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Published ahead of print on November 1, 2006, doi:10.1165/rcmb.2006-0064OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 36, pp. 466-472, 2007
© 2007 American Thoracic Society
DOI: 10.1165/rcmb.2006-0064OC

Epithelial Ion Transport of Human Nasal Polyp and Paranasal Sinus Mucosa

Makoto Yasuda, Naomi Niisato, Hiroaki Miyazaki, Takemitsu Hama, Kenji Dejima, Yasuo Hisa and Yoshinori Marunaka

Department of Molecular Cell Physiology, Department of Otolaryngology–Head and Neck Surgery, and Department of Respiratory Molecular Medicine, Graduate School of Medical Science, Kyoto Prefectural University of Medicine; and Department of Otolaryngology, Kyoto Second Red Cross Hospital, Kyoto, Japan

Correspondence and requests for reprints should be addressed to Dr. Yoshinori Marunaka, Department of Molecular Cell Physiology, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan. E-mail: marunaka{at}koto.kpu-m.ac.jp


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Nasal cavity and paranasal sinus have various functions. However, little information is available on ion transport in these upper airway epithelia. In the present study, we measured the anion secretion and the anion channel activity to characterize the ion transport in epithelial cells prepared from human paranasal sinus mucosa (PSM) and nasal polyp (NP). To estimate the anion secretion and the anion channel activity, we measured the short-circuit current (Isc) and the transepithelial conductance (Gt) sensitive to NPPB (a Cl– channel blocker). The NPPB-sensitive Isc in PSM was larger than that in NP, correlating to the NPPB-sensitive Gt (Cl– channel activity). Forskolin stably elevated the NPPB-sensitive Isc associated with an increase in the NPPB-sensitive Gt in PSM and NP. UTP transiently stimulated the Isc associated with an elevation of Gt in PSM and NP. The stimulatory action of UTP on Isc and Gt was diminished by application of NPPB but not benzamil in PSM and NP, suggesting that UTP induced the NPPB-sensitive Isc (Cl– secretion) and Gt (Cl– channel activity). These observations suggest that in human PSM and NP, cAMP stably stimulates anion secretion by activating the Cl– (anion) channels, and that UTP just transiently elevates anion secretion via activation of some Cl– (anion) channels.

Key Words: epithelium • nasal polyp • paranasal sinus mucosa • CFTR



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
We characterized the ion transport and its regulation in the human tissue, indicating that cAMP and UTP stimulate anion secretion, giving us some information on how to block infection from the upper airway.

 
Airway epithelia play a key role in maintaining the volume and composition of airway surface liquid (1) by regulating transepithelial ion transport, and it is well known that various respiratory tract diseases are caused by impairing its physiologic condition (2). For example, cystic fibrosis (CF), defined as a disease caused by mutation of the cystic fibrosis transmembrane conductance regulator (CFTR), is a lethal inherited disease with progressive and repeatable pulmonary infection (3). CF has varied clinical features, and more than 90% of patients with CF demonstrate clinical and radiologic evidence of chronic rhinosinusitis (4). Chronic rhinosinusitis is one of the most common chronic inflammatory diseases that occur in the nasal and paranasal sinus mucosa. A previous study suggests a possibility that the transepithelial ion transport would play an important role in the development of nasal polyposis that is a special form of chronic rhinosinusitis (5). Although this study reports the bioelectrical properties of nasal polyp and turbinate epithelial cells localized in nasal cavity, little information on epithelial ion transport of paranasal sinus mucosa is available. Furthermore, various hypotheses on paranasal sinus functions have been proposed; for example, paranasal sinuses would play roles in humidifying inhaled air and reducing the weight of skull, but accurate function of paranasal sinuses is still unclear.

In the present study, we tried to characterize the ion transport and study its regulation in human paranasal sinus mucosa compared with those in human nasal polyp to investigate the paranasal sinus function from the view point of epithelial ion transport. The present study reports that in human paranasal sinus mucosa and nasal polyp cAMP stably stimulates anion secretion by activating the Cl– channels, and that UTP just transiently elevates anion secretion via activation of Cl– channels.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
This study was approved by the ethics committees of the Kyoto Prefectural University of Medicine and Kyoto Second Red Cross Hospital, and informed consent was obtained from all patients. Permission numbers were MCHS-487 and S15-10, respectively.

Chemicals
The chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise specified.

Cell Preparation and Culture
Epithelial cells of paranasal sinus mucosa and nasal polyp were obtained from patients with non-CF, nonbronchial asthma requiring surgery for their sinusitis. Each sample was obtained from the paranasal sinus mucosa and nasal polyp from the same patient. Cell culture was performed by modifying the previously described method (6, 7). Briefly, the resected tissues were digested in minimum essential medium containing 0.1% protease (protease type XIV from storeptomyces gryceus) for 1–2 h at 4°C, and epithelial cells were stripped by scraping tissue surface. The cells were centrifuged, plated on collagen-coated dishes, and grown in bronchial epithelial growth media (BEGM). BEGM was composed of LHC basal medium (Biofluids Inc., Rockville, MD) supplemented with 5 µg/ml insulin (Biofluids Inc.), 0.8% bovine pituitary extract (Kurabo, Osaka, Japan), 0.5 mg/ml bovine serum albumin, 25 ng/ml epithelial growth factor (EGF; BD, Bedford, MA), 72 ng/ml hydrocortisone, 0.08 mM CaCl2, 0.6 µg/ml epinephrine, 10 µg/ml transferrin, 0.5 µM phosphorylethanolamine, 0.5 µM ethanolamine, 5 x 10–8 M retinoic acid, 6.5 ng/ml triiodothyronine, trace elements (1x; Biofluids), Stock 4 (1x; Biofluids), Stock 11 (1x; Biofluids), 50 µg/ml gentamycin, 100 unit/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B. Culture medium was changed every other day until cultured cells reached almost confluence. Next, cells were seeded on culture inserts (Costar Corporation, Cambridge, MA) and cultured in air–liquid interface (ALI) medium. The ALI medium was composed of 50:50 LHC basal medium and Dulbecco's modified Eagle's nutrient mixture supplemented with compounds almost identical to those supplied to BEGM except for the concentrations of gentamycin, amphotericin B, and EGF; gentamycin or amphotericin B was not supplied, and EGF of 0.63 ng/ml was supplemented. The cells were seeded at 1 x 105 cells onto Costar wells with 6.5-mm diameter (6.5-mm Transwell filter, tissue culture-treated Transwell; Costar Corporation) for short-circuit current (Isc) and transepithelial conductance (Gt) measurements or at 1 x 106 cells onto Costar well (24-mm diameter, 24-mm Transwell filter, tissue culture-treated Transwell; Costar Corporation) for RNA extraction. Upon reaching confluence, the apical surface of Costar wells was rinsed with PBS, and medium was changed only in the basolateral side of culture. Cultured cells were maintained at 37°C in a 95% air-5% CO2 humidified incubator for 13–15 d before performing the experiments.

Solution
The standard Krebs bicarbonate Ringer (KBR) solution contained (in mM) 115 NaCl, 2.4 K2HPO4, 0.4 KH2PO4, 1.2 MgCl2, 1.2 CaCl2, 25 NaHCO3, and 5.2 D-glucose (5% CO2/95% O2, pH 7.4). For HCO3–-free solution, NaHCO3 was replaced with NaCl (100% O2, pH 7.4).

Transepithelial Conductance and Short Circuit Current
Gt and Isc were measured with an amplifier (VCC-600; Physiologic Instrument, San Diego, CA) in a modified Ussing chamber (Jim's Instrument, Iowa City, IA) as previously reported (8, 9). To block Na+ transport, benzamil of 10 µM was applied to the apical solution (10). The NPPB-sensitive Isc and Gt were respectively used as the anion secretion and the anion channel activity, since NPPB blocks Cl– channels (11). NPPB of 200 µM was applied to the apical solution. We omitted the data if the basal Gt was larger than 2,000 µS/cm2, since the cultured cells might not be confluent. In general, air–liquid interface culture enhances Na+ transport compared with standard submerged culture (12). The difference of tissue resistances between 2- and 3-wk cultures was not significant. The resistance of tissues cultured for 1 wk had a large variation.

Temperature
All experiments were performed at 37–38°C.

Statistics
Results were expressed as the means ± SEM. A value of P < 0.05 was considered statistically significant. The difference between groups was evaluated by a two-way ANOVA. If ANOVA indicated a significant difference, the Dunnett's multiple comparison test was used to identify statistically significant difference versus control group. Correlation coefficient was obtained using simple regression analysis. Statistical analysis was performed using Excel 2003 (Microsoft, Redmond, WA) with the add-in software Statcel 2 and Mini StatMate (13, 14).


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
In the present study, we investigated the ion transport property of upper airway epithelia, especially the property difference between human nasal polyp and paranasal sinus mucosa. Figure 1A shows the time course of Isc in nasal polyp and paranasal sinus mucosa. To block the Na+ current, 10 µM benzamil was applied to the apical solution at time of 0. Benzamil diminished the Isc in nasal polyp and paranasal sinus mucosa. NPPB (200 µM) was applied to the apical solution at 10 min after application of benzamil. NPPB almost abolished the residual Isc. As shown in Figure 1B, the NPPB-sensitive Isc was larger in paranasal sinus mucosa than that in nasal polyp, suggesting that activity of anion secretion into the cavity of paranasal sinus is larger than the nasal cavity. We next tried to clarify the relationship between the NPPB-sensitive Isc and Gt. Figure 2 indicates that the NPPB-sensitive Gt had correlation with the NPPB-sensitive Isc in nasal polyp and paranasal sinus mucosa. In epithelial tissues, the transcellular ionic movement pathway is mediated through two processes; that is, ions should move across the apical and basolateral membranes. Even if we applied NPPB to the apical solution to measure the apical anion conductance, the NPPB-sensitive Gt might not indicate the apical conductance due to the basolateral conductance. To resolve this point, we applied nystatin (100 µM) to the basolateral solution to permeabilize the basolateral membrane. The transepithelial conductances in nasal polyp just before and 30 min after application of nystatin to the basolateral solution were respectively 1,108.7 ± 106.5 and 1,102.5 ± 100.2 µS/cm2 (n = 4, no significant difference). We also estimated the nystatin-induced change in Gt for 30 min application of nystatin (the difference between Gt just before and 30 min after application of nystatin): nystatin, –6.3 ± 34.3 µS/cm2; control (DMSO alone), –0.6 ± 28.2 µS/cm2 (n = 4, no significant difference). These observations suggest that nystatin applied to the basolateral solution had no influence on the Gt or the NPPB-sensitive Gt in nasal polyp. The NPPB-sensitive conductances in nasal polyp without and with treatment of nystatin (basolateral application) were respectively 104.6 ± 10.9 and 99.2 ± 21.6 µS/cm2 (n = 4, no significant difference). Further, we also measured these parameters in paranasal sinus mucosa. The transepithelial conductances in paranasal sinus mucosa just before and 30 min after application of nystatin to the basolateral solution were respectively 1,050.7 ± 133.4 and 1,039.7 ± 120.5 µS/cm2 (n = 4, no significant difference). We also estimated the nystatin-induced change in Gt for 30 min application of nystatin (the difference between Gt just before and 30 min after application of nystatin): nystatin, –11.5 ± 29.5 µS/cm2; control (DMSO alone), –10.9 ± 38.2 µS/cm2 (n = 4, no significant difference). The NPPB-sensitive conductances in paranasal sinus mucosa without and with treatment of nystatin (basolateral application) were respectively 80.6 ± 17.4 and 88.0 ± 13.7 µS/cm2 (n = 4, no significant difference). These observations suggest that nystatin applied to the basolateral solution had no influence on the Gt or the NPPB-sensitive Gt in paranasal sinus mucosa. Since we detected no significant effects of nystatin on the conductance, we tried to confirm whether nystatin is effective as an ionophore by applying nystatin to the apical solution. The transepithelial conductances in nasal polyp just before and 30 min after application of nystatin to the apical solution were respectively 990.9 ± 133.6 and 1,363.6 ± 87.5 µS/cm2 (n = 4, P < 0.05), indicating that nystatin applied to the apical solution increased the conductance. The transepithelial conductances in paranasal sinus mucosa just before and 30 min after application of nystatin were respectively 1,220.1 ± 204.7 and 2,017.4 ± 122.4 µS/cm2 (n = 4, P < 0.01), indicating that nystatin applied to the apical solution increased the conductance. These observations indicate that nystatin has ability to permeabilize the membrane of nasal polyp and paranasal sinus mucosa, and that no significant effects of nystatin applied to the basolateral solution is due to the much larger conductance of the basolateral membrane than that of the apical membrane like other epithelia such as a renal epithelium (8). Since the NPPB-sensitive Gt was not affected by basolateral application of nystatin, the NPPB-sensitive Gt could be estimated by application of NPPB to the apical solution, even under no application of nystatin to the basolateral solution.


Figure 1
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  		Figure 1. The effects of benzamil and NPPB on Isc. (A) Time courses of effects of benzamil and NPPB action on Isc in nasal polyp (open circles) and paranasal sinus mucosa (solid circles). To block the Na+ current, benzamil (10 µM) was applied to the apical solution at time of 0. NPPB (200 µM) was applied to the apical solution at 10 min after application of benzamil. (B) NPPB-sensitive Isc in nasal polyp (open bar) and paranasal sinus mucosa (solid bar). Nasal polyp had larger NPPB-sensitive Isc than paranasal sinus mucosa. *P < 0.05; n = 10.

 

Figure 2
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  		Figure 2. Relationship between NPPB-sensitive Isc and Gt in nasal polyp (A) and paranasal sinus mucosa (B). The NPPB-sensitive Isc had correlation to the NPPB-sensitive Gt in both nasal polyp and paranasal sinus mucosa (n = 10; P < 0.001 in nasal polyp with correlation coefficient [R] of 0.89; P < 0.05 in paranasal sinus mucosa with R of 0. 65).

 
Furthermore, to study the effect of HCO3– on epithelial ion transport, we measured the NPPB-sensitive Isc and Gt in the absence of HCO3–. In nasal polyp, removal of HCO3– diminished the NPPB-sensitive Isc (Figure 3A) without any significant change in the NPPB-sensitive Gt (Figure 3B). On the other hand, in paranasal sinus mucosa, both the NPPB-sensitive Isc and Gt were significantly diminished by removal of HCO3– (Figures 3C and 3D).


Figure 3
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  		Figure 3. Effects of HCO3– on NPPB-sensitive Isc and Gt in nasal polyp and paranasal sinus mucosa. (A) NPPB-sensitive Isc in nasal polyp in the absence (open bar) and presence (solid bar) of HCO3–. *P < 0.05; n = 7 in the absence of HCO3–; n = 12 in the presence of HCO3–. (B) NPPB-sensitive Gt in nasal polyp in the absence (open bar) and presence (solid bar) of HCO3–. *P < 0.05; n = 7 in the absence of HCO3–; n = 12 in the presence of HCO3–. (C) NPPB-sensitive Isc in paranasal sinus mucosa in the absence (open bar) and presence (solid bar) of HCO3–. *P < 0.05; n = 7 in the absence of HCO3–; n = 12 in the presence of HCO3–. (D) NPPB-sensitive Gt in paranasal sinus mucosa in the absence (open bar) and presence (solid bar) of HCO3–. *P < 0.05; n = 7 in the absence of HCO3–; n = 12 in the presence of HCO3–. NPPB (200 µM) was applied to the apical solution to estimate the NPPB-sensitive Isc and Gt.

 
We studied the effect of 10 µM forskolin applied bilaterally, which is known to elevate the cytosolic cAMP. Figure 4A shows the time course of forskolin action on Isc in nasal polyp, indicating that forskolin stably increased Isc at least 130 min after application of forskolin. To test the effects of benzamil and NPPB, we applied benzamil (10 µM) and NPPB (200 µM) to the apical solution before addition of forskolin. Forskolin elevated the Isc even in the presence of benzamil (solid bar in Figure 4B). On the other hand, the forskolin action on Isc was diminished by the presence of NPPB (hatched bar in Figure 4B). Forskolin also induced Gt (Figure 4C), even in the presence of benzamil (solid bar in Figure 4C). On the other hand, the forskolin-induced Gt was significantly diminished by application of NPPB (hatched bar in Figure 4C). These observations suggest that forskolin induced the NPPB-sensitive Isc and Gt in nasal polyp.


Figure 4
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  		Figure 4. Time courses of forskolin action (A), and effects of benzamil and NPPB on forskolin-induced Isc (B) and Gt (C) in nasal polyp. *P < 0.05; n = 6.

 
We further studied the effect of forskolin on ion transport in paranasal sinus mucosa; the time courses are shown in Figure 5A. Forskolin elevated Isc and Gt even in the presence of benzamil (10 µM, apical addition; solid bar in Figures 5B and 5C). On the other hand, the presence of NPPB (200 µM, apical addition) reduced both forskolin-induced Isc (hatched bar in Figure 5B) and Gt (hatched bar in Figure 5C). These observations suggest that a phenomenon responding to forskolin (cAMP) occurs in paranasal sinus mucosa similar to in nasal polyp. Based on these observations, we indicate that forskolin mainly activates apical Cl– (anion) channels in these human nasal polyp and paranasal sinus mucosa.


Figure 5
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  		Figure 5. Time courses of forskolin action (A), and effects of benzamil and NPPB on forskolin-induced Isc (B) and Gt (C) in paranasal sinus mucosa. *P < 0.05; n = 6.

 
We next studied the effect of 100 µM UTP applied to the apical solution, which is known to increase intracellular Ca2+ concentration. Figure 6A shows the time course of UTP action on Isc in nasal polyp, indicating that UTP transiently increased Isc. To study the effects of benzamil and NPPB, we applied benzamil (10 µM, apical application) or NPPB (200 µM, apical application) before addition of UTP. UTP elevated the Isc even in the presence of benzamil (Figure 6A). On the other hand, the UTP action on Isc was diminished by the presence of NPPB (Figure 6A). UTP significantly induced Isc (open bar in Figure 6B). Application of benzamil did not significantly affect the UTP-induced Isc (solid bar in Figure 6B). On the other hand, the UTP-induced Isc was significantly diminished by application of NPPB (hatched bar in Figure 6B). Further, we studied the effects of benzamil and NPPB on the UTP-induced increase in Gt (Figure 6C). UTP significantly increased Gt (open bar in Figure 6C). NPPB significantly diminished Gt (hatched bar in Figure 6C), but benzamil had no effect on Gt (solid bar in Figure 6C). We also studied the effect of UTP on Isc and Gt in paranasal sinus mucosa. Figure 7A shows the time course of UTP (100 µM) action on Isc in paranasal sinus mucosa. UTP significantly increased Isc (open bar in Figure 7B). NPPB significantly diminished the UTP action on Isc (hatched bar in Figure 7B), but benzamil had no significant effect on the UTP action (solid bar in Figure 7B). UTP significantly increased Gt (open bar in Figure 7C). NPPB diminished the UTP-induced Gt (hatched bar in Figure 7C), but benzamil had no significant effect on the UTP-induced Gt (solid bar in Figure 7C). These observations suggest that in both human nasal polyp and paranasal sinus mucosa UTP transiently increases anion secretion by activating the apical anion channel.


Figure 6
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  		Figure 6. Time courses of UTP action (A), and effects of benzamil and NPPB on UTP-induced Isc (B) and Gt (C) in nasal polyp. *P < 0.05 compared with control; n = 11.

 

Figure 7
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  		Figure 7. Time courses of UTP action (A), and effects of benzamil and NPPB on UTP-induced Isc (B) and Gt (C) in paranasal sinus mucosa. *P < 0.05 compared with control; n = 11.

 
We next applied 20 µM BAPTA-AM, a chelator of intracellular Ca2+ before addition of UTP. BAPTA-AM significantly diminished the UTP-induced Isc (solid bar in Figure 8A) and Gt (solid bar in Figure 8B) in nasal polyp. In the same way, BAPTA-AM significantly diminished the UTP-induced Isc (solid bar in Figure 8C) and Gt (solid bar in Figure 8D) in paranasal sinus mucosa.


Figure 8
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  		Figure 8. UTP-induced Isc (A) and Gt (B) in nasal polyp in the absence (open bars) and presence (solid bars) of BAPTA-AM. UTP-induced Isc (C) and Gt (D) in paranasal sinus mucosa in the absence (open bars) and presence (solid bars) of BAPTA-AM. *P < 0.05; n = 7.

 
We applied suramin (200 µM), a nonselective P2 receptor antagonist), to the apical solution 60 min before addition of UTP. The UTP-induced Isc was diminished by suramin both in nasal polyp and in paranasal sinus mucosa: nasal polyp, 6.6 ± 2.9 µA/cm2 versus control 10.8 ± 4.9 µA/cm2 (n = 7; P < 0.05); paranasal sinus mucosa, 4.2 ± 3.2 µA/cm2 versus control 7.3 ± 3.0 µA/cm2 (n = 7; P < 0.05).


   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
In the present study, we show that nasal polyp and paranasal sinus mucosa have some characteristics of Na+ absorption and anion (Cl– and/or HCO3–) secretion by measuring Isc and Gt sensitive to benzamil or NPPB. Especially, it is of interest that the NPPB-sensitive Isc was larger in paranasal sinus mucosa than in nasal polyp. This observation suggests that paranasal sinus mucosa has more activity of anion secretion than nasal polyp. The NPPB-sensitive Isc had correlation to the NPPB-sensitive Gt, suggesting that the apical anion channel plays an essential role in Cl– (and/or HCO3–) secretion. Further, we show that removal of HCO3– diminished the NPPB-sensitive Gt in paranasal sinus mucosa but not in nasal polyp, suggesting that paranasal sinus mucosa would have more permeability to HCO3– than nasal polyp. Since we measured Gt by applying NPPB to the apical membrane, the measured NPPB-sensitive Gt indicates the anion conductance of the apical membrane (i.e., the apical anion conductance [or activity] of anion channels at the apical membrane).

The NPPB-sensitive Isc means anion secretion, which is mediated by two steps: (1) the entry step of anion across the basolateral membrane such as Na+/K+/2Cl– cotransporter, the Na+/HCO3– cotransporter, and the Cl–/HCO3– exchanger at the basolateral membrane; and (2) the releasing step of anion across the apical membrane via anion (Cl–) channels at the apical membrane. As described above, in nasal polyp, the presence of HCO3– elevated the NPPB-sensitive Isc with no influence on the NPPB-sensitive Gt. This observation could be explained as follows: (1) the rate-limiting step of anion secretion in nasal polyp would be the entry step of anion into the cytosolic space across the basolateral membrane (but not apical anion conductance); and (2) even if the apical anion conductance (NPPB-sensitive conductance) is not increased, the amount of anion secretion is elevated by an increase in anion entry (uptake) across the basolateral membrane. In other words, under the condition that the apical ion channel capacity conducting anion (the apical anion conductance) is larger than the amount of anion uptake across the basolateral membrane, the amount of anion secretion can be increased up to the capacity of the apical ion channel conducting anion without any increase in the apical anion channel conductance by an increase in anion uptake across the basolateral membrane. Especially in nasal polyp, removal of HCO3– diminished anion secretion without any influence on apical anion conductance; that is, the presence of HCO3– contributes to anion entry, but does not affect the apical anion channel conductance. In the presence of HCO3–, the Na+/HCO3– cotransporter and Cl–/HCO3– exchanger at the basolateral membrane function to incorporate Cl– into the cytosolic space as follows: the Na+/HCO3– cotransporter contributes to uptake of HCO3– into the cytosolic space, and this cytosolic HCO3– participates in uptake of Cl– via Cl–/HCO3– exchanger, with the result that the presence of HCO3– contributes to entry of Cl– into the cytosolic space across the basolateral membrane via these two processes.

A previous study shows that the epithelium localized in nasal cavity such as nasal polyp or turbinate has much ability to absorb the water (15). However, the source supplying the water to maintain the homeostasis of airway surface liquid in the nasal cavity has not yet been elucidated. Our data suggest one possibility that epithelium of paranasal sinus mucosa would secrete the water and maintain airway surface liquid covering upper airways.

A study in cultured alveolar epithelial type II cells suggests that the cAMP-mediated Na+ uptake across the apical membrane via ENaC (16) might depend on an initial uptake of Cl– (17). A subsequent study in cultured alveolar type II cells under apical air–liquid interface conditions reports that beta-adrenergic agonists produce acute activation of apical Cl– channels associated with enhancement of Na+ absorption (18). These data suggest that activation of apical Cl– channel would increase anion influx, leading the apical membrane to hyperpolarization. This hyperpolarization would increase the ENaC-mediated Na+ absorption via an increase in the driving force for Na+ entry without any change of ENaC activity in alveolar epithelial type II cells. Another in vivo study shows that the cAMP-mediated up-regulation of pulmonary edema fluid clearance might occur across distal airway epithelium as well as at the level of the alveolar epithelium (19). The direction of Cl– movement depending on the electrochemical potential of Cl– would determine the direction of water flux due to activation of Cl– channels at the apical membrane in each airway epithelium. Therefore, we need further studies to confirm the electrochemical potential for Cl– (anion) in the epithelial tissues, leading us to definite understandings on the anion secretion and ENaC function.

We further studied the effect of UTP on ion transport in nasal polyp and paranasal sinus mucosa. The UTP-induced increases in Isc and Gt were diminished by NPPB but not by benzamil, indicating that UTP activates the NPPB-sensitive Isc and Gt, but not the benzamil-sensitive Isc or Gt. UTP is generally reported to induce an increase in the cytosolic Ca2+ concentration (20). Therefore, we considered a possibility that an increase in cytosolic Ca2+ is necessary for the UTP-induced activation of apical Cl– channel. To confirm this point, we applied BAPTA-AM (a chelator of intracellular Ca2+) before addition of UTP. BAPTA-AM significantly diminished the UTP action on Isc and Gt. These observations suggest that UTP stimulates anion secretion by activating the apical Cl– channels via elevation of the cytosolic Ca2+ concentration (21). In airway epithelial cells, extracellular nucleotides show their physiologic functions mainly through P2Y2 and P2Y1 receptors (22, 23). Since the P2Y2 receptor, which responds to extracellular UTP and ATP, is expressed at the apical membrane of human airway epithelial cells (24), we applied 200 µM suramin (a nonselective P2 receptor antagonist) before addition of UTP. Suramin significantly diminished the UTP-induced Isc in both nasal polyp and paranasal sinus mucosa. Our data suggest that UTP would activate PKC via P2Y2 in a Ca2+-dependent manner, increasing anion secretion mediated through the apical Cl– channel such as CFTR Cl– channel (25). This suggests that UTP would affect Na+ transport, since the elevation of the intracellular Ca2+ concentration and PKC activity affects the activity of the benzamil-sensitive Na+-permeable channels including ENaC and nonselective cation channel (26–33). However, the present study indicates that the UTP action on Isc or Gt was not affected by the presence of benzamil, suggesting that UTP would have no effect on the benzamil-sensitive ion transport (Na+ absorption) or channel activity. These observations suggest that the benzamil-sensitive ion channels observed in the present study would be regulated in a manner different from that of the regulatory pathway previously reported (26–33). To confirm the regulatory pathway of the benzamil-sensitive ion channels in human nasal polyp and paranasal mucosa, we need further studies.

A quantity of 200 µM of suramin, which is generally thought to be the sufficient concentration to abolish the UTP action via P2 receptor, did not abolish the UTP action on Isc in the present study, but partially diminished the UTP action. Although the concentration of suramin (200 µM) would be generally sufficient to eliminate the UTP action via P2 receptor, we have no information on the characteristics of P2 receptor in these human tissues. If suramin completely antagonizes P2 receptor in these human tissues, the suramin-resistant component of UTP-induced current would be mediated in a P2 receptor–independent pathway. To confirm this P2 receptor–independent pathway, we need further study to clarify a new type of UTP-binding protein (receptor).

UTP only transiently elevated the Isc, but forskolin stably increased the Isc. To clarify the reason why these agents, forskolin and UTP, showed respectively the stable and transient stimulation in Isc, we compared the time-dependent effects of forskolin and UTP on the NPPB-sensitive Gt. The UTP-induced increase in Gt transiently occurred like Isc, indicating that the UTP-induced transient increase in Isc would be due to the transient activation of the apical anion channels, which could be caused by increase in the cytosolic Ca2+ concentration and activation of PKC. We also show that the NPPB-sensitive Isc and Gt have correlation, suggesting that the rate-limiting step of the transepithelial anion secretion is the releasing step of anions at least in the presence of HCO3–, in which the CFTR Cl– channel or other types of Cl– channels participate. The forskolin (cAMP)-induced activation of anion channels was stable, as shown in the present study. The difference of stimulation of anion secretion in paranasal sinus mucosa and nasal polyp between forskolin and UTP would be due to the stable or transient activation of apical anion channels. Further, the stable stimulation of anion secretion in epithelial tissues requires stable elevation of the anion entry step such as the Na+/K+/2Cl– cotransporter, Na+/HCO3– cotransporter and/or Cl–/HCO3– exchanger in addition to anion channels (34, 35). This suggests that in paranasal sinus mucosa and nasal polyp forskolin (cAMP) activates the anion entry step such as the Na+/K+/2Cl– cotransporter, Na+/HCO3– cotransporter and/or Cl–/HCO3– exchanger.


   Acknowledgments
 
The authors thank Drs. Richard C. Boucher and Scott H. Randell for valuable advice at starting this research and technical assistance in paranasal sinus cell culture.


   Footnotes
 
This work was supported by Grants-in-Aid from Japan Society of The Promotion of Science (17390057, 17590191, 17790154, 8659056), Fuji Foundation for Protein Research, and The Salt Science Research Foundation (0,241), a Research Grant for Nervous and Mental Disorders (15–4) from the Ministry of Health, Labor and Welfare, Japan, and a Leading Project for Biosimulation from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Originally Published in Press as DOI: 10.1165/rcmb.2006-0064OC on November 1, 2006

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form February 9, 2006

Accepted in final form October 9, 2006


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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