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. - 263
카페인이점액분비에미치는영향 Ca 2+ i Ca 2+ i Ca 2+ i - 264 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 0.95 0 300 600 900 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
카페인이점액분비에미치는영향 500% 400% 400% Mucin secretion % 300% 200% 100% Mucin secretion % 200% 0% Control 0 1 10 20 40 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. Ratio352/380 0.1 Units 150 sec Mucin secretion % B 100% 50% 0% Control 1 10 20 40 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. 0 1 0 10 0 20 0 40 100 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
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