Calcium dynamics in the secretory granules of neuroendocrine cells

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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

Calcium

dynamics

in

the

secretory

granules

of

neuroendocrine

cells

Javier

Álvarez

InstitutodeBiologíayGenéticaMolecular(IBGM),DepartamentodeBioquímicayBiologíaMolecularyFisiología,FacultaddeMedicina,UniversidaddeValladolidandConsejo SuperiordeInvestigacionesCientíficas(CSIC),RamónyCajal,7,E-47005Valladolid,Spain

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received28September2011 Receivedinrevisedform 22November2011 Accepted4December2011

Available online 30 December 2011

Keywords:

Ca2+

Secretorygranules Neuroendocrine Chromaffin

Inositoltrisphosphatereceptor Ryanodinereceptor

Ca2+_-ATPase

a

b

s

t

r

a

c

t

CellularCa2+_{signalingresultsfromacomplexinterplayamongavarietyofCa}2+_{fluxesgoingacrossthe} plasmamembraneandacrossthemembranesofseveralorganelles,togetherwiththebufferingeffectof largenumbersofCa2+_{-bindingsitesdistributedalongthecellarchitecture.Endoplasmicandsarcoplasmic} reticulum,mitochondriaandevennucleushaveallbeeninvolvedincellularCa2+_{signaling,andthe} mechanismsforCa2+_{uptakeandreleasefromtheseorganellesarewellknown.Inneuroendocrinecells,} thesecretorygranulesalsoconstituteaveryimportantCa2+_{-storingorganelle,andthepossibleroleof} thestoredCa2+_{asatriggerforsecretionhasattractedconsiderableattention.However,thispossibilityis} frequentlyoverlooked,andthemainreasonforthatisthatthereisstillconsiderableuncertaintyonthe mainquestionsrelatedwithgranularCa2+_{dynamics,e.g.,thefreegranular[Ca}2+_{],thephysicalstateof} thestoredCa2+_{orthemechanismsforCa}2+_{accumulationandreleasefromthegranules.Thisreviewwill} giveacriticaloverviewofthepresentstateofknowledgeandthemainconflictingpointsonsecretory granuleCa2+_{homeostasisinneuroendocrinecells.}

© 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Ca2+_{isanintracellularsecondmessengerabletofulfilla}

vari-ety of different functions in all cell types with no exception, bothexcitableandnon-excitablecells.Letusjustmentionmuscle contraction,neurotransmittersecretion,fertilization,proliferation, development,learning,memoryorcelldeath[1].Thisversatility canonlybeachievedthankstoaveryprecisecontrolofits concen-trationatthesubcellularlevel.Infact,activationoftheseprocesses usuallytakesplaceasaconsequenceoftransientincreasesinthe cytosolic[Ca2+_{] inspecificsubcellularlocations,whichare}

vari-abledepending onthe process. These hot spots or local[Ca2+_]

microdomainshaveacriticalimportanceinCa2+_{signalingandtheir}

sizeanddynamicsdependsonthespatialdispositionofthe differ-entCa2+_{transportsystems(channels,exchangers,pumps,etc.)both}

intheplasmamembraneandinintracellularorganelles.

BecauseofthestrongCa2+_{bufferingofthecytosol(freeCa}2+

con-stitutesonly1%ofthetotalCa2+_{inthecytosol),diffusionofCa}2+

throughthecytosolisseverelylimited,particularlyduringsmall, physiological,cellstimulations.Intracellularorganellesare there-foreessentialtogenerateorcontrollocal[Ca2+_{]microdomainsat}

differentintracellularlocations.Thefirstorganellethatwasknown tohaveanimportantroleinCa2+_{homeostasiswasthe}

endoplas-micreticulum(ER)[2].ThisorganellebehavesasadynamicCa2+

∗Tel.:+34983423085;fax:+34983423588.

E-mailaddress:jalvarez@ibgm.uva.es

store.ItaccumulatesCa2+_{initslumenthroughtheSERCApumpsup}

tonearmillimolarlevels[3],andisabletoinducearapidrelease of Ca2+ _{tothecytosolviaseveralspecificCa}2+ _{channelspresent}

initsmembrane(InsP3andryanodinereceptors,mainly).In

addi-tion,boththeERandthesarcoplasmicreticulumofmusclecells containCa2+_{-bindingproteinswithhigh-capacityandlow-affinity}

thatmakethetotalcalciumcontentoftheERtobeabout10-fold largerthanthefree[Ca2+_]

ER.Themoreabundant,calreticulininthe

ERandcalsequestrinintheSR,haveCa2+_{-affinitiesof1–4mM[2],}

closetothefree[Ca2+_]

ER,thusfacilitatingfastbindingandrelease

ofCa2+_{followingthechangesinfree[Ca}2+_] ER.

AnotherorganellewithanimportantroleincellCa2+

homeo-stasisismitochondria,theorganelleresponsibleofaerobicenergy production in the cell. The ability of mitochondria to take up largeamountsofCa2+_{wasknownsincethesixties.However,the}

low Ca2+_{-affinity of themitochondrial Ca}2+_uniporter(K M above

10␮M,100-foldhigherthantherestingcytosolic[Ca2+_],100nM,

and10-foldhigherthantheusualpeakcytosolic[Ca2+_]found

dur-ingcellstimulation,about1␮M)ledtomostresearchersinthe 1970sand1980stoexcludeasignificantroleinCa2+

homeosta-sisforthisorganelle,atleastunderphysiologicalconditions.Then, inthebeginningofthe1990s,recombinantaequorintargetedto themitochondriashowedthatmitochondrial [Ca2+_{] wasableto}

undergorapidchangesduringcellactivation[4].Thatwasthe ori-ginofaseriesofworksbyseveralresearchgroupsdemonstrating thatmitochondriawereveryactiveplayersinthecontrolofthe globalcellCa2+_{homeostasis.Inparticular,becauseofthelow-Ca}2+

-affinityoftheirCa2+_{uptakemechanism,mitochondriaareverywell}

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

332 J.Álvarez/CellCalcium51 (2012) 331–337

adaptedtotakeupCa2+_{fromlocalhigh-Ca}2+_{microdomains,suchas}

thoseformedaftertheactivationofplasmamembraneor endoplas-micreticulumCa2+_{channels.Mitochondriacanthereforemodulate}

importantcellularfunctionssuchasneurotransmittersecretionby actingaslocalCa2+_sinks[5].

Thesubjectofthisreview,thesecretorygranules,constitutes oneofthelessknowncompartmentsintermsofCa2+_dynamics.

Itiswidelyknownthattheincreaseinintracellular[Ca2+_]levels

triggersexocytosisofapoolofdockedsecretoryvesiclesin neu-ronalandneuroendocrinecells.Thesevesiclesareattachedtothe plasmamembraneandarealsoveryclosetoportionsofERand mitochondria.Thus,itisgenerallyassumedthattheCa2+_required

forexocytosismayenterfromtheextracellularmediumthrough severalkindsofplasmamembraneCa2+_{channels[6]orbereleased}

fromtheendoplasmicreticulumthrougheitherinositol trisphos-phate(InsP3)orryanodinereceptors[7].Nearbymitochondriamay

alsocontributetomodulatethesizeofthelocal[Ca2+_]microdomain

responsibleforvesiclefusionandsecretion.However,itishardly mentionedthatthemainCa2+_{-storeinsomeexcitablecellsresides}

inthesecretoryvesicles. In chromaffincells, secretorygranules containabout60%ofthetotalcellularcalcium[8],andthetotal calciumconcentrationinsidethegranuleshasbeenestimatedtobe around40mM[9],muchhigherthanthatintheER(about5–10mM, see[2]).Ofcourse,anattractivepossibilitywouldbethat intra-granularcalciumcouldcontributetoitsownsecretionbybeing releasedthroughCa2+_{channelsplacedinthegranulemembrane.}

Inthatway,Ca2+_{wouldbereleasedpreciselyintherightplaceto}

triggersecretion.Whatisthenthereasonforthisapparent over-sight?Aswewilldiscussinthisreview,themainreasonforthat isthatthereisstilllittleandsometimescontradictoryinformation onthedynamicsofCa2+_{inthegranules.Thus,themainaspectsof}

Ca2+_{homeostasisinthegranules,thatis,thefree[Ca}2+_]

SGandthe

mechanismsforCa2+_{accumulationandrelease,arestill}

controver-sial.

2. Theproblemofthefreeandtotalcalciumconcentration inthesecretorygranules

Studiesinthe1980sshowedthatthetotalcalcium concentra-tioninthesecretorygranulesis40–80nmol/mgofprotein[10,11], whichconsideringaprotein/waterrelationshipinthegranulesof about0.5mg/␮l[12],impliesthatthetotalcalciumconcentration inthegranulesis20–40mM.WinklerandWesthead[9]calculated atotalof90,000Ca2+_{ionsforavesiclewith270nminternal}

diame-ter,whichmeansaconcentrationof15mM.Studiesoftotalcalcium madebyelectronenergylossimagingspectroscopyinPC12cells showalsoclearlythatdensegranuleshaveahighcalciumcontent (>10mM),withasignalcomparabletothatobtainedintheGolgi complexandsomeregionsoftheER[13].Accordingly,inPC12cells, theacidiccalciumpoolaccountedfor170␮mol/lcellwater,which correspondstoaninternaltotalconcentrationofabout30mM[14]. Thishighcalciumcontentisnotexclusiveofchromaffingranules butinsteaditisalsofoundatsimilarorevenhighervaluesinmost typesofsecretorygranules,e.g.zymogengranulesofpancreatic acinarcells[15],smallsynapticvesiclesoftheneuromuscular junc-tion[13],insulin granules[16] andmany others(see [17]for a review).

Therefore,secretorygranulesappeartobetheorganelleswith thehighercalciumcontentofthecell.Otherorganelleswithhigh calciumcontentaretheendoplasmicreticulumandGolgi appa-ratus.Electronmicroscopytechniqueshaveshownthatthetotal calciumcontentoftheERfromdifferentcelltypesisintherangeof 5–50mM,withthehighestvaluesintheterminalcisternaeofthe sarcoplasmicreticulumandmeanvaluesof5–10mM[2,18],that is,5–10-foldlowerthaninthesecretorygranules.Thefree[Ca2+_]

intheERhasbeenalsomeasuredwithlow-Ca2+_{-affinityaequorin}

andisinthe500–800␮Mrange[7],sothattheCa2+_-bound/Ca2+

-freerelationshipintheERisabout10.Inaddition,thetotalcalcium distributionintheERisheterogeneous,withstronglypositive cis-ternae(includingthenuclearenvelope)lyingintheproximityof orevenindirectcontinuitywithother,apparentlynegative cister-nae[13].Thisheterogeneitymustsurelyreflectthedistributionof theCa2+_{-bindingproteinsinthelumenoftheER,asmostofthe}

calciumcontentoftheERisboundwithlowaffinity(Kd=1–4mM)

toseveralCa2+_{-bindingproteins,mainlycalreticulinintheERand}

calsequestrinintheSR[2,18].TheKdforCa2+bindingofthese

pro-teinsis thereforeclosetothefree[Ca2+_{]in theER,assuring an}

efficientbinding.

Manystudieshavetriedtoestimatethefree[Ca2+_]inthe

secre-torygranules.BulendaandGratzl[10]andHaighetal.[8],using null-pointtitrationtechniques,foundvaluesof24␮Mand5.6␮M, respectively, in isolated acidic chromaffin vesicles. In mast cell granules, studieswithfluorescent dyes showeda resting intra-luminalfree [Ca2+_{]of 25␮M[19].Inthe␤-celllineMIN6,data}

obtainedwithtargetedaequorinindicatedafree[Ca2+_]inthe

vesi-clesaround 50_␮M[20].These values contrastwiththe1.4_␮M [Ca2+_{]valueobtainedbyMahapatraetal.[21]inPC12chromaffin}

granules,usingalsotargetedaequorin.Thediscrepancymay prob-ablybeduetothehigherCa2+_{-affinityoftheaequorintypeused}

inthelaststudy.Becauseaequorinisconsumedfasterinhigh-Ca2+

environments,high-Ca2+_{-affinityaequorinisrapidlyconsumedin}

thoseregionsandfinallytendstoreflectonlythebehaviorofminor compartmentswithlow[Ca2+_{][22].Usinganaequorinwithlower}

Ca2+_{-affinityandcorrectingfortheeffectofacidicpHonaequorin}

luminescence,wehavereportedfree[Ca2+_{]valuesof50–100␮M}

inchromaffingranules[23]and20–40␮MinPC12andINS1cells

[24].Weshouldnotealsothatthesmallsizeofsomeofthe vesi-clesposessomerestrictionstothepossiblevaluesoffreegranular [Ca2+_{].Letusjustmentionthatforasmallsynapticvesicle,with}

adiameterof50nm,25␮M[Ca2+_{]wouldbeobtainedwithonly1}

Ca2+_{ion,andevenforalargedensecorevesicleof200nm}

diame-ter,just2–3Ca2+_{ionspervesiclewouldrepresent1␮M[Ca}2+_](that

wouldgivefiguresof100–300freeCa2+_{ionspervesicle).}

Inconclusion,thefree[Ca2+_{]inseveraltypesofsecretory}

gran-ulesappearstorangebetween20and100␮M.Thesevaluesare significantlylower,nearlybyoneorderofmagnitude,thanthefree [Ca2+_{]intheendoplasmicreticulum,whichisabout500␮M[3,25].}

Giventhatthetotalcalciumcontentislargerinthevesiclesthan intheERalsobynearlyoneorderofmagnitudeintermsof con-centration,thismeansthattheCa2+_bound/Ca2+_{freerelationship}

ismuchhigher(almosttwoordersofmagnitude)inthevesicles thanintheER,fromvaluesof10–20intheERtonearly1000inthe vesicles.ThequestiontheniswhereallthoseCa2+_{-bindingsites}

are.Itisgenerallyassumedthatthelargecalciumstoring capac-ityofthesecretorygranulesismainlyduetothepresenceofhigh concentrations(about2mM)oftheproteinschromograninAand Bandsecretogranin[26].Inbovinechromaffincells,chromogranin Aconstitutesabout90%oftheseCa2+_{-bindingproteins(1.8mM),}

anditisabletobind55molofCa2+_{permolofprotein,withaK} dof

4mM[26].TheCa2+_{-bindingcapacityoftheseproteinsistherefore}

verylargeandcouldstoreupto100mMtotalcalcium.However, theaffinityistoolowifweconsiderthatthefree[Ca2+_]

SGremains

below100␮M.Infact,evenconsideringafreegranular[Ca2+_]of

100␮M,bindingtoaproteinwithaKdof4mMwouldhardlyfill

2.5%oftheavailablebindingsites.Inourcase,thisbindingwould explain2.5mMofthestoredcalcium,thatis,lessthan10%ofthe calciumpresentinthegranules.

HowcanthenweexplainthelargeCa2+_{-storingcapacityofthe}

granules?There areseveralalternatives: (i)theCa2+ _{affinity of}

theseCa2+_{-bindingproteinscouldbedifferentintheconditionsof}

and 125–300mM ATP; (ii)early studiesdescribed a significant numberofhigheraffinityCa2+_{-bindingsitesinvesiclematrix}

pro-teins,thatwouldbeabletobindupto180nmolCa2+_/mgprotein

withaffinitiesinthe100–300␮MrangedependingonthepHand ionicconditions[27,28];and(iii)partofthecalciumcouldalsobe boundtoothervesiclecomponents,suchasnucleotidesorlipids. Regardingthenucleotides,theyarepresentathighconcentrations andcanbindCa2+_withaK

dinthe100–300␮Mrangedepending

onthepHandionicconditions[27].However,catecholaminesand ATPhavebeenshowntointeractattheconcentrationsfoundin thegranulesforminghighlynon-idealsolutionsthatconsiderably reduceosmoticpressure[29].Giventhatthisinteractiondoesnot requirecalcium,andthatmostofvesicleATPandcatecholamines appeartobeinvolvedintheseinteractions,probablyonlyasmall partofthetotalATPisavailabletobindCa2+_{inthevesicles.}

Afinalpointregardstheavailabilityofthestoredcalciumfor exchangefromthebindingsites.IntheERandSR,theCa2+_bound

tocalreticulinorcalsequestrinisinfastequilibriumwiththefree Ca2+_{,anditisthereforereadilyavailableforreleasefromthe}

bind-ingsiteswhentheluminalfree[Ca2+_{]isreduced.Inthatway,when}

theCa2+_{-channelsintheERorSR(e.g.,InsP}

3orryanodine

recep-tors)areactivated,allthestoredCa2+_{cancontributetotherelease}

intothecytosol.Inthecaseofthesecretorygranules,somereports suggestthatatleastpartofthestoredcalciumisrelatively immo-bileorexchangesveryslowlywiththefreeCa2+_{pool.Clementeand}

Meldolesi[15]foundthatzymogengranuleshaveaveryslowrate of45_{Caaccumulationandreleasecomparedwithotherorganelles,}

suggestingthatthestoredCa2+_{couldplaymainlyastructuralrole}

inthe architectureoftheorganelle. Similarly, 45_{Ca flux}

experi-mentsperformedinPC12cellsshowedthatthegranuleCa2+_pool

wasextraordinarilystableinperiodsofhoursinEGTA-containing medium,andveryslowtolabel[14],concludingthatthestored Ca2+_{wouldonlyservetokeepgranulecomponentstogetherand}

tobedischargedtotheextracellularspacebyexocytosis.The ques-tionthenreliesinthenatureandthephysicalstateofthecontents ofthegranule.Evidencefromseveraltechniquesindicatesthatno importantfractionofthesmallmoleculesofthegranuleisinan immobilizedorprecipitatedform(see[9]forareview).However, complexesbetweenATPand catecholaminesandperhaps other componentsmustnecessarilyexisttoexplaintheosmotic prop-ertiesofthegranule[29].Inaddition,chromograninshavebeen showntoaggregateorformtetramersinaCa2+_{-dependentway}

[28,30,31].Thesecomplexescouldperhapstrapinastableform

someofthestoredcalcium.

Asaconclusion,Ca2+_{ionsinthesecretorygranulescanbein}

threedifferentstates:(i)astable,slowlyexchangingpoolwhich maybeimportantforthestructureofthegranularcomponents;(ii) apoolboundtochromograninsorothergranularcomponentswith anaffinityclosetothefree[Ca2+_]

SG,readilyavailableforexchange

and whosesize maydependon[Ca2+_]

c variationsor other

cel-lularconditions;and (iii)thefreeCa2+_{pool.Therelativesizeof}

thesepoolsmaydependonthetypeofgranuleandthecellular conditions.

3. MechanismsofCa2+_{accumulationinthesecretory} granules

Ifthefree[Ca2+_]

SGisinthe20–100␮Mrange,thereshouldbe

inthemembraneofthesecretorygranulesactivemechanismsto accumulatethisioninthematrix,giventhatitsconcentrationin thecytosolisnearly1000-foldlower.Severalcandidateshavebeen proposedforthisrole(seethecartooninFig.1).

Krieger-BrauerandGratzl[32]foundevidenceforthepresence ofa2Na+_/1Ca2+_{exchangeinthemembraneofthesecretory}

gran-ulesofbovinechromaffincells.ThesystemwaspH-independent

andhadhighaffinityforCa2+_{(0.28␮M[33]),sothatitwasstill}

partiallyactiveat100nMextragranular[Ca2+_{],althoughtherate}

increasedathigher concentrations.HaighandPhillips [34] pro-posedthatNa+_/Ca2+_{exchangecouldbecoupledtotheH}+_pumpvia

aNa+_/H+_{exchange,buttheratesofuptaketheyfoundinchromaffin}

granuleghostswereveryslow.Thecapacityofthissystemto accu-mulateCa2+_{inthevesiclesdependsonthemagnitudeoftheNa}+

gradient.Theintragranular[Na+_{]wasestimatedbyKriegler-Brauer}

andGratzl[32,33]about50mM.Haighetal.[8]foundalsothatthe sumoftheintragranular[Na+_]and[K+_{]was50–60mM,theactual}

amountofeachonedependingoftheisolationprocedure.Inany case,thismeansthatthe[Na+_{]gradientbetweenthegranulematrix}

andthecytosolisnolargerthan3-fold.Thus,withastoichiometry of2Na+_/1Ca2+_{,thissystemwouldnotbeabletoaccumulateCa}2+

morethan10-foldinsidethegranules,muchlessthantheexisting gradient.Infact,witha[Ca2+_{]gradientof1000-fold,thissystem}

wouldmainlyworkreleasingCa2+_{fromthevesiclesinexchange}

for Na+_{. It has been actually proposedthat Na}+_/Ca2+ _exchange

maymediate theincreasein intravesicular[Na+_{] thatcoincides}

withthe onset ofsecretion inneurohypophysial nerve endings

[35].

ThepresenceofaCa2+_/H+_{antiporthasbeenreportedin}

synap-ticvesiclesofbraincortex[36].CombinedwiththeH+_-ATPaseand

assumingastoichiometryof1Ca2+_/2H+_{,thissystemwouldbeable}

toaccumulateCa2+_{nearly1000-foldintothevesicles.However,that}

transportsystemshowedverylowCa2+_{affinityforCa}2+_entry,

start-ingtobeactiveatcytosolic[Ca2+_{]around100␮M.Thus,underthese}

conditionsitcouldnotbeinvolvedinphysiologicalCa2+_uptakeinto

thevesicles,althoughitmayperhapsmediateCa2+_{-releasefromthe}

vesiclesfollowingchangesinthe[H+_{]gradient.Infact,the}

disrup-tionofthevesicularpHgradientinducesasignificantleakofCa2+

fromthevesicles[37,38].

Thelastpossibilityisa directATP-dependentmechanism for Ca2+_{accumulation,thatis,aCa}2+_{-ATPase.Thesarco-endoplasmic}

reticulumCa2+_{-ATPase(SERCA)inhibitor2,5-diterbutyl}

benzohy-droquinone (BHQ) hasbeen shown to inhibit Ca2+ _{uptake into}

isolated synapticvesicles [39].In platelets, theacidicCa2+ _pool

wascontrolledbothbyaCa2+_/H+_{exchangeandbyaBHQ-sensitive}

SERCA3[40,41].Similarly,loadingwithCa2+_{ofdense-coreinsulin}

storagegranulesofmousepancreatic␤-cellswassensitivetoboth SERCAinhibitorsBHQandthapsigargin[42].Morerecently,using aequorintomonitorintragranular[Ca2+_{]wehavealsodescribed}

bothinchromaffincellsandinthecelllinesPC12andINS1[23,24], that Ca2+ _{uptakeby thesecretory granuleswasATP-dependent}

and fullysensitivetothapsigarginand BHQ(seeFig.1).Onthe otherhand,in the␤-celllineMIN6,Ca2+_{uptake wasshownto}

beATP-dependentbutinsensitivetothapsigargin[20],suggesting theinvolvementofadifferenttypeofCa2+_{-ATPase,thesecretory}

pathwayCa2+_{-ATPaseorATP2C1,whichisalsopresentintheGolgi}

apparatustogetherwiththeSERCA[43].

Recentdataobtainedfromproteomicstudiesofthesecretory granulesinseveralcelltypesprovidenewimportantinformation onthispoint,thoughdonotclarifyit.Arecentproteomicstudyin thesecretorygranulesofchromaffincellshasidentified371 dis-tinctsolubleproteinsand384distinctmembraneproteins[44]. However,amongthem,theonlymembraneproteinableto accu-mulatecalciuminthevesicles wasasodium/potassium/calcium exchanger.Previousstudiesinpancreaticzymogengranules[45]

(371 proteinsidentified)andcorticotropesdense-core secretory granules[46](150proteinsidentified)hadnotidentifiedany pro-tein relatedwithCa2+_{accumulation.Instead,aproteomicstudy}

thatidentified270proteinsinsecretoryvesiclesfrominsulinoma NIT-1cellsshowedthepresenceofSERCA2inthemembraneofthe vesicles[47],but anotherstudyintheinsulin-secretingcellline INS-1Ethatidentified150vesicularproteinsfailedtofindanyCa2+

334 J.Álvarez/CellCalcium51 (2012) 331–337

Fig.1.MechanismsofCa2+_{accumulationinthegranules.Tracescorrespondto[Ca}2+_]

SGmeasurementsmadewithchromaffincellsexpressingaequorintargetedtothe

secretorygranules(modifiedfromRef.[23]).PanelAshowstheincreasein[Ca2+_]

SGthatoccursuponreadditionofCa2+1mMtotheextracellularmediuminCa2+-depleted

cells,anditsinhibitionbythapsigargin.PanelsBandCshowthattheSERCAinhibitors2,5-diterbutylbenzohydroquinone(BHQ)andcyclopiazonicacid(CPA)bothreversibly inhibitCa2+_{uptakebythegranuleswhenaddedafterrefillingofthegranuleswithCa}2+_{(atthepointmarkedbytheredrectangleinpanelA).Thecartoonresumesthe}

possiblemechanismsforCa2+_{accumulationinthegranules:Ca}2+_{pumps,eitherSERCAorsecretorypathwayCa}2+_{-ATPase(SPCA),Na}+_/Ca2+_{exchangecoupledwithNa}+_/H+

exchange,andCa2+_/H+_{exchange.TheexchangerswouldbeenergizedbytheH}+_{gradientcreatedbytheH}+_-ATPase.Ca2+_{insidethegranulesisrepresentedinthreepossible}

forms,stronglybound(red),boundandreadilyreleasable(blue)andfree(green).

4. MechanismsofCa2+_{releasefromthesecretorygranules}

If theCa2+ _{stored in thegranules hastobe releasedduring}

cellstimulationtocooperatewithCa2+_{comingfromothersources}

intriggering vesiclefusionand secretion, thegranules needto

haveCa2+_{transportsystemsthatareabletoopenoractivate}

dur-ingcell stimulation(seethecartoonin Fig.2).Thefirstsystem tobeproposedforthatrole inchromaffincellswastheinositol 1,4,5-trisphosphatereceptor(InsP3R).Thepresenceof InsP3Rin

secretory granules hasbeena highly conflictingpoint over the

Fig.2.MechanismsofCa2+_{releasefromthegranules.Tracescorrespondto[Ca}2+_]

SGmeasurementsmadewithchromaffincellsexpressingaequorintargetedtothesecretory

granules(modifiedfromRef.[23]).PanelsAandBshowtheCa2+_{releasefromthegranulesinducedbyhistamine(viaInsP}

3production)andbycaffeine(RyRactivator).Panel

Cshowstheeffectofthenaturalagonistacethylcholine,andpanelDshowstheCa2+_{releaseinduceddirectlybyInsP}

3inpermeabilizedcells.Thecartoonresumesthepossible

mechanismsforCa2+_{releasefromthegranules:threeCa}2+_{channels(InsP}

3,ryanodineandNAADPreceptors),and2exchangers,Na+/Ca2+exchangerandH+/Ca2+exchanger,

last20years.In1990,YooandAlbanesi[49]reportedthat inosi-tol1,4,5-trisphosphate(InsP3)releasedCa2+fromchromaffincells

secretoryvesicles.Immunocytochemicallocalization ofallthree typesofInsP3Rinseriallysectionedbovinechromaffincellsshowed

thatthesecretorygranulescontainedabout60%oftheInsP3Rinthe

cell[50],andadditionalevidenceforthepresenceofInsP3Rinthe

granuleswasthefindingthatbothchromograninAandBinteract withInsP3Randmodulateitsactivity[26,51–54].Morerecently,

usingtargetedaequorintomeasure[Ca2+_{]inthesecretorygranules}

ofchromaffincellsandtheneurosecretorycelllinesPC12andINS1

[23,24],wehaveshownthatInsP3andInsP3-releasingagonistscan

releaseCa2+_{fromthegranularcompartment(seeFig.2).}

InsP3Rhavebeenalsodetectedinothertypesofsecretory

gran-ules. Isolated zymogen granules werefound to release Ca2+ _in

thepresenceofInsP3 [55],althoughthesameauthorshavealso

reportedthat inintact cellsmostof theCa2+ _{releasedbyInsP} 3

comesfrom theER [56]. In mast cell granules [19] and mucin granulesofgobletcells[57],InsP3inducedperiodicoscillationsof

luminal[Ca2+_{]bycouplingCa}2+_{releasewiththeactivityofaCa}2+

-dependentK+_{channelinthegranules.Finally,InsP}

3Rhavebeen

alsolocalizedinthesecretorygranulesofinsulin-secreting␤-cells andsomatostatin-secretingcells[58–60],andalsoindense-core vesiclesofastrocytes[61].

However,thereisalsoalargebodyofevidenceagainstthe pres-enceofInsP3Rinsecretorygranules.Inchromaffincells,Endoetal.

[62]foundthatInsP3R-2waspresentintheERbutnotinthe

secre-torygranules.Inratinsulinomacells,InsP3wasfoundtorelease

Ca2+_{fromthemicrosomalfractionbutnotfromthesecretory}

gran-ules[63].Ravazzolaetal.[64]foundthatantibodiesagainstInsP3R

cross-reactedwithinsulin,andthatcouldbeasourceforartifactsin immunostaining(seealso[65]).Measuringtheextragranular[Ca2+_]

withaphogrin–aequorinchimera,Poulietal.[66]foundno dif-ferenceamongtheCa2+_{transientsinthegranulemembraneand}

inthebulkcytosolafterinsulin-secretingMIN6or phaeochromo-cytomaPC12cellsstimulation,indicatingthatnosignificantlocal Ca2+_{releasetakesplaceinthesecells.Similarly,usingatargeted}

aequorintomonitorgranule[Ca2+_{],noeffectofInsP}

3wasobserved

inMIN6cells[20].Regardingzymogengranules,InsP3Rwerenot

foundusinghighlypuregranulepreparations[67]andtheabsence ofzymogengranulesdidnotmodifythecytosolicCa2+_transientsin

ratparotidacinarcells[68].Finally,inplatelets,InsP3Rarepresent

inthedensetubularsystem(theequivalentoftheER)butnotin theacidicgranules[41].

Thereisalsoevidenceforthepresenceofryanodinereceptors insecretorygranules.TheryanodinereceptoractivatorcyclicADP ribose(cADPR)hasbeenreportedtoreleaseCa2+_fromzymogen

granules[55,69].BothcADPRandcaffeinereleasedalsoCa2+_from

thesecretory granulesof insulin-secretingMIN6cells [20],and caffeine,butnotcADPR,releasedCa2+_{fromsecretorygranulesin}

chromaffincells [23].ThelackofeffectofcADPRmayreflectan insensitivitytothisagonistoftheryanodinereceptorsinthesecells, ascADPRwasalsounabletoreleaseCa2+_{fromtheERinchromaffin}

cells[7].Thenovelagonistnicotinicacidadenine dinucleotidephos-phate(NAADP)wasalsoeffectivetorelease Ca2+_fromzymogen

granules[69],plateletacidic stores[41],insulin-secretingMIN6 cells[70]andtheacidicCa2+_{storesofPC12cells[71].Theidentityof}

theNAADPreceptorisstillunknown.Itwasinitiallyassociatedwith theryanodinereceptor,althoughotheralternativeshaverecently appeared(twoporechannels,TPCsorTPCNs,see[72]).

Whatdoproteomicstudiestellusonthispoint?Noneofthe proteomicstudiesmadeuptodatehasdetectedthepresenceof InsP3Rinthesecretorygranulesofchromaffin[44],insulin[47,48],

zymogen[45] orcorticotrope granules[46].Instead,thelargest study[44]foundinchromaffingranulesthepresenceofryanodine receptorstogetherwithseveraltypesofCa2+_channels

(voltage-dependentand transient receptor potential). However, noneof

theseproteinsweredetectedintheotherstudies.Ofcourse,if gran-ular[Ca2+_{]isnearly1000-foldhigherthaninthecytosol,atleast}

somemechanismtoaccumulateCa2+_{inthegranulesmustexist.}

Thismeansthatthesensitivityofthesestudiesmaynotbestill enoughtodetectproteinsatverylowconcentrations,andfurther improvement ofthistechnique mayprovidein thefuture more usefulinformation.

5. Conclusion

Inneuroendocrinecells,thesecretorygranulesconstituteone of thelargestCa2+ _{storesand its totalcalcium concentrationis}

evenhigherthanthatintheER.GiventhatCa2+_{isthekeysignal}

thattriggerssecretionofgranulecontents,thepossiblefunctional roleofthecalciumstoredinthegranuleshasattracted consider-ableattentionformorethanthirtyyears.Theavailableevidence suggestsmoreoverthatintragranularCa2+_{isimportantfor}

secre-tion[37,73–75].However,inspiteoftheeffortsofmanyresearch

groups,mostofthekeyquestionsrelatedwithgranuleCa2+

homeo-stasis,e.g.,thefreegranular[Ca2+_{],thephysicalstateofthestored}

Ca2+_{orthemechanismsforCa}2+_{accumulationandreleasefromthe}

granules,remainobscure.Furtherworkwillbenecessarytoclarify alltheseuncertainties.Besidesthebiochemicalandphysiological approaches,thenewproteomictechniqueslookpromising,butthe dataobtaineduptodateonproteinsrelatedtoCa2+_dynamicsis

stillscarceandtoovariableamongsecretorygranulesofdifferent celltypes.

Acknowledgments

ThisworkwassupportedbygrantsfromMinisterio de Edu-caciónyCiencia(BFU2008-01871)andfromJuntadeCastillayLeón (GR105).

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