Hyun Jae Lee

House dust mite extract activates apical Cl− channels through protease‐activated receptor 2 in human airway epithelia

Adequate fluid secretion from airway mucosa is essential for maintaining mucociliary clearance, and fluid hypersecretion is a prominent feature of inflammatory airway diseases such as allergic rhinitis. House dust mite extract (HDM) has been reported to activate protease‐activated receptors (PARs), which play various roles in airway epithelia. However, the role of HDM in regulating ion transporters and fluid secretion has not been investigated. We examined the effect of HDM on ion transport in human primary nasal epithelial cells. The Ca2+‐sensitive dye Fura2‐AM was used to determine intracellular Ca2+ concentration ([Ca2+]i) by means of spectrofluorometry in human normal nasal epithelial cells (NHNE). Short‐circuit current (Isc) was measured using Ussing chambers. Fluid secretion from porcine airway mucosa was observed by optical measurement. HDM extract (10 µg/Ml) effectively cleaved the PAR‐2 peptide and induced an increase of [Ca2+]i that was abolished by desensitization with trypsin, but not with thrombin. Apical application of HDM‐induced Isc sensitive to both a cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor and a Ca2+‐activated Cl− channel (CaCC) inhibitor. HDM extract also stimulated fluid secretion from porcine airway mucosa. HDM extract activated PAR‐2 and apical Cl− secretion via CaCC and CFTR, and HDM‐induced fluid secretion in porcine airway mucosa. Our results suggest a role for PAR‐2 in mucociliary clearance and fluid hypersecretion of airway mucosa in response to air‐borne allergens such as HDM. J. Cell. Biochem. 109: 1254–1263, 2010.

Thick airway surface liquid volume and weak mucin expression in pendrin‐deficient human airway epithelia

Pendrin is an anion exchanger whose mutations are known to cause hearing loss. However, recent data support the linkage between pendrin expression and airway diseases, such as asthma. To evaluate the role of pendrin in the regulation of the airway surface liquid (ASL) volume and mucin expression, we investigated the function and expression of pendrin and ion channels and anion exchangers. Human nasal epithelial cells were cultured from 16 deaf patients carrying pendrin mutations (DFNB4) and 17 controls. The cells were treated with IL‐13 to induce mucus hypersecretion. Airway surface liquid thickness was measured and real‐time polymerase chain reaction was performed targeting various transporters and MUC5AC. Anion exchanger activity was measured using a pH‐sensitive fluorescent probe. Periodic acid‐Schiff staining was performed on the cultured cells and inferior turbinate tissues. The ASL layer of the nasal epithelia from DFNB4 subjects was thicker than the controls, and the difference became more prominent following IL‐13 stimulation. There was no difference in anion exchange activity after IL‐13 treatment in the cells from DFNB4 patients, while it increased in the controls. Goblet cell metaplasia induced by IL‐13 treatment seen in the controls was not observed in the DFNB4 cells. Furthermore, the periodic acid‐Schiff staining‐positive area was lesser in the inferior turbinate tissues from DFNB4 patients that those from controls. Pendrin plays a critical role in ASL volume regulation and mucin expression as pendrin‐deficient airway epithelial cells are refractory to stimulation with IL‐13. Specific blockers targeting pendrin in the airways may therefore have therapeutic potential in the treatment of allergic airway diseases.

Protease-activated receptor 2 mediates mucus secretion in the airway submucosal gland

Protease-activated receptor 2 (PAR2), a G protein-coupled receptor expressed in airway epithelia and smooth muscle, plays an important role in airway inflammation. In this study, we demonstrated that activation of PAR2 induces mucus secretion from the human airway gland and examined the underlying mechanism using the porcine and murine airway glands. The mucosa with underlying submucosal glands were dissected from the cartilage of tissues, pinned with the mucosal side up at the gas/bath solution interface of a physiological chamber, and covered with oil so that secretions from individual glands could be visualized as spherical bubbles in the oil. Secretion rates were determined by optical monitoring of the bubble diameter. The Ca2+-sensitive dye Fura2-AM was used to determine intracellular Ca2+ concentration ([Ca2+]i) by means of spectrofluorometry. Stimulation of human tracheal mucosa with PAR2-activating peptide (PAR2-AP) elevated intracellular Ca2+ and induced glandular secretion equal to approximately 30% of the carbachol response in the human airway. Porcine gland tissue was more sensitive to PAR2-AP, and this response was dependent on Ca2+ and anion secretion. When the mouse trachea were exposed to PAR2-AP, large amounts of secretion were observed in both wild type and ΔF508 cystic fibrosis transmembrane conductance regulator mutant mice but there is no secretion from PAR-2 knock out mice. In conclusion, PAR2-AP is an agonist for mucus secretion from the airway gland that is Ca2+-dependent and cystic fibrosis transmembrane conductance regulator-independent.

Expression of anion exchangers in cultured human endolymphatic sac epithelia.

Hypothesis Pendrin acts as a Cl-/HCO3- exchanger and is responsible for endolymphatic fluid volume and pH homeostasis in human endolymphatic sac epithelial cells. Background The endolymphatic sac (ES) is part of the membranous labyrinth in the inner ear that plays an important role in maintaining homeostasis of the endolymphatic fluid system. However, the exact mechanism of fluid volume and pH regulation is not fully understood yet. We aimed to demonstrate the expression of various anion exchangers (AEs), including pendrin, in cultured human endolymphatic sac epithelial (HESE) cells. Methods Endolymphatic sac specimens were harvested during acoustic neuroma surgery (n = 24) using the translabyrinthine approach and then subcultured with high epidermal growth factor (EGF) (25 ng/ml) media and differentiated using low-EGF (0.5 ng/ml) media. The cultured cells were classified according to the morphology on TEM. The Cl-/HCO3- exchanger activity was assessed by pHi measurement using pH sensitive dye 2′, 7′-bis (2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF/AM). We performed reverse transcriptase–polymerase chain reaction and immunohistochemical staining for AEs. Results We determined that 7.3 ± 6.7% of cells differentiated into mitochodria-rich cells and 50.2 ± 15.1 of cells differentiated into ribosome-rich cells. bAE3, AE4, SLC26A4, SLC26A6, and SLC26A11 were also expressed in cultured HESE cells. The cultured cells had Cl-/HCO3- and Cl-/formate exchange activity on the luminal membrane, which is sensitive to anion channel inhibitors (DIDS 500 &mgr;M). Furthermore, we showed that pendrin (SLC26A4) was expressed in cultured HESE cell membranes. Conclusion Our results suggest that AEs, including pendrin, are expressed in epithelia of ES and may have role in maintaining ionic homeostasis, and the HESE culture system are useful for uncovering the functional role of ES epithelial cells.

Synergistic mucus secretion by histamine and IL-4 through TMEM16A in airway epithelium

Histamine is an important mediator of allergic reactions, and mucus hypersecretion is a major allergic symptom. However, the direct effect of histamine on mucus secretion from airway mucosal epithelia has not been clearly demonstrated. TMEM16A is a Ca2+-activated chloride channel, and it is closely related to fluid secretion in airway mucosal epithelia. We investigated whether histamine directly induces fluid secretion from epithelial cells or submucosal glands (SMG) and mechanisms related, therewith, in allergic airway diseases. In pig airway tissues from the nose or trachea, histamine was a potent secretagogue that directly induced strong responses. However, gland secretion from human nasal tissue was not induced by histamine, even in allergic rhinitis patients. Histamine type 1 receptor (H1R) and histamine type 2 receptor (H2R) were not noted in SMG by in situ hybridization. Cultured primary human nasal epithelial (NHE) cells were used for the measurement of short-circuit current changes with the Ussing chamber. Histamine-induced slight responses of anion secretions under normal conditions. The response was enhanced by IL-4 stimulation through TMEM16A, which might be related to fluid hypersecretion in allergic rhinitis. Pretreatment with IL-4 augmented the histamine response that was suppressed by a TMEM16A inhibitor. TMEM16A expression was enhanced by 24-h treatment of IL-4 in human nasal epithelial cells. The expression of TMEM16A was significantly elevated in an allergic rhinitis group, compared with a control group. We elucidated histamine-induced fluid secretions in synergy with IL-4 through TMEM16A in the human airway epithelium. In addition, we observed species differences between pigs and humans in terms of gland secretion of histamine.

Vitamin A Deficiency Induces Fluid Hyposecretion from the Airway Submucosal Glands of Mice

Vitamin A deficiency (VAD) alters the phenotype of airway epithelium and attenuates the epithelial defense system, and many studies have reported the association of VAD with respiratory disease. In this study, we investigated changes in submucosal glands (SMG) in a mouse model of VAD. C57BL/6 mice were fed a vitamin A-devoid diet and the others were fed a control diet (1.2 mg retinol/kg). The areas of serous and mucous cells of SMG were measured in 4-, 8-, and 20-wk-old male mice. The volume and lysozyme concentration of glandular secretions were also measured. The 2 groups did not differ in body weight or general morbidity at 3-10 wk of age, although serum retinol concentrations were greater in the control mice than in the VAD mice after 4 wk. Upon histological evaluation, we found that the areal ratio of serous cells:total SMG cells was significantly lower after 8 wk in the VAD mice compared with the control mice, although the total area of SMG did not differ between groups throughout the 20-wk experiment. The number of secretory bubbles did not differ between the groups, but total secretion volume was reduced by 35% in 8-wk-old VAD mice compared with controls. Furthermore, the concentration of lysozyme in secretions from 8-wk-old VAD mice was also less than in controls, compounding the effect of diminished secretion volume. In this study, we found serous cell hypotrophy/hypoplasia and dysfunction in VAD mice, which may contribute to the susceptibility to airway infection linked to VAD.

Functional study of mucus secretion of the eustachian tube in Guinea pigs.

Objectives: Our aim was to verify neural regulation of submucous gland mucus secretions in the Eustachian tubes of guinea pigs. Study Design: Prospective animal study. Methods: Eustachian tubes harvested from 12 guinea pigs were used for this study. For real-time resolution of pure glandular secretion, we used a modified method of single-gland optical measurement. Secretory monitoring was undertaken after each preparation with phenylephrine, isoproterenol, forskolin, and substance P. To confirm the viability of each tissue, we examined glandular secretion after treatment with carbachol. Secretory effects of each agonist were evaluated by comparing with basal secretion using a Students t test (p < 0.01). Results: The Ca2+-elevating agonists carbachol and substance P showed greater effects on submucous gland secretions of the Eustachian tube than the cyclic adenosine monophosphate (cAMP)-elevating agonists forskolin and isoproterenol. However, phenylephrine, although it belongs to the Ca2+-elevating agonist group, did not show any significant secretory effect. Conclusion: The optical measurement method used in this study had the merit of real-time resolution of submucous glandular secretion. Submucous glandular secretion in the Eustachian tube was regulated by both Ca2+- and cAMP-elevating agonists, and Ca2+-elevating agonists seemed to be more potent than cAMP-elevating agonists except phenylephrine. Our results suggest that not only the autonomic nerve system but also the neuropeptides such as substance P are closely related to glandular secretion in the Eustachian tube, and &bgr;-adrenergic receptors seem to be more related to submucous glandular secretion of the Eustachian tube in guinea pig than &agr;-adrenergic receptors.

Integrating TCSC to enhance transmission capability and security: Feasibility studies for Korean electric power system

This paper investigates operational benefits of integrating thyristor controlled series capacitor (TCSC) in Korean electric power system. Critical challenges of balancing the electricity demand rise and deferred transmission construction due to the public opposition and environmental impact as well as unique topological issues of the power grid, endanger reliability of the system. Installation of flexible ac transmission systems (FACTS) is envisioned to be a low-environmental impact technology that effectively resolve these issues. In particular, this research exploits flow control capability of TCSC and examines the benefits of improving transmission capability and security. It presents a series of studies for identifying the adequate location and size of the TCSC and discusses practical issues and lessons learned through studies.

House Dust Mite Nonspecifically Induces Fluid Secretion from Nasal Glands

Objective: Investigate the effects of the house dust mite (HDM) on fluid secretion in submucosal glands in nasal tissue and to compare its effects in control and diseased groups. In addition, we looked to see if PAR2 was involved in the HDM-induced fluid response. Method: Inferior nasal turbinate tissue was harvested from 26 patients with either allergic rhinitis (with HDM-specific IgE) and/or chronic rhinosinusitis, and eight controls. We used a microscope attached to a camera to quantify mucus bubbles secreted from individual submucosal glands in response to HDM extract and PAR2-AP (activating peptide). Results: HDM stimulated fluid secretion, and the diseased group showed a higher secretion rate than did the controls. Interestingly, there was no significant difference between the three diseased groups, and CRS patients without HDM-specific IgE were also stimulated by HDM. The nonspecific response induced by HDM was hypothesized to be through PAR2. The expression of PAR2 was predominant on the basolateral side of gland acini. HDM-induced secretion rates positively correlated with PAR2-AP-induced secretion rates. Carbachol responses and gland density were not different among the three groups. Conclusion: These findings suggest that HDM induces an IgE-independent nonspecific response from nasal submucosal glands, generating fluid secretion in allergic or infective nasal tissue, and PAR2 is at least partially involved in this process.