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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+signalingresultsfromacomplexinterplayamongavarietyofCa2+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[Ca2+],thephysicalstateof thestoredCa2+orthemechanismsforCa2+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(freeCa2+
con-stitutesonly1%ofthetotalCa2+inthecytosol),diffusionofCa2+
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+ tothecytosolviaseveralspecificCa2+ 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[Ca2+] 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 Ca2+uniporter(K M above
10M,100-foldhigherthantherestingcytosolic[Ca2+],100nM,
and10-foldhigherthantheusualpeakcytosolic[Ca2+]found
dur-ingcellstimulation,about1M)ledtomostresearchersinthe 1970sand1980stoexcludeasignificantroleinCa2+
homeosta-sisforthisorganelle,atleastunderphysiologicalconditions.Then, inthebeginningofthe1990s,recombinantaequorintargetedto themitochondriashowedthatmitochondrial [Ca2+] wasableto
undergorapidchangesduringcellactivation[4].Thatwasthe ori-ginofaseriesofworksbyseveralresearchgroupsdemonstrating thatmitochondriawereveryactiveplayersinthecontrolofthe globalcellCa2+homeostasis.Inparticular,becauseofthelow-Ca2+
-affinityoftheirCa2+uptakemechanism,mitochondriaareverywell
0143-4160/$–seefrontmatter© 2011 Elsevier Ltd. All rights reserved.
332 J.Álvarez/CellCalcium51 (2012) 331–337
adaptedtotakeupCa2+fromlocalhigh-Ca2+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[Ca2+]
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, theacidiccalciumpoolaccountedfor170mol/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
andisinthe500–800Mrange[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,foundvaluesof24Mand5.6M, respectively, in isolated acidic chromaffin vesicles. In mast cell granules, studieswithfluorescent dyes showeda resting intra-luminalfree [Ca2+]of 25M[19].Inthe-celllineMIN6,data
obtainedwithtargetedaequorinindicatedafree[Ca2+]inthe
vesi-clesaround 50M[20].These values contrastwiththe1.4M [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+]valuesof50–100M
inchromaffingranules[23]and20–40MinPC12andINS1cells
[24].Weshouldnotealsothatthesmallsizeofsomeofthe vesi-clesposessomerestrictionstothepossiblevaluesoffreegranular [Ca2+].Letusjustmentionthatforasmallsynapticvesicle,with
adiameterof50nm,25M[Ca2+]wouldbeobtainedwithonly1
Ca2+ion,andevenforalargedensecorevesicleof200nm
diame-ter,just2–3Ca2+ionspervesiclewouldrepresent1M[Ca2+](that
wouldgivefiguresof100–300freeCa2+ionspervesicle).
Inconclusion,thefree[Ca2+]inseveraltypesofsecretory
gran-ulesappearstorangebetween20and100M.Thesevaluesare significantlylower,nearlybyoneorderofmagnitude,thanthefree [Ca2+]intheendoplasmicreticulum,whichisabout500M[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
below100M.Infact,evenconsideringafreegranular[Ca2+]of
100M,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
withaffinitiesinthe100–300MrangedependingonthepHand ionicconditions[27,28];and(iii)partofthecalciumcouldalsobe boundtoothervesiclecomponents,suchasnucleotidesorlipids. Regardingthenucleotides,theyarepresentathighconcentrations andcanbindCa2+withaK
dinthe100–300Mrangedepending
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 of45Caaccumulationandreleasecomparedwithotherorganelles,
suggestingthatthestoredCa2+couldplaymainlyastructuralrole
inthe architectureoftheorganelle. Similarly, 45Ca 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+]
SGisinthe20–100Mrange,thereshouldbe
inthemembraneofthesecretorygranulesactivemechanismsto accumulatethisioninthematrix,giventhatitsconcentrationin thecytosolisnearly1000-foldlower.Severalcandidateshavebeen proposedforthisrole(seethecartooninFig.1).
Krieger-BrauerandGratzl[32]foundevidenceforthepresence ofa2Na+/1Ca2+exchangeinthemembraneofthesecretory
gran-ulesofbovinechromaffincells.ThesystemwaspH-independent
andhadhighaffinityforCa2+(0.28M[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+,thissystemwouldnotbeabletoaccumulateCa2+
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+affinityforCa2+entry,
start-ingtobeactiveatcytosolic[Ca2+]around100M.Thus,underthese
conditionsitcouldnotbeinvolvedinphysiologicalCa2+uptakeinto
thevesicles,althoughitmayperhapsmediateCa2+-releasefromthe
vesiclesfollowingchangesinthe[H+]gradient.Infact,the
disrup-tionofthevesicularpHgradientinducesasignificantleakofCa2+
fromthevesicles[37,38].
Thelastpossibilityisa directATP-dependentmechanism for Ca2+accumulation,thatis,aCa2+-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[Ca2+]
SGmeasurementsmadewithchromaffincellsexpressingaequorintargetedtothe
secretorygranules(modifiedfromRef.[23]).PanelAshowstheincreasein[Ca2+]
SGthatoccursuponreadditionofCa2+1mMtotheextracellularmediuminCa2+-depleted
cells,anditsinhibitionbythapsigargin.PanelsBandCshowthattheSERCAinhibitors2,5-diterbutylbenzohydroquinone(BHQ)andcyclopiazonicacid(CPA)bothreversibly inhibitCa2+uptakebythegranuleswhenaddedafterrefillingofthegranuleswithCa2+(atthepointmarkedbytheredrectangleinpanelA).Thecartoonresumesthe
possiblemechanismsforCa2+accumulationinthegranules:Ca2+pumps,eitherSERCAorsecretorypathwayCa2+-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[Ca2+]
SGmeasurementsmadewithchromaffincellsexpressingaequorintargetedtothesecretory
granules(modifiedfromRef.[23]).PanelsAandBshowtheCa2+releasefromthegranulesinducedbyhistamine(viaInsP
3production)andbycaffeine(RyRactivator).Panel
Cshowstheeffectofthenaturalagonistacethylcholine,andpanelDshowstheCa2+releaseinduceddirectlybyInsP
3inpermeabilizedcells.Thecartoonresumesthepossible
mechanismsforCa2+releasefromthegranules:threeCa2+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+]bycouplingCa2+releasewiththeactivityofaCa2+
-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+orthemechanismsforCa2+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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