常氧和急性低氧下大鼠傳導(dǎo)性肺動(dòng)脈平滑肌細(xì)胞的電生理特性

1、生理學(xué)報(bào)ActaPhysiologicaSinicaOctober25200658(5):477482.cn477研究論文Received20051121Accepted20060710ThiswkwassupptedbytheNationalNaturalScienceFoundationofChina(No.3039313330260034).Crespondingauth.Tel:869716142063Fax:869716142

2、063Email:.常氧和急性低氧下大鼠傳導(dǎo)性肺動(dòng)脈平滑肌細(xì)胞的電生理特性常氧和急性低氧下大鼠傳導(dǎo)性肺動(dòng)脈平滑肌細(xì)胞的電生理特性胡穎1，鄒飛2，蔡春青2，吳航宇2，韻海霞1，陳云天1，靳國(guó)恩1，格日力11青海大學(xué)高原醫(yī)學(xué)研究中心，西寧810001；2南方醫(yī)科大學(xué)公共衛(wèi)生與熱帶醫(yī)學(xué)學(xué)院，廣州510515摘要：要：本文旨在研究大鼠傳導(dǎo)性肺動(dòng)脈平滑肌細(xì)胞(pulmonaryarterysmoothmusclecellsPASMCs)的電生理特

3、征及對(duì)急性低氧的反應(yīng)。用酶解法急性分離出1~2級(jí)分支的PASMCs，通過(guò)全細(xì)胞膜片鉗方法研究常氧及急性低氧狀況下細(xì)胞鉀電流的差異，并在常氧下先后使用iBTX和4AP阻斷大電導(dǎo)鈣激活鉀離子(largeconductanceCaactivatedKBKCa)通道及延遲整流性鉀離子(delayedrectifierKKDR)通道后，觀察細(xì)胞鉀電流特征。根據(jù)細(xì)胞的大小、形態(tài)及電生理特征可將PASMCs分為Ⅰ、Ⅱ、Ⅲ類。iBTX對(duì)Ⅰ類細(xì)胞幾乎無(wú)作

4、用，而4AP幾乎完全阻斷它的鉀電流；Ⅱ類細(xì)胞的鉀電流在加入iBTX后大部分被抑制，其余的對(duì)4AP敏感；Ⅲ類細(xì)胞的鉀電流對(duì)iBTX及4AP均敏感。急性低氧對(duì)三類細(xì)胞的鉀電流均有不同程度的抑制，并使Ⅰ類細(xì)胞的膜電位顯著升高，而Ⅱ、Ⅲ類細(xì)胞膜電位升高的程度不如Ⅰ類顯著。結(jié)果表明，傳導(dǎo)性肺動(dòng)脈有3種形態(tài)及電生理特性不同的PASMCs，在急性低氧時(shí)其鉀電流不同程度地受到抑制，同時(shí)靜息膜電位也有不同程度去極化，這些可能參與急性低氧時(shí)傳導(dǎo)性肺動(dòng)脈舒縮

5、反應(yīng)的調(diào)節(jié)。KDR及BKCa通道在3種細(xì)胞中的比例不同可能是急性低氧對(duì)3種PASMCs影響不同的離子基礎(chǔ)。關(guān)鍵詞：關(guān)鍵詞：電生理；肺動(dòng)脈；平滑肌細(xì)胞；低氧；鉀通道中圖分類號(hào)：中圖分類號(hào)：Q25ElectrophysiologicalstudyonratconduitpulmonaryarterysmoothmusclecellsundernmoxiaacutehypoxiaHUYing1ZOUFei2CAIChunQing2WUHang

6、Yu2YUNHaiXia1CHENYunTian1JINGuoEn1GERiLi11ResearchCenterFHighAltitudeMedicineofQinghaiUniversityXining810001China2SchoolofPublicHealthTropicalMedicineSouthernMedicalUniversityGuangzhou510515ChinaAbstract:Thepresentstudyw

7、asdesignedtoinvestigatetheelectrophysiologicalacteristicsofratconduitpulmonaryarterysmoothmusclecells(PASMCs)theresponsetoacutehypoxia.PASMCsofthe1stto2ndderbranchesintheconduitpulmonaryarterieswereobtainedbyenzymaticiso

8、lation.ThePASMCsweredividedintoacutehypoxiapreconditionedgroupnmoxiagroup.Hypoxiasolutionswereachievedbybubblingwith5%CO2plus95%N2fatleast30minbefecellperfusion.Potassiumcurrentswerecomparedbetweenthesetwogroupsusingwhol

9、ecellpatchclamptechnique.ThetotaloutwardcurrentofPASMCswasmeasuredundernmoxiaconditionwheniBTX[specificblockingagentoflargeconductanceCaactivatedK(BKCa)channel]4AP[specificblockingagentofdelayedrectifierK(KDR)channel]wer

10、eaddedconsequentlyintobathsolution.PASMCswereclassifiedintothreetypesaccdingtotheirsizeshapeelectrophysiologicalacteristics.TypeIcellsarethesmallestwithspindleshapesmoothsurfacediscreteperinuclearbulge.TypeIIcellsshowthe

11、biggestsizewithbananalikeappearance.TypeIIIcellshavethesimilarsizewithtypeIpresentintermediaryshapebetweentypeItypeII.iBTXhadlittleeffectonthetotaloutwardcurrentintypeIcellswhile4APalmostcompletelyblockedit.Mostofthetota

12、loutwardcurrentintypeIIcellswasinhibitedbyiBTXtheremainingwassensitiveto4AP.IntypeIIIcellsthetotaloutwardcurrentwassensitivetobothiBTX4AP.Acutehypoxiareducedthecurrentinallthreetypesofcells:(1614.862.5)pAto(892.433.6)pAf

13、typeIcells(P0.01)(438.342.8)pAto(277.544.7)pAftypeIIcells(P0.01)(1042.037.2)pAto(613.623.8)pAftypeIII(P0.01)raisedtherestingmembranepotentials(Em)inallthesethreetypesofcells:(–41.61.6)mVto(–18.61.5)mV(P0.01)(–42.33.8)mVt

14、o479胡穎等：常氧和急性低氧下大鼠傳導(dǎo)性肺動(dòng)脈平滑肌細(xì)胞的電生理特性錄鉀電流。電流值均取膜除極至60mV的值。1.4統(tǒng)計(jì)處理統(tǒng)計(jì)處理常氧組與低氧組之間的數(shù)據(jù)比較采用獨(dú)立樣本的t檢驗(yàn)。三類細(xì)胞之間的數(shù)據(jù)比較采用OneWayANOVA檢驗(yàn)。實(shí)驗(yàn)數(shù)據(jù)均以meansSEM表示，取P0.05作為差異顯著的水平。2結(jié)果結(jié)果2.1傳導(dǎo)性傳導(dǎo)性PASMCs的形態(tài)學(xué)特點(diǎn)的形態(tài)學(xué)特點(diǎn)根據(jù)細(xì)胞的大小及形態(tài)可分三類[5]:Ⅰ類細(xì)胞體形較小，平均膜電容為(10

15、.20.8)pF，細(xì)胞呈梭形，兩端尖，細(xì)胞中部明顯膨大，表面比較光滑(圖1A)Ⅱ類細(xì)胞體形較大，平均膜電容(17.91.1)pF，呈香蕉狀，細(xì)胞兩端較鈍，形態(tài)較均勻，細(xì)胞中部無(wú)明顯的膨大(圖1B)Ⅲ類與Ⅰ類細(xì)胞大小接近，平均膜電容(12.01.1)pF，形態(tài)居于Ⅰ、Ⅱ類細(xì)胞之間(圖1C)。2.2三類細(xì)胞外向電流的特點(diǎn)三類細(xì)胞外向電流的特點(diǎn)三類細(xì)胞之間電流值差異顯著(P0.01，表1)，iBTX及4AP的對(duì)它們電流的阻斷程度也不相同。Ⅰ類

16、細(xì)胞的電流值最大，電流曲線光滑，無(wú)波動(dòng)性，電流基本不受iBTX影響，而幾乎完全被4AP所抑制(圖2A、B)。Ⅱ類細(xì)胞的電流值最小，電圖1.三類平滑肌細(xì)胞的照片F(xiàn)ig.1.RepresentativeimagesofthreetypesofPASMCs.A:TypeIcell.B:TypeIIcell.C:TypeIIIcell.Scalebar25μm.圖2.iBTX及4AP對(duì)Ⅰ、Ⅱ、Ⅲ類細(xì)胞外向電流的影響Fig.2.Effectsof