세포배양 방 TPH 효소활성측정 g 법 Korean J Psychopharmacol 2004;15(4):

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현희 필
6 years ago
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1 KISEP Original Article RBL-2H3 세포에서 Fluoxetine에의한 Tryptophan Hydroxylase 활성도변화 백승연 1 정경화 1 최미란 1 양병환 2 김석현 2 최인근 3 채영규 1 한양대학교분자생명과학부, 1 한양대학교의과대학신경정신과학교실및정신건강연구소, 2 한림대학교의과대학신경정신과학교실 3 ABSTRACT Fluoxetine-induced Changes on Activity of Tryptophan Hydroxylase at RBL-2H3 Cells Seung-Youn Baik, MS, 1 Kyoung-Hwa Jung, MS, 1 Mi-Ran Choi, MS, 1 Byung-Hwan Yang, MD, 2 Suk-Hyun Kim, MD, 2 Ihn-Geun Choi, MD 3 and Young-Gyu Chai, PhD 1 1 Division of Molecular and Life Sciences, Hanyang University, Ansan, 2 Department of Neuropsychiatry, College of Medicine & Institute of Mental Health, Hanyang University, Seoul, 3 Department of Neuropsychiatry, College of Medicine, Hallim University, Seoul, Korea Objectives:The aim of the present study is to explore the effect of fluoxetine on transcription, translation and activity of tryptophan hydroxylase (TPH), and intracellular level of serotonin. Methods:The expression level of the TPH mrna and the protein, the TPH enzyme activity, and the intracellular level of serotonin were explored at the fluoxetine-treated RBL-2H3 cells. Real-time RT-PCR and immunoblotting analysis confirmed changes in the expression of TPH mrna and protein. The activity of TPH was measured using [ 3 H]tryptophan. The intracellular level of serotonin was measured by HPLC. Results:The TPH activity was gradually increased on time from 24hr to 72hr. The real-time RT-PCR also revealed that the TPH mrna was increased at 12, 24 and 72hr in the fluoxetine-treated RBL-2H3 cells. The immunoblotting analysis also revealed that the TPH protein was decreased at 72hr in the fluoxetine-treated RBL-2H3 cells. The intracellular level of serotonin was increased at 48hr after treatment of fluoxetine. Conclusion:Fluoxetine induced the increases of the TPH mrna, the TPH enzyme activity and intracellular level of serotonin, and the decrease of the TPH protein expression at the RBL- 2H3 cells. (Korean J Psychopharmacol 2004;15(4): ) KEY WORDS:Antidepressant Selective serotonin reuptake inhibitor Fluoxetine Tryptophan hydroxylase Serotonin RBL-2H3 cell. 서 론 교신저자 449

2 세포배양 방 TPH 효소활성측정 g 법 Korean J Psychopharmacol 2004;15(4):

3 세포내세로토닌농도측정 - 4. Real-Time RT-PCR C C C C C C C C C 5. Immunoblot analysis g 451

4 통계학적분석 결 과 1. Fluoxetine 에의한 TPH 효소활성증가 - CPM Figure 1. Measurement of TPH activity in the fluoxetinetreated RBL-2H3 cells. RBL-2H3 cells were treated by 10 M fluoxetine at 0 con, 24, 48 and 72h. TPH activity was measured as produced 3 HH 2O using 3 Htryptophan. The 3 HH 2O was measured by liquid scintillation. All data are mean S.D. values expressed as CPM per 400 g of protein by liquid scintillation. According to an one-way ANOVA method with post hoc Tukey s HSD n3, all p0.05 were considered statistically significant vs. control. TPHtryptophan hydroxylase, CPMcounts per minute. 2. Fluoxetine 에의한세포내세로토닌농도증가 Con 24 h 48 h 72 h - 3. Fluoxetine 에의한 TPH mrna 발현증가 452 Korean J Psychopharmacol 2004;15(4):

5 60 TPH 50 Fluoxetine Control Percent fold change Oh 48 Time Figure 2. Measurement of intracellular level of serotonin in the fluoxetine-treated RBL-2H3 cells. RBL-2H3 cells were cultured in the presence of 10 M fluoxetine or 10% ethanol control for 0 and 48 h. The level of serotonin was measured by high-performance liquid chromatography coupled with an electrochemical detector. Analysis of data was performed by comparision of the difference between each control group and treated group. C 0 Con 12 h 24 h 48 h 72 h Figure 3. Real-time RT-PCR of TPH gene in the fluoxetinetreated RBL-2H3 cells. RBL-2H3 cells were cultured in the presence or absence of 10 M fluoxetine for 0 con, 12, 24, 48 and 72 h. Total RNA of the cells was prepared and each cdna was synthesized. Glyceraldehyde-3- phosphate dehydrogenase was used as an internal control for each reactions. All Data are mean S.D. vaues expressed the 2 - CT that shows the difference beween each the control group and the treated group. The value of 2 - CT is the fold change of TPH mrna. According to post hoc Tukey s HSD n3, all p0.05 were considered statistically significant vs. control. TPH tryptophan hydroxylase. 4. Fluoxetine 에의한 TPH 단백질발현감소 - 453

TPH Relative density - 454 Flu 0 48 72 2 1 0 Con 48 h 72 h Figure 4. Immunoblotting of TPH protein in the fluoxetinetreated RBL-2H3 cells.

6 TPH Relative density Flu Con 48 h 72 h Figure 4. Immunoblotting of TPH protein in the fluoxetinetreated RBL-2H3 cells. RBL-2H3 cells were cultured in the presence of 10 M fluoxetine for 0 con, 48 and 72 h. Total protein was prepared and 10 g of the protein was run at 12 % SDS-PAGE. The mouse anti-tph antibody Sigma was used for the immunoblot of TPH. Experiments were performed on five independent culture preparations. According to post hoc Tukey s HSD n5, p0.05 was considered statistically significant vs. control. TPHtryptophan hydroxylase. 고 찰 Korean J Psychopharmacol 2004;15(4):

7 요약 목적 방법 455

8 - 결과 결론 중심단어 참고문헌 1) Meltzer CC, Smith G, DeKosky ST, Pollock BG, Mathis CA, Moore RY, et al. Serotonin in aging, late-life depression, and Alzheimer s disease: the emerging role of functional imaging. Neuropsychopharmacology 1998;18: ) Huh SO, Park DH, Cho JY, Joh TH, Son JH. A 6.1 kb 5 upstream region of the mouse tryptophan hydroxylase gene directs expression of E. coli lacz to major serotonergic brain regions and pineal gland in transgenic mice. Brain Res Mol Brain Res 1994;24: ) Delgado PL, Charney DS, Price LH, Aghajanian GK, Landis H, Heninger GR. Serotonin function and the mechanism of antidepressant action. Reversal of antidepressant induced remission by rapid depletion of plasma tryptophan. Arch Gen Psychiatry ;47: ) Fuller RW, Wong DT. Serotonin reuptake blockers in vitro and in vivo. J Clin Psychopharmacol 1987;7: ) Mockus SM, Vrana KE. Advances in the molecular characterization of tryptophan hydroxylase. J Mol Neurosci 1998;10: ) Blier P, Montigny CD. Current advances and trends in the treatment of depression. Trends Pharmacol Sci 1994;15: ) Zill P, Baghai TC, Zwanzger P, Schule C, Eser D, Rupprecht R, et al. SNP and haplotype analysis of a novel tryptophan hydroxylase isoform (TPH2) gene provide evidence for association with major depression. Mol Psychiatry 2004;9: ) Harvey M, Shink E, Tremblay M, Gagne B, Raymond C, Labbe M, et al. Support for the involvement of TPH2 gene in affective disorders. Mol Psychiatry 2004;9: ) Kim SW, Park SY, Hwang O. Up-regulation of tryptophan hydroxylase expression and serotonin synthesis by sertraline. Mol Pharmacol 2002;61: ) Vaswani M, Linda FK, Ramesh S. Role of selective serotonin reuptake inhibitors in psychiatric disorders: a comprehensive review. Prog Neuropsychopharmacol Biol Psychiatry 2003;27: ) Hasegawa H, Kojima M, Iida Y, Oguro K, Nakanishi N. Stimulation of tryptophan hydroxylase production in a serotoninproducing cell line (RBL-2H3) by intracellular calcium mobilizing reagents. FEBS Lett 1996;392: ) Vrana SL, Vrana KE, Koves TR, Smith JE, Dworkin SI. Chronic cocaine administration increases CNS tyrosine hydroxylase enzyme activity and mrna levels and tryptophan hydroxylase enzyme activity levels. J Neurochem 1993;61: ) Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2 (-Delta Delta C(T)) Method Methods 2001;25: ) Petroni A, Cappa M, Blasevich M, Solinas M, Uziel G. New findings on X-linked Adrenoleukodystrophy: 5alpha-reductase isoform 2 relative gene expression is modified in affected fibroblasts. Neurosci Lett 2004;367: ) Koubi D, Bezin L, Cottet-Emard JM, Gharib A, Bobillier P, Sarda N. Regulation of expression and enzymatic activities of tyrosine and tryptophan hydroxylases in rat brain after acute electroconvulsive shock. Brain Res 2001;905: ) Joh J, Huh SO, Son JH. Gene expression of serotonergic neurons in the central nervous system: molecular and developmental analysis. Prog Brain Res 1995;105: ) Nibuya M, Nestler EJ, Duman RS. Chronic antidepressant administration increases the expression of camp response element binding protein (CREB) in rat hippocampus. J Neurosci 1996; 16: ) Newman ME, Gur E, Shapira B, Lerer B. Neurochemical mechanisms of action of ECS: evidence from in vivo studies. J ECT 1998;14: ) Ehret M, Cash CD, Hamon M, Maitre M. Formal demonstration of the phosphorylation of rat brain tryptophan hydroxylase by Ca2+/calmodulin-dependent protein kinase. J Neurochem 1989; 52: ) Iida Y, Sawabe K, Kojima M, Oguro K, Nakanishi N, Hasegawa H. Proteasome-driven turnover of tryptophan hydroxylase is triggered by phosphorylation in RBL2H3 cells, a serotonin producing mast cell line. Eur J Biochem 2002;269: Korean J Psychopharmacol 2004;15(4):

Pharmacotherapeutics Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee Univ

Pharmacotherapeutics Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee Univ Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee University College of Medicine & Hospital E mail : ycell2@yahoo.co.kr Abstract