세포배양 실험재료및방법 2. 전기생리적실험 Korean J Psychopharmacol 2001;123:

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1 KISEP Original Articles RINm5F 인슐린종세포에서막전압및이온전류들에미치는 Fluoxetine의효과 ABSTRACT 박기창 1* 오국택 1 차승규 2 이건일 2 박규상 2 정성우 2 공인덕 2 이중우 2 Effects of Fluoxetine on Membrane Potential and Ionic Currents in RINm5F Insulinoma Cells Ki Chang Park, MD, 1 Guk Taek Oh, MD, 1 Seung Kyu Cha, MS, 2 Keon Il Lee, MD, 2 Kyu Sang Park, MD, 2 Seong Woo Jeong, PhD, 2 In Deok Kong, MD 2 and Joong Woo Lee, PhD 2 1 Department of Psychiatry, 2 Physiology, Yonsei University Wonju College of Medicine, Wonju, Korea ObjectiveThe purpose of this study was to investigate the effects of fluoxetine Prozac on membrane potential and ionic currents in RINm5F insulinoma cells. MethodsMembrane potential and ionic currents in RINm5F cell were recorded by using whole-cell and perforated-patch clamp techniques. ResultsUnder current clamp conditions, diazoxide 200 M, an activator of K ATP channels, induced a hyperpolarization of the resting membrane potential mv, n8, which was accompanied by a abolition of action potential firing. This diazoxide-induced hyperpolarization was blocked by glibenclamide 10 M. Fluoxetine produced significant depolarization of membrane potential mv, n5 and blocked diazoxide-induced hyperpolarization. Diazoxide activated inward currents in the presence of high external K 90 mmat a holding potential of 60 mv. Fluoxetine suppressed diazoxide-activated currents in a concentration-dependent IC M manner. However, the inhibitory action of fluoxetine was not specific to K ATP currents because it also inhibited both voltage-activated K and Ca 2 currents in a concentration-dependent manner. K ATP currents were more sensitive to fluoxetine block than both voltage-activated K and Ca 2 currents. ConclusionOur results indicate that fluoxetine increased excitability of RINm5F cells mainly by the preferential block of K ATP currents. Fluoxetine-induced depolarization may influence insulin secretion in insulinoma cells. Korean J Psychopharmacol 2001;123: KEY WORDSFluoxetine K ATP channel Membrane potential Ionic current Insulin secretion. 서 론 233

2 세포배양 실험재료및방법 2. 전기생리적실험 Korean J Psychopharmacol 2001;123:

3 3. 세포외및전극내용액 4. 약물 5. 자료분석 235

4 결과 1. RINm5F 세포에서안정막전압에대한 ATP-민감성 K + 통로효현제및차단제의효과 2. Fluoxetine이안정막전압및 diazoxide에의해유발된과분극에미치는효과 3. RINm5F 세포에서 K ATP 전류의특성 Figure 1. Effects of fluoxetine on membrane potential of RINm5F cell. AUnder current-clamp mode, application of diazoxide a KATP opener, 200 M induced hyperpolarization and inhibited slow waves and spikes. Glibenclamide a KATP blocker, 10 M blocked diazoxide-induced hyperpolarization and restored spontaneous spikes. B Fluoxetine 10 M induced significant depolarization, and decreased slow waves and action potentials. CFluoxetine also blocked diazoxide-induced hyperpolarization. 236 Korean J Psychopharmacol 2001;123:

5 4. Fluoxetine이막전압의존적 K + 통로에미치는영향 Figure 2. Effects of fluoxetine on inward KATP currents in RINm5F cell. AChanging the external solution from 5 mm K to 90 mm K elicited an inward current. Addition of diazoxide 200 M activated a steady-state current that was blocked largely by glibenclamide 10 M. BThe diazoxide-activated current was blocked by fluoxetine in concentation-dependent manners. Figure 3. Effects of fluoxetine on voltage-activated K currents. AWhole-cell outward currents K elicited by 200 ms test-pulses to potentials between 80 mv and 70 mv in steps 10 mv under control condition uppermost and after the addition of 10 M fluoxetine lowermost. BCurrent-voltage relationships were measured at the peak of the currents in the absence and presence of fluoxetine. CTime course of fluoxetine effect was shown. 237

6 5. Fluoxetine이막전압의존적 Ca 2+ 전류에미치는효과 Figure 4. Effects of fluoxetine on Ca 2 currents. Ba 2 was used a charge carrier to record Ca 2 currents. ACa 2 current records elicited from a holding potential of 80 mv by 200 ms depolarizing steps to the indicated test potentials under control condition uppermost, and after the addition of 10 M fluoxetine lowermost. BCurrent-voltage relationship in the absence and presence of 10 M fluoxetine. Peak Ca 2 currents were plotted at the test potential as a normalized value. C Time course of fluoxetine effect was shown. Figure 5. Effects of fluoxetine on KATP and both voltage-activated K and Ca 2 current. AConcentration dependency of the inhibition of the KATP and both voltage-activated K and Ca 2 current by fluoxetine. IC50 concentration for fluoxetine was 0.87 M, 4.03 M, and 4.26 M, respectively. BPercent inhibition of KATP, K Kv and Ca 2 currents compared at a concentration of 1 M fluoxetine, that is therapeutic concentration of fluoxetine. Each bar represents the means.e. **p Korean J Psychopharmacol 2001;123:

7 6. 혈중농도하에서 fluoxetine이이온통로에미치는영향비교 고 찰 239

8 요약 목적 : 방법 : 결과 : 결론 : 중심단어 참고문헌 1) Charney DS. Monoamine dysfunction and the pathophysiology and treatment of depression. J Clin Psychiatry 1998;59: ) Stark P, Fuller RW, Wong DT. The pharmacologic profile of fluoxetine. J Clin Psychiatry 1985;46: ) Wong PT, Horng JS, Bymaster FP, Hauser KL, Molloy BB. A selective inhibitor of serotonin uptake; Lilly , 3 (ptrifluoromethyl phenoxy)-n-methyl-3-phenyl propylamine. Life Sci 1974;15: ) Rae JL, Rich A, Zamudio AC, Candia OA. Effect of prozac on whole cell ionic currents in lens and corneal epithelia. Am J Physiol 1995;269:C250-C256. 5) Park KS, Kong ID, Park KC, Lee JW. Fluoxetine inhibits L- type Ca 2+ and transient outward K + currents in rat ventricular myocytes. Yonsei Med J 1999;40: ) Choi JS, Hahn SJ, Rhie DJ, Yoon SH, Jo YH, Kim MS. Mechanism of fluoxetine block of cloned voltage-activated potassium channel Kv1.3. J Pharmacol Exp Ther 1999;291 (1):1-6. 7) Hahn SJ, Choi JS, Rhie DJ, Oh CS, Jo YH, Kim MS. Inhibition by fluoxetine of voltage-activated ion channels in rat PC12 cells. Eur J Pharmacol 1999;367(1): ) Tytgat J, Maerteus Ch, Daeneus P. Effect of fluoxetine on a neuronal, voltage-dependent potassium channel (Kv1.1). Br J Pharmacol 1997;122: ) Aguilar-Bryan, Bryan J. Molecular biology of ATP sensitive potassium channels. Endocrine Rev 1999;20(2): ) Malaisse WJ. Metabolic signaling of insulin secretion. Diabetes Rev 1996;4: ) Newgard CB, McGarry JD. Metabolic coupling factors in pancreatic β-cell signal transduction. Annu Rev Biochem 1995;64: ) Wollhein CB, Lang J, Regazzi R. The exocytotic process of insulin secretion and its regulation by Ca 2+ and G-proteins. Diabetes Rev 1966;4: ) Dukes ID, Philipson LH. K + channels: generating excitement in pancreatic β-cells. Diabetes 1996;45: ) Panten U, Schwanstecher M, Schwanstecher C. Sulfonylurea receptors and mechanism of sulfonylurea action. Exp Clin Endocrinol Diabetes 1996;104: ) Goodrick PJ, Henry JH, Buki UMV. Treatment of depression in patients with D.M. J Clin Psychiatry 1995;56: ) Wilson GA, Furman BL. Effects of inhihitors to 5-HT uptake 240 Korean J Psychopharmacol 2001;123:

9 on plasma glucose and their interaction with 5-HT in producing hypoglycemia in mice. Eu J Pharmacol 1982;78: ) Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch-clamp techinques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch 1981;391: ) Gembal M, Gilon P, Henquin JC. Evidence that glucose can control insulin release independently from its action on ATPsensitive K + channels in mouse β-cells. J Clin Invest 1992; 89(4): ) Schofl C, Borger J, Mader T, Waring M, von zur Muhlen A, Brabant G. Tolbutamide and diazoxide modulate phospholipase C-linked Ca 2+ signaling and insulin secretion in betacells. Am J Physiol Endocrinol Metab 2000;278(4):E ) Altamura AC, Moro AR, Percudani M. Clinical pharmacokinetics of fluoxetine. Clin Pharmacokinet 1994;26: ) Lustman PJ, Freedland KE, Griffith LS, Clouse RE. Fluoxetine for depression in diabetes: a randomized doubleblind placebo-controlled trial. Diabetes Care 2000;23(5): ) Goodnick PJ. Use of antidepressants in treatment of comorbid diabetes mellitus and depression as well as in diabetic neuropathy. Ann Clin Psychiatry 2001;13(1): ) Deeg MA, Lipkin EW. Hypoglycemia associated with the use of fluoxetine. West J Med 1996;164(3): ) Gomez R, Huber J, Tombini G, Barros HM. Acute effect of different antidepressants on glycemia in diabetic and nondiabetic rats. Braz J Med Biol Res 2001;34(1): ) Yamada J, Sugimoto Y, Inoue K. Selective serotonin reuptake inhibitors fluoxetine and fluvoxamine induce hyperglycemia by different mechanisms. Eur J Pharmacol 1999;382(3): ) Potter van Loon BJ, Radder JK, Frolich M, Krans HM, Zwinderman AH, Meinders AE. Fluoxetine increases insulin action in obese type II (non-insulin dependent) diabetic patients. Int J Obes Relat Metab Disord 1996;16(4):S ) Maheux P, Ducros F, Bourque J, Garon J, Chiasson JL. Fluoxetine improves insulin sensitivity in obese patients with non-insulin-dependent diabetes mellitus independently of weight loss. Int J Obes Relat Metab Disord 1997;21(2): ) Scherubl H, Hescheler J, Bychkov R, Cuber JC, John M, Riecken EO, et al. Electrical activity and calcium channels in neuroendocrine cells. Ann N Y Acad Sci 1994;15(733):

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