Cytosolic organelles shape calcium signals and exo–endocytotic responses of chromaffin cells

Texto completo

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ContentslistsavailableatSciVerseScienceDirect

Cell

Calcium

jo u r n al h om ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / c e c a

Cytosolic

organelles

shape

calcium

signals

and

exo–endocytotic

responses

of

chromaffin

cells

Antonio

G.

García

a,b,c,∗,1

,

Fernando

Padín

a,b,1

,

José

C.

Fernández-Morales

a,b

,

Marcos

Maroto

a,b

,

Javier

García-Sancho

d

aInstitutoTeófiloHernando,InstitutodeInvestigacionesSanitariasdelHospitaldelaPrincesa,FacultaddeMedicina,UniversidadAutónomadeMadrid,Madrid,Spain bDepartamentodeFarmacologíayTerapéutica,InstitutodeInvestigacionesSanitariasdelHospitaldelaPrincesa,FacultaddeMedicina,UniversidadAutónomadeMadrid,

Madrid,Spain

cServiciodeFarmacologíaClínica,InstitutodeInvestigacionesSanitariasdelHospitaldelaPrincesa,FacultaddeMedicina,UniversidadAutónomadeMadrid,Madrid,Spain dInstitutodeBiologíayGenéticaMolecular(IBGM),UniversidaddeValladolidandCSIC,c/SanzyForés,3,47003Valladolid,Spain

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received13October2011

Receivedinrevisedform2December2011 Accepted5December2011

Available online 29 December 2011

Keywords: Calciumtetrads Chromaffincells Calciumsignalling Calciummicrodomains Exocytosis

Endocytosis Mitochondria Endoplasmicreticulum Ca2+cycling

a

b

s

t

r

a

c

t

Theconceptofstimulus–secretioncouplingwasbornfromexperimentsperformedinchromaffincells50 yearsago.Stimulationofthesecellswithacetylcholineenhancescalcium(Ca2+)entryandthisgeneratesa

transientelevationofthecytosolicCa2+concentration([Ca2+]

c)thattriggerstheexocytoticreleaseof

cat-echolamines.Thecontrolofthe[Ca2+]

csignaliscomplexanddependsonvariousclassesofplasmalemmal

calciumchannels,cytosoliccalciumbuffers,theuptakeandreleaseofCa2+fromcytoplasmicorganelles,

suchastheendoplasmicreticulum,mitochondria,chromaffinvesiclesandthenucleus,andCa2+

extru-sionmechanisms,suchastheplasmamembraneCa2+-stimulatedATPase,andtheNa+/Ca2+exchanger.

ComputationoftheratesofCa2+fluxesbetweenthedifferentcellcompartmentssupporttheproposal

thatthechromaffincellhasdevelopedfunctionalcalciumtetradsformedbycalciumchannels,cytosolic calciumbuffers,theendoplasmicreticulum,andmitochondrianearbytheexocytoticplasmalemmalsites. ThesetetradsshapetheCa2+transientsoccurringduringcellactivationtoregulateearlyandlatestepsof

exocytosis,andtheensuingendocytoticresponses.Thedifferentpatternsofcatecholaminesecretionin responsetostressmaythusdependonsuchlocal[Ca2+]

ctransientsoccurringatdifferentcell

compart-ments,andgeneratedbyredistributionandreleaseofCa2+bycytoplasmicorganelles.Inthismanner,

thecalciumtetradsservetocouplethevariableenergydemandsduetoexo–endocytoticactivitieswith energyproductionandproteinsynthesis.

© 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Stressful conflicts trigger a surge of the catecholamines adrenalineandnoradrenalinethatmobilizethebodytosurviveby combatinganenemyortofleefromdanger,theso-called“fight orflight”response.Thisresponseistheendresultofasecretory eventthattakesplacein theadrenal medulla,theinnerpartof thetwoadrenalglandslocatedjustabovethekidneys.Theadrenal medullaiscomposedofchromaffincellsthatsecreteadrenaline andnoradrenaline.Thesecellsareofinterestnotonlytoexplore themechanismsunderlyingthe“fightorflight”response,butalso becausetheyhavebeenusedfordecadesasexcellentmodelsto studytheworkingofothersecretorycells,inparticularneurons.

Correspondingauthorat:InstitutoTeófiloHernando,FacultaddeMedicina,

Uni-versidadAutónomadeMadrid,C/ArzobispoMorcillo,4,28029Madrid,Spain. Tel.:+34914973120.

E-mailaddress:agg@uam.es(A.G.García).

1Equalcontributors.

Acetylcholine, the physiological neurotransmitter at the splanchnicnerve-chromaffincellsynapse[1],causesthereleaseof catecholaminesfromtheadrenalgland.Thissecretoryresponseis suppressedintheabsenceofextracellularcalcium(Ca2+)[2].Also,

acetylcholineenhancesCa2+entryintoadrenalmedullary

chromaf-fincells[3].Onthebasisoftheseandotherpioneeringexperiments William W. Douglas coined the expression “stimulus–secretion coupling”asthebasic mechanisminvolved inneurotransmitter andhormonesecretion;Ca2+wasthecouplingionbetweenthe

stimulusandtheexocytoticresponse[4].Sincethen,adrenal chro-maffin cells from various mammalian species but mostly from bovine,ratsandmicehaveextensivelybeenusedtostudythe rela-tionshipbetweenthechangesofcytosolicconcentrationsoffree Ca2+ionsinthecytosol([Ca2+]

c),itsredistributionintoorganelles,

itsclearancefromthecytosolandtheexocytoticandendocytotic responsestriggeredbyacetylcholineandothernicotinicand mus-carinicreceptoragonists,variousagonistsforG-proteincoupled receptorsand differentdepolarising stimuliincludinghigh con-centrationsofpotassium(K+),squaredepolarisingpulsesoraction

potentials.

0143-4160/$–seefrontmatter© 2011 Elsevier Ltd. All rights reserved.

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Chromaffincells areexcitablecellsand fireaction potentials thatopenvariousoftheneuronal-typevoltage-dependentcalcium channels(VDCCs)andproduceCa2+entry;theresulting[Ca2+]

c

sig-naltriggers exocytosis.Becausecytoplasmicorganellescantake upandreleaseCa2+tothecytosol,understandingthe[Ca2+]

c

sig-nalrequiresunderstandingoftheCa2+redistributionbetweenthe

cytosolandthedifferentorganelles.Thecodingofthe photopro-tein aequorin gene [5] made it possible to introduce targeting sequences, and measuring selective [Ca2+] changes in different

organelles[6].Thismethodologyhasbeenappliedduringthelast decadetogaininsightintotheroleoforganellesinshaping[Ca2+]

c

signallingandexocytosisinchromaffincells.Thisreviewfocuseson thepathwaysforCa2+entryintothechromaffincell,onthe

intra-cellularorganellesthatcontributetotheredistributionoftheCa2+

enteringthecell,andonthemechanismsthatterminatethe[Ca2+] c

signalsandextrudethecationoutsidethecell.Wealsoanalysethe influenceofthisCa2+traffickingbetweenthedifferentorganelles

ontheexocytoticresponses.Finally,weanalysethekineticsofCa2+

handlingatdifferentcellcompartments,tryingtoobtaina uni-fiedpictureofCa2+handlingandtheexo–endocytoticresponsesof

chromaffincells.Severalreviewsofsomeofthesequestionshave beenpublished[7–11].

2. Calciuminflux

ThemostrelevantCa2+entrypathwaysinchromaffincellsare

VDCCs,store-operatedCa2+channels(SOCCs)andligand-gated

cal-ciumchannels.Thecharacteristicsand regulationofthevarious VDCCsubtypeswillextensivelybedescribedbyE.Carboneinthis specialnumberofCellCalcium.So,wewillonlymakeabrief men-tiontothem.

2.1. Voltage-dependentcalciumchannels

Asinneurons[12],multipleVDCCsareexpressedinchromaffin cells[13].Significantdifferencesexistinthedensitiesofeach chan-nelsubtypeincellsfromdifferentspecies.Forinstance,Lchannels (␣1D,Cav1.3)carrynear50%ofthewhole-cellcurrentincat,rat

andmousechromaffincells.Incontrast,P/Qchannels(␣1A,Cav2.1)

accountfor50–60%ofthecurrentinbovineandhuman chromaf-fincells.N-typechannels(␣1B,Cav2.2)contribute80%inpig,45%

incatand30%inbovine,rat,mouseandhumanchromaffincells. Finally,R-typechannels(␣1E,Cav2.3)arepresentonlyinmouse

chromaffincells[9].

2.2. Store-operatedcalciumchannels

Inmanynon-excitablecells,inositol1,4,5-trisphosphate(InsP3)

generated byagoniststimulationcauses a biphasicelevationof [Ca2+]

c. Theinitialpeak isdue toERCa2+ release viatheInsP3

receptorchannelwhilethesubsequentmaintainedplateauphase isassociated toCa2+entrythrough SOCCs[14–16].Theplateau

phaseisproducedbyasmall-conductance,voltage-independent Ca2+releaseactivatedCa2+current(I

CRAC),thatservestoreplenish

theCa2+store[17–19].HavingmultipletypesofVDCCs,excitable

cellscouldberefillingtheirdepletedERCa2+storebyCa2+entering

throughthosehigh-conductancechannels.Thishasbeenshownto applyforsomeneurosecretorycells[20,21]includingbovine chro-maffincells;inthesecellsloadedwithER-targetedaequorin,high K+acceleratestheERCa2+storerefillinguponCa2+reintroduction

[22].

Early experiments demonstrated Ca2+ influx through SOCCs

uponERCa2+depletionofbovinechromaffincells[23];thiswas

corroboratedbylaterexperiments[22,24–28].Adirectprooffor thepresenceofSOCCswasobtainedfromvoltage-clampedbovine chromaffin cells where a small-amplitude,voltage-independent

ICRACcarriedbyCa2+andNa+,wascharacterisedunderconditions

ofCa2+storedepletion[29].ACa2+entrypathwaytriggeredby

his-tamineandindependentoftheERCa2+storeisalsopresentinthese

cells[25,30].

A few studieshave explored therole of Ca2+ entrythrough

SOCCsin triggeringexocytosisin bovinechromaffincells. Thus, histamineandangiotensinIIstimulateexocytosisbya combina-tionofERCa2+releaseandadditionalCa2+entrythroughSOCCs [24].Moreconvincingevidencearisesfromexperimentsperformed involtage-clampedcells,whereangiotensinII-inducedexocytosis wasassociatedwithanuncharacterisedleakcurrent[27].In addi-tion,exocytosiscouldbeelicitedintheabsenceofdepolarisationby photolysisofcagedInsP3[31]orbybradykinin[32].Butthemost

convincingevidencecomesfromexperimentsdonewith stimula-tionofCa2+entrythroughSOCCsbystoredepletionthatproduces

exocytosisatnegativemembranepotentialsthatmaintainclosed theVDCCs[29].

Whya bovinechromaffincellexpressingL,Nand P/Q high-conductanceVDCCs[9]shouldstill requireadditional pathways forCa2+entryispuzzling.Thefactsuchpathwayscanbe

physio-logicallyactivatedbyactionpotentialsorsustaineddepolarisation triggered by acetylcholine is even more puzzling. Combining aequorins and confocal microscopy, Ca2+-induced Ca2+ release

(CICR)wasshown tobeactivated byK+ or 50-msdepolarising

pulsesinbovinechromaffincells[22].Uponrepetitivestimulation withbursts ofactionpotentialsunderstress,CICR mayproduce partialERCa2+depletionandgiverisetoSOCCactivation.A

modula-toryroleofthiscapacitativeCa2+entryonexocytosisinchromaffin

cellshasbeensuggested,butotherpathwaysforCa2+entrywere

notundercontrolintheseexperiments[26].Later,direct experi-mentsdemonstratedthatreceptor-freeactivationofCa2+entryvia

SOCCsissufficienttotriggerandorfacilitateexocytosisinthese cells[29].Inthiscontext,itisinterestingthathyperpolarisationis associatedwithhistaminereceptorstimulationthatiscoupledto ERCa2+releaseandactivationofsmall-conductanceCa2+-activated

K+ channels[33].ThismechanismcouldamplifyCa2+ influxvia

SOCCs,thusfacilitatingtheexocytosistriggeredbyburstsofaction potentials, ina kindof long-lasting modulatory mechanism for stimulus–secretioncoupling.

2.3. Ligand-gatedcalciumchannels

Nicotinicreceptorsforacetylcholine(nAChRs),aswellas recep-tors forglutamate and ATP,underlieexcitatory transmissionat centralandperipheralsynapses.Thesereceptorsareionchannels permeabletocations.Thefractionoftheinwardcationcurrent car-riedbyCa2+,triggeredbyagonistsinvariouscelltypesisabout

5%fornAChRsandATPreceptorsandaround10%for N-methyl-d-aspartate(NMDA)receptors[34,35].Inbovinechromaffincells the fractionof acetylcholine-elicited inward current carried by Ca2+accountsfor about5%[36]. Ca2+entering throughnAChRs

maycontributetoaugmentvesiclemovementandthesizeofthe ready-releasevesiclepool[37,38].Furthermore,glutamate recep-torsseemtomediateanincreaseof[Ca2+]

candexocytosisinbovine

chromaffincells[39].Ontheotherhand,variouspurinoceptor sub-typesthatrespondtoATPwitha[Ca2+]

c increasehavealsobeen

foundinthesecells[40].Remarkabledifferencesamongspecies havebeenfound.Forinstance,ratchromaffincellslackP2X recep-tors while in the guinea-pig, ATP generatesan inward current that seemsto be associated toP2X2 receptors[41].Na+ influx

throughP2Xchannelscauses depolarisationofbovine chromaf-fincells,enhancesCa2+entrythroughVDCCsandcatecholamine

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receptorsexertanautocrineregulatoryinhibitionofinwardCa2+

currentsthroughVDCCsofbovinecells[44,45].

GABAAreceptoragonistsalsocausecelldepolarisation,an

ele-vationof[Ca2+]

c,likelyduetoopeningofVDCCsandthereleaseof

catecholamines[46–48].Furthermore,GABAenhancesthe[Ca2+] c

elevationelicitedbylow-frequencyelectricalfieldstimulationof perfusedratadrenals[49].ItisstillunclearhowGABAcanexert thosemodulatoryeffects;aparacrinerolehasbeensuggestedfor GABAco-storedandco-releasedwithcatecholaminesduring elec-tricalstimulationofthesplanchnicnervesattheadrenalmedulla

[49].

3. Calciumredistribution

Theabrupt[Ca2+]

ctransientgeneratedbydepolarisingstimuli

elicitedbyeitheractionpotentialsorsustaineddepolarisations,are controllednotonlybythedifferentsubtypesofVDCCsexpressed by chromaffin cells, but also bycytosolic calciumbuffers, Ca2+

sequestrationorreleasebycytoplasmicorganellesandextrusion byplasmalemmalcalciumtransporters.Wewillseparatelyanalyse thesecalciumregulatoryelements(Fig.1).

3.1. Cytosoliccalciumbuffers

Ca2+ buffering and diffusion in bovine chromaffin cells has

beenstudiedextensivelybyNeherandcoworkers(seeSection5). However,themolecular natureof thecytosolic calciumbuffers isunknown.Onlyafewreportshavebeendevotedtothestudy ofcalcium-bindingproteinsin chromaffincells.For instance,in

Fig.1.Calcium(Ca2+)cyclinginthechromaffincell.Uponcelldepolarisation,

extra-cellularCa2+entersthecellthroughvoltage-dependentCa2+channels(1,VDCCs).

ThisgeneratesalocalcytosolicCa2+transient([Ca2+]

c),withactivationand

clear-ancephasesexhibitingspatialandtemporalpatternsthataretightlyregulatedby nearbypoorlydefinedimmobilecytosoliccalciumbuffers(2,CCB),theendoplasmic reticulum(3,ER)andthemitochondrion(4,MIT).Ca2+takenupbyorganellesand

cytosoliccalciumbuffersisreleasedbackintothecytosolallowingitsredistribution towardsthecellcore(5).Finally,tore-establishthecellCa2+balance,the

plas-malemmalCa2+pump(6,PMCA)andNa2+/Ca2+exchangerNCX(7)driveCa2+efflux

backtotheextracellularspace.Thenucleus(8)andchromaffinvesicles(9,CV)may alsocontributetoCa2+redistribution.PathwaysforCa2+entryotherthanVDCCs,

suchasstore-operatedcalciumchannels(SOCCs),nicotinicacetylcholinereceptors, purinergicreceptors,GABAandglutamatereceptorshavealsobeenreportedtobe presentinchromaffincells;theyarenotrepresentedforthesakeofsimplicity.Being anexcitablecelldrivenbythesympatheticnervoussystem,theCa2+cyclingmust

becontinuouslygoingonintheintactadrenalmedullarytissue.Thevelocityofsuch Ca2+cycling(10),dependsontherateofactionpotentialfiringandthesympathetic

cholinergicinputatdifferentstresssituations.SuchvariationsinthevelocityofCa2+

cyclingservetoadaptthebioenergeticneedsofthecell,inordertosecuretherapid releaseofcatecholaminesintothecirculation,topreparethebodyforthefightor flightresponse.

bovinecellsparvalbumincontainsCa2+/Mg2+mixedsitesthatshow

slowCa2+-bindingkineticsunderphysiologicalconditions.

Parval-bumin actsas a Ca2+ sourceduring relaxation of[Ca2+] c peaks

andextendsthe[Ca2+]

c transientbyconversionofa

monoexpo-nentialdecayinabiexponentialone[50].Anotherstudyreported thatcalbindin-D28kishomogeneouslydistributedinthecytosol of bovinecells while itsdistributionwaspreferentially concen-tratedatsubmembranesitesinmousecells.Theclearanceofthe K+-evoked[Ca2+]

c transientswasslowerinbovinecells, butthe

initialquantalsecretoryresponsewasfasterin mouse chromaf-fincells. Thus,thedifferentdistributionofcalbindin-D28kdoes certainly affectCa2+signalling andexocytosisinbothcelltypes

[51].

3.2. Nucleus

Ca2+hasrelevantfunctionsintheregulationofgene

expres-sioninthenucleus.In addition,afew studieshaveapproached the nuclearCa2+ kinetics[52]. For instance,there is consensus

thatthenuclearenvelopemaysomewhatdelaythepropagation ofCa2+wavesfromthecytosoltothenucleus[53–55].InPC12

andothercelltypes,half-equilibriumtimesforCa2+fluxesthrough

thenuclearenvelopeareintherangeofseconds[54].Underthese conditions,strongstimuli such asK+ depolarisation or

stimula-tionwithUTPorbradykiningenerateCa2+signalsthatarequickly

transmittedtothenucleus. Onthe contrary,theprogression of high-frequency[Ca2+]

c oscillationstothenucleusmaybe

damp-enedbythenuclearenvelope[54].Thenuclearmatrixalsodiffer fromthecytosolinhavinga largerCa2+-bufferingcapacity[56],

whichwouldalsoresultinanobviousslowingintheprogression oftheCa2+wave.Itisinterestingtonotethatselectivenuclear

sig-nallingmightbeachievedbyCa2+releasefromnuclearstoresin

certaincells[57].

3.3. Chromaffinvesicles

Chromaffinvesiclesofbovinechromaffincellscontainasmuch as 40mMcalcium [58].Most ofthis calcium(>99.9%) is bound tochromograninsandthefreeCa2+concentrationisabout40M

[59,60].AtpH7.5,chromograninAbinds32molofCa2+/mol

pro-tein,withaKDof4mM;thebindingcapacityincreasesto55molof

Ca2+/molproteinwithaK

Dof2.7mMattheintravesicularpHof5.5 [61].Thus,anincreaseofintravesicularpHincreasesthefreeCa2+

concentration,therebyfacilitatingitsreleaseintothecytosol.This hasbeenexperimentally demonstrated withalkalinisingagents andprotonophores,whichenhancevesicularCa2+release,vesicle

motionandexocytosis[62–66].Asmuchas20–30%ofthebasal chromaffincellsvolumeisoccupiedbyabout20,000chromaffin vesicles[67]thatstorearound60%oftotalcellCa2+[59,68];

how-ever,scarcedataareavailabletosupporttheoriginalhypothesis statingthatintravesicularCa2+couldbeinvolvedintheexocytotic

process [69].Experiments withalkalinisingagentsare certainly interesting;but it isdifficult toenvision thephysiological con-textthattheycouldmimic.ThepresenceofInsP3receptorsinthe

chromaffinvesiclemembrane [70,71]andInsP3-induced

vesicu-larCa2+release[60,72,73]suggestthattheInsP

3pathwaymaybe

physiologicallyrelevant.ItseemslikelythatvesicularCa2+release

couldbeinvolvedinslowpre-exocytoticstepsaimedat mobiliz-ingvesiclesfromareservepooltoa ready-releasablepool,asit isthecasefor Ca2+releasefromtheER(seeSection3.4).

How-ever,itisunlikelythatthisslowCa2+releasecancompetewiththe

rapidhigh-Ca2+microdomains(HCMDs)formedat

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tosuchHCMDsmayenlightenthecontributionofvesicularCa2+

releaseinthevariousstepsofexocytosis.Forfurtheranalysisof thistopic,seetworecentreviews[74,75].

3.4. Endoplasmicreticulum

EarlierobservationsestablishedthatCa2+uptakeoccurredin

thesarcoplasmic reticulumofskeletalmuscle[76,77]througha Mg2+-andATP-dependentP-typetransportCa2+ATPase,the

sarco-endoplasmicreticulumCa2+-ATPase(SERCA)[78].Thisledtothe

conceptofintracellularcalciumstoresthatwassoonextendedto mostnonexcitableandexcitablecellsincludingneuronsand neu-rosecretory cells [79]. Twochannelsare mainlyresponsible for thereleaseofCa2+fromtheERstorenamely,theInsP

3 receptor

channel,whichisactivatedbytheInsP3generatedasaresultof

G-proteincoupledmembranereceptor activation,andthe ryan-odinereceptorchannel(RyR)thatisactivatedbyenhanced[Ca2+]

c,

caffeineandryanodine. Ca2+ bindingtoRyRopensthechannel,

therebytriggeringthereleaseofCa2+intothecytosolthroughthe

Ca2+-inducedCa2+releasemechanism(CICR).

Considerableeffortshavebeendevotedtoclarifythekinetics ofCa2+fluxesoftheERCa2+store,anditsroleincontrolling

pre-exocytoticand thelast exocytoticstepsin chromaffincells. For instance,histamine,angiotensinII,bradykininandcarbacholhave beenshowntoaugmenttheproductionofInsP3inbovine

chro-maffincells[80–82].AparallelincreaseofInsP3and[Ca2+]coccurs

uponchallengingthesecellswithhistamineorangiotensinII[83]. Theaugmentationof[Ca2+]

celicitedbystimulationwithhistamine

ismimickedbydirectstimulationwithInsP3,suggestingthat

stim-ulationofhistaminereceptorsiscoupledtoInsP3generationand

thesubsequentstimulationof InsP3 receptorsto causeERCa2+

release[84,85].Pituitaryadenylatecyclase-activatingpolypeptide (PACAP)hasalsobeenshowntoenhancebothInsP3 production

andenhanced[Ca2+]

c [86].Inratchromaffincells,stimulationof

muscarinicand␤2adrenergicreceptorsmodulatestheamplitude

of[Ca2+]

coscillations[87];suchCa2+oscillationsaredependenton

ERCa2+releasefromheparin-sensitiveCa2+stores[88].

Thefunctionalcorrelateofhistamine-elicitedERCa2+release

hasalsobeenstudied.For instance,this [Ca2+]

c signalactivates

small-conductanceCa2+-activatedK+channelsleadingto

hyperpo-larisationofbovinechromaffincells[33].Inthislineisthefinding thatmuscarineproducesa[Ca2+]

celevationandanoutwardK+

cur-rent,duetoactivationofCa2+-activatedK+channelsinguinea-pig

chromaffincells[89].Thesechannelsareregulatingthenicotinic andmuscarinicsecretoryresponseofcatandbovinechromaffin cells[90–92].WhileERCa2+releasebyhistaminecausesa mild

and transientcatecholaminerelease response [93],a more sus-tainedapplicationcausesalongereffect[93–95].Thisgreatereffect couldbeexplainedby thefacthistamine-elicited[Ca2+]

c

eleva-tionshastwo components:aninitialtransientphase duetoER Ca2+releaseand alate moresustainedphasedue toCa2+entry

[30,83,96,97].Thesecondcomponenthasbeenassociatedto

inhi-bitionofanM-currentbysustainedhistamineapplication,leading tocelldepolarisation,dischargeofactionpotentialsandopening ofVDCCsinbovinechromaffincells[98],althoughstimulationof SOCCsbyERemptyingcouldalsocontributetothiseffect(see Sec-tion2.2).Finally,itisinterestingthathistaminehasbeenusedas atooltoelicitsubthreshold[Ca2+]

celevationsinvoltage-clamped

bovinechromaffincells.This[Ca2+]

csignaldoesnotelicit

exocyto-sisbyitself,butpotentiatesthesubsequentexocytoticresponseto adepolarisingstimulus,likelyduetoanaccelerationoftheflowof newvesiclestowardsexocytoticsubplasmalemmalsites[99].Also, angiotensinIIaugments[Ca2+]

candsecretioninbovinechromaffin

cellsbuttoalesserextentthanhistamine[24,100].Ontheother hand,thenicotinicresponseseemstohaveacomponentlinkedto ERCa2+release[101].

ConcerningRyRchannels,ithasbeenknownforlongthatbovine chromaffincellspossessapowerfulcaffeine-sensitivecalciumstore

[102].ThereleaseofERCa2+bycaffeinewaslatershowntofollowa

quantalpattern,suggestingthatthecaffeine-sensitiveCa2+poolis

composedoffunctionallydiscretestoreswithheterogeneous sensi-tivitiestocaffeine[103,104].Additionally,thepresenceofseparate oroverlappingCa2+poolsresponsivetoeithercaffeine,InsP

3 or

cyclicADPribose,theirdifferentialsensitivitytoSERCAinhibitors suchasthapsigargin,andthephysiologicalsignificanceorthe dif-ferentCa2+releasemechanisms,havebeensubjectofdebatefor

manyyears[85,105–108].

Direct monitoring of changes in the ER Ca2+ concentration

([Ca2+]

ER)inbovinechromaffincellstransfectedwithER-targeted

aequorin,permittedclarificationofsomeofthoseissues[22,109]. Thus, Ca2+ entry elicited by depolarisation triggers a transient

Ca2+ release from the ER that is highly dependent on[Ca2+] ER

andsensitisedbylowcaffeineconcentrations.Ontheotherhand, caffeine-inducedCa2+releasewasquantalinnaturedueto

mod-ulationby[Ca2+]

ER.Whereascaffeinereleasesessentiallyallthe

Ca2+fromtheER,InsP

3-producingagonistsreleaseonly60–80%.

However,indigitonin-permeabilisedcellsbothInsP3andcaffeine

emptiedcompletelythecalciumstorewhilecyclicADPribosehas noeffect.Finally,thewaveofCa2+elicitedby100msdepolarising

pulsesmeasuredwithconfocalmicroscopy,isdelayedandreduced inintensityinryanodine-treatedcells.Thesedatasuggestthatthe ERof bovinechromaffincells behavesasa single thapsigargin-sensitivecalciumpoolthatcanreleaseCa2+bothviaInsP

3receptors

orCICR.Alaterreportshowedthatmousechromaffincellsinthe intactglandexhibitedasmallerornonexistentCICR[110]. How-ever,inarecentstudyperformedonculturedmousechromaffin cellstheexpressionofRyRsandafunctionalCICRmechanismwas shown[111].

Inisolatedbovinechromaffincells,caffeinecausesamild secre-toryresponse[102],andthiseffectisalsoobservedintheabsence ofextracellularCa2+[112,113].ActivationofCICRduringcell

depo-larisationmayhavefunctionalconsequencesforthecontrolofthe exocytoticprocess.Inthiscontext,itisinterestingthatwhenthe Ca2+storehasbeendepletedbysustainedcaffeinestimulation,a

subsequentdepolarisationbyhighK+ elicitsasmallersecretion.

Consistently,afterfullERCa2+depletion,thefirsttwoorthreeinitial

depolarisationscontributetorefilltheERwithCa2+andtherefore,

theERbehavesasasink,reducingtheamountofCa2+availablefor

secretion[113].

Involtage-clampedbovinechromaffincells,exocytosisis unaf-fectedbypreviousERCa2+depletionwiththapsigargin[114,115];

however,alaterstudyshowdepressedsecretion[115].Inbovine chromaffin cells stimulated with acetylcholine, severe ER Ca2+

depletionwith a mixture of caffeine,ryanodine and thapsigar-ginhalvesthecatecholaminereleaseresponses.However,theK+

responses are little affected. This may be due to thefact that acetylcholine elicits discrete and more localised [Ca2+]

c

eleva-tions, whereasK+ pulsesproduce higher[Ca2+]

c transients that

spreadquicklythroughoutthecytosol[116].Thisdifferencemay beexplainedconsideringthatacetylcholineevokesaction poten-tials[117]whileK+producessustainedcelldepolarisation[118]in

bovinechromaffincells.Thus,itisplausiblethatthecontribution ofCICRtotheexocytoticresponseismorevisibleunderconditions ofphysiologicalstimulationofchromaffincellswithacetylcholine.

3.5. Mitochondria

Mitochondriaarethemainenergy-producingcentresof eukary-oticcells[119,120].Theyarecapableofaccumulatingvastamounts ofCa2+intheirmatrixthroughtheirCa2+uniporter,thatusesthe

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withalargetransmembranepotentialdifference(near−180mV) thatisgeneratedbytherespiratorychainorbyATPhydrolysis.Ca2+

accumulatedinmitochondriaisthenreleasedbackintothe cyto-solbyelectroneutralantiportersthatexportCa2+fromthematrix

byswappingoneCa2+ionfortwoNa+throughthemitochondrial

Na+/Ca2+exchanger(mNCX).ANa+/H+exchangemechanismdoes

alsoexist,butitislessactivethanmNCX[122,123].Additionally, mitochondrialCa2+-inducedCa2+releasemediatedbythecalcium

uniporterhasalsobeenobserved[124].

Duringcellactivation,somemitochondriatakeupCa2+from

cytosolicHCMDsthatarecreatedbytheopeningofnearbyVDCCs

[109,125].Inratchromaffincells,mitochondriaactasrapidand

reversibleCa2+buffersduringcellstimulation[126,127];theyalso

contributetotheclearanceoflargeCa2+loadsinbovinechromaffin

cells[109,128].However,earlymeasurementsof[Ca2+]changesin

themitochondrialmatrix([Ca2+]

M)providesvaluesonlyinthelow

micromolarrange[126],mostprobablybecauseunderestimation bysaturationofthemeasuringfluorescentCa2+probe.Byusing

mitochondriallytargetedaequorinswithdifferentCa2+affinities,

whichhaveamuchwiderdynamicrange[52,129],itwaslateron shownthatbovinechromaffincellmitochondriaexhibit surpris-inglyrapidmillimolarCa2+transientsuponcellstimulationwith

acetylcholine,caffeineorK+[130].

Thisavid Ca2+ uptake bymitochondriasurelyhasfunctional

consequences. For instance, dissipation of the proton gradi-ent by protonophores decreases the Ca2+ buffering capacity of

mitochondria[130,131]and drasticallyaugmentstheexocytotic responsein voltage-clampedbovinechromaffincells stimulated withdepolarisingpulses[132].Inperifusedpopulationsofbovine chromaffin cells stimulated with acetylcholine, caffeine or K+,

mitochondrialprotonophoresenhance3–5foldthereleaseof cat-echolamines[116,130,133].Blockadeofthemitochondrialcalcium uniporteralsoenhancestheK+-evokedsecretioninsinglebovine

chromaffin cells [131]. K+-elicited secretionis particularly

aug-mentedbyprotonophoreswhen Ca2+entryviaL-typeVDCCsis

enhancedbyFPL64176[134].Inmousechromaffincellshowever, protonophoreshalved theK+-evoked [Ca2+]

c and catecholamine

releaseresponses[135];thiscouldbeexplainedbydifferencesin theexpressionofVDCCsubtypesinbovineversusmouse chromaf-fincells,and/ordifferentratesofinactivationofVDCCsubtypes dur-ingblockadeofmitochondrialCa2+uptakebyprotonophores[136].

4. Calciumefflux

ThemaintransportersusedbycellstoextrudeCa2+fromthe

intracellulartotheextracellularcompartmentarethe plasmalem-malCa2+pumporCa2+-ATPaseandtheNa+/Ca2+exchanger(NCX)

(Fig.1).Bothtransporterscontributetomaintainthelong-term Ca2+ homeostasisthrougha wellbalancedCa2+ influxand Ca2+

efflux activities. The functional expression of these two trans-porterswasfirstdemonstratedusingplasmamembranevesicles from bovine adrenal medulla [137]. The plasmalemmal Ca2+

-ATPase has a high Ca2+ affinity (K

D in the 10−7M range) and

operates asan electrogenic Ca2+/H+ exchanger witha 1:1

stoi-chiometry[138].

TheNCXusestheenergyprovidedbytheNa+gradienttoachieve

an electrogenic exchange of 3 Na+ ions for 1 Ca2+ ion. Under

physiologicalconditionsNa+istransportedintothecellandCa2+

isextrudedfromthecytosol[139].However,whenthe electro-chemicalgradientforNa+isreversed,suchasduringmembrane

depolarisationortheopeningofgatedNa+channels,theexchanger

movesNa+outofthecellandCa2+intothecell[140].TheCa2+

exitmodeisreferredtoastheforwardmode,andtheCa2+entry

modeasthereversemodeoftheNCX[141].Bovinechromaffincells expressthemajorisoformoftheNCX,namelyNCX1[142],which

canmediateNa+-dependentCa2+influx[143]orCa2+export[144],

dependingonthecircumstances.

Thecardiotonicsteroidouabain,theclassicalinhibitorof the plasmalemmalNa+/K+-ATPase(NKA)orNa+pump[145]hasbeen

widely used toinfer therole of theNCX in various celltypes. AlthoughouabainupsetsprimarilytheNa+andK+gradientsacross

theplasmamembrane,thecollapseoftheNa+gradientcan sec-ondarily drive Ca2+ entry though NCX. This is the mechanism

underlyingtheheartinotropiceffectofcardiacglycosides.Onthe otherhand,repeatedactionpotentialfiringleadingtoNa+

accu-mulation,canalsoforceNCXtoworkinreversemode, thereby increasing[Ca2+]

c andfavouringthereplenishmentwithCa2+of

thesarcoplasmicreticulum.Duringsubsequentactionpotentials, augmentedCICR,whichispotentiatedbytheincreased[Ca2+]

ER,

leadstoenhancedcardiaccontraction[146].

Sincelongweknowthatouabainenhancesboththe sponta-neous[147–149]andtheK+-evokedcatecholaminereleasefrom

catchromaffincells[150]andbovinechromaffincells[151,152]. Theseeffectswereinitiallyinterpretedasasecondaryactivationof theNCXbyouabain[143,153,154],throughamechanismsimilar tothatoccurringintheheart.However,analternativeexplanation canbeinferredfromtherecentobservationthatNKAco-localises withsubplasmalemmalregionsoftheER[155,156].Recentdata onbovinechromaffincellsshowthatouabaincausestherelease ofCa2+ fromtheERand augmentsthecatecholaminesecretory

responsestosequentialK+pulses.ERCa2+depletionpreventssuch

potentiationandcausesagradualdecreaseoftheresponsestoK+.

Furthermore,ouabainenhancesthenumberofdockedvesiclesat subplasmalemmalregions,asrevealedwithTIRFmicroscopy[152]. AllthesedatasupportearliersuggestionsthattheERCa2+store

contributes tomaintainhealthy secretory responseselicited by depolarisingpulsesappliedtobovinechromaffincells[115,116]. Thefactthatendogenousouabainhasbeenidentifiedinhuman plasma [157,158]and thatbovineadrenal cortexis particularly richinendogenousouabain[159],suggestsaphysiologicalroleof thismediatorinthecontrolofCa2+-dependentvesicleflowfroma

reservepooltowardsready-releaseandimmediate-releasevesicle poolsatsubplasmalemmalsites[7].

Bovine chromaffin cells express the major isoform of the

NCX, NCX1 [160]. In bovine chromaffin cells NCX1 can favour

Na+-dependentCa2+ influx [143]or Ca2+ export [144]and has

been proposed to participate in the regulation of [Ca2+]

c and

exocytosis in cat [150,161,162] and bovine chromaffin cells

[142,143,153,163–165]. In addition,chromaffin cells co-express

NCXandtheretinalrod-typeK+-dependentNa+/Ca2+exchanger

[166].AttemptstoclarifytheparticipationoftheNCXin

physio-logicalandpathologicalprocesseshavebeenhamperedbythelack ofpotentandselectiveblockers.TheantagonistKB-R7943 pref-erentiallyinhibits,atlowmicromolarconcentrations,thereverse

modeoftheNCX[167].Unfortunately,thiscompoundalsoblocks

othertransporters,suchasthemitochondrialuniporter[168],and thenicotinicreceptorsofbovinechromaffincells[169].Noveland moreselectiveinhibitorssuchasSEA0400,SN-6andYM-244769

[170]shouldhelptofurtherclarifytheroleoftheNCXinCa2+

sig-nallingandexocytosisinchromaffincells.Infact,SEA0400inhibits Na+-dependentCa2+uptakeandcatecholaminereleaseinbovine

chromaffincells,withIC50 of40 and100nM,respectively,

com-paredwithIC50of1.8and3.7␮MforKB-R7943,thatwas40-fold

lesspotent[171].

5. Afunctionaltetradshapescalciumgradientsand calciummicrodomains

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Fig.2.Functionaltetradstoshapethehigh-Ca2+microdomains(HCMDs)an

low-Ca2+microdomains(LCMDs)thatdetermine,respectively,thefastexocytosis(FE)

releaseofadrenaline(AD)andnoradrenaline(NA),fromanimmediatelyreleasable vesiclepool(IRP),andchromaffinvesiclesmovement(CVM)fromareservepool (RP).Tetradsareformedbyvoltage-dependentCa2+channels(1,VDCCs),cytosolic

Ca2+buffers(2,CCB),theendoplasmicreticulum(3,ER)andthemitochondrialpool1

(MIT1)locatednearbytheplasmalemma(4).Thistetrad(redline)isresponsiblefor generatingandshapingtheHCMDtransients(10–100␮M),nearby subplasmalem-malexocytoticsites,totriggerfastcatecholaminereleasefromtheIRP,thatcan bemonitoredatthesingle-vesiclelevelasamperometricspikes(AS)withacarbon fibremicroelectrode(5).TheLCMD(<1␮M)islocatedatcytosolicsitesawayfrom theplasmalemma,andfacilitatetheCa2+-dependentCVM.CrosstalkbetweenHCMD

andLCMDisneededtosecurethesupplyofnewvesiclestothesecretorymachinery underdifferentstimulationratesofchromaffincells(seetextforfurtherdetails).

synapseofthesplanchnicnerve andchromaffincells[1].Direct membranedepolarisationoractionpotentialsfiredbythe inter-actionofacetylcholinewithnicotinicreceptorsonthesurfaceof chromaffincells[117]islikelytheprimarystimulusthatinduces the[Ca2+]

c transient,thustriggeringthedischargeofadrenaline

and noradrenalineintothe circulation[38].Ca2+ entrythrough

thevarioussubtypesofVDCCsistheprimarydeterminantforthe extentandshapeoftheinitial[Ca2+]

ctransient.However,cytosolic

calciumbuffers,Ca2+sequestrationorreleasefromthecytoplasmic

organelles,andplasmalemmalCa2+extrussionhaveaprominent

roleinthefinetuningoftheCa2+signal.Ontheotherhand,correct

Ca2+signallingiscriticaltowarranttheadaptationoftheentire

organismtoastressresponsewhichdeterminesitssurvival.We willemphasisehereourpresentintegrativeviewofthebiophysics ofCa2+redistribution,whichistheultimateregulatorofthe

exo-cytoticresponse(Fig.2).

EssentialtotheunderstandingofCa2+functioninchromaffin

cellsistheconceptthatorganellesandcytosoliccalciumbuffers shape [Ca2+]

c transients atdifferentcelllocations,theso-called

HCMDs,thatdonotnecessarily crosstalk.Severalkindsofthese HCMDshavebeendescribed indifferentcellsystemsand given evocative names, such as sparks, puffs, sparklets and syntillas

[172,173]. Syntillasare brieffocal [Ca2+]

c transients elicited by

localisedERCa2+releaseviaRyRchannels,firstreportedin

neuro-hypophysialterminalsatmagnocellularneurons[174].Thesefocal Ca2+transientswerelateronfoundinmousechromaffincells[175]

and,paradoxically,theyseemtoblockspontaneousexocytosisin thesecells[176].BecauseCICRispresentinbovinechromaffincells, itcouldbeofinteresttoinvestigatewhethertheCa2+wavethat

extendsfromsubplasmalemmalsitestotheinnercytosolfollowing a100msdepolarisingpulseandCICRactivation[22],iscomposed ofelementarysyntillas.Wehaverecentlyfoundthatnanomolar concentrationsofthewinegrapepolyphenolresveratrolcausesER Ca2+releaseinbovinechromaffincellsand,atthesametime,it

blocksthequantalcatecholaminereleaseresponse[177].Itwould beinterestingtoclarifywhethertheseeffectsofresveratrol are

linkedtotheproductionofCa2+syntillas.Itseemshoweverthat

thepresenceand functionalrole forCa2+syntillasare seriously

questionedandcontroversial.Infact,caffeineorryanodinedonot augment[Ca2+]

c and neuropeptide release atneurohypophysial

terminals[178,179].

The rate of Ca2+ fluxes between different chromaffin cell

compartments have been estimated using more or less direct approaches,andundertemperatureconditions(i.e.room tempera-ture)thatmightaffecttheactivityofsomeCa2+transporters.Even

withtheselimitations,puttingtogethertheestimatesofthe dif-ferentfluxesallowsforseveralinterestingpredictions[109].For instance,fora15-␮mdiameterbovinechromaffincell,arateofCa2+

entryof700␮molLcells−1s−1canbecomputedfromthemeasured

Ca2+inwardcurrent[180].Asimilarvalue(400molLcells−1s−1)

wasestimatedby measuring45Ca2+ uptake intoK+ depolarised

bovinechromaffincells[181].Ca2+entrywouldbefocusedatthe

channelslocationandthendiffusethroughthesurroundingcytosol. RegardingprogressionoftheCa2+wavegeneratedbyCa2+entry

throughplasmamembraneCa2+channels,bindingtocytosolic

cal-ciumbuffersisamostimportantdeterminant.Thecytosolofbovine chromaffincellshasaCa2+bindingcapacityof4mmol/L cells.

Thecytosoliccalciumbuffersarescarcelymobileandhavealow Ca2+affinity(K

D∼100␮M)withanactivitycoefficientof∼1/40

[128,180].Thetwo-dimensionaldiffusioncoefficientis∼40␮m2/s

and showsinhomogeneitiesat thenuclearenvelopeand atthe plasmamembrane[53].BriefopeningsofVDCCsgenerateHCMDs nearthechannelmouththatcanbedetectedinCa2+imaging

mea-surements[182].TheseHCMDscanreachconcentrationsashighas 10–100␮M[7,182]BecauseofrapiddiffusionofCa2+towardsthe

surroundingcytosol,theHCMDsarehighlyrestrictedintimeand space[7,183].Thepresenceofmobilecalciumbuffersaccelerates diffusionandopposesthedevelopmentofHCMDs[180,184–186]; for example, at concentrations of 50␮M, fura-2 increases the apparentrateofCa2+diffusionfourtimes[180].

Ca2+enteringthecellredistributesamongthedifferentcell

com-partments.Theincreaseof[Ca2+]

cactivatestheSERCAandtheER

avidlytakesupCa2+fromthecytosol.Forexample,during

stimula-tionofbovinechromaffincells[22,109,128]andratchromaffincells

[187],themaximalCa2+uptakebytheERrangesbetween40and

80␮molLcells−1s−1.Atrest,therateofCa2+exchangebetweenER

andcytosolatsteadystateis2–3␮molLcells−1s−1.ThenetCa2+

influxuponmaximalstimulationwithcaffeineorInsP3-producing

agonistsis10–20timesfaster[22].

Concerningmitochondria,itisnotoriousthattheCa2+activity

coefficient(freeCa2+/bound calcium)in thematrix isvery low,

in the 1/1000 range [109,126]. Mitochondriaare very effective intheclearingof[Ca2+]

c transients,althoughdrasticdifferences

have been reported between bovine and rat chromaffin cells. For instance, in experiments with photorelease of caged Ca2+

in bovinechromaffin cells, ratesof [Ca2+]

M increase as highas

4800␮molLcells−1s−1,atsaturating[Ca2+]

c(200␮M),werefound

[128]. Incontrast, inrat chromaffincells, mitochondrial uptake

rates are150–300 fold slowerbut at [Ca2+]

c of only0.2–2␮M,

werefound[187].Thesedifferences areconsistentwith depen-denceoftherateofuptakethroughtheuniporteronthesecond powerof[Ca2+]

c[123,124,130,188].Usingmitochondria-targeted

aequorin to specifically monitor [Ca2+]

M, we found that

mito-chondria took up about 1100␮molLcells−1s−1 upon maximal

stimulationofCa2+entryintobovinechromaffincellsdepolarised

withK+[109,124,130];thisvalueiscomparablewiththerateof

Ca2+entrythroughVDCCs.ThemaximalrateofCa2+releasefrom

mitochondriatroughthemNCXat37◦Cinbovinechromaffincells isabout800␮molLcells−1s−1.Regardingthekineticsofthis

mito-chondrialCa2+efflux,thedependenceon[Ca2+]

Misexponential

andK50approaches200␮M[109].Transportthroughtheuniporter

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completelydepolarised,theuniportermayallowCa2+exitfromthe

matrixinasortofmitochondrialCICRmechanism[124].

Ca2+extrusionfromthecelltotheextracellularmediumisdue

tojointoperationofbothCa2+-ATPaseandNCX.Thejointactionof

bothtransportsystemshasbeenestimatedtodecrease[Ca2+]

ctoa

maximalrateof20␮molLcells−1s−1,inratchromaffincellsat27C

[127,187].At37◦Ctheratecanbecloseto100␮molLcells−1s−1

[109].

Ateachandeverymomentthe[Ca2+]

c isdefinedbytherate

ofCa2+ redistributionintochromaffin cellcompartmentswhich

inturndependsonfluxesbetweentheextracellularmedium,the cytosol,cytosoliccalciumbuffersandorganelles.Atrest,asteady state withCa2+ exchange rates below 10molLcells−1s−1 and

[Ca2+]

c near0.1␮Mis established;[Ca2+]M issimilar to[Ca2+]c

while [Ca2+]

ER is much higher, reaching 500–1000␮M.

Conse-quently,thereareenormouselectrochemicalgradientsfavouring Ca2+diffusiontothecytosolfromboth,theERandtheextracellular

mediumwheretheCa2+concentrationisabove1mM.

Atlow-frequencystimulationwithactionpotentials,therateof Ca2+diffusionthroughthecytosolandbindingbytheendogenous

Ca2+buffersarethemaindeterminantsofthe[Ca2+]

csignal[7,183].

Under these conditions, global [Ca2+]

c goes up to about 1␮M

andCa2+clearanceisprimarilyachievedthroughthehigh-affinity

Ca2+-ATPaseandSERCA.Uponstrongstimulation(high-frequency

actionpotentialsorprolongeddepolarisation),global[Ca2+]

cmay

approach 10␮M, a concentrationhigh enough toactivate Ca2+

uptakethroughthemitochondrialuniporter.Underthese condi-tions, mostoftheCa2+ thatenterschromaffin cellsis takenup

bymitochondria[109,127,130,187].Forexample, mitochondria-targetedaequorinrevealedthat90%oftheCa2+thatentersabovine

chromaffincell stimulatedwitha 10-sK+ pulseistaken upby

mitochondria.Later,whenthestimulationceases,theCa2+

accu-mulatedinmitochondriaisreleasedbacktothecytosolduringa periodofsecondsorevenminutes[109].TheCa2+accumulatedin

mitochondriastimulatesrespirationuntilCa2+extrusionfromthe

mitochondrialmatrixiscomplete[109].Itcanbespeculatedthat theextraenergyprovidedinthiswaymaybeusedforclearingthe Ca2+loadandrestoringCa2+homeostasisaftertheactivityperiod.

In bovine chromaffin cells, the opening of VDCCs gener-ates HCMDs of about 0.3␮m diameter and 10␮M [Ca2+]

c

[99,182,189–191]. Building of HCMDs may be favoured by

co-localisationofVDCCclustersandchromaffinvesicles[31,192,193]. Evanescentmicroscopyhasshownfast(t1/2∼100ms)andlocalised

(∼350nm)HCMDsbeneaththeplasmamembraneofstimulated chromaffincells[194].TheseHCMDsselectivelytriggertherelease ofvesiclesdockedwithin300nm,indicatingthatsomevesiclesare dockedbutnot primed.Itis interestingthatHCMDsreducethe distancebetweendocked vesiclesandCa2+entrysites,

suggest-ingaroleforstimulation-dependentfacilitationofexocytosisin chromaffincells[193,194].

Mitochondria located nearby VDCCs at subplasmalem-mal sites can sense HCMDs during physiological stimulation

[109,127,130,187,195].Through measurementsof aequorin

con-sumptionuponrepeatedstimulationofbovinechromaffincells, thecumulativehistoryofCa2+uptakemaybetraced.Usingthis

approach, two pools ofmitochondria withdifferentsubcellular distributionwereevidenced.PoolM1,locatednearbyexocytotic sites,accumulates[Ca2+]

catarateof2000␮molLcells−1s−1,while

poolM2locatedatinnercytosolicareastakesupCa2+atamuch

lowerrate,12␮molLcells−1s−1[109,130].Theseratesarereached

atconcentrationsof20and2␮M[Ca2+]

c respectively,whichare

coincidentwiththeconcentrationsreachedatsubplasmalemmal sitesandthecellcoreduringcellstimulation.TheM1poolwould tunethemitochondrialfunctiontomatchthelocalenergyneeds forexocytosisandCa2+redistributionwhereastheM2pool,located

atthebulkcytosol,couldservetoredistributeCa2+andcanalize

ittowardsinnercytosolicregionstoserveothercellfunctions,i.e. transport ofnewsecretory vesiclestoplasmalemmalexocytotic sites.

ERCa2+fluxescouldalsocontributetotheregulationofHCMDs

formedduringcell stimulation.For instance,underK+

depolar-isation of bovinechromaffin cells transfected withER-targeted aequorin,reductionsof60–100␮M[Ca2+]

ERareobserved(about

10–15%ofthetotalERCa2+content)[22],suggestingCa2+-induced

Ca2+release. Althoughthedecreaseof [Ca2+]

ER mayseem quite

small,itcouldcorrespondtolargereleaseatcertainsubcellularER locationscompensatedbystronguptakeinothers.CICRsitesseem toco-localisewithplasmalemmalVDCCsandtheM1mitochondrial pool.Thus,complexfunctionaltetradsincludingVDCCs,cytosolic calciumbuffers,themitochondrialuniporterandtheRyRare essen-tialfortheefficaciousregulationofadequatelocal[Ca2+]

ctransients

tocontroltherateandextentofexocytoticcatecholaminerelease (Fig.2).

6. Relationshipbetweencalciumandtheexo–endocytotic responses

A few studies have addressedthe question of the quantita-tiverelationshipbetweenCa2+andtheexo–endocytoticresponses

triggered bychromaffincell stimulation.Oneapproach consists inthedialysisofbovinechromaffincellswithsolutions contain-ingknown[Ca2+] toelicitsecretion,measuredasanincreaseof

membranecapacitance(Cm)[32].Alsocaffeineisusedto aug-ment[Ca2+]

c andmeasureCm[196].Bothapproacheslead to

a[Ca2+]

c-exocytosisrelationshipthatscaledtoapowerfunction

withanexponentof3.Stillotherstudiesusevoltagesteps(square depolarisingpulses)toboostCa2+influx(Q

Ca)andexocytosis;they

foundaQCa/Cmrelationshipthatfittedapowerfunctionwithan

exponentof1.5[197,198].Thereareadditionalstudiesinbovine chromaffincellsstimulatedwithsingleortrainsofdepolarising pulses[199]oractionpotentialwaveformtrainsaswellas100ms depolarisingpulses[132].Thelongerdepolarisingpulsesproduced

QCa/Cmrelationshipsthatfittedtopowerfunctionsof1.2–2.In

linewiththeseconclusionsistheobservationinratchromaffin cellsstimulatedwithsingledepolarisingpulsesofincreasinglength (10–150ms),showingalinearQCa/Cmrelationship[200].Flash

photolysisofcagedCa2+hasalsobeenusedtostudythekinetic

componentsofafastexocytoticburst[201].Depolarisingpulsesare knowntobemuchlessefficientthanCa2+photoreleaseintriggering

exocytosis[202,203].

Otherstudies have used action potentialwaveforms to cor-relatethestimulationfrequencyinbovinechromaffincellswith amperometricspikesecretioninratchromaffincells[192]orwith capacitance increase in bovine chromaffin cells [204]. In addi-tion, depolarisingpulses have been used in transgenicmice to study the role of exocytotic proteins on the kinetics of Cm

[203].Ontheotherhand,astudycomparingdepolarisingpulses

ofincreasinglengthwithacetylcholine-typeactionpotentialsin voltage-clampedbovinechromaffincells,foundlessCa2+entryand

sloweractivationof[Ca2+]

ctransientswithfasterdelayeddecay.

WithactionpotentialsalinearrelationshipisfoundbetweenQCa

andstimulusduration,capacitanceincreaseandstimulusduration andQCaandcapacitanceincrease.Theserelationshipsare

nonlin-ear withdepolarising pulses.Furthermore,capacitance increase responseselicitedbyactionpotentialtrainsarefollowedbylittle slowendocytosis,whilethoseinducedbydepolarisingpulsesare followedbyapronouncedendocytosis,particularlyatthelonger pulses[205].

Controversy exists over the manner in which membrane retrieval during endocytosis is affected by Ca2+. For instance,

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chromaffincells[204,206].TheexistenceofthesetwoCa2+sensors

isconsistentwiththefactthatCa2+andBa2+supportexcessive

membraneretrievalinbovinechromaffincells[207].Incontrast, apreviousstudyinthesamecells showedthatrapid endocyto-siswassupportedbyCa2+butnotbySr2+orBa2+[208].Arecent

studyshowsalinearcorrelationbetweenendocytosisandQCa in

voltage-clampedbovinechromaffincells[209].

ItisinterestingthatCa2+-dependentendocytosistriggeredby

singlelongdepolarisingpulses involtage-clampedbovine chro-maffincellsseemstobecoupledtoL-typeVDCCs,whereasN-or PQ-type of calciumchannelsseem toplay littlerole [210,211]. Lackof co-localisationbetween VDCC subtypesand clathrinor dynamin suggests a functional, rather than physical coupling betweenL-type calciumchannels and theendocytotic machin-ery.Inbovinechromaffincells,L-typecalciumchannelsundergo aCa2+-dependentinactivationslowerthanN-orPQ-typeof

cal-ciumchannels[136,212].Itisthereforeplausiblethataslowerbut moresustainedCa2+entrythroughslowlyinactivatingL-type

cal-ciumchannels,ratherthanthroughhigherbutfast-inactivating N-andPQ-typeofcalciumchannels,isarequirementtotrigger endo-cytosisefficiently,atleastinbovinechromaffincells[211].This Ca2+-dependentendocytoticresponseisenhancedbysphingosine

dialysis,thatseemstoplayapermissiveroleforendocytosisby act-ingonanendocytoticpathwaydifferenttothoseofdynamin-and calmodulin-signallingpathways[209].

7. Conclusionsandperspectives

Anumberofstudieshaveclarifiedtheroleofseveralfamiliesof ionchannelsandtransportersinshapingthe[Ca2+]

c signalsand

theexo–endocytoticresponsesoccurringduringchromaffincell stimulation.Fromthe1970s onwardsmostofthestudieswere performedinreadyavailablebovinechromaffincells.Duringthe lasttwodecades,however,chromaffincells fromratshavealso been thoroughlyused. It is surprising, however, that only few studies onCa2+ handling in mouse chromaffin cells have been

performed.Transgenic mice lacking or over-expressinga given proteinhaveextensivelybeenusedtoclarifymolecular mecha-nismsof thesecretory machinery. It would bevery interesting tousechromaffincellsasmodelstoidentifyalterations ofCa2+

homeostatic mechanisms and the release of catecholamines in mousemodelsofdisease.Forinstance,intransgenicmousemodels ofAlzheimer’sdisease,amyotrophiclateralsclerosis,Parkinson’s disease and other neurodegenerative diseases, the expectation is that highand low Ca2+ microdomains maydifferently affect

pre- and exocytotic steps,which could bea peripheralmarker of a brain synaptic dysfunction. There is increasing concern on the involvement of Ca2+ dyshomeostasis in these diseases

[213–216].

WhetherthelargeCa2+concentrationsinchromaffinvesicles

playafunctionotherthanthemerepackingofcatecholaminesuch asforinstance,contributingtoregulationofthelaststepsof exo-cytosis,requiresfurtherclarification.Wealsoknowlittleonthe roleofCa2+fluxesinthechromaffincellnucleus,althoughtheyare

likelyinvolvedinthecontrolofgeneexpression.Itwouldbeniceto know,forexample,whethersuchnuclearCa2+signallingisinvolved

intheexpressionoftheenzymesofcatecholaminesynthesisand degradation.

Effortsshouldalsobedonetoextrapolatethenumerousdata obtained in cultures of chromaffin cells to more physiological preparationssuchasadrenalslicesoreventheintactadrenal,using electricalstimulationofthesympatheticcholinergicnerve termi-nalsthatinnervatechromaffincellstoregulatesecretion.Attempts toestablishorganotypicculturesofadrenalslicesshouldalsobe pursued,asthiscouldfacilitatechronictreatmentstostudynovel

aspectsofcatecholaminesynthesis,storageandreleaseandonthe roleofCa2+signallingunderthesemorephysiologicalconditions

ofpreservationoftissuestructure.

Acknowledgements

Theworkoftheauthor’slaboratorieshasbeensupportedby the following institutions.To AGG: (1) SAF2010-21795, Minis-terio de Ciencia eInnovación (MCINN);(2) RETICS RD06/0009, InstitutodeSaludCarlosIII(MICINN);(3)S-SAL-0275-2006, Comu-nidadAutónomadeMadrid;(4)NDG07/9yNDG09/8,AgenciaLaín Entralgo,ComunidadAutónomadeMadrid,Spain.(5)Fundación TeófiloHernando.ToJGS:grantsfromtheEU-ERA-Netprogramme, theSpanishMinisteriodeCienciaeInnovación(MICINN; SAF2008-03175-E and BFU2010-17379), the Instituto de SaludCarlos III (RD06/0010/0000)andtheJuntadeCastillayLeón(gr175).

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Figure

Fig. 1. Calcium (Ca 2+ ) cycling in the chromaffin cell. Upon cell depolarisation, extra- extra-cellular Ca 2+ enters the cell through voltage-dependent Ca 2+ channels (1, VDCCs)
Fig. 1. Calcium (Ca 2+ ) cycling in the chromaffin cell. Upon cell depolarisation, extra- extra-cellular Ca 2+ enters the cell through voltage-dependent Ca 2+ channels (1, VDCCs) p.3
Fig. 2. Functional tetrads to shape the high-Ca 2+ microdomains (HCMDs) an low- low-Ca 2+ microdomains (LCMDs) that determine, respectively, the fast exocytosis (FE) release of adrenaline (AD) and noradrenaline (NA), from an immediately releasable vesicle
Fig. 2. Functional tetrads to shape the high-Ca 2+ microdomains (HCMDs) an low- low-Ca 2+ microdomains (LCMDs) that determine, respectively, the fast exocytosis (FE) release of adrenaline (AD) and noradrenaline (NA), from an immediately releasable vesicle p.6

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Related subjects : Calcium channels