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1 KISEP Otology Korean J Otolaryngol 2006;49:263-8 사람중이점막상피세포에서 Uridine-5 Triphosphate 에의한칼슘이동과점액분비에미치는카페인의효과 최재영 김성헌 정상호 손은진 박헌이 신중욱 윤주헌 Effect of Caffeine on UTP-induced Ca 2+ Mobilization and Mucin Secretion in Human Middle Ear Epithelial Cells Jae-Young Choi, MD, Sung Huhn Kim, MD, Sang Ho Jung, MD, Eun Jin Son, MD, Hun Yi Park, MD, Joong Wook Shin, MD and Joo-Heon Yoon, MD Department of Otorhinolaryngology, Yonsei University College of Medicine, Seoul, Korea ABSTRACT Background and Objectives:Puringeric receptors and their agonists like uridine-5-triphosphate UTP and adenosine triphosphate ATP, regulate mucin secretion in middle ear epithelial cells. In the present study, we examined the effects of purinergic agonists on Ca 2+ influx Ca 2 i in normal human middle ear epithelial NHMEE cells. We also examined the effect of caffeine, an inositol 1, 4, 5-triphosphate IP 3 inhibitor, on UTP induced Ca 2 i and mucin secretion in NHMEE cells. Materials and Method:NHMEE cells were stimulated with various purinergic agonists, such as UTP, and Ca 2 i was measured using a miniature double perfusion chamber. UTP-induced mucin secretion was quantitated by immunoblotting assay. Results:The determined order of purinergic agonist potency with respect to Ca 2 i was ATPUTP2-MeSATPADP adenosine. UTP-induced mucin secretion was inhibited when the intracellular Ca 2+ was removed with 2-bis 2-aminophenoxy ethane-n, N, N, N -tetraacetic acid-acetoxymethyl ester. Caffeine suppressed UTP-induced Ca 2 i, and but inhibited UTPinduced and constitutional mucin secretion. Conclusion:Our results suggest that caffeine may have a therapeutic effect in mucoid otitis media by suppressing mucin secretion. (Korean J Otolaryngol 2006;49:263-8) KEY WORDS:Mucin Caffeine Calcium

2 카페인이점액분비에미치는영향 Ca 2+ i Ca 2+ i Ca 2+ i i Ca 2+ i - Ca 2+ i Korean J Otolaryngol 2006;49:263-8

최재영외 t i Ca 2+ i Ca 2+ i Ca 2+ i Ca 2+ i Ca 2+ i Ca 2+ i Ratio352/380 0.1 Units A 1.15 1.05 B 100 MATP 100 MUTP 100 MUDP 100 M 100 M Adenosine 2MeATP 0.

by purinergic agonists in cultured normal human middle ear epithelial cells. Fig. 2.

3 최재영외 t i Ca 2+ i Ca 2+ i Ca 2+ i Ca 2+ i Ca 2+ i Ca 2+ i Ratio352/ Units A B 100 MATP 100 MUTP 100 MUDP 100 M 100 M Adenosine 2MeATP Sec Fig. 1. Mobilization of intracellular Ca 2+ by purinergic agonists in cultured normal human middle ear epithelial cells. Fig. 2. Immunohistochemical analysis of normal human middle ear epithelial NHMEE cells using antibody against the P2Y 2 receptor. NHMEE cells showed positive immunofluorescent reactions in the apical and basal cell layers A, whereas, negative controls, which were treated with anti-sense peptides, showed no immunoreactivity B. 265

4 카페인이점액분비에미치는영향 500% 400% 400% Mucin secretion % 300% 200% 100% Mucin secretion % 200% 0% Control Caffeine mm 0% UTP BAPTA-AM A 100 M of UTP Fig. 3. Effect of intracellular calcium depletion on mucin secretion in NHMEE cells. Intracellular Ca 2+ was depleted by adding 50 M 2-bis 2-aminophenoxy ethane-n, N, N, N -tetraacetic acid-acetoxymethyl ester. This treatment suppressed both UTPinduced mucin secretion and constitutional mucin secretion. Ratio352/ Units 150 sec Mucin secretion % B 100% 50% 0% Control Caffeine mm Fig. 5. Effect of caffeine on mucin secretion in NHMEE cells. Caffeine suppressed UTP-induced mucin secretion in NHMEE cells in a dose- dependent manner A. More than 10 mm of caffeine also suppressed constitutional mucin secretion B M UTP mm caffeine Fig. 4. Effect of caffeine on UTP-induced Ca 2+ mobilization in NHMEE cells. Caffeine suppressed UTP-induced mucin Ca 2+ influx in a dose-dependant manner. Ca 2+ i Ca 2+ i 0 in vivoin vitro Ca 2+ i Ca 2+ i Ca 2+ i 266 Korean J Otolaryngol 2006;49:263-8

5 최재영외 Ca 2+ i Ca 2+ i - Ca 2+ i Ca 2+ i Ca 2+ i REFERENCES 1) Alessandra R, Alfio F. The pathology and clinical features of glue ear: A review. Eur Arch Otorhinolaryngol 2000;257: ) Kim KC, Park HR. Shin CY, Akiyama T, Ko KH. Nucleotide-induced mucin release from primary hamster tracheal surface epithelial cells involves the P2u purinoreceptor. Eur Respir J 1996;9: ) Choi JY, Namkung W, Shin JH, Yoon JH. UTP and ATPrS induce mucin secretion via Ca 2+ dependent pathways in human nasal epithelial cells. Acta Otolaryngol 2003;123: ) Choi JY, Cho KN, Yoon JH. Effect of Uridine 5 -Triphosphate on Mucin and Lysozyme Expression in Human Middle Ear Epithelial Cells. Acta Otolaryngol 2003;123: ) Ko KH, Jo M, McCracken K, Kim KC. ATP-induced mucin release from cultured airway goblet cells involves, in part, activation of protein kinase C. Am J Respir Cell Mol Biol 1997;16: ) Chen Y, Zhao YH, Wu R. Differential regulation of airway mucin gene expression and mucin secretion by extracellular nucleotide triphosphates. Am J Respir Cell Mol Biol 2001;25: ) Dartt DA, Rios JD, Kanno H, Rawe IM, Zieske JD, Ralda N, et al. Regulation of conjunctival goblet cell secretion by Ca(2+) and protein kinase C. Exp Eye Res 2000;71: ) Dray-Charier N, Paul A, Combettes L, Bouin M, Mergey M, Balladur P, et al. Regulation of mucin secretion in human gallbladder epithelial cells: Predominant role of calcium and protein kinase C. Gastroenterology 1997;112: ) Brewer GJ. The methylxanthine beverages and foods: Chemistry, consumption, and health effects. Prog Clin Biol Res 1984;158: ) Osipchuk YV, Wakui M, Yule DI, Gallacher DV, Petersen OH. Cytoplasmic Ca 2+ oscillations evoked by receptor stimulation, G-protein - activation, internal application of inositol trisphosphate or Ca 2+ : Simultaneous microfluorimetry and Ca 2+ dependent Cl - current recording in single pancreatic acinar cells. EMBO 1990; 9: ) Hamada E, Nakajima T, Hata Y, Hazama H, Iwasawa K, Takahashi M, et al. Effect of caffeine on mucus secretion and agonist-dependent Ca 2+ mobilization in human gastric mucus secreting cells. Biochim Biophys Acta 1997;24;1356: ) Choi JY, Kim CH, Lee WS, Kim HN, Song KS, Yoon JH. Ciliary and secretory differentiation of normal human middle ear epithelial cells. Acta Otolaryngol 2002;122: ) Yoon JH, Kim KS, Kim SS, Lee JG, Park IY. Secretory differentiation of serially passaged normal human nasal epithelial cells by retinoic acid: Expression of mucin and lysozyme. Ann Otol Rhinol Laryngol 2000;109: ) Ko WH, Law VW, Wong HY, Wilson SM. The simultaneous measurement of epithelial ion transport and intracellular free Ca 2+ in cultured equine sweat gland secretory epithelium. J Membr Biol 1999; 170: ) King BF, Townsend-Nicholson A, Burnstock G. Metabotropic receptors for ATP and UTP: Exploring the correspondence between 267

6 카페인이점액분비에미치는영향 native and recombinant nucleotide receptors.trends Pharmacol Sci 1998;19: ) Lazarowski ER, Paradiso AM, Watt WC, Harden TK, Boucher RC. UDP activates a mucosal-restricted receptor on human nasal epithelial cells that is distinct from the P2Y2 receptor. Proc Natl Acad Sci USA 1997;94: ) Yen PT, Herman P, Van den Abbeele T, Tan CT, Bordure P, Marianowski R, et al. Extracellular ATP modulates ion transport via P2Y purinoceptors in a middle-ear epithelial cell line. ORL J Otorhinolaryngol Relat Spec 1997;59: ) Soltoff SP. Related adhesion focal tyrosine kinase and the epidermal growth factor receptor mediate the stimulation of mitogen-activated protein kinase by the G-protein-coupled P2Y2 receptor. Phorbol ester or [Ca 2+ ]i elevation can substitute for receptor activation. J Biol Chem 1998;273: ) Malgaroli A, Fesce R, Meldolesi J. Spontaneous [Ca 2+ ]i fluctuations in rat chromaffin cells do not require inositol 1, 4, 5-trisphosphate elevations but are generated by a caffeine- and ryanodine-sensitive intracellular Ca 2+ store. J Biol Chem 1990;265: Korean J Otolaryngol 2006;49:263-8

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Microsoft Word doc KISEP Rhinology Korean J Otolaryngol 2006;49:307-12 사람정상코점막상피세포에서겔형성점소 (Gel-Forming Mucin) 분비에대한 Prostaglandin E 2 의효과 김진국 1 김창훈 2,3 김규보 2 장현종 1 김현직 2 윤주헌 2,3,4 Effect of Prostaglandin E 2 on Gel-forming