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钙通道阻塞剂对大鼠肝细胞钙释放激活的钙电流的影响

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钙通道阻塞剂对大鼠肝细胞钙释放激活的钙电流的影响 ISSN C~&53-9756 ALIaPhamocol Sin中国药理学报 1999May;舯 (5):415—418 trap://www mm ac cn; chir~atb V.ca/l~ioclical 415 Effects of calcium channel blockers on calcium release-activated calcium currents in rat hepatocytes1 cui Gui-ying2,LJ Jin-Ming.CUI Hon ·HAO Lj-Ying...
钙通道阻塞剂对大鼠肝细胞钙释放激活的钙电流的影响
ISSN C~&53-9756 ALIaPhamocol Sin中国药理学报 1999May;舯 (5):415—418 trap://www mm ac cn; chir~atb V.ca/l~ioclical 415 Effects of calcium channel blockers on calcium release-activated calcium currents in rat hepatocytes1 cui Gui-ying2,LJ Jin-Ming.CUI Hon ·HAO Lj-Ying,uu Dong-Ju3,zHANG Ke—Yi ()6epart.~t ofPharmacalogy,China Medical University,Shenyang 110301,China; 。Department ofGeneral Surgery, ClinicalInstitute,China MedicalUniversity,Shenyang110001,China) KEY WORDS calcium channels:calcium channel solution with Ca2 10 mmol·L‘ ). CONCLU- blockers;patch—clamp techniques; liver;verapamil; SION:The three calcium antagonists inhibited lc~nc diltiazem;nifedipine;cultured cells effectively and protected hepatocytes from calcium overload viatheinhibition oflcRAC. ABSTRACT AIM:T0咖dy tIleⅫ ue口ces of calcium channe1 TRoDlUcⅡ0N blockers on calcium release-activated calcium currents (』 )in rat hepatocytes ME咖 DS:Whole—cell patch.clam p technique was used. RESULTS: Ⅱ】e peak amplitude of was 一0.41 nA±0.09 nA(n = 15). its reveisal potential was about 0 mV. Verapamil(Ver),diltiazem (Dil),and nifedipine (Nif)decreased』cRAC strikingly,without affecting its reversalpo tentia1 Theinhibitory rate ofVer 5tanol- L_。was 40% ±12% 【 =3),Ver 50 tmaol·L-。 reducedthe peak amplitudeoflCRACfrom 一0 49 nA± 0.12 nA to 一 0.20 nA ± 0.09 nA 【P < 0.01 contro1.n=5). The inhibitory rate was 57% ± l5% . D|1.50t-~mol。L and Nif reducedlcRACfrom 一 0 43 nA± 0 10 nA to 一0.29 nA ±0 07 nA (P < 0 01懈 contro1, =5)。from 一0.32 nA ±0 08 nA to 一 0.27 nA ± 0.08 nA (P < 0.01 c~tro1. =5). Theinhibitory ratewas 31% ±11% .19% ±7 % .respectively. The am plitude of lcRAC was dependent on ex~ lltflar C concentration. The peakam plitude of』cRAcwas一0.21 nA±0 o8 nA t =3)inTyrode’s solutionwithCa2 1.8mlllO1.LI1(P <0.01 the pe am plitude of lcxac in external 。Project silppo~ bytheNational Nanral Science Foundation of Oaiila. № 39400138. Con'eslxmdencetoMs CUI Gui—Ying. Nowin Laboratory"of Neurophyslology, Brain Research !nsttuae, Cldna:Medical UniversiO,,Shenyang1f0001,China Phn 86-24-Z386-3731+extj54。. Fax86-24-2322.4417. E-malllib~ y@iris clnu edu∞ Received19o~4)5-18 Accepted19v~-10-27 The drastic increase of calcium in hepatoc ytes caused by the hepat~oxic substance diamidinothiona- phthene(98/202)killed the cell Vempmnil(Ver), mfedipine(Nif),and衄tiazem(Dil)were effectivein preventing the cell death caused by substance 98/ 2O2一J . Mora NP had a transplantation experiment of liver,which had been protected hypothermallywi th Ver for 24 h andVer had protecting function~ TheCa2 influxwas mainlymediated by voltage- dependent Ca2 channels and receptor—mediated Ca2 channels. Most scholars held that store depletion - dependent Ca2 channel makdy acted to regulate Ca2 influx in nonexcitable cells,nam ely,by the calcium release.activated calcium current, which was called receptor-mediated Ca2 channels generally 3 Hepatocytes as the nonexcitable cellswere short of the voltage—dependent Ca2 channels【 . Calcium influx in isolated hepatocytes w∞ studied by indirect methods and it mainly depended on receptor-mediated Ca2 end/ .CalciumantagonistsVer,Nif,andDil principally affected the voltage—dependent C channels. Then how did calcium antagonists decrease the cytosolic free Ca2 concentration and protect hepatocytes? The purpose ofthis paperwas to observe the effects of calcium antagonists on calcium release- activated calcium cutrent(』cR^c) _n isolated rat hepatocytes and into initially the mechanism of protection from cytosolic calcium overload. 维普资讯 http://www.cqvip.com 416 ISSN 0~53-9756 Acta Foanmcal Sin E-mail aps@server sht:llC ac 1311 中国药理学报 1999M ;20(5) Hu RⅨ8昏2l—邮 4一 M A皿 RIALS AND M匝T】日[0DS Isolation of single hepatocytes Hepato~ytes were jsolmedL . Briefly.adult Wistar rats of either sex(n=43,Grade lI,Certificate№ O34,weighing 175 g±25 g)were anesthetized byinhalation of ethel" and ip pent@barbital sodinliq 3o rrIg‘ ~ . The portal vein was cannulated and perfused with oxygenated C 一freeHanks’solution 25mL·mill_。at 37℃ for 4 — 5 mill f0llowed by l~n'fusion with 一free Hanks’ solution containing collagenase(Type IV,Sigmaj(0.5 叠·LI1)for10—15rain. 1heliver w鹅 chop~ in Caz .freeHanks’~lution10mL . rnle cell susperhsion was filtered through gauze t0 ILffilTIove fibrous ssues. CellswereincubatedinKB(Kmbs-Heuseleit)~ tlln for 2 —3 h and preserved in DMEM (Dulbecco's and compared by ttest Voltage pulses were applied every 5 s at holding potential of 0mV.command potential of一100协 + 80mV,will1 2OmV increments. The口eak amplitude 0f was 一0.41 nA±0.09 nA (n=15),its reversal pomntial was about 0 mV (Fig 1). The currentwas steady andwithout rundownwithin 5min modified Eagle’smedium)at 4℃. { Drllgs and solutions Ca2+-free Hanks’ solufion wa$prepared without Ca2 and M . 1be KB solution was composed of glutamie acid 70.tlll'ille l5,KCl 130,KH2PO4 1O,蛐BH 10,glucose 11, eddic acid 0 5 mmol·L_。. e extelTla]solufion con tained NaC1140,KC1 2.8,CaCI210,MgC12 0.5, glucose l1. H臼mS 10 mmol·L一 . 1be internal solution contained po tassium-glutamate 145.NaC1 8. MgC12l,Mg-A1]P O.5,egtazie acid 10,唧 S lO mmol·LI1). TheTyrode’s solution was composed of NaC1 l44, KC1 4.0, CaC12 1.8, MgCh 0.53, NaI~PO4 0.33,冒uo3se 5.5,and卸 S 5 mmol‘ L~,pH 7.2. Ver,Nif(Bering Tianfeng Pharma- ceutical Factory),Di1(Sigma). Eleclrophysiologic recording rnle recording cham~r(1.5mL)w船 perfusedwith ex~frnal solution 2—3 mL·mill一 at 22±2 ℃ . Membrane jonie cun'cnts were measured in a standard whole,cell patch clamp configuration wi th all Axopatch-lD ~aaplifier tAxon Instruments,USA). nle pipettes pulled intwo stagesfrom hard glass capillaries using a vertical microeleetrode puller(Narishige,Japan). Elecaode had a resistance of3—5Mn forwhole-cell~ rding. k町1brane potential and currant signal were mom~red with a dual beammemory oscilloscope(VC.10,Nil~,n Kohden)and stored in a computer. For the currant measurement,the holdingpo tential was kept at 0mV, the command po tential was 一10o mV. and the duration was 2OO ms Data were presented as j± ⋯ 霍 : B I-·_ 0 / ● l50 -50/ 0 50 1 1 口 舢 . 0 . 0.6 Fi叠1. Cun眦 ol cald1Ⅱn relem e·铆 嘧耐 caleirm current{Jm c^)in rat hepatooytes. A) J : voltage pulses we argued every 5 s at h0】mlIg Imtea~al of 0mV.c0nI哪 dImtea~al of一邶 to+ 80 mV,in 20 mV iner~aents. B)cun 吐·voltage relationship I the data .m A. Measured at the be rIrIjl1gpoint 0£thepulse. Ver,Dil,andNif decreased 】^ ,and did not affect its reversal potentia1. The hthibitory rate ofVer 5/tlTiOl_L.1 was 40% ±12% (n=4).Ver 50 pmol‘L reduced the peal(amplitude of cRAc from 一 0.49 nA±0.12 nA 一0.∞ ±0.09 nA (P(0.01 control,n=5),the inhibitory rate was 57% ± 15% (Fig 2). EitherDilo1"Nif50 加01.L reduced jcR^cfrom 一 0.43 nA ±0.10 nA 协 一0.29 nA ±0.07 nA (P < 0.O1 contro1.n=5)and from 一0.32 nA±0.惦 nA 协 一0.27 nA±0.惦 nA (P<0.01 contro1. 维普资讯 http://www.cqvip.com 工sSN 53_9756 Acta Phattm~1 Sin E-marl s@㈣ .cn 中国药理学报 l999 May;舯 ‘5) PW R 86-21—6474-2629 417 40m s 0.3 0.1 一 。 50 I d 3 n —--C卜一c0小r nA —._Verapamil Fig2. j inisolated rat hepatocytes befoce【A and after cB】Vex 50 pmol-L~. ”=5),respectively. Theinhibitory ratewas31%± l1%,l9% ±7%,respectivdy(Fig 3) The amplitude of ICeAC was dependent on extracellular Ca2 concentration . Th e peak amplitude ● O.2 : 0 铷 I _0.4 - -o-- C0n d 0flcRACwas一0.21 nA±0啤 nA f =3)in Tyrode’ solutionwith Ca2 1 .8 mmol-L。。(P <0.OlⅧ 山e peak amplitude oflcRACin external solution with Ca2 10mmol。L ) Ver 5~zmol’L。。decreased ,cRAc from 一0.21 nA ±0.08 nA to 一0 14 nA ±0.05 nA (H=3). Calcium influxdepended oilthe activatiOil ofCa2 currents causedby山e depletionofintracellular stores in nonexcitable cells ,which had been named ,@ c^ 耵1e existence of loo , c—type currents has been recently demon strated in many differentcells The experiments had been carded out in hie.h ex~x:elltdarCaz concentrationin orderto ob~rve山e effect of drugs Oil七RAc.1 results showed thatthe reversal potential of,cRAc in rat hepatocytes was about 0 mV and the holding potential was 0 mV appear~ . although the gating of lcp . m was independent of membrane voltage,there was,nevertheless,a strong dependence ofC influx oilthe drivingforce exerted by the membrane petention. ie, 山e influx mte increased wi山 hyperpolm'ization and decreased with de polarization . Our leSt]Its were the same as 0 . 0.2 0 50 1 ,/ --o--c0mtrd 一 叽6 Fig 3. ICr~C in rat hepatoeytes. A,B)control and蹦 50 I_L一 :C)current-voltage relationship of I吣 c obtainedfromA and B:D,E】control andNit:50 fn0l-L一 ;F)cta-t-e~t-voltage relationship ofIot^c obtainedh蚵I cta-t-e~ts 0£D andE. 维普资讯 http://www.cqvip.com ]SSN 0'253 97,56.Acta Phi l Sin 中国药理学报 1999May;20(5) E-amiI aps@s~1%,el-shcnc ac cⅡ Pht~:'Fax 21—“ 14 documents. The amplitude of Ic~,Ac depended on Calcium influx and its control by calcium re[ease fCa2 ] in rat hepatocytes.The amplitude of,cR^c Q盯呻 N㈣ Dl ;3:368—74 s ngIv I。wcr{n Tvmde,s s。luIi。n(cach⋯1 . 8 ‰ c· d B—P0zz叩 T R唧 acti 删 一 l_ ) than that .n h ⋯ igh extema] Ca2 :。嚣 .一 concentrnfion(CaC12 10 mmol。L一 j 9 He舒P . L抛l鲫【IaI1 JB. erIRW. Di r£mm0des 0fca2 Ca]dam antagonists were mainly used in channel gating beha-douxfa,ioured 幽 di 。~ m ’ 一 删 甜 嘶 ag oni clinic as antihypertension antiangina pectoris 删 · ~ “ :黜 , , —单 antiarrhytlunic drugs and organ transplantation for its pro~ecfion of histocyte by inhibiting calcium overload. Ca]cium antagonists inhibited ca]cium overload by decreasing calcium influx, which blocked voltage- operated calcium channels(VOC) in cardiac muscle "and smoothmuscleL9J . Three calcium antagonistsVer. Dil.and Nif decreased the amplitude of lcRAC at 50 tzmol·L i11the exl~Tililents.the effect ofVerwas the most distinct and that of Dil and Nifthe second place among them. As the nonexcitable cell mt tepa~ ytes js without VOC.the experiment results indicated that the 3 calcium antagonists could protect tepat~ytes by inhibiting , to decrease intracellular Ca2 concentrati0n REFERENCES l SippeI H.Stauffer~I. Estier CI. Protective effect of ~arious calcium antagonists agaimt an experimentally induced calcium overl~d jn isolreed hepatocyms. Biocliem Pl',4mmicol l993;46:lg 一44 2 MoraNP, n 酗 JA,Bemaldo de QIl L,孙 di11o F.Pereira F,Fores R.et a1. Successful liver allograf~hmctioa after 24-hour preservatioa: cum ulative effects of p~-tacyclin plos verar~mU. Tmrtsolant Proc l987.3932—6 3 Sm~anoboti T Takanashi H.Himoka M ,lida Y.Kamisaka K.Maezawa H,eI a1. Electrophysiological prope~ies of isolated ratliver cells JCell Physio1]909;139:j踯 一5. 4 Slriggow F. ~tmensack R. Verapamil a¨d dilliazem inl'fibit cd) operated calcium channels ar-d intracellulat" calcium o~-illati0砸 in rat hepatocytes Fl三Bs Leltl993;3l8:341—4 5 Seglen p0 Ple帅 邮 of isolated rat U哪 celIs. Methods Cdl Blol l976:I3 29一端 6 14oth /vI.Penner R D州 of inWacellul~ calcitall stores activates a calcium currentinill~t cells Nature l992;355: 一6. 7 Pen~r R.F~olatoC,Holh M 钙通道阻滞剂对大鼠肝细胞钙释放激活的 钙电流的影响 y72 崔桂 李金 崔 都丽 崔桂英 ,主 崔 宏 ,都丽莫,⋯ 剐东举 ,张克义 (中国医科大学药理教研室,。中国医科大学第一临 床学院普外科,沈阳110001,中国) 关键词 钙通道;钙通道阻滞剂;膜片箝技术; 肝;维拉帕采■ 磊蕈;硝苯地平;培养的细胞 ’ - — ’ 一 - ‘ 。 — ’ 。 ‘ ’ 一 目的:研究钙通道拮抗剂对大鼠肝细胞钙释放激 活的钙电流(, )的影响.方法:应用全细胞膜 片箝技术. 结果:lce.~c的电流峰值是(一0.41± 0.09)nA(n=15),反转电位约为0mV.维拉帕 米(Vet),地尔硫革(DiD和硝苯地平(Nif)显著降低 ,cRAc,不影响它的反转电位. Ver 5~zrnol·L 的 抑制率是40%±12% (n=3),Vet 50“mol-L 使 ,cRAc的幅值从(一0.49±0.12)nA降到(一0.20 ±0.09)nA(n=5,P<0 01),抑制率为 57%± 15%. Dil和 Njf 50 InD1.L-。分别从(一0.48± 0.10)nA(n=5),(一0 32±0.o8)nA(n=5)降到 (一O.29±O.07)nA(P<0.01),(一0.27±O.08) nA(P<0.01). 抑制率分别为 31%±11%, l9%±7%. ,cRAc的幅值大小依赖细胞外钙浓 度. ,cRAc在含台氏液 1.8 rmnol‘L 中电流幅值 为(一0.21±0.08)nA(n=3,P<0.()1). 结论: 这三种钙通道拮抗剂有效抑制 ICRAC并且通过抑制 lce.~c减少肝细胞钙超载. (责任编辑 李 颖) 维普资讯 http://www.cqvip.com
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