Unco ec ed p oo
Modula ion o sec e ion by he endoplasmic e iculum in
mouse ch oma ®n cells
Rica do Rigual, May e Mon e o, Albe o J. Rico, Jesu
Âs P ie o-Llo e , Ma õ
Âa Te esa Alonso and Ja ie Al a ez
Ins i u o de Biologõ
Âa y Gene
 ica Molecula (IBGM), Depa amen o de Bioquõ
Âmica y Biologõ
Âa Molecula y Fisiologõ
Âa,
Facul ad de Medicina, Uni e sidad de Valladolid and Consejo Supe io de In es igaciones Cien õ
®cas (CSIC),
Ramo
Ân y Cajal, 7, E-47005 Valladolid, Spain
Keywo ds: ad enal gland, aequo in, Ca
2+
,Ca
2+
channels, ca eine, ca echolamine, ciclopiazonic acid, yanodine, hapsiga gin
Abs ac
The endoplasmic e iculum (ER) has been sugges ed o modula e sec e ion ei he beha ing as a Ca
2+
sink o as a Ca
2+
sou ce
in neu onal cells. Wo king as a Ca
2+
sink, h ough ER-Ca
2+
pumping, i may educe sec e ion induced by di e en s imuli.
Ins ead, wo king as a Ca
2+
sou ce h ough he Ca
2+
induced Ca
2+
elease (CICR) phenomenon, i may po en ia e sec e ion
igge ed by ac i a ion o plasma memb ane Ca
2+
channels. We ha e p e iously demons a ed he p esence o CICR in bo ine
ch oma ®n cells, bu we now ®nd ha mouse ch oma ®n cells almos lack unc ional ca eine-sensi i e yanodine ecep o s in he
ER and, consis en ly, no CICR om he ER could be obse ed. In addi ion, inhibi ion o ER Ca
2+
pumping wi h ciclopiazonic acid
o hapsiga gin s ongly s imula ed high-K
+
-e oked ca echolamine sec e ion and cy osolic [Ca
2+
] ([Ca
2+
]
c
) ansien s. Su p isingly,
5m
Mca eine educed high-K
+
-induced [Ca
2+
]
c
peaks bu conside ably po en ia ed sec e ion induced by high-K
+
s imula ion.
Howe e , his po en ia ion was insensi i e o yanodine and addi i e o ha induced by emp ying he ER o Ca
2+
wi h
hapsiga gin, sugges ing ha i is un ela ed o he ac i a ion o yanodine ecep o s. We conclude ha , in mouse ch oma ®n cells,
CICR is no unc ional and he ER s ongly inhibi s sec e ion by ac ing as a dampe o he [Ca
2+
]
c
signal.
In oduc ion
Ca
2+
binding o yanodine ecep o s (RyR) p esen in he endoplas-
mic e iculum (ER) may lead o he opening o hese ecep o s,
igge ing Ca
2+
elease in o he cy osol (Be idge, 1998). This
phenomenon, known as Ca
2+
induced Ca
2+
elease (CICR), is a well-
cha ac e ized mechanism in hea cells, bu i s p esence and
unc ional ole in o he exci able cells, in pa icula neu onal cells,
is unclea . Th ee iso o ms o RyR a e exp essed in mammalian
issues. RyR1 and RyR2 a e exp essed p edominan ly in sa coplasmic
e iculum o skele al muscle and hea , espec i ely (Su ko & Ai ey,
1996; Zucchi & Ronca-Tes oni, 1997), bu all h ee RyR ha e been
also ound in b ain (McPhe son & Campbell, 1993; So en ino &
Volpe, 1993; Giannini e al., 1995; Ma in e al., 1998; Mo i e al.,
2000; Fau e e al., 2001) and o he issues, including he ad enal
gland (Giannini e al., 1995; Mack ill e al., 1997; Jime
Ânez &
He na
Ândez-C uz, 2001). The p esence o RyR in di e en neu onal
issues, oge he wi h he demons a ion o a unc ional ole o
ca eine- o yanodine-sensi i e Ca
2+
s o es in neu onal Ca
2+
ansien s o synap ic ansmission (Peng, 1996; Smi h & Cunnane,
1996; Mo he e al., 1998; Na i a e al., 1998, 2000; Llano e al.,
2000; Emp age e al., 2001; Ca e e al., 2002) has led o sugges an
impo an ole o CICR in Ca
2+
signalling, Ca
2+
homeos asis and
ac i i y-dependen synap ic plas ici y in neu ons (Rose & Konne h,
2001).
Measu ing CICR in small neu onal cells has p o en elusi e
because o he di ®cul y o dis inguishing Ca
2+
elease om Ca
2+
en y when only cy osolic [Ca
2+
] ([Ca
2+
]
c
) is moni o ed. In bo ine
ch oma ®n cells, we ha e shown unequi ocally he p esence o CICR
by measu ing di ec ly he [Ca
2+
] in he ER ([Ca
2+
]
ER
). S imula ion
wi h high-K
+
medium p oduced a la ge inc ease in [Ca
2+
]
c
in pa allel
wi h a apid dec ease o [Ca
2+
]
ER
media ed by RyR (Alonso e al.,
1999). Howe e , he impo ance o his phenomenon o ca echola-
mine sec e ion is unclea . Capaci ance measu emen s in bo ine
ch oma ®n cells ha e shown a dep ession in he sec e o y esponse
a e ER Ca
2+
deple ion wi h hapsiga gin (Molla d e al., 1995; Pan
& Fox, 2000). In he same cells, measu emen s in cell popula ions
ha e sugges ed ha he ER could beha e ei he as a Ca
2+
sou ce
(enhancing sec e ion) when i was ull o as a Ca
2+
sink ( educing
sec e ion) when i was ully emp ied by ca eine (La a e al., 1997).
In con as , i has been shown ecen ly (Cuchillo-Iba
Ân
Äez e al., 2002)
ha i e e sible emp ying o he ER wi h hapsiga gin modi®es li le
he sec e o y esponse induced by high-K
+
. Al hough mos o hese
da a sugges a ole o he ER in he modula ion o he sec e o y
esponse, he p ecise mechanism(s) o his modula ion emain
obscu e. We ha e used he e mouse ch oma ®n cells o s udy his
phenomenon and show ha hey beha e in a e y di e en way
ega ding he CICR phenomenon. They almos lack unc ional RyR
in he ER and bo h he [Ca
2+
]
c
inc ease and he sec e o y esponses
induced by high-K
+
we e s ongly po en ia ed by inhibi ing ER Ca
2+
pumping. In e es ingly, he sec e o y esponse was also po en ia ed
by low concen a ions o ca eine, bu his e ec was un ela ed o
CICR, as i was insensi i e o yanodine and addi i e o ha ob ained
a e ER Ca
2+
deple ion.
ejn2244
Co espondence: Ja ie Al a ez, as abo e.
E-mail: [email p o ec ed]
Recei ed 27 May 2002, e ised 25 July 2002, accep ed 23 Augus 2002
Q1
Eu opean Jou nal o Neu oscience, Vol. 16, pp. 1±8, 2002 ãFede a ion o Eu opean Neu oscience Socie ies
Unco ec ed p oo
Ma e ials and me hods
Animal and su gical p ocedu es
Expe imen s we e pe o med using ad enal medulla om mice o 6±
8 weeks. Mice we e anaes he ized wi h sodium pen oba bi one
adminis e ed in ape i oneally (60 mg/kg body weigh ). A e a
longi udinal incision in he abdomen, ad enal glands we e emo ed
and placed in a luci e chambe ®lled wi h ice cold Ty ode-bica bona e
solu ion (116 mMNaCl, 5 mMKCl, 2 mMCaCl
2
; 1.1 mMMgCl
2
,
5m
Mglucose, 23 mMNaHCO
3
) equilib a ed wi h 95%O
2
: 5%CO
2.
Unde a dissec ing mic oscope, ad enal glands we e gen ly
decapsula ed wi h ®ne o ceps (Dumon n°5) and s o ed in esh ice
cold Ty ode un il issues we e p ocessed o measu ing ca echola-
mine con en , ca echolamine sec e ion o moni o ing calcium le els
in dissocia ed cells om p ima y issue cul u es using ecombinan
aequo in.
Measu emen o ca echolamine con en
Ad enal medulla we e homogenized (glass/glass) in ice-cold
(200 mL) pe chlo ic acid 0.2 Mcon aining 0.01% EDTA and
cen i uged (10 000 3g o 10 min). Aliquo s om supe na an
(25 mL) we e injec ed di ec ly in o a high-pe o mance liquid
ch oma og aphy-elec ochemical de ec ion (HPLC-ED) sys em.
HPLC-ED sys em was composed o a Mil on Roy CM 400 pump
(Ri ie a, FL, USA), a Wa e c18 (pa icle size 4 mm) column, a
Wa e U6K injec o (Mil o d, MA, USA) and a Bioanali ical Sys em
(Wes La aye e, IN, USA) LC-4 elec ochemical de ec o (holding
po en ial o 0.65 V). Iden i®ca ion and quan i®ca ion o endogenous
con en we e pe o med agains ex e nal s anda ds (PeakSimple
Ch oma og aphy Da a Sys em; SRI ins umen s, Eas No walk, CT,
USA).
Ampe ome ic measu emen s o ca echolamine sec e ion
Ad enal medulla we e incuba ed in a dilu ed enzyma ic solu ion o
20 min a 37 °C wi h gen le agi a ion in o de o acili a e pene a ion
o he ca bon ®b e elec ode in o he issues. The dilu ed enzyma ic
Ty ode-bica bona e solu ion equilib a ed wi h 95%O
2
: 5%CO
2
con-
ains collagenase (0.1%, Wo hing on ype I, Lakewood, NJ, USA)
and ypsin (0.01% Sigma g ade IX). A e washing in esh ice-cold
Ty ode, issues p e iously subjec ed o he dilu ed enzyma ic
solu ion, we e ans e ed o a he mos a ized luci e eco ding
chambe (37 °C, olume 200 mL) and supe used by g a i y wi h
Ty ode bica bona e solu ion equilib a ed wi h 20% O
2
:5%
CO
2
: 75% N
2
(3±4 mL/min). The pe usion sys em had se e al
lines and swi ching om one o ano he was made by an elec onic
al e sys em. Bo h saline ese oi bo les and he supe usion ube
lines we e imme sed in a ci cula ing wa e ba h a 37 °C.
F ee ca echolamines we e measu ed using ca bon ®b e elec odes
composed o a single 5-mm ca bon ®b e insula ed, excep o he ip,
wi h a polye hylene ube (P oCFE, DAGAN Ins umen s,
Minneapolis, MN, USA). The elec odes we e a ached o an EI-
400 po en ios a (Ensman Ins umen a ion, Blooming on, IN, USA).
Reco dings we e unde aken wi h a ®xed ol age (0.6 V ampe o-
me ic mode) agains an AgCl e e ence elec ode. This ol age is
op imum o measu ing no epineph ine and epineph ine ( he main
ca echolamine con ained in mice ad enal medulla), as was demon-
s a ed by he HPLC-ED measu emen s o he ca echolamine con en
in mice ad enal medulla. Cu en s co esponding o ee ca echola-
mine measu emen s we e sampled a 4 Hz, eco ded by compu e
(Da a sponge, WPI Ins umen s, Sa aso a, FL, USA) and la e
analyzed o ¯ine (Mic ocal O igin, No hamp on, MA, USA).
Tissues we e impaled wi h a single 5-mm ca bon ®b e unde
mic oscope. A e a a iable pe iod (60±90 min) o supe usion in
con ol condi ions, he ampe ome ic eco dings became s able and
p epa a ions we e subjec ed o di e en expe imen al p o ocols, as
de ailed in he Resul s sec ion. In mos cases, p io o eco ding om
he issues he ca bon ®b e elec ode was ad anced in o he ba h
chambe and calib a ed by swi ching be ween Ty ode solu ion wi h o
wi hou 10 mMepineph ine. A he end o he expe imen , he
elec ode was wi hd awn om he issue and calib a ed again.
Compa ison o he calib a ion be o e and ollowing he s udy was
used o assess whe he he elec ode sensi i i y changed o e he
eco ding pe iod. As ca eine was used a high concen a ion (5 mM),
he possible e ec o he d ug on ou ampe ome ic eco ding sys em
was checked. I was ound ha ca eine, unde ou condi ions, nei he
gene a es any signal no in e e es in he measu emen o he
ca echolamine sec e o y esponse.
Measu emen s o [Ca
2+
]
c
and [Ca
2+
]
ER
. Decapsula ed ad enal
glands we e dissocia ed as desc ibed p e iously (He na
Ândez-Guijo
e al., 1998) by incuba ion o 40 min a 37 °C in Locke's medium
(154 mMNaCl, 5.6 mMKCl; 3.6 mMNaHCO
3
; 5.6 mMglucose,
10 mMHepes, pH 7.2) con aining 1.3 mg/mL collagenase
(Ho mann-La Roche, Basel, Swi ze land) and 3 mg/mL bo ine
se um albumin, wi h gen le pe iodic agi a ion. The suspension was
cen i uged o 10 min a 100 3gand esuspended in Locke's
medium. Cells we e hen pla ed on o 13 mm glass poly D-lysine-
FIG. 1. E ec s o ciclopiazonic acid on ca echolamine sec e ion and [Ca
2+
]
c
inc ease induced by high K
+
. In panel A, ca echolamine sec e ion induced
by ei he 5 s o 10 s pulses (ma ked by mo dsymbols a he bo om) o
medium con aining 35 mMKCl was measu ed in a whole mouse ad enal
medulla. When indica ed, 10 mMciclopiazonic acid (CPA) was pe used. In
panel B, [Ca
2+
]
c
peaks induced by 10 s pulses (d) o medium con aining
35 mMKCl we e measu ed in dissocia ed mouse ch oma ®n cells
exp essing cy osolic aequo in. When indica ed, 10 mMCPA was pe used.
Q2
2 R. Rigual e al.
ã2002 Fede a ion o Eu opean Neu oscience Socie ies, Eu opean Jou nal o Neu oscience, 16, 1±8
Unco ec ed p oo
coa ed co e slips and cul u ed in Dulbecco's modi®ed Eagle's
medium (DMEM) supplemen ed wi h 5% oe al cal se um, 50 IU/
mL penicillin and 50 mg/mL s ep omycin. A e allowing 6 h o
comple e cell a achmen , hey we e in ec ed wi h de ec i e he pes
simplex i uses ype 1 ca ying he co esponding cons uc o ei he
he cy osolic- o he ER- a ge ed aequo in chime a. P epa a ion o
mu a ed aequo in a ge ed o he endoplasmic e iculum has been
desc ibed be o e (Mon e o e al., 1995). Cy osolic aequo in cDNA
was ob ained om Molecula P obes and cloned in he pHSVpUC
plasmid. Vi us packaging and i eing ha e been p e iously desc ibed
(Alonso e al., 1998). Ch oma ®n cell cul u es we e ou inely in ec ed
wi h 2 310
3
in ec ious i us uni s 12±24 h p io o measu emen s.
Fo aequo in econs i u ion, cells exp essing wild- ype aequo in
a ge ed o he cy osol we e incuba ed o 1±2 h a oom empe a u e
wi h 1 mMo wild- ype coelen e azine in medium con aining 145 mM
NaCl, 5 mMKCl, 1 mMMgCl
2
,1mMCaCl
2
,10mMglucose, and
10 mMHEPES, 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. In
he case o aequo in a ge ed o he ER, Ca
2+
deple ion o he ER was
equi ed p io o econs i u ion. Fo his pu pose, cells we e incuba ed
o 10 min in medium con aining 145 mMNaCl, 5 mMKCl, 1 mM
MgCl
2
, 0.5 mMEGTA, 10 mM2,5-di- e -bu hyl-hyd oquinone (an
inhibi o o ER Ca
2+
ATPase), 10 mMglucose, and 10 mMHEPES,
pH 7.4. Then, a e washing once, cells we e placed in he same
medium in he p esence o 1 mMcoelen e azine n o 1±2 h. Be o e
s a ing he expe imen , cells we e placed in he pe usion chambe o
a luminome e (composed o a 9789-A pho omul iplie connec ed
h ough an ampli®e -disc imina o AD 2 o a PCB CT1 ca d in he
compu e , all componen s om Elec on Tubes, Ruislip, UK) and
pe used wi h he same medium wi hou 2,5-di- e -bu hyl-hyd o-
quinone o a leas 5 min. Expe imen s wi h ER- a ge ed aequo in
we e pe o med a 22 °C o educe he a e o aequo in consump ion.
Ma e ials
Wild- ype coelen e azine and coelen e azine n we e ob ained om
Molecula P obes Eu ope BV, Leiden, The Ne he lands.
Ciclopiazonic acid and yanodine we e om Sigma, Mad id.
Thapsiga gin was om Alomone Labo a o ies, Is ael. O he eagen s
we e om Sigma, Mad id o Me ck, Da ms ad .
Resul s
Cha ac e iza ion o he supe used in i o p epa a ion o mice
ad enal medulla
A new in i o supe used p epa a ion o ad enal medulla om mice
is epo ed in his wo k. This p epa a ion is use ul o s udy he
sec e ion o ca echolamines om he ad enal medulla as i is easy o
ob ain and allows moni o ing he sec e o y esponses e oked by
di e en s imuli in he in ac o gan wi h a good ime esolu ion.
Reco dings o he ca echolamine e ¯ux om he glands we e ini ia ed
a e a 60±90-min pe iod o allow s abiliza ion o he p epa a ion. As
shown below, he esponses e oked by sho pulses (5±10 s) o
depola izing s imuli we e qui e as , had a sho la ency and emained
cons an wi hou signi®can a enua ion o se e al hou s. Ano he
aspec ha we ha e cha ac e ized in ou p epa a ion was he con en
FIG. 2. E ec o ca eine, CPA and high K
+
on [Ca
2+
]
ER
. Dissocia ed mouse ch oma ®n cells exp essing ER- a ge ed aequo in we e deple ed o Ca
2+
and
econs i u ed wi h coelen e azine n. Then, when indica ed in he panels, medium con aining 1 mMCa
2+
was pe used o e®ll he ER wi h Ca
2+
. Once
[Ca
2+
]
ER
was almos a he s eady-s a e le el, ei he 50 mMca eine (panel A), 10 mMCPA (panel B), 10 s pulses o medium con aining 70 mMKCl (panels
C and D) o 5 mMca eine (panel D) we e pe used as indica ed.
Con ol o sec e ion by he endoplasmic e iculum 3
ã2002 Fede a ion o Eu opean Neu oscience Socie ies, Eu opean Jou nal o Neu oscience, 16, 1±8
Unco ec ed p oo
o ca echolamines. Using HPLC-ED, he alues ob ained om hese
measu emen s, exp essed in nmol/gland (mean 6SEM, n= 6), we e
1.08 60.30, 2.16 60.65 and 0.60 60.10 o no epineph ine,
epineph ine and dopamine, espec i ely. These esul s a e qui e
simila o hose p e iously epo ed by o he au ho s (Bo ns ein e al.,
1999; Bland e al., 2000). All o hese p ope ies make he in i o
p epa a ion o ad enal medulla om mice a good model o s udy he
modula ion o he elease esponse e oked by di e en s imuli.
Cyclopiazonic acid po en ia es bo h sec e ion and [Ca
2+
]
c
peaks e oked by high-K
+
Using he p epa a ion desc ibed abo e, we ha e ® s s udied he
con ibu ion o calcium s o es o he ca echolamine elease esponse
e oked by depola izing s imuli. Fi s , we emp ied he ER-Ca
2+
s o e
by inhibi ing he sa co-endoplasmic e iculum Ca
2+
ATPase
(SERCA) wi h cyclopiazonic acid (CPA). In his way bo h CICR
and Ca
2+
up ake by he ER we e abolished. Figu e 1a shows ha
pe usion o CPA 10 mMmodi®ed li le he es ing ca echolamine
elease bu p oduced a la ge inc ease in he sec e o y esponse e oked
when 5 o 10 s pulses o medium con aining 35 mMKCl we e
applied. The elease esponses we e inc eased o 148 65% o he
con ol alue (mean 6SEM, n= 9) when 10 s depola izing pulses
we e used and much mo e (273 636%, mean 6SEM, n= 6) o
sho e depola izing pulses. This e ec was no due o sa u a ion o
he sec e o y esponse, as s onge s imula ion (i.e. 60 mMK
+
,10s)
p oduced a much la ge sec e ion peak (up o 6- old highe ). The
same e ec s we e ob ained using hapsiga gin o emp y he ER o
Ca
2+
(Fig. 4a). These esul s sugges ha CICR is no unc ional in
hese cells and ha he ER beha es mo e as a sink o Ca
2+
han as a
sou ce unde es ing condi ions (wi h he ER ull o Ca
2+
). I his is
he case, inhibi ion o Ca
2+
pumping o he ER would be expec ed o
inc ease he [Ca
2+
]
c
le els ob ained a e he depola izing s imulus.
Figu e 1b shows ha his was he case. Pe usion o CPA p oduced a
small inc ease in he es ing [Ca
2+
]
c
le el, om 102 66nM o
157 615 nM(mean 6SEM, n= 7), bu conside ably inc eased he
[Ca
2+
]
c
peaks induced by high-K
+
ea men . In se e al simila
expe imen s, high-K
+
-induced [Ca
2+
]
c
peaks we e inc eased o
150 614% (mean 6SEM, n= 9) o he con ol alue in he
p esence o CPA.
Absence o CICR induced by high-K
+
and poo Ca
2+
elease
esponse o ca eine
Ano he pha macological manoeu e usually employed o ac i a ed
RyR is ca eine. In bo ine ch oma ®n cells, s imula ion wi h 50 mM
ca eine p oduces a e y as and ull emp ying o he ER (
1
2
<10s,
Alonso e al., 1999), and a signi®can elease esponse (Mon e o e al.,
2000). Hal -maximal e ec was ob ained wi h 5 mMca eine, and
lowe concen a ions (1±2 mM) p oduced li le e ec by hemsel es,
bu po en ia ed CICR induced by high-K
+
s imula ion (Alonso e al.,
1999). In con as , 50 mMca eine p oduced a e y small Ca
2+
elease om he ER in mouse ch oma ®n cells. Figu e 2a shows ha
50 mMca eine p oduced only a small ini ial Ca
2+
elease ha
educed he [Ca
2+
]
ER
by app oxima ely 50 mM(50 67mM,
mean 6SEM, n= 8), ollowed by a much slowe dec ease in he
ER Ca
2+
con en . Consis en ly, 50 mMca eine p oduced only a small
sec e o y esponse (da a no shown). Figu e 2b shows he e ec o
FIG. 3. E ec s o 5 mMca eine on high-K
+
-induced ca echolamine
sec e ion and [Ca
2+
]
c
peaks. Panel A shows a eco d o ca echolamine
sec e ion ob ained om a whole mouse ad enal medulla. Sec e ion was
s imula ed by pulses o 7 s o medium con aining 35 mMKCl (d). When
indica ed, 5 mMca eine (`Ca 5 mM') was pe used. Panel B shows a
eco d o [Ca
2+
]
c
in dissocia ed mouse ch oma ®n cells exp essing cy osolic
aequo in. Cells we e s imula ed by 5 s pulses o medium con aining 35 mM
KCl (d). When indica ed, 5 mMca eine was pe used (`Ca 5 mM').
FIG. 4. E ec s o ca eine, hapsiga gin and yanodine on high-K
+
-induced
ca echolamine sec e ion. The ®gu e shows ca echolamine sec e ion eco ds
ob ained om whole mouse ad enal medulla. Sec e ion was s imula ed by
ei he 10 s (panel A) o 7 s (panel B) pulses o medium con aining 35 mM
KCl (d). When indica ed, ei he 5 mMca eine (`Ca 5 mM'), 1 mM
hapsiga gin (`Tg 1 mM') o 10 mM yanodine + 50 mMca eine (`Ry
10 mM+ Ca 50 mM') we e pe used.
4 R. Rigual e al.
ã2002 Fede a ion o Eu opean Neu oscience Socie ies, Eu opean Jou nal o Neu oscience, 16, 1±8
Unco ec ed p oo
CPA on [Ca
2+
]
ER
o compa ison. Inhibi ing SERCA p oduces a
ela i ely slow elease o Ca
2+
om he ER, ha was in his case
much as e han ha induced by ca eine, and comple e. Gi en he
poo e ec o ca eine on ER-Ca
2+
elease, we expec ed ha CICR
could no be obse ed in hese cells. Figu e 2c shows ha his was he
case. In con as o he esul s ob ained in bo ine ch oma ®n cells
(Alonso e al., 1999), s imula ion wi h high-K
+
o mouse ch oma ®n
cells p oduced a apid inc ease in [Ca
2+
]
ER
, p obably because o he
ac i a ion o SERCA a e he high-K
+
-induced [Ca
2+
]
c
inc ease. The
p esence o low concen a ions o ca eine (2±5 mM) did no modi y
he e ec o high-K
+
on [Ca
2+
]
ER
(Fig. 2d). This panel shows also
ha a concen a ion o ca eine o 5 mM, ha p oduced a hal -
maximal Ca
2+
elease esponse in bo ine ch oma ®n cells (Alonso
e al., 1999) had no e ec on [Ca
2+
]
ER
in mouse ch oma ®n cells.
Ca eine po en ia es he sec e o y esponse e oked by
depola izing s imuli by a mechanism un ela ed o ER-Ca
2+
elease
The esul s o Figs 1 and 2 clea ly sugges ha CICR is no ope a i e
in mouse ch oma ®n cells. I was he e o e a su p ise o ®nd ha low
concen a ions o ca eine, which do no a ec he basal ca echola-
mine esponse, s ongly po en ia es he elease esponse p omo ed by
35 mMKCl. Figu e 3a shows ha pe usion o 5 mMca eine did no
modi y he sec e o y esponse by i sel , bu la gely inc eased ha
induced by high-K
+
s imula ion. In se e al simila expe imen s, high-
K
+
-e oked ca echolamine sec e ion was inc eased o 224 615%
(mean 6SEM, n= 12) o he con ol alue in he p esence o 5 mM
ca eine. Howe e , his e ec was no due o an inc ease in he high-
K
+
-induced [Ca
2+
]
c
peaks. Figu e 3b shows ha , ins ead, he [Ca
2+
]
c
ansien s induced by depola iza ion we e educed in he p esence o
ca eine 5 mM. In se e al simila expe imen s, [Ca
2+
]
c
peaks e oked
by high-K
+
in he p esence o ca eine we e educed o 66 63%
(mean 6SEM, n= 19) wi h espec o he con ols.
Fu he e idence in a ou o he sugges ion ha he e ec o
ca eine was un ela ed o CICR was ob ained by s udying i s e ec
a e emp ying he ER o Ca
2+
. Figu e 4a shows ha ca eine s ill
po en ia ed sec e ion a e ull and i e e sible deple ion o Ca
2+
om
he ER. Taking ad an age o he long pe iod o ime ha he in i o
p epa a ion o he mice ad enal medulla emains s able, we es ed
whe he ca eine s ill po en ia es he sec e o y esponse e oked by
10 s pulses o 35 mMKCl once he ER had been comple ely emp ied
o Ca
2+
wi h 1 mM hapsiga gin, a powe ul, selec i e and i e e sible
inhibi o o SERCA. In he expe imen shown in Fig. 4a, he ini ial
pa demons a es again he po en ia ion by ca eine o he sec e o y
esponse e oked by 35 mMKCl. A e washing away ca eine, he
con ol sec e o y esponses o high-K
+
s imula ion we e eco e ed.
Then, a 5 min supe usion wi h hapsiga gin was applied o
i e e sibly emp y he ER o Ca
2+
. I can be obse ed ha he
esponses o high-K
+
became po en ia ed a e his ea men ( o
166 613% o he con ol, mean 6SEM, n= 6), p obably by he
same mechanism ha in he case o CPA (Fig. 1). Finally, pe usion
o 5 mMca eine s ongly po en ia ed again he sec e o y esponses o
high-K
+
( o 269 627% o he esponse in he p esence o
hapsiga gin, mean 6SEM, n= 6). A e ea men wi h hapsiga -
gin, he esponse e oked by he depola izing s imulus in he p esence
o ca eine was app oxima ely he sum o he indi idual esponses
ob ained in he p esence o ei he hapsiga gin alone o ca eine
alone. The po en ia ing e ec o ca eine was he e o e addi i e o
ha o emp ying he ER o Ca
2+
, indica ing ha i is no due o
ac i a ion o CICR om he ER.
The p esence o RyR in subcellula loca ions o he han he ER is
con o e sial. I has been sugges ed, o example, ha RyR may be
p esen in he memb ane o he sec e o y esicles, and ha i may
media e Ca
2+
elease om he esicle (Ge asimenko e al., 1996;
Mi chell e al., 2001). In his way, hese RyR could con ibu e o he
elease o he Ca
2+
equi ed o he sec e ion o ha esicle. To
in es iga e i he e ec s o ca eine could be due o s imula ion o
RyR loca ed ou side he ER, we decided o use yanodine o block
RyR esponses independen ly o i s subcellula loca ion. Figu e 4b
shows a sec e ion expe imen in which we ® s demons a e he
e e sible po en ia ing e ec o 5 mMca eine. Once he high-K
+
-
induced sec e o y peaks had almos e u ned o he con ol le els, we
supe used he mice ad enal medulla wi h yanodine, he i e e sible
an agonis o yanodine ecep o s. Ryanodine (10 mM) was applied in
he p esence o high doses o ca eine o assu e he i e e sible
an agonis e ec o yanodine ha equi es he channel o be open in
o de o block i ( he so-called use-dependence o yanodine, Cheek
e al., 1994; Eh lich e al., 1994; Alonso e al., 1999). A long pe iod
o washing was allowed a e ha , du ing which he esponse o high-
K
+
s imula ion slowly dec eased, p obably as a esul o he slow
washing o he po en ia ing e ec o ca eine. A e ha , pe usion o
5m
Mca eine inc eased again he sec e o y esponse e oked by he
depola izing s imulus. O e all, hese esul s s ongly a gue agains
he in ol emen o RyR in he po en ia ion o sec e ion by ca eine.
The ac ual mechanism o his po en ia ion may hen ely on a
di e en p ope y o ca eine, e.g. he inhibi ion o phosphodies e -
ase. In ac , pe usion o 1 mM3-isobu yl-1-me hylxan hine, a
compound ha inhibi s phosphodies e ase wi hou ac i a ing RyR,
s imula ed sec e ion induced by high-K
+
o a simila ex en as
ca eine (da a no shown).
Discussion
In his pape we p o ide new e idence o he in ol emen o he ER
in he modula ion o ca echolamine sec e ion in ch oma ®n cells.
Blocking Ca
2+
pumping in o he ER, ei he wi h CPA o wi h
hapsiga gin, p oduced a s ong s imula ion o ca echolamine sec e-
ion, which was mo e e iden when sho high-K
+
s imula ions we e
used. Consis en ly, high-K
+
s imula ion p oduced a as inc ease in
[Ca
2+
]
ER
. These ®ndings sugges ha , a e cell depola iza ion, he
ER akes up a signi®can p opo ion o he Ca
2+
en e ing he cell
h ough ol age-ope a ed Ca
2+
channels, damping he sec e o y
esponse. This modula ion appea s o be pa icula ly impo an o
s imuli o small in ensi y. This seems qui e easonable, because when
he s imulus is oo in ense, he la ge inc ease in [Ca
2+
]
c
p obably
sa u a es ER Ca
2+
up ake, so ha he amoun o Ca
2+
aken up by he
ER becomes a small ac ion o he o al Ca
2+
en y. Unde hese
condi ions, Ca
2+
up ake by he ER canno be e ec i e o modula e
sec e ion. Ins ead, Ca
2+
up ake by he ER appea s o be p epa ed o
play a ole in he con ol o sec e ion induced by low-in ensi y, mo e
physiological, s imuli. In his case, ER egions close o he plasma
memb ane Ca
2+
channels may be able o seques e a signi®can
p opo ion o he Ca
2+
en e ing he cell h ough hose channels. O
cou se, Ca
2+
up ake by he ER is no he only mechanism esponsible
o cy osolic Ca
2+
clea ance. Mi ochond ia and plasma memb ane
Ca
2+
pump and Na
+
/Ca
2+
exchange also con ibu e o damp he
[Ca
2+
]
c
signal. On he o he hand, we should no e ha deple ion o
Ca
2+
om he ER may ha e addi ional e ec s, such as ac i a ion o
s o e-ope a ed Ca
2+
channels, which may con ibu e o he acili a ion
o exocy osis (Fomina e al., 1999). Howe e , blocking ER-Ca
2+
up ake wi h CPA o hapsiga gin p oduced only a small and ansien
inc ease in [Ca
2+
]
c
. This sugges s ha he di e en Ca
2+
clea ance
Q3
Con ol o sec e ion by he endoplasmic e iculum 5
ã2002 Fede a ion o Eu opean Neu oscience Socie ies, Eu opean Jou nal o Neu oscience, 16, 1±8

Unco ec ed p oo
mechanisms apidly ex ude he Ca
2+
eleased om he ER and also
ha s o e-ope a ed Ca
2+
channels a e no e y ac i e in hese cells.
The absence o CICR and he small e ec o ca eine on [Ca
2+
]
ER
in mouse ch oma ®n cells sugges ha hese cells possess only a
sca ce numbe o unc ional RyR in he ER. This came as a su p ise,
gi en he high ac i i y obse ed in bo ine ch oma ®n cells (Alonso
e al., 1999), whe e hey clea ly pa icipa e in CICR induced by high-
K
+
. Ch oma ®n cells ob ained om 19 o 31-day-old- a s a e also
e y sensi i e o ca eine, bo h in e ms o sec e ion and [Ca
2+
]
c
esponse (Finnegan e al., 1996; Guo e al., 1996). In e es ingly,
cul u es ob ained om 10-day-old a s lack a signi®can esponse o
ca eine, and only eco e i a e 15 days in cul u e in he p esence
o ne e g ow h ac o , in pa allel wi h an inc eased exp ession o
RyR ype 2 (Jime
Ânez & He na
Ândez-C uz, 2001). CICR appea s
he e o e no o be an essen ial mechanism o he de elopmen o he
sec e o y esponse in ch oma ®n cells, al hough i may ha e
physiological signi®cance when i is p esen . Rega ding he modu-
la ion by ER Ca
2+
up ake, deple ion o Ca
2+
om he ER in bo ine
ch oma ®n cells p oduced li le e ec (o pe haps a sligh enhance-
men ) on he sec e o y esponse induced by high-K
+
(Cuchillo-Iba
Ân
Äez
e al., 2002), bu educed sec e ion induced by cell depola iza ion
(Pan & Fox, 2000) and ha induced by ac i a ion o nico inic
ecep o s (Molla d e al., 1995; Cuchillo-Iba
Ân
Äez e al., 2002). Pe haps
he mix u e o ER-Ca
2+
up ake and CICR in hese cells may p oduce
compensa ing e ec s, ha could be a iable depending on he
s imulus and e en he e ogeneous a he single-cell le el. This could
also explain he con o e sial ®ndings men ioned abo e. In he
mouse, he absence o CICR helps o obse e uncon amina ed he
e ec s o ER-Ca
2+
up ake on he sec e o y esponse. We should
men ion also ha inosi ol 1,4,5- isphospha e ecep o s migh
pa icipa e as well in CICR phenomena. Mouse ch oma ®n cells
possess his kind o ecep o s, which can be ac i a ed by agonis s
such as ca bachol p oducing la ge [Ca
2+
]
c
peaks (1.70 60.14 mM,
mean 6SEM, n= 6). Howe e , his esponse equi es he p esence
o inosi ol 1,4,5- isphospha e, and is no expec ed o occu a e
high-K
+
s imula ion.
In he absence o unc ional RyR in he ER, he po en ia ing e ec
o low ca eine concen a ions on ca echolamine sec e ion igge ed
by cell depola iza ion was puzzling. This e ec pe sis ed a e ull
ER-Ca
2+
deple ion wi h hapsiga gin and also a e ea men wi h
yanodine. The e o e, we can conclude ha RyR a e no in ol ed in
ha po en ia ion. Addi ional e ec s o ca eine, such as phospho-
dies e ase inhibi ion and he subsequen inc ease o cyclic nucleo ide
le els, may be esponsible o he enhancemen o he sec e o y
esponse. Po en ia ion o ca echolamine sec e ion by an inc ease in
he le els o cyclic nucleo ides has been epo ed p e iously (Mo i a
e al., 1985; S a ke e al., 1989), and ou ®nding ha an inhibi o o
phosphodies e ase, 3-isobu yl-1-me hylxan hine, s imula es sec e ion
simila ly o ca eine, a gues also in a ou o his hypo hesis. On he
o he hand, he educ ion obse ed in he size o he high-K
+
-induced
[Ca
2+
]
c
peaks in he p esence o ca eine may be a ibu ed o he
inhibi ion o ol age-ope a ed Ca
2+
channels by his compound
(Villalobos & Ga cõ
Âa-Sancho, 1996). Mo eo e , hese ®ndings
indica e ha he e ec s o ca eine should no be aken as
unequi ocally due o ac i a ion o RyR o as an equi alen o CICR.
The di e en ial beha iou ega ding CICR o mouse and bo ine
ch oma ®n cells is a he su p ising. I has been epo ed p e iously
ha ch oma ®n cells om di e en species (cow, pig, ca , a o
mouse) show la ge di e ences in he ela i e exp ession o plasma
memb ane high- h eshold Ca
2+
channel sub ypes (He na
Ândez-Guijo
e al., 1998). We show he e ha ex eme di e ences (all-o -none) in
exp ession among di e en species can also be ound ega ding
in acellula Ca
2+
channels. Knowledge o hese p ope ies o mouse
ch oma ®n cells is in e es ing, because o he possibili y o gene a ing
ansgenic mice o s udy speci®c p oblems o Ca
2+
homeos asis and
sec e ion. On he o he hand, in e species di e ences obse ed in his
s udy aise ques ions as o he physiological impo ance o mechan-
isms such as CICR on he sec e o y esponse o ad enal cells, i i is
no obse ed in all species.
Acknowledgemen s
Financial suppo om Minis e io de Ciencia y Tecnologõ
Âa (BFI2002/01397)
and Jun a de Cas illa y Leo
Ân (VA 005/02) o JA, and om Fondo de
In es igaciones Sani a ias (FIS 01/0728) and Jun a de Cas illa y Leo
Ân (VA 46/
00B) o RR a e g a e ully acknowledged. We hank Da id Donnelly o
help ul discussions and commen s on he manusc ip .
Abb e ia ions
[Ca
2+
]
c
, cy osolic [Ca
2+
]; [Ca
2+
]
ER
, endoplasmic e iculum [Ca
2+
]; ER,
endoplasmic e iculum; CICR, Ca
2+
induced Ca
2+
elease; RyR, yanodine
ecep o s; SERCA, sa coendoplasmic e iculum Ca
2+
ATPase; CPA, ciclo-
piazonic acid; DMEM, Dulbecco's modi®ed Eagle medium; HPLC-ED, high-
pe o mance liquid ch oma og aphy-elec ochemical de ec ion.
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Q4
Q5
Q6
Con ol o sec e ion by he endoplasmic e iculum 7
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Que y
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Q1 Pan e al. 2000 has been changed o
Pan & Fox 2000 so ha his ci a ion
ma ches he lis
Q2 He nandez-Guijo e al. 1998 has been
changed o He na
Ândez-Guijo e al.
1998 so ha his ci a ion ma ches he
lis
Q3 Fomina & Nowycky 1999 has been
changed o Fomina e al. 1999 so ha
his ci a ion ma ches he lis
Q4 Ka hapaa & To nquis 1997 has no
been ound in he ex
Q5 K izaj e al. 1999 has no been ound
in he ex
Q6 Please supply jou nal, olume numbe
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8 R. Rigual e al.
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