細(xì)胞鈣信號(hào)轉(zhuǎn)導(dǎo)-WLL-2014

IntracellularCa2+SignalingWangLinlin

Ph.DDepartmentofPhysiologyZhejiangUniversitySchoolofMedicinePhysiologicalFunctionofCa2+PhysiologicalFunctionof

Ca2+參與神經(jīng)肌肉的反應(yīng)過(guò)程鎮(zhèn)靜作用：缺鈣可引起神經(jīng)的興奮性增高調(diào)節(jié)細(xì)胞功能，激活蛋白激酶，促進(jìn)蛋白質(zhì)磷酸化促進(jìn)內(nèi)、外分泌腺的分泌，神經(jīng)介質(zhì)的分泌參與血液的凝固維持細(xì)胞膜的通透性及完整性是十分必要的參與免疫反應(yīng)其他Ca2+參與的遞質(zhì)的釋放Ca2+參與的肌肉收縮Ca2+參與的細(xì)胞連接Ca2+的測(cè)定方法（1）血清鈣的測(cè)定5種方法：即滴定法、比色法、火焰法、離子選擇電極法、活化分析法。

（2）細(xì)胞內(nèi)鈣的測(cè)定：光譜熒光法（fluoromotric）利用單波長(zhǎng)或雙波長(zhǎng)熒光分光光度計(jì)，應(yīng)用熒光指示劑Quin-2／AM或Fura-2／AM測(cè)定細(xì)胞內(nèi)Ca2+濃度，這是目前研究Ca2+在生命科學(xué)中的生理生化功能的有力武器。血Ca2+濃度與Ca2+的轉(zhuǎn)運(yùn)血漿Ca2+濃度正常維持在8.5-11.1mg％之間，其中46％與血漿蛋白特別是白蛋白結(jié)合，47.5％為游離鈣(Ca2+)，僅有6.5％的鈣以磷酸鈣、檸檬酸鈣、重碳酸鈣、酒石酸鈣、棕櫚酸鈣等復(fù)合物形式存在。只有血漿離子鈣才具有生理活性。正常人的血漿Ca2+不是固定的，每日可有

0.1mg／dl的變化范圍。影響血Ca2+的因素來(lái)源：食物三個(gè)主要靶器官：腎、腸、骨其他影響因素

激素對(duì)Ca2+轉(zhuǎn)運(yùn)的作用3種激素：PTH,l，25-(OH)2-D3,CT此外生長(zhǎng)激素、甲狀腺素、性激素、前列腺素等也有調(diào)節(jié)血漿Ca2+的作用細(xì)胞內(nèi)Ca2+的分布胞內(nèi)鈣的分布極不均勻細(xì)胞內(nèi)總鈣濃度約為lmmol／L50％存在于細(xì)胞核線粒體占30％，濃度為0.6mmol／L內(nèi)質(zhì)網(wǎng)占14％，約0.28mmoI／L質(zhì)膜(外層)占5％，約0.1mmol／L而細(xì)胞溶質(zhì)中僅占總鈣的0.5％(結(jié)合態(tài))或0.005％(離子態(tài))

儲(chǔ)存Ca2+的肌漿網(wǎng)/內(nèi)質(zhì)網(wǎng)BerridgeMJetal.,Nature.1998;395:645-648.

Ca2+

–alifeanddeathsignalCalciumisSparkofLife,KissofDeathforNerveCells靜息狀態(tài)下的Ca2+梯度[Ca2+]o10-3M[Ca2+]i10-7M

HOW?1.Sarcolemma

impermeabletoCa2+

Ca2+pump(Ca2+-ATPase)Na+-Ca2+exchanger2.Endoplasmic/sarcoplasmicreticulum(ER/SR)

Ca2+-ATPase3.MitochondriaInitiationofintracellularCa2+signal[Ca2+]o10-3M[Ca2+]i10-7M

HOW?Twoprimarysources:1.ExternalExtracellularCa2+2.InternalER/SR[Ca2+]i10-5M1.External胞外Ca2+內(nèi)流途徑

:(1)VOC:電壓門控Ca2+

通道(2)ROC:受體操作Ca2+

通道(3)SOC:store-operatedCa2+channels(4)LeaksystemVoltage-GatedCalciumChannels

SIGMA-ALDRICH電壓依賴性鈣通道類型:

(1)L-type(long-lasting)(Nowycky,1985)

(2)T-type(transient)(Nowycky,1985)

(3)N-type(neitherL-typenorT-type)

(Nowycky,1985)

(4)P-type(Purkinje)(Llinas,1992)

(5)F-type(fetal)(Tohse,1992)

Ca2+channelsDurationofcurrent long-lasting transientActivationkinetics slower faster Inactivationkinetics slower fasterThreshold high(-35mV) Low(-60mV)cAMP/cGMP-regulated Yes NoPhosphorylation-regulated Yes NoOpeners Bay-K-8644 -Blockers

varapamil

Tetramethrin

nifedipine,diltiazem

Ni2+

Inactivationby[Ca2+]i

Yes slightPatch-clamprecording run-down relativelystableL-type

T-type

Inmusclecells,includingskeletalmuscle,cardiacmuscleandmosttypesofsmoothmuscle,themajorCa2+

inwardcurrentisthatthroughtheL-typeCa2+channels.

L-typeCa2+

channelPKAphosphorylationL-typeCa2+channels

的調(diào)節(jié):Increasesin: number probability opentimeROC:Areceptorproteinwhichhasanionchannel,asanintegralpartofitsstructurethatisgated,whenthenormalligandbindstothereceptor.(2)ROC (1)VOC2.InternalCa2+releasefromER/SRRyanodinereceptor(2)IP3receptorCa2+wavesinhepatocytes

(1)Ryanodinereceptor(RyR): mediatestheeffluxofCa2+

fromtheSRandplaysacentralroleinexcitation-contraction(E-C)coupling.MechanismsofE-Ccoupling興奮-收縮耦聯(lián)機(jī)制

RyR異構(gòu)體RyR1:SkeletalmuscleRyR2:CardiacmuscleRyR3:Brain(Coronado,1994)

SRRegulationofRyRActivation:Caffeine咖啡因ryanodine(nM)CICR鈣誘導(dǎo)鈣釋放Phosphorylation磷酸化…Inhibition:ryanodine(M)rutheniumred釕紅…鈣火花（calciumsparks）進(jìn)入胞內(nèi)的少量鈣離子激活RyRs，導(dǎo)致大量的鈣離子涌出SR的過(guò)程被稱為鈣誘導(dǎo)性鈣釋放(CICR,calciuminducedcalciumrelease)。肌漿內(nèi)的鈣離子濃度瞬時(shí)增高，這種現(xiàn)象被稱為鈣火花(calciumsparks)。鈣火花第一次被人們發(fā)現(xiàn)便是在靜息心肌細(xì)胞中。這項(xiàng)工作于1993年由MarkB.Cannell和PeaceCheng（程和平）揭示的。心肌細(xì)胞鈣火花是肌漿網(wǎng)上一個(gè)或幾個(gè)成簇狀分布的RyR所產(chǎn)生的局部鈣釋放,可通過(guò)單個(gè)L型鈣通道開(kāi)放所觸發(fā)或自發(fā)發(fā)放鈣火花呈隨機(jī)發(fā)放,出現(xiàn)在Z線附近,其動(dòng)力學(xué)特征受多種因素的調(diào)節(jié)T型鈣電流、Na+/Ca2+交換、ICa(TTX)和IP3等也是鈣火花觸發(fā)的來(lái)源(2)IP3receptor(IP3R):IP3R1:brain,nonexcitablecells(ubiquitous)IP3R2:cardiacmuscle,nonexcitablecells,liverIP3R3:brain,nonexcitablecells胞內(nèi)Ca2+回復(fù)到靜息水平1.Sarcolemma

Ca2+-ATPaseNa+-Ca2+exchanger2.Endoplasmic/sarcoplasmicreticulum(ER/SR)

Ca2+-ATPase3.Mitochondria*Sarco-endoplasmicreticulumCa2+-ATPase(SERCA)

SERCA1fast-twitchskeletalmuscle(SM) adult(SERCA1a) neonatal(SERCA1b)SERCA2cardiacmuscle(SERCA2a) slow-twitchSM(SERCA2b)SERCA3muscleandnonmusclecells(Arai,1994)

RegulationofSERCAbyphospholamban

Phospholamban:low-molecular-weightprotein(52-amino-acid)SERCAPhospholamban磷酸化:phospholambaninhibitstheSERCAbydecreasingitsaffinityforCa2+

去磷酸化:phospholambanenhancestheactivityofSERCAbyincreasingitsaffinityforCa2+calcium/calmodulin-dependentproteinkinaseandcAMP-dependentproteinkinaseA采用轉(zhuǎn)基因動(dòng)物研究研究phospholamban

對(duì)心臟的作用Phospholamban-deficientmice敲除小鼠IncreaseinSERCAaffinityforCa2+withenhancedratesofcontractionandrelaxationaswellasincreasedpumprate(Luo,1994,1996)

Phospholamban-overexpressingmice過(guò)度表達(dá)DepressedSRSERCAaffinityforCa2+andmyocardialdysfunction (Kadambi,1994,1996)

Ca2+研究背景蛙心灌流(Ringer,1883)跨膜電流(Heillbriunn&Kamada,1953)

膜片鉗單通道技術(shù)(Neher&Sakmann,1978)Fura-2熒光探針(Tsien,1985)VoltageClampHowtostudy?NobelPrizeinPhysiologyorMedicine1963"fortheirdiscoveriesconcerningtheionicmechanismsinvolvedinexcitationandinhibitionintheperipheralandcentralportionsofthenervecellmembrane"Eccles Hodgkin HuxleyPatchClampNobelPrizeinPhysiologyorMedicine1991"fortheirdiscoveriesconcerningthefunctionofsingleionchannelsincells"ErwinNeher BertSakmann

激光共聚焦CalciumsparkCalciumhomeostasis[Ca2+]i[Ca2+]iExternalVOC,ROC,SOC,Na+-Ca2+exchangerInternalRyRIP3RSERCASarcolemma

Ca2+-ATPaseNa+-Ca2+exchangerMitochondria****CalciumsignalpathwayCa2+

穩(wěn)態(tài)與病理生理學(xué)Hypertension原發(fā)性高血壓hyper/hypo-thyroidism

甲亢/甲低ischemia/reperfusioninjury缺血復(fù)灌損傷Cardiomyopathy心肌病heartfailure心衰其他EctopicCalcification膀胱結(jié)石ApoptosisandCa2+

abnormity細(xì)胞凋亡（apoptosis）：由體內(nèi)外因素觸發(fā)細(xì)胞內(nèi)預(yù)存的死亡程序而導(dǎo)致的細(xì)胞死亡過(guò)程稱