2005, Vl. 49, N. 5 Printed in the Republic f Krea 디지토닌으로투과성을높인 Ver 76 세포의포스포리파제 D 특성 š * w yw (2005. 7. 27 ) Characterizatin f Phsphlipase D Activity in Ver 76 Cells Permeabilized by Digitnin Eun-Hie Kh* Department f Chemistry, Duksung Wman s University, Seul 132-714, Krea (Received July 27, 2005). s s q D y s sww w s y mw. Ver 76 s( v s) s s q D w m w mw. Ver 76 s s s q D m ƒ 30 µg/ml s 37 C 30 w y 9 ƒw. w m w s n ƒ xÿ x y w. m w s s q D y EGTA w 70% w w, wì ƒ jù w w PD98059 R32-0432 m w» s s q D y j w w. w jù C w, PMA 24 w s m w y w. m w Ver 76 s w, s s q D1 D2 Ver 76 s x g s s q D w m w mw. : s s q D, m, Ver76 s ABSTRACT. Phsphlipase D (PLD) activity is knwn t be mdulated thrugh cellular signaling pathways including membrane perturbatin. In this study, we examined effect f digitnin n the PLD activity in mnkey kidney epithelial Ver76 cells. At 30 µg/ml dsage f digitnin, the PLD activity was induced apprximately 9 flds within 30 min at 37 C. Under the same cnditin the increased membrane permeability culd be verified by flurescence micrscpe. The digitnin-induced PLD activity was inhibited as much as 70% by EGTA and pretreatment with varius prtein kinase inhibitrs such as PD98059 and R32-0432 significantly decreased the digitnin-induced PLD activity. The results f inhibitr studies suggest an invlvement f prtein kinase C. This finding was further verified by blcking digitnin effect by the pretreatment with PMA fr 24 hurs. Additinally we examined the viability f Ver76 cells under the digitnin influence and tested the digitnin effect n the Ver76 cells transiently transfected with PLD1 and PLD2 cdnas. Keywrds: Phsphlipase D, Digitnin, Ver76 cells s š s s» š. ù d š s ƒ š. s 1 y s sw w s x w 473
474 š e. yw ù p w s ƒ yw x k x š. s desaturase n y w z x y ƒw s ƒ š. n ù 2 UV HeLa s ù mk ƒ x j c-jun NH 2-terminal prtein kinase y y k. 3 s k y ù» s jš ƒ j. s x w s» wš df w. s w s n k v, s ƒ ƒ m ù v v y w. 5 m l g t s n. m s ethidium brmide(etbr) š LDH sw ƒ sü ü. m s n 6 w ƒ. w m thymcytes phsphlipase D(PLD) y y jš. 7 PLD xk s w y y. ƒ y y 8 G-prtein p jù ƒ. w PLD y prtein kinase C(PKC) wš. phsphlipase C(PLC) x g diacylglycerl PKCƒ wì PLDy ƒ jš. PLD y PKC w yƒ PKC PLDƒ y ƒ w šƒ. 9 w m s j ƒ s x w eš. X-ray w w PLDƒ y y Ver76 s m w wš w. x PLD y d w š, ƒ w s y k w» w ƒ PKC w mw. dw Ver76 s m w w PLDy y PKC ƒ w. x. [9,10-³H]palmitic acid (5mCi/mL) Dupnt NEN (Wilmingtn, DEL). l w. Phsphatidylbutanl(PBt) š x PLD w phsphatidylchline l. 10 Digitnin, U731221-[6-((17β-3-methxyestra-1,3,5(10)- trien-17-yl)amin)hexyl]-1h-pyrrle-2,5-dine, PD98059 SB203580 Calbichem (San Dieg, CA) l w. Bisnidlylmaleimide (R32-0432), 3-[4,5-dimethyl thiazl-2-yl]-2,5-diphenyltetrazlim brmide(mtt) genistein Sigma (St. Luis, MO, USA) l, š Enhanced chemilumine-scence (ECL) kit Amersham (Aylesbury, England) l w. Cell culture mediums, DMEM, fetal bvine serum (FBS), Lipfect AMINE PLUS reagent, Dulbecc s phsphate-buffered saline Gibc BRL (Gaithersburg, MD) t. Precated silica gel TLC plate (Kieselgel 60 F254) Merck (Damstadt, Germany) l w w. s t. v s(ver76) 0.12 mg/ml penicillin G, 0.2 mg/ml streptmycin, 2 mg/ml sdium bicarbnate, 20 mm HEPES, ph7.3 10% FBS ƒw DMEM ¼. s 37 C 5% CO 2 95% œ»»ƒ» w. t w Ver76 s x w q, w 0.3% BSA 2 µci/ml [³H]palmitic acidƒ sw x š 37 C 3 w t phsphatidyl chline(pc). PLD y d. t Ver76 s û [³H]palmitic acid w» w DMEM w. PLD y d Lee» w ww. 11 [³H] t s DMEM(2 mg/ml sdium
bicarbnate, 20 mm HEPES, ph7.3) ƒ š j g. t 37 C 30 w, PLD 1 ml methanl g. 2mL chlrfrm 0.8 ml 0.1 N KCl š w. sww chlrfrm SpeedVac œ» g. [³H]Phsphatidic butanl (PBt) w» w w PBt ƒ š chlrfrm þ. ethylacetate/ isctane/acetic acid/water (13:2:3:10;v/v) yw w TLCq w. TLC q» g ùkù [³H]PBt (Rf value; ~0.47) q þ. PBt ethanl/1 N HCl (100:1;v/v) g liquid scintillatin cunter. PLD y d 3 w, v w 2 3 w. m n. Ver76 s x w q w. s 30 µg/ml digitnin 37 C 30 w z 10 µg EtBr š xÿ x w. s viability m(mtt d ). MTT d w Ver76 s w 96 q w. m w» w s 37 C m w w. w ó 50 µl MTT(2 mg/ml) ƒ š q 37 C 5 w. z» ƒ w, 150 µl DMSO þ. Ÿw Ÿw»» 550 nm. Ver76 s s s q D 475 z œ š s 10% FBS sww w DMEM 48 j. PLDƒ x s DMEM z PLD y x ww. š m w PLD y. Ver76 s m w» w [³H]palmitic acid t s 0.3% n-butanl w m y j w. PLD y transphsphatidylatin w x [³H]PBt d w w (Fig. 1). m ƒ [³H]PBt 30 m PLD x. Ver76 s PLD isfrm cdnas x LipfectAMINE PLUS reagent t mg ww. w 100 mm 70% s 4 µg DNA, 30 µl LipfectAMINE PLUS reagent, š 20 µl PLUS reagent swwš x w 6.5 ml DMEM 4 w. Fig. 1. Dse- and time-dependence f digitnin stimulated PLD activity in Ver76 cells. (A) The [ 3 H]palmitic acid labeled Ver76 cells were treated with indicated dse f digitnin and incubated fr 30 min in the presence f 0.3% f n-butanl; (B) cells treated with 30 µg/ml digitnin were incubated fr indicated time. 2005, Vl. 49, N. 5
476 고은희 Fig. 2. Effect f digitnin n the mrphlgy f Ver76 cells. The cells were visualized under the nrmal r flurescence micrscpe with 10 µg EtBr. (A) and (B), cntrlled Ver76 cells; (C) and (D) treated cells with 30 µg/ml digitnin fr 30 min at 37C (B) and (D), the flurescence visualizatin. 지토닌양이 30 µg/ml일 때 거의 최고의 값을 보여주 고 있다(Fig. 1A). 이 조건에서 디지토닌에 의해 활성 화된 PLD활성은 원래 Ver76세포에 있던 PLD 활성 보다 거의 9배 증가한 것이다. 이에 따라 시간변화에 대한 실험은 30 µg/ml의 디지토닌 농도에서 실행하 였다. 시간에 따라 PLD활성은 60분까지 증가 하는 것 으로 보였으나 30분 전후해서 증가폭이 줄어드는 것 으로 나타났다(Fig. 1B). 이들 결과를 감안 하여 디지 토닌에 의한 PLD활성실험은 30 µg/ml 디지토닌 농 도 조건에서 30분간 배양하여 수행하였다. 디지토닌 유도 세포막투과성. 디지토닌에 의한 세 포막 투과성을 관찰하기위해 EtBr를 이용해 Ver76세 포의 흡수여부를 검토하였다. 실험은 디지토닌에 의 해 PLD가 최고로 활성화되는 조건에서 Ver76세포 의 모양을 형광현미경으로 관찰하였다(Fig. 2). 계면 활성제 디지토닌의 존재하에서 세포막의 투과성이 증 가한 결과 세포내 EtBr에 의한 형광이 선명하게 나타 났다(Fig. 2D). 이조건에서 대부분의 세포에 EtBr이 투과 된 것으로 보여진다. 따라서 디지토닌이 Ver76 세포의 투과성을 높이는 과정에서 Ver76세포의 PLD 가 활성화 된다고 사료된다. PLD활성화와 세포밖 칼슘. 칼슘은 여러 종류의 세 포주에서 PLD활성을 좌우하는 주요한 조절인자로 알 려져 있다. 따라서 본 실험에서도 디지토닌에 의한 PLD활성과 Ca² 사이의 관계를 검토하였다(Fig. 3). 디지토닌 효과는 2 mm EGTA로 세포밖 Ca 을 제거 하면 PLD활성화가 현저하게 줄어들었다. 이 결과는 세포밖 칼슘이 디지토닌에 의한 PLD 활성화에 주요 12 + 2+ Fig. 3. Effect f extracellular calcium n the PLD activity by digitnin. The [3H]palmitic acid labeled Ver76 cells were preincubated with EGTA (2 mm) and then incubated fr 30 min in the presence f 0.3% n-butanl after digitnin treatment.
Ver76 s s s q D 477 w w w š. jù w w. m w PLDy yƒ s y ƒ» w Ver76 s ƒ jù w w. Fig. 4. PD98059 (MEK w ), SB203059 (MAPK w ), R32-0432 (PKC w ) w w z ù, wrtmannine(pi3-kinase w ) genistein(prtein tyrsine kinase w ) z ƒ ùkû. jù w z p PKC w R32-0432ƒ ƒ z, PKCƒ PLDy ƒ š ewš. 13 wr w PLDƒ PKC w serine/ threnine»ƒ y š. 14 MEK MAPK w w m w PLD y y s y ƒ ƒ w. Fig. 4. Effect f kinase inhibitrs n the digitnin-stimulated PLD activity in Ver76 cells. The [ 3 H]palmitic acid labeled cells were preincubated with PD98059 (25 µm), SB 203581 (10 µm) fr 1 h in the presence f 0.3% f n-butanl after 30 µg/ml digitnin treatment. The preincubatin time fr Wrtmannin (10 µm), R32-0432 (10 µm), Genistein (100 µm), U73122 (10 µm) were 30 min. Fig. 5. Effect f PKC dwn regulatin with PMA n digitninstimulated PLD activity in Ver76 cells. The [ 3 H]palmitic acid labeled cells were preincubated with PMA fr 24 h and then incubated fr 30 min in the presence f 0.3% n-butanl after digitnin stimulatin (30 µg/ml). PMA w Ver76 s m w. s PKC phbl ester(pma) w s PKC y r ƒ w š. m 9» PLD y PKC y wš PMA ƒ š PKC ww w (Fig. 5).» w PMA w Ver76 s m PLD y 60%. m y y PLD y PKCƒ sw š ù, y w. s m. m w PLD y y x m w s n ƒ w s y w v. w MTT w s (viability) mw. Fig. 6 ùkû 30 30 µg/ml m 54% š. š 50 µg/ml m 30 ü 70%ƒ sƒ w w ùkû. Fig. 1 w 30 µg/ml m ƒ { PLDy w w s PLDy y» w w q. x PLD m w. m w PLDy y z ƒ s ü w PLD w 2005, Vl. 49, N. 5
478 š Fig. 6. Identificatin f cell viability thrugh the MTT assay. Results are expressed as percentage f cntrl. Data represent the mean ± S.D. f at least three experiments. Cncentratins f digitnin were ( ) 0, ( ) 30, ( ) 50 µg/mlu š x PLD z ƒ m wƒ w wš. x Ver76 s PLDƒ s n j m w j ƒw š. y y PLD p s e s y PKC. š m s j w PLD y y j ùkû. s w PLD y s y w ƒ v w. Ver76 s m m s n w w. 2004 w w w, w x w w yw yw x «x. x Fig. 7. Effect f digitnin n the PLD activity f Ver76 cells expressing PLD isfrms. The transient transfectin f 4 µg PLD1, PLD2 cdnas was cnducted by using LipfectAMINE PLUS reagents int Ver76 cells. Labeled cells were stimulated by 30 µg/ml digitnin fr 30 min at 37 C. PLD activity was measured by determining the frmatin f [ 3 H]PBt frm the cells labeled with [ 3 H]palmitic acid in the presence f 0.3% n-butanl. GDigitnin (0 µg/ml), GDigitnin (30G µg/ml)u y w» w PLD cdna Ver76 s x g m z mw. x k PLD 1 š PLD 2 Lipfect AMINE PLUS reagent w x g. x Western bltting mw, PLDƒ x s m w PLDy y Fig. 7 ùkþ. PLD 2ƒ x Ver76 s 4 y yƒ ùkû PLD 1 1.4 y yƒ ùkû. x PLD y y š ù y y ü w PLD q. 1. Singer, S.J.; Niclsn, G.L. Science. 1972, 175, 720. 2. Ls, D. A.; Murata, N. Science s stke. 2000, 62, 1. 3. Rsette, C.; Karin, M. Science. 1996, 274, 1194. 4. Raucher, D.; Sheetz, M. P. J. Cell Bil. 1999, 154, 535. 5. Park, H.; G, Y. M.; St. Jhn, P. L.; Maland, M. C.; Lisanti, M. P.; Abrahamsn, D. R.;J, H. J. Bil. Chem. 1998, 273, 32304. 6. Vitale, M. L.; Castill, R. D.; Trifar, J.-M. J. Neurchem. 1992, 59, 1717. 7. Lee, Y. K. M.S. dissertatin, Department f Chemistry, Seul Natinal University. 1998. 8. Extn, J. H. Bichim Biphys Acta. 1994, 1212, 26. 9. Extn, J. H. FEBS Letters. 2002, 531, 58. 10. Jung, K.; Kh, E.; Chi, M-U. Bull. Krean. Chem. Sc. 1989, 10, 585. 11. Lee, S.Y.; Park, N.G.; Chi, M-U. FEBS Lett. 1998, 432, 50. 12. Extn, J. H. Physil. Rev. 1997, 77, 303. 13. Min, D. S.; Kim, E. G.; and Extn, J. H. J. Bil. Chem. 1998, 273, 29986. 14. Kim, Y.; Han, J.M.; Han, B. R.; Lee, K. A.; Kim, J. H.; Lee, B. D.; Jang, I. H., Suh, P. G.; Ryu, S. H. J. Bil Chem. 2000, 275, 13621.