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

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

a_{InstitutoTeófiloHernando,InstitutodeInvestigacionesSanitariasdelHospitaldelaPrincesa,FacultaddeMedicina,UniversidadAutónomadeMadrid,Madrid,Spain} b_{DepartamentodeFarmacologíayTerapéutica,InstitutodeInvestigacionesSanitariasdelHospitaldelaPrincesa,FacultaddeMedicina,UniversidadAutónomadeMadrid,}

Madrid,Spain

c_{ServiciodeFarmacologíaClínica,InstitutodeInvestigacionesSanitariasdelHospitaldelaPrincesa,FacultaddeMedicina,UniversidadAutónomadeMadrid,Madrid,Spain} d_{InstitutodeBiologíayGenéticaMolecular(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([Ca}2+_]

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).

1_{Equalcontributors.}

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([Ca}2+_]

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.

Chromaffincells areexcitablecellsand fireaction potentials thatopenvariousoftheneuronal-typevoltage-dependentcalcium channels(VDCCs)andproduceCa2+_{entry;theresulting[Ca}2+_]

c

sig-naltriggers exocytosis.Becausecytoplasmicorganellescantake upandreleaseCa2+_{tothecytosol,understandingthe[Ca}2+_]

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+_{releaseactivatedCa}2+_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+_{acceleratestheERCa}2+_{storerefillinguponCa}2+_{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].ACa}2+_{entrypathwaytriggeredby}

his-tamineandindependentoftheERCa2+_{storeisalsopresentinthese}

cells[25,30].

A few studieshave explored therole of Ca2+ _entrythrough

SOCCsin triggeringexocytosisin bovinechromaffincells. Thus, histamineandangiotensinIIstimulateexocytosisbya combina-tionofERCa2+_{releaseandadditionalCa}2+_{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 Ca}2+ _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-conductanceCa}2+_-activated

K+ _{channels[33].ThismechanismcouldamplifyCa}2+ _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]. Ca}2+_{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}

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-dependentCa}2+_{channels(1,VDCCs).}

ThisgeneratesalocalcytosolicCa2+_{transient([Ca}2+_]

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)andNa}2+_/Ca2+_{exchangerNCX(7)driveCa}2+_efflux

backtotheextracellularspace.Thenucleus(8)andchromaffinvesicles(9,CV)may alsocontributetoCa2+_{redistribution.PathwaysforCa}2+_{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[Ca}2+_] 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+_{concentrationisabout40␮M}

[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}

tosuchHCMDsmayenlightenthecontributionofvesicularCa2+

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

3.4. Endoplasmicreticulum

EarlierobservationsestablishedthatCa2+_{uptakeoccurredin}

thesarcoplasmic reticulumofskeletalmuscle[76,77]througha Mg2+_{-andATP-dependentP-typetransportCa}2+_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+_{fluxesoftheERCa}2+_{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-sensitiveCa}2+_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 toCa}2+_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[Ca}2+_] 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[Ca}2+_]

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+_{intheirmatrixthroughtheirCa}2+_{uniporter,thatusesthe}

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 [Ca}2+_]

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 wellbalancedCa}2+ _{influxand Ca}2+

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 Ca}2+ _{ion. Under}

physiologicalconditionsNa+_{istransportedintothecellandCa}2+

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

depolarisationortheopeningofgatedNa+_{channels,theexchanger}

movesNa+_{outofthecellandCa}2+_{intothecell[140].TheCa}2+

exitmodeisreferredtoastheforwardmode,andtheCa2+_entry

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

canmediateNa+_-dependentCa2+_{influx[143]orCa}2+_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 Ca}2+ _{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

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−1_s−1_{canbecomputedfromthemeasured}

Ca2+_{inwardcurrent[180].Asimilarvalue(400␮molLcells}−1_s−1₎

wasestimatedby measuring45_Ca2+ _{uptake intoK}+ _depolarised

bovinechromaffincells[181].Ca2+_{entrywouldbefocusedatthe}

channelslocationandthendiffusethroughthesurroundingcytosol. RegardingprogressionoftheCa2+_{wavegeneratedbyCa}2+_entry

throughplasmamembraneCa2+_{channels,bindingtocytosolic}

cal-ciumbuffersisamostimportantdeterminant.Thecytosolofbovine chromaffincellshasaCa2+_{bindingcapacityof∼4mmol/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−1_s−1_{.Atrest,therateofCa}2+_{exchangebetweenER}

andcytosolatsteadystateis2–3␮molLcells−1_s−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−1_s−1_{,atsaturating[Ca}2+_]

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−1_s−1 _{upon maximal}

stimulationofCa2+_{entryintobovinechromaffincellsdepolarised}

withK+_{[109,124,130];thisvalueiscomparablewiththerateof}

Ca2+_{entrythroughVDCCs.ThemaximalrateofCa}2+_releasefrom

mitochondriatroughthemNCXat37◦Cinbovinechromaffincells isabout800␮molLcells−1_s−1_{.Regardingthekineticsofthis}

mito-chondrialCa2+_{efflux,thedependenceon[Ca}2+_]

Misexponential

andK50approaches200␮M[109].Transportthroughtheuniporter

completelydepolarised,theuniportermayallowCa2+_exitfromthe

matrixinasortofmitochondrialCICRmechanism[124].

Ca2+_{extrusionfromthecelltotheextracellularmediumisdue}

tojointoperationofbothCa2+_{-ATPaseandNCX.Thejointactionof}

bothtransportsystemshasbeenestimatedtodecrease[Ca2+_]

ctoa

maximalrateof20␮molLcells−1_s−1_{,inratchromaffincellsat27}◦_C

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

[109].

Ateachandeverymomentthe[Ca2+_]

c isdefinedbytherate

ofCa2+ _{redistributionintochromaffin cellcompartmentswhich}

inturndependsonfluxesbetweentheextracellularmedium,the cytosol,cytosoliccalciumbuffersandorganelles.Atrest,asteady state withCa2+ _{exchange rates below 10␮molLcells}−1_s−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[Ca}2+_]

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+_{loadandrestoringCa}2+_{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−1_s−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],suggestingCa}2+_-induced

Ca2+_{release. Althoughthedecreaseof [Ca}2+_]

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,}

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).

References

[1]W.Feldberg,B.Mintz,Diewirkungvonazetylcholinaufdienebennieren, Arch.Exp.Pathol.Pharmakol.168(1932)287–291.

[2]W.W.Douglas,R.P.Rubin,Theroleofcalciuminthesecretoryresponseofthe adrenalmedullatoacetylcholine,J.Physiol.159(1961)40–57.

[3]W.W.Douglas,A.M.Poisner,Onthemodeofactionofacetylcholineinevoking adrenalmedullarysecretion:increaseduptakeofcalciumduringthe secre-toryresponse,J.Physiol.162(1962)385–392.

[4]W.W.Douglas,Stimulus–secretioncoupling:theconceptandcluesfrom chro-maffinandothercells,Br.J.Pharmacol.34(1968)451–474.

[5] O.Shimomura,Thediscoveryofaequorinandgreenfluorescentprotein,J. Microsc.217(2005)1–15.

[6]R.Rizzuto,M.Brini,T.Pozzan,Intracellulartargetingofthephotoprotein aequorin:anewapproachformeasuring,inlivingcells,Ca2+_{concentrationsin}

definedcellularcompartments,Cytotechnology11(Suppl.1)(1993)S44–S46. [7] E.Neher,VesiclepoolsandCa2+_{microdomains:newtoolsforunderstanding}

theirrolesinneurotransmitterrelease,Neuron20(1998)389–399. [8]R.D.Burgoyne,A.Morgan,Secretorygranuleexocytosis,Physiol.Rev.83

(2003)581–632.

[9] A.G.Garcia,A.M.Garcia-De-Diego,L.Gandia,R.Borges,J.Garcia-Sancho, Cal-ciumsignalingandexocytosisinadrenalchromaffincells,Physiol.Rev.86 (2006)1093–1131.

[10] J.Garcia-Sancho,A.Verkhratsky,Cytoplasmicorganellesdetermine com-plexityandspecificityofcalciumsignallinginadrenalchromaffincells,Acta Physiol.(Oxf.)192(2008)263–271.

[11]A.M.deDiego,L.Gandia,A.G.Garcia,Aphysiologicalviewofthecentraland peripheralmechanismsthatregulatethereleaseofcatecholaminesatthe adrenalmedulla,ActaPhysiol.(Oxf.)192(2008)287–301.

[12]B.M.Olivera,G.P.Miljanich,J.Ramachandran,M.E.Adams,Calciumchannel diversityandneurotransmitterrelease:theomega-conotoxinsand omega-agatoxins,Annu.Rev.Biochem.63(1994)823–867.

[13]E.Garcia-Palomero,I.Cuchillo-Ibanez,A.G.Garcia,J.Renart,A.Albillos,C. Montiel,GreaterdiversitythanpreviouslythoughtofchromaffincellCa2+

channels,derivedfrommRNAidentificationstudies,FEBSLett.481(2000) 235–239.

[14]M.J.Berridge,Inositoltrisphosphateandcalciumsignaling,Ann.N.Y.Acad. Sci.766(1995)31–43.

[15]J.W.Putney,Thephysiologicalfunctionofstore-operatedcalciumentry, Neu-rochem.Res.36(2011)1157–1165.

[16]J.W.PutneyJr.,Capacitativecalciumentryrevisited,CellCalcium11(1990) 611–624.

[17]I.M.Manjarres,M.T.Alonso,J.Garcia-Sancho,Calciumentry-calciumrefilling (CECR)couplingbetweenstore-operatedCa(2+)entryandsarco/endoplasmic reticulumCa(2+)-ATPase,CellCalcium49(2011)153–161.

[18]I.M. Manjarres,A. Rodriguez-Garcia, M.T. Alonso, J. Garcia-Sancho, The sarco/endoplasmicreticulumCa(2+)ATPase(SERCA)isthethirdelementin capacitativecalciumentry,CellCalcium47(2010)412–418.

[19]R.Penner,G.Matthews,E.Neher,Regulationofcalciuminfluxbysecond messengersinratmastcells,Nature334(1988)499–504.

[20]A.F.Fomina,E.S.Levitan,ThreephasesofTRH-inducedfacilitationof exocy-tosisbysinglelactotrophs,J.Neurosci.15(1995)4982–4991.

[21] Y.X.Li,S.S.Stojilkovic,J.Keizer,J.Rinzel,Sensingandrefillingcalciumstores inanexcitablecell,Biophys.J.72(1997)1080–1091.

[22]M.T.Alonso,M.J.Barrero,P.Michelena,E.Carnicero,I.Cuchillo,A.G.Garcia, J.Garcia-Sancho,M.Montero,J.Alvarez,Ca2+_-inducedCa2+_releasein

chro-maffincellsseenfrominsidetheERwithtargetedaequorin,J.CellBiol.144 (1999)241–254.

[23]I.M.Robinson,T.R.Cheek,R.D.Burgoyne,Ca2+_{influxinducedbythe}

Ca(2+)-ATPaseinhibitors2,5-di-(t-butyl)-1,4-benzohydroquinoneandthapsigargin inbovineadrenalchromaffincells,Biochem.J.288(Pt2)(1992)457–463. [24]T.R.Cheek,A.Morgan,A.J.O’Sullivan,R.B.Moreton,M.J.Berridge,R.D.