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.
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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.
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中国药理学报 l999 May;舯 ‘5)
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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.
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]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)
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