical G_Cl in T84 cells was 30 µM. To directly compare the effects of NS004 and 8-MOP, we chose to utilize 10 µM 8-MOP for our HBE studies. As shown in Fig. 4, 8-MOP induced a sustained, bumetanide-sensitive increase in Cl secretion across wt HBE. In five monolayers, 8-MOP increased I_sc from 10.0 ± 2.3 to 20.0 ± 0.4 µA/cm². Addition of bumetanide reduced this current to 9.7 ± 1.4 µA/cm². These results confirm that, following hyperpolarization of the apical membrane with amiloride, Cl channel agonists are capable of stimulating Cl secretion across wt HBE. In addition, we determined the effect of 8-MOP on I_sc in the absence of amiloride. 8-MOP had no significant effect on Na⁺ transport (see Effect of NS004, 8-MOP, 1-EBIO, and Genistein on Na⁺ Absorption Across F-HBE), suggesting any increase in I_sc was due to anion secretion. Similar to NS004, 8-MOP induced a small, albeit significant increase in I_sc of 3.7 ± 1.3 µA/cm² (n = 5, P < 0.05) in the absence of amiloride.

View larger version (9K): [in this window] [in a new window]   Fig. 4.   Effect of 8-methoxypsoralen (Methoxsalen) on secretion across wt HBE. Subsequent to inhibition of basal Na+ absorption by amiloride (10 µM), Methoxsalen (10 µM) induced a sustained increase in Isc that was inhibited by bumetanide (20 µM). Dashed line, zero current level.

Genistein. Genistein has been shown to stimulate Cl secretion across both T84 (28) and Calu-3 (26) cells, and more recently has been demonstrated to directly activate both wt and F508 CFTR in excised patches (25, 54). We therefore determined whether genistein would stimulate transepithelial Cl secretion across wt CFTR-expressing HBE. As shown in Fig. 5A, subsequent to amiloride, genistein (50 µM) induced an initial peak increase in I_sc followed by a sustained, bumetanide-sensitive plateau. This concentration of genistein was chosen based on previous studies (27, 45) as well as on the K_s determined in our own studies (see below). In 17 monolayers, genistein induced an initial peak increase in I_sc from 11.0 ± 1.3 to 18.9 ± 1.2 µA/cm² followed by a sustained plateau at 16.6 ± 1.3 µA/cm². The addition of bumetanide in nine of these experiments reduced I_sc to 5.4 ± 0.7 µA/cm². In an additional six monolayers a complete concentration-response relationship was generated for genistein and the data fitted to a Michaelis-Menten function having an apparent K_s of 1.9 ± 0.4 µM (Fig. 5B). We were unable to evaluate the effects of genistein on anion secretion in the absence of amiloride as we have demonstrated that genistein stimulates a significant increase in Na⁺ absorption across HBE (12) (see Effect of NS004, 8-MOP, 1-EBIO, and Genistein on Na⁺ Absorption Across F-HBE). Thus changes in I_sc could not be reliably attributed to either cation absorption or anion secretion.

View larger version (11K): [in this window] [in a new window]   Fig. 5.   Effect of genistein (50 µM) on ion transport across wt CFTR-expressing HBE. A: in the presence of mucosal and serosal NaCl, genistein induced a sustained increase in Isc that was inhibited by bumetanide (20 µM). B: concentration-response curve for the genistein-induced Isc response in wt HBE, subsequent to amiloride. Data were fitted to a Michaelis-Menten function with an apparent Ks of 1.9 ± 0.4 µM. C: in the absence of mucosal and serosal Cl, genistein stimulated a sustained increase in Isc that was inhibited by both serosal DNDS (3 mM) and acetazolamide (100 µM). Dashed lines, zero current level.

Effect of the Ca²+-Dependent K⁺ Channel Opener 1-EBIO on Cl Secretion Across wt HBE

View larger version (8K): [in this window] [in a new window]   Fig. 6.   Effect of 1-EBIO (1 mM) and forskolin (10 µM) on Isc in wt CFTR-expressing HBE. A: subsequent to amiloride (10 µM), forskolin induced a sustained increase in Isc that was further potentiated by the addition of 1-EBIO. Addition of CTX (50 nM, serosal) and bumetanide (20 µM) inhibited the forskolin and 1-EBIO-induced Isc. B: subsequent to amiloride, 1-EBIO induced a sustained increase in Isc that was further increased by the addition of forskolin. Addition of CTX (50 nM, serosal) and bumetanide (20 µM) inhibited the forskolin and 1-EBIO-induced Isc. Dashed lines, zero current level.

Our results above suggest that forskolin, as well as the Cl channel openers, genistein and NS004, stimulate HCO₃ secretion across wt HBE, in addition to their effects on Cl secretion. Because 1-EBIO activates both CFTR and K_Ca (15), we determined whether 1-EBIO would similarly stimulate I_sc in a Cl-independent manner, suggestive of HCO₃ transport. In the absence of mucosal and serosal Cl, 1-EBIO (1 mM) increased I_sc from 4.4 ± 0.3 to 8.1 ± 1.1 µA/cm² (n = 3). Addition of serosal DNDS (3 mM) and acetazolamide (100 µM) reduced I_sc to 6.3 ± 0.3 and 5.1 ± 0.2 µA/cm², respectively, suggesting that 1-EBIO stimulates HCO₃ secretion across wt HBE.

Effect of Mucosal UTP on Cl Secretion Across HBE

View larger version (7K): [in this window] [in a new window]   Fig. 7.   Effect of mucosal UTP on Isc across wt HBE. Subsequent to amiloride (10 µM), mucosal UTP (100 µM) induced a transient increase in Isc followed by a sustained plateau that was sensitive to inhibition by bumetanide (20 µM). Dashed line, zero current level.

In the T84 colonic cell line the response to the Ca²⁺-mediated agonist, carbachol, is potentiated by both Cl channel activators, including forskolin (17), NS004 and 1-EBIO (13), and psoralens (14), as well as compounds that impinge on second messenger pathways such as wortmannin (phosphatidylinositol 3-kinase) (52) and arachidonyl trifluoromethyl ketone (AACOCF₃) [phospholipase A₂ (PLA₂)] (2, 11). Indeed, we demonstrated that NS004 (13) and the psoralens (14) not only potentiated the initial increase in I_sc across T84 cells but also induced a sustained phase to the carbachol response. Potentiation of either the transient or sustained phase of a Ca²⁺-mediated response in HBE would be expected to be of therapeutic benefit. Therefore, we evaluated the effect of these compounds on the I_sc response to mucosal UTP across wt HBE. In contrast to previous results from T84 cells, the effect of UTP on Cl secretion across wt HBE was unchanged in the presence of NS004 (10 µM; I_sc = 10.9 ± 2.7 µA/cm²; n = 4), 1-EBIO (1 mM; I_sc = 5.0 ± 0.2 µA/cm²; n = 9), AACOCF₃ (100 µM; I_sc = 8.9 ± 1.6 µA/cm²; n = 6) or wortmannin (100 nM, I_sc = 10.1 ± 2.3 µA/cm²; n = 7). These results suggest that the pharmacological potentiation of Ca²⁺-mediated agonists observed in other model Cl secretory systems cannot be readily extrapolated to human bronchial epithelia.

The response to mucosal UTP (100 µM) in the presence of forskolin is shown for one experiment in Fig. 8A. Subsequent to forskolin, addition of mucosal UTP induced a further increase in I_sc that was followed by a decline to below the initial forskolin plateau level. In a total of 30 monolayers, mucosal UTP increased I_sc by only 7.0 ± 1.2 µA/cm², with the plateau current level being 4.9 ± 0.6 µA/cm² below the sustained forskolin-induced current level. These results demonstrate that the response to mucosal UTP is not potentiated by forskolin. Indeed, the mucosal UTP-induced increase in I_sc in the presence of forskolin is smaller than that observed in the absence of forskolin (P < 0.001). Also, the net effect of UTP is a decrease in total outward current. The net decrease in outward current observed could be due to an inhibition of Cl secretion or a stimulation of K⁺ secretion. Indeed, we have previously shown that mucosal UTP inhibits the Ba²⁺- and quinine-sensitive basolateral G_K likely responsible for maintaining the driving force for Cl secretion in the presence of forskolin (12) (Fig. 2). To determine whether the decrease in I_sc observed was specific for UTP, we determined the effect of the Ca²⁺-ATPase inhibitor, thapsigargin. Similar to mucosal UTP, following establishment of a sustained forskolin response, thapsigargin (1 µM) induced an initial small increase in I_sc (I_sc = 3.9 ± 0.8 µA/cm²; n = 10) followed by a decline in current to 4.3 ± 0.9 µA/cm² below the sustained forskolin current level. This result indicates the decrease in current observed is independent of receptor-mediated effects.

View larger version (11K): [in this window] [in a new window]   Fig. 8.   Effect of mucosal UTP on basolateral K+ conductance. A: subsequent to amiloride (Amil; 10 µM), forskolin (Forsk) induced a sustained increase in Isc. Addition of mucosal UTP (100 µM) induced a further transient increase in Isc followed by a decline to below the forskolin-induced Isc level. B: addition of CTX (50 nM, serosal) had no effect on the forskolin-dependent increase in Isc, although it completely inhibited the UTP-dependent increase in Isc. CTX had no effect on the UTP-dependent decrease in Isc. In both cases, bumetanide (20 µM) inhibited the forskolin-dependent increase in Isc. Dashed lines, zero current level.

We next determined whether the initial increase in I_sc induced by UTP is due to the activation of a CTX-sensitive basolateral membrane K⁺ channel. As shown in Fig. 8B, CTX (50 nM) completely inhibited the initial increase in I_sc induced by mucosal UTP, whereas the apparent inhibitory phase was unaffected. In seven monolayers, the initial increase induced by UTP in the presence of CTX averaged 0.4 ± 0.4 µA/cm², whereas in seven paired experiments, carried out on the same day on monolayers from the same patient, UTP induced a significantly greater increase in I_sc, averaging 12.9 ± 3.9 µA/cm² (P < 0.001). These results demonstrate that the initial increase in Cl secretion induced by UTP is due to the activation of a basolateral membrane, CTX-sensitive K⁺ channel. Prior microelectrode studies confirm that mucosal ATP induces a large decrease in basolateral membrane resistance with no change in the electromotive force of the basolateral membrane, consistent with activation of both basolateral G_K and G_Cl (6). In this case, inhibition of G_K with CTX would result in a depolarization of the basolateral membrane toward the Cl reversal potential (E_Cl) such that I_sc is decreased.

Ca²+-Dependent K⁺ Secretion Across wt and F-508 HBE

View larger version (8K): [in this window] [in a new window]   Fig. 9.   Effect of mucosal UTP (100 µM, A) and thapsigargin (1 µM, B) on K+ secretion across wt CFTR (A)- and F508 CFTR (B)-expressing HBE. After establishment of a serosa-to-mucosa K+ gradient across the HBE monolayer in the absence of Cl (120 mM potassium gluconate:5 mM potassium gluconate) and inhibition of Na+ absorption with amiloride (10 µM), both mucosal UTP (A) and thapsigargin (B) induced an inward K+ secretory current. Addition of glibenclamide (Gliben; 100 µM) to the mucosal solution inhibited this K+ current in both wt CFTR (A)- and F508 CFTR (B)-expressing HBE. The subsequent addition of Ba2+ (5 mM) to the mucosal solution resulted in a further decrease in K+ current. Dashed lines, zero current level.

The observation that glibenclamide inhibited the Ca²⁺-dependent K⁺ secretory response led us to determine whether increased Ca²⁺ would similarly stimulate K⁺ secretion across F508 HBE in a glibenclamide-dependent fashion. As shown in Fig. 9B, thapsigargin stimulated K⁺ secretion across F508 HBE that was sensitive to block by mucosal glibenclamide (100 µM). The subsequent addition of mucosal Ba²⁺ (5 mM) further inhibited I_sc. In six experiments, thapsigargin increased I_sc from 56 ± 12 to 235 ± 28 µA/cm². Addition of glibenclamide and Ba²⁺ reduced I_sc to 56 ± 6 and 18 ± 1 µA/cm², respectively. These results demonstrate that Ca²⁺-dependent agonists similarly activate an apical membrane, glibenclamide-sensitive G_K in F508 HBE. Also, these results suggest that the magnitude of the UTP-dependent Cl secretory response observed may be underestimated, as any simultaneous UTP-dependent K⁺ secretory response would produce a current of opposite polarity.

Effect of Pharmacological Modulators on Cl Secretion Across F-HBE

1-EBIO. Before evaluating the effects of 1-EBIO on transepithelial Cl secretion across F-HBE, we determined whether the basolateral membrane K⁺ conductance in F-HBE would respond to pharmacological (1-EBIO) modulation. The mucosal membrane was permeabilized with nystatin and a serosa-to-mucosa K⁺ gradient established across the epithelium to measure serosal membrane K⁺ currents (I_K; see METHODS). After nystatin permeabilization, 1-EBIO (1 mM) increased I_K from 11.3 ± 1.2 to 31.0 ± 2.8 µA/cm² (n = 8), and this was inhibited by CTX (50 nM; 13.8 ± 1.2 µA/cm²), consistent with activation of a basolateral membrane Ca²⁺-dependent K⁺ conductance (10, 15). This response to 1-EBIO was similar in magnitude to that induced by mucosal UTP (I_K = 31.2 ± 6.9 µA/cm², n = 9).

8-MOP. We next evaluated the effect of 8-MOP on ion transport across F-HBE, subsequent to inhibition of Na⁺ absorption with amiloride. Similar to 1-EBIO, 8-MOP failed to significantly increase I_sc, subsequent to amiloride. In four monolayers, amiloride reduced I_sc from 44.1 ± 5.1 to 1.8 ± 0.2 µA/cm², with the subsequent addition of 8-MOP (10 µM) increasing I_sc to only 2.2 ± 0.4 µA/cm².

NS004. Because NS004 stimulates Cl secretion across HBE-expressing wt CFTR (Fig. 4), we determined whether this proposed CFTR activator would stimulate Cl secretion in F-HBE. As shown in Fig. 10A, subsequent to amiloride, NS004 (10 µM) had little effect on I_sc. The subsequent addition of forskolin (10 µM) induced an increase in I_sc, which was sensitive to bumetanide. In 13 experiments NS004 failed to significantly increase I_sc (0.4 ± 0.3 µA/cm²), whereas the subsequent addition of forskolin increased I_sc (2.3 ± 0.3 µA/cm²). The effect of NS004 on I_sc was also evaluated subsequent to forskolin addition (Fig. 10B). Forskolin increased I_sc an average of 1.4 ± 0.3 µA/cm². In contrast to NS004 alone, addition of NS004 subsequent to forskolin induced a significantly greater, bumetanide-sensitive, increase in I_sc (2.1 ± 0.3 µA/cm², n = 21, P < 0.001). These results suggest that forskolin and NS004 act in a synergistic fashion to stimulate Cl secretion across F-HBE (Fig. 10C).

View larger version (12K): [in this window] [in a new window]   Fig. 10.   Effect of NS004 and forskolin on Isc in HBE expressing F508 CFTR. A: NS004 (10 µM) induced a small increase in Isc subsequent to amiloride, and this Isc response was further increased by the addition of forskolin (10 µM) in a bumetanide (Bumet)-dependent fashion. B: subsequent to amiloride, forskolin (10 µM) induced a small increase in Isc that was further potentiated by the addition of NS004 (10 µM) in a bumetanide-dependent manner. C: average changes in Isc (Isc) for forskolin alone (Forsk), NS004 alone (NS004), or the combined Isc response elicited by forskolin followed by NS004 (Forsk + NS004). Numbers in parentheses indicate the numbers of experiments.

Genistein. The effect of genistein (50 µM) on Cl secretion across F-HBE is shown in Fig. 11. Subsequent to amiloride, genistein induced a small, bumetanide-sensitive increase in I_sc. In contrast to our results with NS004, addition of forskolin caused no further increase in I_sc following genistein. In 11 experiments, genistein increased I_sc an average of 2.5 ± 0.5 µA/cm² (P < 0.01), with the subsequent addition of forskolin increasing I_sc by only an additional 0.4 ± 0.5 µA/cm². In an additional nine experiments carried out on monolayers from the same culture, the order of forskolin and genistein addition were reversed. In these monolayers forskolin increased I_sc by 0.5 ± 0.2 µA/cm², with genistein further increasing I_sc by only 0.7 ± 0.5 µA/cm².

View larger version (13K): [in this window] [in a new window]   Fig. 11.   Effect of genistein (Gen; 50 µM) and forskolin (10 µM) on Isc in HBE-expressing F508 CFTR. Subsequent to amiloride, genistein induced a sustained, bumetanide-sensitive increase in Isc that was not further increased by forskolin.

Effect of Pharmacological Activators on F-HBE Cultured at 26°C

View larger version (16K): [in this window] [in a new window]   Fig. 12.   Effect of 1-EBIO (1 mM, A), NS004 (10 µM, B) and genistein (50 µM, C) on Isc in F508 CFTR-expressing HBE after incubation at 26°C for 24 h. All compounds were evaluated after inhibition of basal Na+ absorption with amiloride (10 µM). D: average data.1-EBIO, NS004, and genistein all induced significantly greater increases in Isc after culture at 26°C compared with results at 37°C (* P < 0.05).

Effect of 1-EBIO and Forskolin on F508/2789 +5GA HBE