Cell Calcium 37 (2005) 555–564
Calcium dynamics in ca echolamine-con aining sec e o y esicles
Al edo Mo enoa, Ca men D. Loba ´
ona, Jaime San oDomingoa, Lau a Vaya, Es he
He n´
andez-SanMiguela, Rosa io Rizzu ob, May e Mon e oa, Ja ie Al a eza,∗
aIns i u o de Biolog´ıa y Gen´e ica Molecula (IBGM), Depa amen o de Bioqu´ımica y Biolog´ıa Molecula y Fisiolog´ıa, Facul ad de Medicina,
Uni e sidad de Valladolid and Consejo Supe io de In es igaciones Cien ´ıficas (CSIC), Ram´on y Cajal 7, E-47005 Valladolid, Spain
bExpe imen al and Diagnos ic Medicine Sec ion o Gene al Pa hology, Uni e si y o Fe a a, 44100 Fe a a, I aly
Recei ed 19 Oc obe 2004; ecei ed in e ised o m 17 Feb ua y 2005; accep ed 21 Feb ua y 2005
A ailable online 8 Ap il 2005
Abs ac
Weha eusedanaequo inchime a a ge ed o hememb aneo hesec e o yg anules omoni o he ee[Ca2+]inside heminneu osec e o y
PC12 cells. Mo e han 95% o he p obe was loca ed in a compa men wi h an homogeneous [Ca2+] a ound 40␮M. Cell s imula ion wi h
ei he ATP, ca eine o high-K+depola iza ion inc eased cy osolic [Ca2+] and dec eased sec e o y g anule [Ca2+] ([Ca2+]SG). Inosi ol-(1,4,5)-
isphospha e, cyclic ADP ibose and nico inic acid adenine dinucleo ide phospha e we e all ine ec i e o elease Ca2+ om he g anules.
Changes in cy osolic [Na+] (0–140mM) o [Ca2+] (0–10␮M) did no modi y ei he ([Ca2+]SG). Ins ead, [Ca2+]SG was highly sensi i e o
changes in he pH g adien be ween he cy osol and he g anules. Bo h ca bonyl cyanide 4-( i luo ome hoxy)phenylhyd azone (FCCP) and
nige icin, as well as cy osolic acidi ica ion, e e sibly dec eased [Ca2+]SG, while cy osolic alcaliniza ion e e sibly inc eased [Ca2+]SG. These
esul s a e consis en wi h he ope a ion o a H+/Ca2+ an ipo e in he esicula memb ane. This an ipo e could also media e he e ec s
o ATP, ca eine and high-K+on [Ca2+]SG, because all o hem induced a ansien cy osolic acidi ica ion. The FCCP-induced dec ease in
[Ca2+]SG was e e sible in 10–15min e en in he absence o cy osolic Ca2+ o ATP, sugges ing ha mos o he calcium con en o he esicles
isbound oaslowlyexchangingCa2+ bu e . Thisla ges o ebu e s[Ca2+]SG changesin helong- e m bu allowshighly dynamic ee[Ca2+]SG
changes o occu in seconds o minu es.
© 2005 Else ie L d. All igh s ese ed.
Keywo ds: Calcium; Sec e o y esicles; PC12; Ca echolamine; Aequo in
1. In oduc ion
Sec e o y esicles cons i u e one o he less known
in acellula Ca2+ pools. I has been known o mo e han
20 yea s ha hey con ain la ge amoun s o o al calcium
(20–60mM), in pa icula ch oma in g anules o he ad enal
medulla and zymogen g anules o he exoc ine panc eas
[1–3]. In addi ion, ch oma in g anules con ain also la ge
amoun s o p o eins (100mg/ml, o which 40% is ch omo-
Abb e ia ions: [Ca2+]SG, sec e o y g anule [Ca2+]; [Ca2+]c, cy osolic
[Ca2+]; cADPR, cyclic ADP ibose; EGFP, enhanced g een luo escen p o-
ein; FCCP, ca bonyl cyanide 4-( i luo ome hoxy)phenylhyd azone; InsP3,
inosi ol-(1,4,5)- isphospha e; NAADP, nico inic acid adenine dinucleo ide
phospha e; VAMP, esicle-associa ed memb ane p o ein; VAMP-mu aeq,
VAMP-mu a ed aequo in chime a
∗Co esponding au ho . Tel.: +34 983 423085; ax: +34 983 423588.
E-mail add ess: [email p o ec ed]a.es (J. Al a ez).
g anin A), ca echolamines (0.5–0.6M) and ATP (130mM)
[2,4]. The physiological signi icance o in ag anula Ca2+
([Ca2+]SG) wi hin hese densely packed esicles, ei he o
play a ole in sec e ion, o be des ined o sec e ion o jus
s uc u al o keep g anule con en s oge he , is s ill obscu e.
Simila ly, he mechanisms o Ca2+ up ake and elease om
he g anules a e poo ly known o con o e sial and may be
di e en depending on he cell ype [5–9].
Recen ly, using a esicle-associa ed memb ane p o ein
(VAMP)–aequo in chime a a ge ed o he memb ane o he
sec e o y esicles, i was possible o he i s ime o moni-
o hein ag anula [Ca2+]inMIN6␤-cells[8].In ag anula
ee [Ca2+] was a ound 50␮M, Ca2+ was accumula ed in o
he esicles ia a P- ype Ca2+-ATPase and eleased h ough
yanodine ecep o s and nico inic acid adenine dinucleo ide
phospha e (NAADP) ecep o s [8,9]. We ha e used he e he
same VAMP–aequo in chime a o moni o he dynamics o
0143-4160/$ – see on ma e © 2005 Else ie L d. All igh s ese ed.
doi:10.1016/j.ceca.2005.02.002
556 A. Mo eno e al. / Cell Calcium 37 (2005) 555–564
[Ca2+] in heca echolamine-con ainingsec e o y g anules o
neu osec e o y PC12 cells. Ou esul s show ha , al hough
he ee [Ca2+] is simila o ha ound in MIN6 ␤-cells, he
mechanisms o Ca2+ up ake and elease and he esponse o
ex acellula s imuli a e la gely di e en .
2. Expe imen al p ocedu es
2.1. Cell cul u e and ans ec ion
The cons uc ion s a egy o he 2-synap ob e in-mu a ed
aequo in (VAMP-mu aeq) chime a has been desc ibed p e-
iously [8]. PC12 a pheoch omocy oma cells we e g own
in Dulbecco’s Modi ied Eagle’s Medium supplemen ed wi h
7.5% e al cal se um, 7.5% ho se se um and 2mM glu-
amine.Thecons uc o cy osolicaequo inhasbeenalsode-
sc ibed p e iously [10]. The VAMP-enhanced g een luo es-
cen p o ein(EGFP)cons uc wasmadebyinse ingin ame
he VAMP sequence in he HindIII si e o he pEGFP-N1
plasmid. T ans ec ions we e ca ied ou using Me a ec ene
(Bion ex, Ge many) o ExGen500 (Fe men as, Ge many).
2.2. [Ca2+]Mand [Ca2+]cmeasu emen s wi h aequo in
PC12 cells pla ed on o 13mm plas ic ound co e slips
we e ansien ly ans ec ed wi h he co esponding plasmid.
Fo aequo in econs i u ion, PC12 cells exp essing cy osolic
aequo in we e incuba ed o 1–2h a oom empe a u e
wi h 1␮M o wild- ype coelen e azine, and cells exp essing
VAMP-mu aeq we e incuba ed o 1–2h a oom empe -
a u e wi h 1␮M o coelen e azine n, in s anda d medium
(145mM NaCl, 5mM KCl, 1mM MgCl2, 1mM CaCl2,
10mM glucose and 10mM Hepes, pH 7.4). Cells we e
hen placed in he pe usion chambe o a pu pose-buil
luminome e he mos a ized a 37◦C. Fo expe imen s wi h
pe meabilized cells, cells exp essing VAMP-mu aeq we e
econs i u ed wi h 1␮M coelen e azine n o 1–2h and hen
placed in he luminome e as abo e. Then, s anda d medium
con aining 0.5mM EGTA ins ead o Ca2+ was pe used o
1min, ollowed by 1min o in acellula medium (130mM
KCl, 10mM NaCl, 1mM MgCl2, 1mM K3PO4, 0.5mM
EGTA, 1mM ATP, 20␮M ADP, 2mM succina e and 20mM
Hepes,pH7)con aining20␮Mdigi onin.Then,in acellula
medium wi hou digi onin and con aining 100nM [Ca2+]
(p epa ed wi h an EGTA/Ca2+ mix u e) was pe used.
Con ol expe imen s pe o med in PC12 cells exp essing
mi ochond ially a ge ed mu a ed aequo in showed ha his
pe meabiliza ion p ocedu e ende ed all he mi ochond ial
space accessible o he pe usion medium (da a no shown).
Calib a ion o he luminescence da a in o [Ca2+] was made
using an algo i hm as p e iously desc ibed [11].
2.3. Con ocal measu emen s
PC12 cells exp essing VAMP-EGFP we e imaged on a
BioRad Radiance 2000 con ocal spec opho ome e using a
60×oil imme sion objec i e moun ed in a Nikon Eclipse
TE2000mic oscope.Cellswe ealsoloadedwi h50nMlyso-
acke ed o 1mina oom empe a u eandimagedsequen-
ially o bo h EGFP and lyso acke ed. EGFP was exci ed
wi h he 488nm line o he A gon lase , and he luo escence
emi ed be ween 500 and 530nm was collec ed. In he case
o lyso acke ed, he luo escence exci ed wi h he 543nm
line o he g een He–Ne lase and emi ed be ween 570 and
700nm was measu ed.
2.4. Measu emen o cy osolic pH
pH measu emen s we e ca ied ou by mic o luo ime y
o bcec -loaded HeLa cells. Cells we e loaded wi h bcec by
incuba ionwi h0.8␮M bcec -AM o 30min a 30◦C.Then,
he dye was washed ou and cells we e incuba ed o 30min
a 30◦C be o e moun ing in o he pe usion chambe o an
Axio e 200 mic oscope. Single cell luo escence was ex-
ci eda 490and440nmusingaCai nmonoch oma o .Apai
o images was aken e e y 2s by a Hamama su ORCA-ER
came a and a ioed a e backg ound subs ac ion using he
Me a luo p og am (Uni e sal Imaging). Expe imen s we e
pe o med a 37◦C. Fo calib a ion o pH measu emen s, so-
lu ions con aining 150mM KCl, 10mM Hepes, 10mM glu-
cose,1mMCaCl2,1mMMgCl2,5␮Mnige icin andpH6.6,
7.0 o 7.4 we e pe used a he end o e e y expe imen .
2.5. Ma e ials
Wild- ype coelen e azine, coelen e azine n, lyso acke
ed and bcec -AM we e ob ained om Molecula P obes,
OR, USA. Ba ilomycin A was om LC labo a o ies,
Wobu n, MA, USA o om Sigma, Mad id. Inosi ol-(1,4,5)-
isphospha e (InsP3), cyclic ADP ibose (cADPR), nico inic
acid adenine dinucleo ide phospha e (NAADP), ca bonyl
cyanide 4-( i luo ome hoxy)phenylhyd azone (FCCP) and
nige icin we e om Sigma, Mad id. O he eagen s we e
om Sigma, Mad id o Me ck, Da ms ad .
3. Resul s
3.1. Ta ge ing o VAMP-mu aeq in PC12 cells
The speci ici y o a ge ing o he VAMP-mu aeq chime a
o sec e o y esicles has been p e iously shown by im-
muno luo escence and immunoelec on mic oscopy in in-
sulin con aining sec e o y esicles o MIN6 ␤-cells [8].We
ha e used he e a VAMP-EGFP chime a o in es iga e he a -
ge ing o he p obe in PC12 cells. Fig. 1 (le panels) shows
con ocalimageso he luo escenceo aPC12cellexp essing
VAMP-EGFP a h ee di e en planes. The pa e n is ypi-
cally g anula and consis en wi h he a ge ing o he sec e-
o y g anules. In addi ion, VAMP-EGFP colocalizes wi h he
ma ke o acidic compa men s lyso acke ed. The middle
panels show he luo escence o lyso acke ed ob ained in
A. Mo eno e al. / Cell Calcium 37 (2005) 555–564 557
Fig. 1. Con ocal luo escence co-localiza ion o VAMP-EGFP and lyso acke ed in PC12 cells. PC12 cells exp essing VAMP-EGFP we e loaded wi h
lyso acke ed as desc ibed in Sec ion 2. The le panels show h ee di e en con ocal planes o he luo escence o VAMP-EGFP. The middle panels show he
luo escence o lyso acke ed in he same con ocal planes. The igh panels show he o e lap o bo h luo escence images.
he same con ocal planes and he igh panels show ha he
luo escences o bo h VAMP-EGFP and lyso acke ed a e
mos ly o e lapping.
Al hough he VAMP-EGFP luo escence images sugges
ha he p obe is co ec ly a ge ed o sec e o y g anules also
in PC12 cells, i is di icul o es ima e om hese images i
he e may be a small pe cen age o he chime a in a di e en
loca ion. To in es iga e his poin , we ha e used he p op-
e ies o aequo in o de e mine he deg ee o homogenei y
o he [Ca2+] in he compa men exp essing VAMP-mu aeq.
Emission o ligh by aequo in is i e e sible om an expe -
imen al poin o iew. Thus, aequo in is being con inuously
consumed along he expe imen s a a a e which is highly
dependen on he local [Ca2+]. When aequo in is dis ibu ed
in compa men s wi h di e en [Ca2+], i will be consumed
as e in hose compa men s wi h highe [Ca2+]. This can be
easily de ec ed and quan i ied in he aequo in luminescence
eco ds, and his p ope y has been used p e iously o es i-
ma e he p esence and size o compa men s wi h di e en
[Ca2+] in mi ochond ia and endoplasmic e iculum [12,13].
Fig.2 (uppe panel) showsac ude eco do luminescence
in PC12 cells exp essing VAMP-mu aeq. As in he p e ious
558 A. Mo eno e al. / Cell Calcium 37 (2005) 555–564
Fig. 2. S eady-s a e [Ca2+] in he sec e o y g anules. PC12 cells exp essing
VAMP–aequo in we e econs i u ed wi h coelen e azine n as desc ibed in
Sec ion 2and hen he aequo in luminescence was moni o ed unde con-
inuous pe usion wi h ex e nal medium. Uppe panel: c ude luminescence
eco d. When indica ed “cell lysis”, s anda d medium con aining 100␮M
digi onin and 10mM Ca2+ was pe used. Middle panel: calib a ed [Ca2+]
alues om he same expe imen . Lowe panel: calib a ed [Ca2+] alues
om he same expe imen ob ained no aking in o accoun he inal 3.5% o
he emi ed luminescence. This expe imen is ep esen a i e o se en simila
ones.
wo k in MIN6 ␤-cells, we ha e econs i u ed VAMP-mu aeq
exp essingcellswi h coelen e azinen ino de omeasu e he
high-[Ca2+] p esumablycon ainedwi hin he sec e o y g an-
ules. Howe e , we ha e p e e ed o a oid Ca2+-deple ion o
he sec e o y g anules p io o he measu emen s, by wo ea-
sons: (i) he a e o aequo in consump ion a he [Ca2+] mea-
su ed allowed moni o ing he [Ca2+]SG du ing easonable
imes (20–30min) and (ii) i has been desc ibed ha e illing
o hesec e o y g anule Ca2+ s o einPC12 cellsisex emely
slow [3]. Because o he lack o p e ious Ca2+-deple ion, he
Ca2+ con en o hesec e o yg anulesishigha hebeginning
o he expe imen , and he eco d o luminescence shows a
con inuously dec easing end due o aequo in consump ion
in a high-[Ca2+] en i onmen . Once he luminescence had
eached nea ly ze o alues, cells we e pe meabilized wi h
digi onin o e eal all he aequo in luminescence s ill p esen
in he sample. In case a signi ican p opo ion o aequo in
was p esen (due o mis a ge ing) in compa men s ha ing
low es ing [Ca2+], i s luminescence should be eleased he e.
Howe e , only a small pe cen age o he o al luminescence,
below 5% (4.3±0.3%, mean±S.E.M., n=7), was eleased
a his poin .
Fig.2(middlepanel)shows he eco do [Ca2+]calcula ed
om he same expe imen . I shows a s eady-s a e [Ca2+]
le el a ound 35␮M, which keeps s able o 15–20min and
hen s a s o dec ease slowly. This inal dec ease is due o
he p esence o a small pe cen age o aequo in in a compa -
men wi h low-[Ca2+]. As mos o he aequo in p esen in
he high-Ca2+ compa men is being consumed, he ela i e
size o he low-Ca2+ compa men inc eases p og essi ely
and his is e lec ed as a dec ease in he calib a ed [Ca2+] al-
ues. The pe cen age o aequo in p esen in ha low-[Ca2+]
compa men can be es ima ed om he ela i e amoun o
luminescence emi ed a e cell pe meabiliza ion, ha is, less
han 5%. In ac , i ha pe cen age o luminescence was no
akenin oaccoun du ing hecalib a iono luminescencein o
[Ca2+] alues, he eco d o calib a ed [Ca2+] emained s a-
ble o nea ly 1h a ound 40␮M(Fig. 2, lowe panel). In his
case, calcula ion o [Ca2+] alues was made no aking in o
accoun he inal3.5%o heluminescence. Inse e alsimila
expe imen s, his pe cen age was always below 5%. The e-
o e, hese esul s cons i u e unc ional e idence ha mo e
han95%o ou VAMP-mu aeqp obeismeasu ing [Ca2+]in
a compa men wi h an homogeneous [Ca2+] a ound 40␮M
(43±3␮M, mean±S.E.M., n=7).
3.2. Dynamic measu emen s o [Ca2+] in he sec e o y
esicles
Weha enow in es iga ed he e ec on hesec e o yg an-
ule [Ca2+] o a se ies o agen s known o induce cy oso-
lic Ca2+ ansien s by di e en mechanisms: ATP, ac ing on
pu ine gic ecep o s, igge sInsP3p oduc ionand elease o
Ca2+ om he ER; ca eine, ac ing on yanodine ecep o s,
induces also Ca2+ elease om he ER; and high-K+,byde-
pola izing hecells,inducesCa2+ en y iaplasmamemb ane
ol age-dependen Ca2+ channels. Fig. 3 shows he e ec s o
hese s imuli on bo h cy osolic and sec e o y g anule [Ca2+].
As expec ed, all o he s imuli induced ansien [Ca2+] in-
c eases in he cy osol. Howe e , i came as a su p ise o see
ha allo heminducedalsoa ansien dec easein he[Ca2+]
o sec e o y g anules. These esul s sugges ed ha sec e o y
g anules could elease Ca2+ du ing he p ocess o s imula ed
sec e ion. To in es iga e he mechanism o s imula ed Ca2+
elease om he g anules, we used pe meabilized cells o es
he e ec s o se e al second messenge s: InsP3, cADPR and
NAADP.Fig.4shows ha noneo hemp oducedanychange
in he[Ca2+]SG.Simila ly,pe usiono a10␮M[Ca2+]bu e
A. Mo eno e al. / Cell Calcium 37 (2005) 555–564 559
Fig. 3. E ec s o ATP, ca eine and high-K+on bo h [Ca2+]cand [Ca2+]SG. PC12 cells we e ans ec ed wi h ei he cy osolic aequo in (le panels) o
VAMP–aequo in ( igh panels), and econs i u ed wi h coelen e azine as desc ibed in Sec ion 2. The le panels show he cy osolic [Ca2+] peaks induced by
1min s imula ion wi h 100␮M ATP o 10mM ca eine, o 30s s imula ion wi h a solu ion con aining 70mM K+. The igh panels show he e ec o he same
s imuli on [Ca2+]SG. These expe imen s a e ep esen a i e o 5–10 simila ones o each kind.
omimic localinc easesin he[Ca2+] a ound he esicleshad
no e ec on [Ca2+]SG. The e o e, hese second messenge s
a e appa en ly no in ol ed in he mechanism o he dec ease
in [Ca2+]SG induced by he agonis s.
Rega ding al e na i e Ca2+ anspo sys ems in he g an-
ules, he p esence o ei he Ca2+-ATPases, Na+/Ca2+ o
H+/Ca2+ exchange sys ems ha e been p oposed in di e en
p epa a ions [14–18]. Inhibi ion o sa coendoplasmic e icu-
lumCa2+-ATPasewi h 10␮M benzohyd oquinonep oduced
li le e ec s on [Ca2+]SG (da a no shown), sugges ing ha
his ype o Ca2+-ATPase was no in ol ed. On he o he
hand, in expe imen s pe o med wi h pe meabilized cells,
changes in he concen a ion o Na+ion om 10 o 0mM
o om 10 o 140mM p oduced also no change in he
[Ca2+]SG (da a no shown), sugges ing ha Na+/Ca2+ ex-
change is no ope a ing in hese g anules. Ins ead, changes
in he pH in he cy osolic side o he esicles induced as
and e e sible changes in [Ca2+]SG consis en wi h he op-
e a ion o a H+/Ca2+ exchange sys em. Fig. 5 shows expe i-
men s pe o med in pe meabilized PC12 cells pe used wi h

560 A. Mo eno e al. / Cell Calcium 37 (2005) 555–564
Fig. 4. E ec o InsP3, cADPR, NAADP and Ca2+ on [Ca2+]SG. PC12 cells
exp essing VAMP–aequo in we e econs i u ed wi h coelen e azine n and
pe meabilized wi h digi onin as desc ibed in Sec ion 2immedia ely p io
o he expe imen . Then, he e ec s o ei he 5␮M InsP3,5␮M cADPR,
10␮MNAADPo 10␮MCa2+ we e es ed, as indica ed. Theseexpe imen s
a e ep esen a i e o i e simila ones o each kind.
bu e s con aining 100nM [Ca2+] and di e en pH. I can be
obse ed ha a dec ease in pH om 7.0 o 6.6 induced a de-
c ease in [Ca2+]SG om 40␮M o below 30␮M, e e sible
a e e u ning he cy osolic pH o 7.0. Vice e sa, an in-
c ease in pH om 7.0 o 7.4 induced an inc ease in [Ca2+]SG
om40␮M omo e han50␮M, e e siblea e e u ning o
pH 7.0. The e ec s o nige icin, an ionopho e ha ca alizes
H+/K+exchange, we e also consis en wi h he p esence o a
H+/Ca2+ exchange. Fig. 5 (lowe panel) shows ha nige icin
induced a dec ease in [Ca2+]SG, which was e e sible a e
washing he ionopho e wi h a 0.1% albumin con aining so-
lu ion.
The dec ease in [Ca2+]SG induced by ATP, ca eine and
high-K+could be also media ed by a esicula H+/Ca2+ an-
ipo e in case hese s imuli induce a cy osolic acidi ica ion.
This was he case. Fig. 6 shows he mean esponse o all
he cells p esen unde he mic oscope ield in expe imen s
pe o med wi h each agonis . Mos o he cells esponded o
he s imuli wi h a ansien o p olonged acidi ica ion and he
mean esponse was in all he cases an acidi ica ion o a ound
0.1 pH uni .
Fig. 7 (uppe panel) shows ha he p o onopho e FCCP
p oduced also a dec ease in [Ca2+]SG, which was e e sible
a e washing he ionopho e. The e ec o FCCP is again
consis en wi h he p esence o a H+/Ca2+ exchange ope a -
ing in he memb ane o he sec e o y g anules. In case his
sys em was esponsible o he accumula ion o Ca2+ in o he
esicles, we easoned ha he e e sibili y o he e ec o
FCCP on [Ca2+]SG should depend on he es o a ion o he
esicula H+g adien ia he ba ilomycin-sensi i e acuola
H+-ATPase. Howe e , as shown in he lowe panel o Fig. 7,
ba ilomycin had no e ec on his p ocess. Simila ly, incuba-
ion wi h 200nM ba ilomycin o 15min p oduced no e ec
on he es ing [Ca2+]SG (da a no shown).
The e o e, es o a ion o he esicula H+g adien was
no equi ed o e e he changes in [Ca2+]SG induced by
Fig. 5. E ec o pH changes and nige icin on [Ca2+]SG. PC12 cells exp ess-
ing VAMP–aequo in we e econs i u ed wi h coelen e azine n. In he uppe
andmediumpanels, heywe ealsope meabilizedwi hdigi oninasdesc ibed
in Sec ion 2immedia ely p io o he expe imen . Then, solu ions con ain-
ing 100nM Ca2+ and he di e en pH indica ed in he igu e we e pe used.
In he lowe panel, in ac PC12 cells we e ea ed wi h 2␮M nige icin as
indica ed. The ionopho e was hen washed by pe using medium con aining
0.1% bo ine se um albumin. These expe imen s a e ep esen a i e o ou
o eigh simila ones o each kind.
FCCP. Al e na i ely, he p esence o P- ype Ca2+-ATPases,
as sugges ed o he insulin con aining esicles o MIN6 ␤-
cells [8], canno be excluded. To ob ain u he e idence on
hispoin ,we pe usedin acellula medium con ainingADP
(and 5␮M oligomycin o abolish in e nal ATP p oduc ion)
ins ead o ATP in pe meabilized cells, bu we did no ob-
se e any changes in [Ca2+]SG. In addi ion, Fig. 8 (uppe and
A. Mo eno e al. / Cell Calcium 37 (2005) 555–564 561
Fig. 6. E ec s o ATP, ca eine and high-K+on cy osolic pH. PC12 cells
we e loaded wi h bcec as desc ibed in Sec ion 2and hen s imula ed wi h
ei he 100␮M ATP o 1min, 10mM ca eine o 1min o wi h a solu ion
con aining 70mM K+ o 30s, as indica ed in he igu e. T aces shown
co espond o he mean o all he cells p esen unde he mic oscope ield
(high-K+, 20 cells; ca eine, 19 cells; ATP, 40 cells). These expe imen s a e
ep esen a i e o a leas h ee sepa a e expe imen s o each kind.
Fig.7. E ec o FCCPon[Ca2+]SG.PC12cellsexp essingVAMP–aequo in
we e econs i u ed wi h coelen e azine n as desc ibed in Sec ion 2. Then,
2␮M FCCP was pe used as indica ed. In he lowe panel, 200nM
ba ilomycin was also p esen be o e, du ing and a e FCCP addi ion, as
indica ed. These expe imen s a e ep esen a i e o six simila ones o each
kind.
Fig. 8. E ec o FCCP on [Ca2+]SG in pe meabilized cells. PC12 cells
exp essing VAMP–aequo in we e econs i u ed wi h coelen e azine n and
pe meabilized wi h digi onin as desc ibed in Sec ion 2immedia ely p io
o he expe imen . Then, in he uppe and medium panels, solu ions con-
aining 100nM Ca2+ and ei he s anda d in acellula medium con aining
1mM ATP (uppe panel) o in acellula medium con aining 1mM ADP
and 5␮M oligomycin ins ead o ATP (medium panel) we e pe used. In he
lowe panel, s anda d in acellula medium con aining 2mM EGTA and no
Ca2+ was pe used. When indica ed, 2␮M FCCP was pe used in he same
medium used o each expe imen . These expe imen s a e ep esen a i e o
h ee o i e simila ones o each kind.
562 A. Mo eno e al. / Cell Calcium 37 (2005) 555–564
medium panels) shows ha he eco e y om he [Ca2+]SG
dec ease induced by FCCP occu ed simila ly in pe meabi-
lized cells pe used wi h medium wi h o wi hou ATP, sug-
ges ing ha ATP hyd olysis was also no necessa y o es o e
he [Ca2+]SG le els. Finally, Fig. 8 (lowe panel) shows ha
eco e y o [Ca2+]SG a e FCCP ac ion ook place unmodi-
ied e en in pe meabilized cells pe used wi h medium in he
absence o Ca2+ (EGTA 2mM).
4. Discussion
We show in his pape dynamic measu emen s o [Ca2+]
in he ca echolamine-con aining sec e o y g anules o PC12
cellsusingaVAMP–aequo inchime a.Rega ding hequali y
o he a ge ing o he sec e o y g anules, we p o ide unc-
ionale idence ha ou p obe(a leas ha able o econs i u e
wi h coelen e azine n in he absence o Ca2+-deple ion) is
mo e han 95% loca ed in a compa men con aining a ound
40␮M[Ca2+]unde s eady-s a econdi ions.Noo he subcel-
lula compa men known has [Ca2+] le els in his ange. En-
doplasmic e iculum and Golgi appa a us ha e [Ca2+] 5–10-
old highe [19,20], while cy osol, nucleus and mi ochond ia
s ay unde es ing condi ions a [Ca2+] below 1␮M. Ou da a
o es ing [Ca2+]SG a e consis en wi h hose ob ained wi h
he same p obe in MIN6 ␤-cells (a ound 50␮M, see e [8])
and con i m ha sec e o y g anules a e he o ganelle wi h
he la ges bound/ ee Ca2+ ela ionship. Es ima ions o he
o al calcium con en o sec e o y g anules in ch oma in o
PC12 cells ange be ween 20 and 40mM [3,21]. Mos o
his Ca2+ mus be bound o p o eins such as ch omog anin
A, he majo in a esicula p o ein o ad enal ch oma in
g anules. This p o ein, which cons i u es 40% o he solu-
ble in a esicula p o eins [22], is known o bind 152nmol
o Ca2+/mg o p o ein wi h a Kdo 54␮M, apa o se e al
addi ional low a ini y (millimola ) binding si es [4,21,23].
Gi en ha he ee [Ca2+]SG is a ound 40␮M, only he high
a ini y si es can bind signi ican amoun s o Ca2+.Inad-
di ion, a signi ican p opo ion o Ca2+ mus be bound o
ATP, as ch oma in g anules may con ain up o 130mM ATP
[2].
The apid elease o Ca2+ om he sec e o y esicles in-
duced by he di e en agonis s sugges s ha Ca2+ elease
om he g anules du ing cell s imula ion could play a ole
in sec e ion, in pa icula i he obse ed global dec ease in
[Ca2+]SG induced by he agonis s could be es ic ed o a
small popula ion o esicles unde going la ge changes in
hei [Ca2+].Howe e ,noneo heknownsecondmessenge s
es ed (InsP3, cADPR, NAADP o Ca2+) p oduced any e ec
on [Ca2+]SG in pe meabilized cells. Thus, unless addi ional
cellula componen s (pe haps los du ing pe meabiliza ion)
maybe equi ed,ou da a sugges ha nei he InsP3 ecep o s
no yanodine ecep o s (a leas hose ac i a ed by cADPR
o Ca2+) o NAADP-ac i a ed channels a e in ol ed in he
mechanism o agonis -induced Ca2+ elease om PC12 se-
c e o y g anules.
Ou da a show also ha changes in he pH g adien be-
ween he cy osol and he sec e o y g anules immedia ely
igge changes in [Ca2+]SG ha a e consis en wi h he op-
e a ion o a H+/Ca2+ exchange. These e ec s canno be a -
ibu ed o hee ec so in ag anula pHchangesinaequo in
luminescence,becauseaequo inisli lesensi i e opHin his
ange[8].Inaddi ion, i shouldbe ema ked ha bo hlow pH
andnige icino FCCPinduceddec easesin[Ca2+]SG (Fig.5),
al hough hei e ec s in in ag anula pH should be oppo-
si e. pH-dependen Ca2+ luxes hus appea o be he only
mechanism able o gene a e as changes in [Ca2+]SG unde
ou condi ions, and could be esponsible o agonis -induced
Ca2+ elease om he g anules. We show he e ha cell s im-
ula ion wi h ATP, ca eine o high-K+induces a ansien
acidi ica ion o he cy osol o abou 0.1 pH uni . I is di icul
oassessi hisacidi ica ionisenough oinduce hemeasu ed
dec eases in [Ca2+]SG, bu ob iously he changes go in he
igh di ec ion.Inaddi ion,localpHchangesin he icini yo
he sec e o y g anules close o he plasma memb ane may be
la ge han he global changes measu ed he e. Consis en ly,
i has been shown ha s imula ion o PC12 cells wi h high-
K+depola iza ioninducesalcaliniza iono sec e o y esicles
[24]. A simila inding has been desc ibed in mas cells [25].
Ope a ion o a H+/Ca2+ exchange has been p e iously de-
sc ibed in synap ic esicles [18,26] and yeas acuoles [27].
As men ioned abo e, ou da a in PC12 sec e o y g anules
sha e wi h hose desc ibed in MIN6 ␤-cells [8,9] he simila
s eady-s a e [Ca2+] le els. Howe e , he e a e many di e -
ences in he dynamics, such as he lack o e ec o FCCP on
MIN6 ␤-cells [Ca2+]SG, he inc ease in [Ca2+]SG induced by
an InsP3-p oducing agonis in hose cells and he elease o
Ca2+ om he MIN6 ␤-cells h ough bo h yanodine ecep-
o s and NAADP ecep o s. On he con a y, none o hese
ecep o sappea s obe p esen in hePC12 sec e o y esicles
and he p o onopho e FCCP, he H+/K+exchange ionopho e
nige icine, and he di e en ex acellula s imuli induced all
o hem apid dec eases o [Ca2+]SG in PC12 cells. These
di e ences p obably esul om he p esence o a H+/Ca2+
exchange sys em in PC12 g anules.
Rega ding he mechanism o accumula ion o Ca2+ in o
PC12 g anules, ou da a on he es o a ion o [Ca2+]SG le -
els a e FCCP ac ion a e in iguing. In pe meabilized cells,
FCCP induced a dec ease in [Ca2+]SG ha was comple ely
e e sible e en in he absence o Ca2+ and ATP in he pe u-
sion medium. The only explana ion o hese esul s would be
he p esence in he sec e o y g anules o a la ge capaci y, low
a ini y and slow-exchanging Ca2+ bu e , ha would be able
o elease Ca2+ du ing he ollowing 10–15min a e FCCP
ac ion o es o e [Ca2+]SG le els in he absence o any Ca2+
in lux om he cy osol. This possibili y is consis en wi h
p e ious da a showing ha calcium binds and p ecipi a es
ch omog anins a a ini ies compa ible wi h hei physiolog-
ical concen a ions and ha his igh binding could accoun
o mo e han 90% o in a esicula calcium [28]. The pic-
u e ha eme ges nowis ha o a esiclecon aining ela i ely
small amoun s o ee o apidly exchanging bound Ca2+ and
A. Mo eno e al. / Cell Calcium 37 (2005) 555–564 563
a la ge s o e o igh ly bound slow-exchanging Ca2+. Ac-
co dingly, dis up ion o he esicula pH g adien wi h weak
amines induces a e y slow elease o calcium om he esi-
cles, abou 20% in 15min [29]. The s eady-s a e Ca2+ ac-
cumula ion in o he esicles may be due o he ope a ion o
H+/Ca2+ exchangecombinedwi h heene gyp o idedby he
H+g adien gene a ed by he acuola H+-ATPase. Howe e ,
hep esenceo P- ypeCa2+-ATPasescanno beexcluded,and
bo h sys ems could in ac coexis , as has been desc ibed in
synap ic esicles [30] and yeas acuoles [27].
In conclusion, he main indings o his wo k a e: (i)
ca echolamine-con aining sec e o y g anules ha e an homo-
geneous s eady-s a e [Ca2+] a ound 40␮M; (ii) cell s im-
ula ion by di e en mechanisms, ei he media ed by InsP3
ecep o s, yanodine ecep o s o plasma memb ane Ca2+
channels, induces a ansien dec ease in sec e o y g anule
[Ca2+] ha occu s simul aneously o he inc ease in cy oso-
lic[Ca2+];(iii)changesin cy osolicpHo aboli ion o hepH
g adien be ween he cy osol and he g anules induce apid
changes o [Ca2+] in he sec e o y esicles ha a e consis en
wi h he ope a ion o a H+/Ca2+ an ipo e in he esicula
memb ane; (i ) e e sibili y o FCCP-induced [Ca2+]SG de-
c easeoccu ssimila lyei he in hep esenceo in heabsence
o Ca2+ o ATP in he cy osol, sugges ing he p esence o a
la ge and slow-exchanging Ca2+ bu e in he esicle ma ix.
This la ge s o e o igh ly bound calcium bu e s [Ca2+]SG
changes in he long- e m, bu allows apid changes in ee
[Ca2+]SG occu ing in imes o seconds o minu es. Sec e-
o y g anules a e he e o e a much mo e dynamic o ganelle
in e ms o ee [Ca2+]SG han p e iously hough . The unc-
ional signi icance o hese Ca2+ changes is s ill unknown,
bu e idence o a ole o esicula Ca2+ in exocy osis has
been p o ided in se e al ypes o sec e o y esicles [31–33].
A pape ecen ly published by Mahapa a e al. [34] ex-
plo es also he dynamics o [Ca2+] in he sec e o y g anules
o PC12 cells using a ch omog anin–aequo in chime ic p o-
ein. Howe e , hei esul s a e la gely di e en om ou s
bo h in e ms o he ee [Ca2+] in he sec e o y g anules
(only 1.4␮M) and he esponse o di e en s imuli. The dis-
c epancyisclea lydue o hedi e en Ca2+ a ini y o he ae-
quo inp obesinbo hcases.Mahapa ae al.usedanaequo in
chime a wi h much highe Ca2+ a ini y, which is unable o
econs i u e wi h coelen e azine in a eas whe e he [Ca2+]is
abo ea ewmic omola .Thus, hei measu emen s e lec he
beha io o he small compa men (less han 5% o he o-
alspace)ha inglow es ing[Ca2+]and unc ionalp ope ies
comple elydi e en om hoseshownhe e o hebulko he
g anula space. We ha e epo ed be o e simila disc epan-
cies in he dynamics o [Ca2+] in he endoplasmic e iculum
using aequo in chime as wi h di e en Ca2+ a ini ies [12].
Acknowledgemen s
This wo k was suppo ed by g an s om Di ecci´
on Gen-
e aldeEnse˜
nanzaSupe io (BFI2002-01397)and omJun a
deCas illayLe´
on(VA005/02).J.S.andE.H.-S.holdFPIand
FPU ellowships, espec i ely, om he Spanish Minis e io
de Educaci´
on y Ciencia.
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