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. 2011 Sep;121(9):1929-34.
doi: 10.1002/lary.21921. Epub 2011 Aug 16.

Transepithelial ion transport is suppressed in hypoxic sinonasal epithelium

Angela Blount  1 , Shaoyan Zhang, Michael Chestnut, Brian Hixon, Daniel Skinner, Eric J Sorscher, Bradford A Woodworth
Affiliations

Transepithelial ion transport is suppressed in hypoxic sinonasal epithelium

Angela Blount et al. Laryngoscope. 2011 Sep.

Abstract

Objectives/hypothesis: Sinonasal respiratory epithelial mucociliary clearance is dependent on the transepithelial transport of ions such as Cl(-) . The objectives of the present study were to investigate the role of oxygen restriction in 1) Cl(-) transport across primary sinonasal epithelial monolayers, 2) expression of the apical Cl(-) channels cystic fibrosis transmembrane conductance regulator (CFTR) and transmembrane protein 16A (TMEM16A), and 3) the pathogenesis of chronic rhinosinusitis.

Study design: In vitro investigation.

Methods: Murine nasal septal epithelial (MNSE), wild type, and human sinonasal epithelial (HSNE) cultures were incubated under hypoxic conditions (1% O(2) , 5% CO(2) ). Cultures were mounted in Ussing chambers for ion transport measurements. CFTR and TMEM16A expression were measured using quantitative reverse-transcription polymerase chain reaction (RT-PCR).

Results: The change in short-circuit current (ΔI(SC) in microamperes per square centimeter) attributable to CFTR (forskolin-stimulated) was significantly decreased due to a 12-hour hypoxia exposure in both MNSE (13.55 ± 0.46 vs. 19.23 ± 0.18) and HSNE (19.55 ± 0.56 vs. 25.49 ± 1.48 [control]; P < .05). TMEM16A (uridine triphosphate-stimulated transport) was inhibited by 48 hours of hypoxic exposure in MNSE (15.92 ± 2.87 vs. 51.44 ± 3.71 [control]; P < .05) and by 12 hours of hypoxic exposure in HSNE (16.75 ± 0.68 vs. 24.15 ± 1.35 [control]). Quantitative RT-PCR (reported as relative mRNA levels ± standard deviation) demonstrated significant reductions in both CFTR and TMEM16A mRNA expression in MNSE and HSNE owing to airway epithelial hypoxia.

Conclusions: Sinonasal epithelial CFTR and TMEM16A-mediated Cl(-) transport and mRNA expression were robustly decreased in an oxygen-restricted environment. These findings indicate that persistent hypoxia may lead to acquired defects in sinonasal Cl(-) transport in a fashion likely to confer mucociliary dysfunction in chronic rhinosinusitis.

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Conflict of interest statement

Conflict of Interest: Bradford Woodworth M.D. is a consultant for Arthrocare ENT and Gyrus ENT. Dr. Sorscher and Dr. Woodworth are inventors on a patent submitted regarding the possible activity of chloride secretagogues for therapy of sinus disease (Provisional Patent Application Under 35 U.S.C. ξ111(b) and 37 C.F.R ξ.53 (c) in the United States Patent and Trademark Office.

Figures

Figure 1
Figure 1
A. Representative ISC tracing of wild type murine nasal septal epithelial (MNSE) cultures. Wild type MNSE cells grown on transwell permeable supports and incubated in 1% O2 or physiologic O2 (control, 21%) were mounted in modified Ussing chambers under short-circuit conditions and sequentially exposed to amiloride (100µM); forskolin (20 µM); INH-172 (10 µM) and uridine triphosphate (UTP) (150 µM). By convention, a positive deflection in the tracing represents the movement of anion in the serosal to mucosal direction. Note the decrease in ISC in hypoxic MNSE (lower tracing) in all conditions compared to control (upper tracing). B. Change in short-circuit current (ΔISC) in murine nasal septal epithelial cultures after 12, 24, and 48 hours under hypoxic conditions. Forskolin-stimulated ΔISC and CFTR blockade by INH-72 were significantly decreased compared to control (n ≥ 6 per condition). UTP-stimulated ΔISC was significantly inhibited at 48 hours.
Figure 1
Figure 1
A. Representative ISC tracing of wild type murine nasal septal epithelial (MNSE) cultures. Wild type MNSE cells grown on transwell permeable supports and incubated in 1% O2 or physiologic O2 (control, 21%) were mounted in modified Ussing chambers under short-circuit conditions and sequentially exposed to amiloride (100µM); forskolin (20 µM); INH-172 (10 µM) and uridine triphosphate (UTP) (150 µM). By convention, a positive deflection in the tracing represents the movement of anion in the serosal to mucosal direction. Note the decrease in ISC in hypoxic MNSE (lower tracing) in all conditions compared to control (upper tracing). B. Change in short-circuit current (ΔISC) in murine nasal septal epithelial cultures after 12, 24, and 48 hours under hypoxic conditions. Forskolin-stimulated ΔISC and CFTR blockade by INH-72 were significantly decreased compared to control (n ≥ 6 per condition). UTP-stimulated ΔISC was significantly inhibited at 48 hours.
Figure 2
Figure 2
A: Representative ISC tracing of human sinonasal epithelial (HSNE) cultures. HSNE cells grown on transwell permeable supports and incubated in 1% O2 or physiologic O2 (control, 21%) were mounted in modified Ussing chambers under short-circuit conditions and sequentially exposed to amiloride (100µM); forskolin (20 µM); INH-172 (10 µM) and uridine triphosphate (UTP) (150 µM). Note the relative increase in ΔISC attributable to sodium blockade with amiloride (negative deflection) and decrease in forskolin-stimulated ΔISC at 24 hours under hypoxic conditions. B. Change in short-circuit current (ΔISC) in human sinonasal epithelial cultures after 12 and 24 hours under hypoxic (1% O2) conditions and following 24 hour recovery period under physiologic conditions (21% O2). Forskolin-stimulated ΔISC was sensitive to hypoxic stress, and demonstrated significant reduction in CFTR-mediated Cl− transport (n ≥ 6 per condition). Decreased ΔISC from INH-172 blockade verified the contribution of CFTR to the ISC. Conversely, CaCC-mediated ΔISC (UTP-stimulated) was more susceptible to oxygen restriction compared to MNSE and demonstrated early inhibition at 12 hours. Sodium absorption (amiloride blockade) was significantly increased at 12 hours and returned back to baseline by 24 hours. HSNE demonstrated significant recovery of Cl− transport following 24 hours in a physiologic O2 (21%) environment.
Figure 2
Figure 2
A: Representative ISC tracing of human sinonasal epithelial (HSNE) cultures. HSNE cells grown on transwell permeable supports and incubated in 1% O2 or physiologic O2 (control, 21%) were mounted in modified Ussing chambers under short-circuit conditions and sequentially exposed to amiloride (100µM); forskolin (20 µM); INH-172 (10 µM) and uridine triphosphate (UTP) (150 µM). Note the relative increase in ΔISC attributable to sodium blockade with amiloride (negative deflection) and decrease in forskolin-stimulated ΔISC at 24 hours under hypoxic conditions. B. Change in short-circuit current (ΔISC) in human sinonasal epithelial cultures after 12 and 24 hours under hypoxic (1% O2) conditions and following 24 hour recovery period under physiologic conditions (21% O2). Forskolin-stimulated ΔISC was sensitive to hypoxic stress, and demonstrated significant reduction in CFTR-mediated Cl− transport (n ≥ 6 per condition). Decreased ΔISC from INH-172 blockade verified the contribution of CFTR to the ISC. Conversely, CaCC-mediated ΔISC (UTP-stimulated) was more susceptible to oxygen restriction compared to MNSE and demonstrated early inhibition at 12 hours. Sodium absorption (amiloride blockade) was significantly increased at 12 hours and returned back to baseline by 24 hours. HSNE demonstrated significant recovery of Cl− transport following 24 hours in a physiologic O2 (21%) environment.
Figure 3
Figure 3. RT-PCR demonstrating presence of TMEM16A mRNA in murine nasal septal and human sinonasal epithelial cultures
PCR products of the expected sizes were amplified using TMEM16A-specific primer pairs in both species and are consistent with the expression of the calcium-activated chloride channel in sinus and nasal respiratory epithelium.
Figure 4
Figure 4. Effect of 48 hours hypoxia on TMEM16A and CFTR gene expression
Quantitative RT-PCR (reported as relative mRNA levels +/− S.D.) was performed on MNSE (A) and HSNE (B) incubated in 1% O2 for 48 hours (n ≥ 6 per condition). CFTR and TMEM16A mRNA were robustly decreased following incubation in this oxygen restricted environment.
Figure 4
Figure 4. Effect of 48 hours hypoxia on TMEM16A and CFTR gene expression
Quantitative RT-PCR (reported as relative mRNA levels +/− S.D.) was performed on MNSE (A) and HSNE (B) incubated in 1% O2 for 48 hours (n ≥ 6 per condition). CFTR and TMEM16A mRNA were robustly decreased following incubation in this oxygen restricted environment.
Figure 5
Figure 5. A (top) and B (bottom): Confirmation and localization of the TMEM16A channel protein
Western blotting confirmed that the TMEM16A channel was present in MNSE identifying the protein with a molecular weight of approximately 114 kDa (A). Co-localization of TMEM16A (red) and type IV Beta-tubulin (cilia – green) was also performed in HSNE to determine whether the channel is present on the apical membrane. Apical localization of TMEM16A was verified in HSNE at the base of the cilia (B).
Figure 5
Figure 5. A (top) and B (bottom): Confirmation and localization of the TMEM16A channel protein
Western blotting confirmed that the TMEM16A channel was present in MNSE identifying the protein with a molecular weight of approximately 114 kDa (A). Co-localization of TMEM16A (red) and type IV Beta-tubulin (cilia – green) was also performed in HSNE to determine whether the channel is present on the apical membrane. Apical localization of TMEM16A was verified in HSNE at the base of the cilia (B).
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