Functional measurements of [Ca2+] in the endoplasmic reticulum using a herpes virus to deliver targeted aequorin

Resea ch Cell Calcium (1999) 24(Z), 87-96 8 Ha cou l B ace &Co. L d 1998 Func ional measu emen s o CCa2+1 in -~ he endoplasmic e iculum u&g d he pes i us o deli e a ge ed aequo in Ma ia Te esa Alonso, Ma ia Jose Ba e o, Es ela Ca nice o, May e Mon e o, Ja ie Ga cia-Sancho, Ja ie Al a ez Depa amen o de Bioquimica y Biologia Molecula y Fisiologia, lns i u o de Biologia y Gene ica Molecula (IBGM), Facul ad de Medicina, Uni e sidad de Valladolid y CSIC, Valladolid, Spain Summa y Changes in he ee calcium concen a ion o he endoplasmic e iculum ([Ca’+],,) play a cen al ole con olling cellula unc ions like con ac ion, sec e ion o neu onal signaling. We ecen ly epo ed ha ecombinan aequo in a ge ed o he endoplasmic e iculum (ER) [Mon e o M., B ini M., Ma saul R. e al. Moni o ing dynamic changes in ee Ca2+ concen a ion in he endoplasmic e iculum o in ac cells. EMBO J 1995; 14: 5467-5475, Mon e o M., Ba e o M.J., Al a ez J. [Ca2+] mic odomains con ol agonis -induced Ca2+ elease in in ac cells. FASEB J 1997; 11: 881-8861 can be used o moni o selec i ely [Ca2+le, in in ac HeLa cells. He e we ha e used a he pes simplex i us ype 1 (HSV-1) based sys em o deli e a ge ed aequo in in o a numbe o di e en cell ypes including bo h pos mi o ic p ima y cells (an e io pi ui a y cells, ch oma in cells and ce ebella neu ons) and cell lines (HeLa, NIH3T3, GH, and PC12 cells). Func ional s udies showed ha he s eady s a e lumenal [Ca*+],, anged om a ound 300 pM in g anule cells o 800 ). M in GH,cells. InsP,-coupled ecep o s imula ion wi h agonis s like his amine (in HeLa, NIH3T3 and ch oma in cells), UTP and b adykinin (in PC12 cells) o hy o opin- eleasing ho mone (TRH, in GH,cells) p oduced a e y apid dec ease in lumenal [Ca’+],,. Ca eine caused a apid Ca2+ deple ion o he ER in ch oma in cells, bu no in he o he cell ypes. Depola iza ion by high K+ p oduced an immedia e and e e sible inc ease o [Ca2+le in all he exci able cells (an e io pi ui a y, GH,, ch oma in cells and g anule neu ons). We conclude ha deli e y o ecombinan aequo in o he ER using HSV amplicon p o ides he i s di ec quan i a i e and dynamic measu emen s o [Ca2+le, in se e al p ima y non-di iding cells. INTRODUCTION Changes in he concen a ion o cy osolic Caz+ ([Ca”],) a e known o egula e many physiological esponses o di e en ex acellula s imuli, bo h in exci able and non- Recei ed 5 Ma ch 1998 Re ised 12 June 1998 Accep ed 16 June 1998 Co espondence o: D Ma ia Te esa Alonso, Depa amen o de Bioquimica y Biologia Molecula y Fisiologia. Facul ad de Medicina, Uni e sidad de Valladolid, 47005 Valladolid, Spain Tel: +34 983 423085; Fax: +34 983 423588 E-mail: [email p o ec ed] exci able cells. Recep o s imula ion can lead o a a ie y o complex spa io empo al pa e ns o inc eases in [Ca’+], like Ca2+ wa es and Ca2+ oscilla ions. The mechanisms o Ca2+ elease om he ER, o Ca2+ en y ac oss he plasma memb ane, and o seques e ing eleased Ca2+ unde lie hese complex esponses 13-51. The ER, o some o i s subcompa men s, is belie ed o be he majo cellula Ca2+ s o e, eleasing Ca2+ upon s imula ion o InsP,-coupled plasma memb ane ecep o s o ia Ca2+- induced Ca2+- elease (CICR), o cause ises in [Ca’+], [6]. Howe e , in spi e o e o s ocused on s udying he di e en componen s o calcium signaling, di ec and speci ic measu emen o [Ca2+] inside he ER o in ac cells has been a di icul ask. Recen ly, he cloning o he gene encoding o he pho op o ein aequo in has allowed a7 88 M T Alonso, M J Ba e o, E Ca nice o, M Mon e o, J Ga cia-Sancho, J Al a ez i s exp ession in mammalian cell cul u es. Fu he mo e, he aequo in gene has been modi ied by including de ined a ge ing signals. Thus, a numbe o aequo in chime as a ge ed o di e en subcellula compa men s such as mi ochond ia [7l, he nucleoplasm [8], cy osol [9], ER [l,lO,ll] and sa coplasmic e iculum [12] ha e been gene a ed. The a ge ing s a egy was ully success ul in e e y case, bu measu emen o [Ca*+], was pa icula ly di hcul because o he high [Ca2+] in ha compa men , which p oduced a apid consump ion o aequo in [1,13]. We ecen ly sol ed his p oblem by using coelen e azine n, a modi ied o m o he p os he ic g oup o aequo in which educes he a e o luminescence and allows he pe o - mance o long-las ing measu emen s o [caZ+],, in in ac cells [2,14]. Howe e , al hough he use o hese chime as has ep esen ed a landma k in he ield o subcellula [Ca2+] in es iga ion, mos s udies ha e been pe o med in one pa icula cell line (HeLa). This es ic ion is due o he di hcul y in in oducing plasmid DNA in o p ima y pos mi o ic cells by non- i al me hods such as Ca2+ phospha e, elec opo a ion o liposomes. To o e come his limi a ion, we decided o de elop a deli e y sys em capable o di ec ing he exp ession o ER- a ge ed aequo in in di e en cell ypes and pa icula ly in p ima y cells, he mos in e es ing ones om a physiological poin o iew. He pes simplex i us ype 1 (HSV-1) is a neu o opic i us wi h many unique ea u es ha make i sui able as a gene ans e ec o o mammalian cells, bo h in i o and in i o [ 151. I s cha ac e is ics include wide hos cell ange, e icien in ec ion, abili y o deli e genes o bo h di iding and non-di iding cells, long- e m pe sis ence and he capaci y o accommoda e la ge molecules o o eign DNA. In pa icula , he na u al opism o he pes i us o in ec pos mi o ic neu ons is an ad an age ha o e comes he di icul y o ans ec hese cells. In his epo , we desc ibe an amplicon ha e icien ly exp esses aequo in a ge ed o he ER (PHSVe AEQ) in a a ie y o cell lines and non di iding p ima y cells, in pa icula an e io pi ui a y cells (AP), ch oma in cells and g anule neu ons. The co ec subcellula aequo in loca ion was es ablished by immunocy ochemis y and by di ec displaying o Ca-sensi i e luminescence in each in ec ed cell ype. Ou esul s show ha deli e y o ecombinan aequo in o he ER using HSV amplicon is a simple and e icien me hod o moni o di ec ly [Ca2+],, in adul p ima y cells such as AP cells, ch oma in and neu ons, as well as in se e al cell lines in cul u e. MATERIALS AND METHODS Cell cul u e NIH3T3, wild ype HeLa cells and he e AEQmu -HeLa cell clone EM26 [l] we e g own in DMEM supplemen ed Cell Calcium (1998)24(2), 67-96 wi h 10% FCS, 2 mM glu amine, 100 U/ml penicillin and 100 mg/ml s ep omycin. The 2-2 cell line which con ains he HSV- 1 IE2 gene [ 161 was g own in he same medium. PC12 and GH, cells we e main ained in DMEM wi h 10% e al cal se um and 5% ho se se um. An e io pi ui a y (AP) cells we e isola ed om adul a s as p e iously desc ibed [17]. Bo ine ad enal ch oma in cells we e kindly p o ided by P o . An onio Ga cia, Uni e sidad Au dnoma de Mad id, Spain [ 181. Ce ebella g anule cells we e p epa ed om 4-7 day-old a s as p e iously epo ed [ 191. Exp ession o aequo in in cell cul u es using de ec i e HSV ec o s Plasmid cons uc ion The chime& low-a ini y mu an o aequo in e AEQmu has been desc ibed p e iously [l]. The e AEQmu cDNA was isola ed om he o iginal ec o by EcoRI diges ion and cloned in o he ecipien ec o pHSVpuc [20] o gene a e he pHSVe AEQ. The pHSVlac amplicon ec o which exp esses he P-galac osidase gene o Esche idzia coli has been desc ibed p e iously [2 11. Packaging o HSV- 1 ec o s pHSVe AEQ DNA was packaged in o HSV-1 pa icles using a dele ion mu an packaging sys em [22]. In b ie , 3 x lo5 2-2 cells we e seeded on 60 mm dishes and ans ec ed wi h 6 pg o pHSVe AEQ o pHSVlac using he lipo ec amine p ocedu e acco ding o he manu- ac u e ’s p o ocol (Gibco, BRL, Mad id, Spain). One day la e he medium was eplaced wi h 3 ml DMEM, 5% FBS and cells we e in ec ed wi h -2 x lo6 plaque- o ming uni s o 5dll.2 helpe i us which con ains a dele ion in he IE2 gene o HSV-1 s ain KOS [23]. On he ollowing day, i us was ha es ed and subsequen ly passaged on esh 2-2 cells 3 imes o inc ease bo h he a io o ec o o helpe and he o al amoun o i us. Vec o i e ing Ti e s we e measu ed in PC12 as desc ibed p e iously [22]. Cells we e seeded in o poly-L-lysine coa ed 24-well pla es a 5 x lo5 cells/well. The nex day, cells we e in ec ed wi h a ious dilu ions o each i us and 24 h la e he cells we e ixed in 4% pa a o maldehyde o 30 min. pHSVlac in ec ed cells we e de ec ed as p e iously desc ibed [24]. pHSVe AEQ in ec ed cells we e isualized by using a mouse an i-HA1 p ima y an ibody 1:200 (Boeh inge , Ge many) ollowed by an AP-conjuga ed an i-mouse seconda y an ibody (Sigma, Spain). Alkaline phospha ase was isualized using ni oblue e azolium and 5-b omo-4-chlo o-3-indolyl phospha e acco ding o he manu ac u e ’s ins uc ions (Sigma, Spain). S ained 0 Ha cou B ace & Co. L d 1998 Vi al ans e o ER- a ge ed aequo in 89 cells we e coun ed by mic oscopy and i e s o in ec ious pa icles we e calcula ed. Ti e s o ec o we e exp essed as in ec ious ec o pa icles (i p) pe ml. In ec ion o cell cul u es To assay o he capabili y o he HSV i uses o in ec he di e en cell ypes, cul u es we e i s in ec ed wi h pHSVlac. Cells we e seeded a semicon luency on o a 13 mm co e slip and in ec ed 24 h p io o expe imen s. HeLa cells and NIH3T3 mouse ib oblas s we e in ec ed wi h 1.2 x IO4 i phnl. PC12, GH, and AP cells we e in ec ed wi h 4.8 x lo4 i phnl, ch oma lim cells wi h 3.6 x lo4 i p/ml and g anule cells wi h 72 x 104 i phnl. Using an an ibody agains HA1 we ou inely obse ed a ange be ween 5-30% o cells exp essing aequo in. Con ol expe imen s we e pe o med o es o cy o oxici y by compa ing he e ec o ex acellula agonis s on [Caz+], in bo h in ec ed and non in ec ed PC 12 cells (see below, Fig. 4B). Mo eo e , measu emen s o ca echolamine sec e ion om in ec ed bo ine ch oma in cells s imula ed wi h ca eine o 70 mM K+ we e i ually iden ical o he ones ob ained in unin ec ed cells (P. Michelena and A. Ga cia, unpublished da a). Judging om hese wo unc ional analyses, i al in ec ion did no p oduce signi ican cy o oxici y in he es ed cell ypes. lmmuno luo escence Cells we e ixed 24 h a e in ec ion wi h 4% pa a o m- aldehyde in PBS o 20 min, washed wice wi h PBS and once wi h TBS (100 mM T is-HCI pH 75, 150 mM NaCl). Cells we e hen blocked o 5 min in TST (0.1% T i on X- 100, 1% FCS in TBS) and incuba ed wi h he an i-HA1 monoclonal an ibody 12CA5 (Boeh inge , dilu ion 1:200) dilu ed in TST o e nigh a 4°C. A e wo washes in TBS, samples we e incuba ed wi h an i-mouse IgG an ibody FITC conjuga ed (dilu ion 1:200) o l-4 h a oom empe a u e. Fluo escence was analyzed ou inely wi h a Nikon Diapho mic oscope. Con ocal mic oscopy analysis was pe o med in a Zeiss ISCM 3 10 appa a us wi h a 488 nm band. In he [Caz+], expe imen s pe o - med in he in ec ed PC12 cells (Fig. 4B), an abb e ia ed immuno luo escence p o ocol was ca ied ou a he end o he [Ca2+], measu emen s [25]. [Ca*+&, measu emen s Cells we e seeded on o 13 mm poly-L-lysine- ea ed glass co e slips and in ec ed 24 h p io o he expe imen s. Be o e econs i u ing aequo in, [Ca2+],, was educed by incuba ing he cells o 10 min a 37°C wi h he sa coplasmic and endoplasmic e iculum Ca2+-ATPase (SERCA) inhibi o 2,5-d& e -bu yl-benzohyd oquinone A lg hea y chain gene AEQ cDNA * I I L VDJ CHl I;IBABI AEQ e AEQ , lkb , B EcoR I Fig. 1 (A) Schema ic map o he chime ic e AEQ cDNA. In he lg moie y, coding and in onic egions a e ep esen ed by shaded boxes and hick lines, espec i ely; in he aequo in moie y, coding and non-coding sequences a e indica ed by a whi e box and a hin line, espec i ely. The sho sequence encoding he HA1 ag and he poin mu a ion (Asp11 9 + Ala, as e isk) a e indica ed. (B) Schema ic diag am o he amplicon pHSVe AEQ plasmid. The ansc ip ional uni con ains he IE 4/5 p omo e , he e AEQ chime ic gene and a polyadenyla ion signal. Two gene ic elemen s om HSV-1, he o i, and he HSV packaging sequences, allow eplica ion and packaging o he amplicon. The p oka yo ic sequences con ain a bac e ial o igin o eplica ion and an ampicillin selec ion ma ke ha allow p opaga ion and ampli ica ion in E. co/i. A simila ec o , ca ying he E. co/i /acZ gene in place o e AEQ was used as a con ol. (BHQ) 10 PM in KRB (K ebs-Ringe modi ied bu e : 125 mM NaCl, 5 mM KCl, 1 mM Na,PO,, 1 mM MgCl,, 5.5 mM glucose, 20 mM HEPES, pH 74) supplemen ed wi h 3 mM EGTA. Cells we e hen incuba ed o 1 h a oom empe a u e in KRB con aining 0.5 mM EGTA, 10 j&l BHQ and 0.5 mM coelen e azine n. The co e slip was hen washed o 5 min in KRB con aining 0.5 mM EGTA, 5% bo ine se um albumin and 10 j. M BHQ and inally placed in he pe usion chambe o a pu pose-buil he mos a ed luminome e . Expe imen s we e pe o med ei he a 37°C 0 Ha cou B ace & Co. L d 1998 Cell Calcium (1998) 24(2), 67-96 90 M T Alonso, M J Ba e o, E Camice o, M Mon e o, J Ga cia-Sancho, J Al a ez Fig. 2 lmmunolocaliza ion o he e AEQ in he in ec ed cells. Con ocal images o HeLa cells (A) and GH, pi ui a y cells (B) in ec ed wi h he pHSVe AEQ i us 24 h p io o he s aining wi h he an i-HA1 an ibody e ealed wi h an FITC-labeled an i-mouse seconda y an ibody. o a 22°C. [Ca2+],, alues we e calcula ed om he luminescence eco ds using an algo i hm which ollows he calib a ion cu es p e iously epo ed [2,9,14]. [Ca*+], measu emen s The cells we e seeded on o 12 mm poly-L-lysine ea ed co e slips and in ec ed 24 h p io o he expe imen s. Cells we e loaded wi h 4 @VI Fu a- and single-cell [Ca”+], measu emen s and ime- esol ed digi al image analysis we e pe o med as p e iously desc ibed [25]. RESULTS P oduc ion o pHSVe AEQ i us and exp ession o a ge ed aequo in The s uc u e o he chime ic cDNA coding o aequo in a ge ed o he ER has been p e iously desc ibed [l] and is depic ed in Figu e 1A. This chime a esul s om he usion o aequo in wi h he C- e minus o se e al domains o he Ig@b hea y chain esponsible o a ge ing he gene o he ER. The cons uc also includes an HA1 epi ope ag o allow de ec ion by immunocy ochemis y and aequo in cDNA ca ies a mu a ion (Asp1 19 + Ala) in one o he h ee Caz+ binding si es o educe he a ini y o Ca2+ o he pho op o ein. The pHSVe AEQ plasmid ec o is shown schema ically in Figu e 1B. The e AEQ chime ic cons uc is cloned in he EcoRI si e in o he pHSVpuc amplicon [20] unde he con ol o he HSV-1 IE (immedia e ea ly gene) 4/5 p omo e and hen packaged in o i us pa icles as ou lined in Ma e ials and me hods. Cell Calcium (1998) 24(2), 87-96 In o de o e i y he co ec exp ession and localiza ion o he a ge ed p o ein deli e ed by he i al ec o , cell cul u es we e in ec ed wi h he pH5Ve AEQ i us and he ansduced p o ein was de ec ed by immunochemis y using a monoclonal an ibody o he HA1 epi ope o he aequo in moie y. Figu e 2 shows he con ocal luo escence images o aequo in dis ibu ion in wo ep esen a i e cell ypes: HeLa (Fig. 2A) and pi ui a y GH, cells (Fig. 2B). The images show a clea pe inuclea e icula e s aining ex ending o he pe iphe y o he cell which is cha ac e is ic o a p o ein localized in he ER. Simila pa e ns o localiza ion we e obse ed in he es o he s udied cell ypes (da a no shown). De ailed e idence showing ha he exp essed e AEQ usion p o ein localizes speci ically in he lumen o he ER has been epo ed p e iously in HeLa cells, i s om s udies o colocaliza ion wi h he ER ma ke ERp72 [l] and mo e ecen ly om elec on mic oscopy s udies [ 131. Func ional measu emen s in cell lines To in es iga e whe he he exp essed aequo in was ully unc ional, we measu ed changes in he [Ca2+],,in in ec ed HeLa cells and compa ed hem wi h hose ob ained using a s able ans ec ed clone o HeLa cells (clone EM26, [l]) exp essing he same chime& aequo in cloned in he mammalian exp ession ec o pcDNA unde he con ol o he cy omegalo i us p omo e . Figu e 3 shows he ime cou se o Ca2+ eloading and elease o he ER in pHSVe AEQ in ec ed HeLa cells (Fig. 3A) and in he HeLa EM26 clone (Fig. 3B). Bo h cell ypes we e deple ed o Caz+ and econs i u ed wi h coelen e azine n as desc ibed in 0 Ha cou B ace & Co. L d 1998 Vi al ans e o M- a ge ed aequo in 91 B 01 - Fig. 3 Compa ison be ween he e ec o his amine on [Ca2+le in pHSVe AEQ-in ec ed HeLa ceils (A) and in he HeLa EM26 clone (B). Wild ype HeLa cells we e in ec ed wi h 1.2 x lo4 i plml24 h p io o measu emen s. Bo h cells we e deple ed o CaZ+ and econs i u ed wi h coelen e azine n o 1 h p io o measu emen s. Whe e indica ed, medium con aining ei he only 1 mM CaCI, (Ca*+) o wi h 100 FM his amine (His) was pe used. Measu emen s we e pe o med a 37°C. The o al luminescence coun s pe co e slip we e 0.36 0.13 x 10” coun s/s (cps, n = 3) in (A) and 12.8 2 1 .l x 1 O6 cps (n = 17) in (6). These expe imen s a e ep esen a i e o a leas 3 simila ones. ‘Ma e ials and me hods! The addi ion o 1 mM ex a- cellula Ca2+ p oduced a smoo h inc ease in [Ca2+le leading o a s eady-s a e o 540 * 40 PM (mean SD, n = 3) wi hin 2 min in he pHSVe AEQ in ec ed HeLa cells (Fig. 3A). This alue o [Ca2+],, was e y simila o 550 70 @VI (n = 45, [Z]), ob ained in he EM26 clone (Fig. 3B). A subsequen challenge wi h 100 @I his amine e oked an immedia e dec ease o [Ca2+],, in bo h cell ypes wi h simila kine ics. His amine is known o mobilize in acellula Ca2+ ia gene a ion o InsP, in hese cells 1261. Al hough he o al aequo in luminescence was much lowe in he in ec ed HeLa cells han in he s able clone (see igu e cap ion), he calib a ed signal showed no signi ican di e ences be ween he dynamics o [Ca2+],, in bo h ypes o cells. A simila pa e n o [Ca2+],, esponse was ob ained when he ela ed ib oblas cell line NIH3T3 was in ec ed (da a no shown). Ra pheoch omocy oma PC12 cells a e a neu o- sec e o y cell line e y equen ly used as a ne e cell model because hey sha e a a ie y o p ope ies wi h neu ons. Cell cul u es we e exposed o UTP and b adykinin, agonis s coupled o polyphosphoinosi ide A 350 ca=+ UTP BK 300 - - 5 250 - z 200 - 5 150 - 100 - 50 - o- - Con ol ---- HSV in ec ed 600 i :: h 1 min - Fig. 4 E ec s o UTP, b adykinin (BK) and high K+ on [Ca*+],,(A) and ICa2+1- Bj in oHSVe AEQ-in ec ed PC12 cells. Cells we e in ec ed & 4.8 ;( lo4 i p/ml24 h p io o measu emen s, deple ed o CaZ+ and ei he econs i u ed wi h coelen e azine n ( o measu ing [Ca*+],,, A) o loaded wi h Fu a- ( o measu ing Ca*+l~. B). 100 uM UTP. 1 uM b ad kinin and 70 mM K+ we e addez’when indica ed. in (B), an immuno luo escence assay using he an i-HA1 an ibody was ca ied ou ‘on line’ a he end o he [Ca2+], measu emen s o iden i y he in ec ed cells. The solid line is he a e age o 57 con ol (non-in ec ed) cells and he dashed line he a e age o 10 in ec ed cells in he same mic oscope ield. The [Ca*+], inc emen s (measu ed a he [Cap+lc peak) we e (mean SE): 435 24 nM (con ol) compa ed o 531 73 nM (in ec ed) o UTP; 452 * 17 nM (con ol) and 575 46 nM (in ec ed) o BK; and 519 * 20 nM (con ol) compa ed o 430 * 62 nM (in ec ed) o high K+. The o al luminescence coun s pe co e slip we e 1.86 0.63 x lo6 cps (n = 5). These expe imen s a e ep esen a i e o a leas 3 simila ones. hyd olysis h ough he P2x pu icep o [27] and he B2 ecep o [28], espec i ely. Figu e 4 shows he esul s ob ained in PC12 cells in ec ed wi h pHSVe AEQ 24 h p io o he measu emen s. The s eady-s a e le el o [Ca2+le was 370 * 70 PM (n = 3) and addi ion o 100 PM UTP igge ed a e y apid Caz+ elease (Fig. 4A). [Ca”],, e u ned o he le el p io o s imula ion wi hin 2 min and subsequen s imula ion wi h 1 l&i b adykinin (BK) p oduced a u he e e sible dec ease in [Ca*+],,. These indings a e consis en wi h he esul s in Figu e 4B 0 Ha cou B ace & Co. L d 1998 Cell Calcium (1998) 24(2), 87-96 92 M T Alonso, M J Ba e o, E Ca nice o, M Mon e o, J Ga cia-Sancho, J Al a ez Ca B 1600- 1400 - Ca * K = 600. C 1000 I 800 - OL E 600 ,u +o * 200 0- Ca T= /’ Ca Ca TX T= 800. 2 & 600. 3 4*- 200 - D 700. 600 - 2 500- l 400 2 300 3 2oo. IOO- O- F 800 - 5600. Ca TX Fig. 5 E ec s o high K+ and TRH on [Ca2+le,and [Ca’+], in pHSVe AEQ-in ec ed GH, cells (A,C,E) and AP cells (B,D,F). Cells we e in ec ed wi h 4.8 x lo4 i p/ml 24 h p io o measu emen s, deple ed o Ca2+and ei he econs i u ed wi h coelen e azine n ( o measu ing [Ca*+]-., A-D) o loaded wi h Fu a- ( o measu ing [Ca*+],, Eand F)yThe EP was e illed by incuba ion wi h 1 mM- CaCI. (Ca) and ei he 50 mM K+ (A and B) o 100 nM TRH [C-F) we e’ac ded when indica ed. The- o al lum nescence coun s’pe ’ co e slip we e 0.98 * 0.09 x 10” cps (n = 18) o GH,cells and 0.68 k 0.10 x lo6 cps (n = 19) o AP cells. T aces (E) and (F) ep esen he a e age o 67 GH,and 57 AP ceils, espec i ely, p esen in he same mic oscope ield. All he measu emen s we e pe o med a 22°C. Each expe imen is ep esen a i e o a leas 3 simila ones. showing he cy osolic Ca2+ inc ease by he same concen a ions o UTP and b adykinin in Fu a- loaded PC12 cells. Depola iza ion wi h high K+ (70 mM) also elici ed a [Ca2+], ise. In his igu e, he [Ca2+lc esponses in con ol cells (a e age o 57 cells, solid line) and in in ec ed cells (a e age o 10 cells, dashed line) a e also di ec ly compa ed. The in ec ed cells we e iden i ied by immuno- luo escence a he end o he expe imen in he same mic oscope ield used o he Ca2+ imaging expe imen (see ‘Ma e ials and me hods’). Bo h he maximal alue and he Cell Calcium (1998) 24(2), 87-96 in eg a ed a ea unde he peak o each agonis we e no signi kan ly di e en in con ol and in ec ed cells. These esul s indica e ha he ansduced aequo in was ully unc ional and ha i al in ec ion did no modi y unc ional pa ame e s such as agonis -induced Caz+ elease, sugges ing ha Ca2+ egula ion was essen ially unal e ed in pHSVe AEQ in ec ed cells. Func ional measu emen s in p ima y cells To exploi he capaci y o he pes i us o in ec a wide ange o cells, we nex exp essed a ge ed aequo in and measu ed [Ca2+le in di e en p ima y cul u e cells. Mos o he expe imen s we e ca ied ou a 22°C because he a e o aequo in consump ion a his empe a u e is one o de o magni ude lowe han a 37”C, and his allows moni o ing [Ca2+],,du ing longe imes [14]. Pi ui a y cells a e a model o s udying he s imulus- sec e ion coupling o pi ui a y ho mones. They espond o hy o opin- eleasing ho mone (TRH) wi h a apid inc ease in [Ca2+lc due bo h o elease o Ca2+ om in acellula s o es and o s imula ed Ca2+ in lux om he ex acellula medium [29]. They possess ol age- dependen Caz+-channels ha ac i a e on depola iza ion o he plasma memb ane, p oducing a as peak in cy osolic Ca2+ [17,25]. In Figu e 5 we compa ed he e ec s o bo h TRH and depola iza ion wi h 50 mM K+ on [Caz+],, and [Ca’+], in he GH, a pi ui a y cell line (Fig. 5A,C,E) and in p ima y AP cells (Fig. 5B,D,F), om which he cell line GH, de i es. Bo h cell ypes we e in ec ed wi h pH.SVe AEQ i us du ing 24 h. Cells we e deple ed o Ca2+ as desc ibed in Ma e ials and me hods and hen eloaded by addi ion o 1 mM ex acellula Ca2+ as indica ed in he igu e. [Ca2+],, inc eased smoo hly du ing 4-5 min and eached a s eady-s a e a 780 + 70 @VI and 600 + 150 @I in GH, and AP cells (mean SD, n = 7) espec i ely. Depola iza ion wi h 70 mM KC1 induced a simila esponse in bo h pi ui a y cul u es, esul ing in a apid o e shoo o [Ca2+],, om 780 @I up o abou 1.3 mM in GH, cells (Fig. 5A) and nea 1.5 mM in p ima y AP cul u es (Fig. 5B). In con as , s imula ion wi h TRH esul ed in a apid and sus ained dec ease o [Ca2+le , (by 470 150 )&l, n = 3) in GH, cells (Fig. 5C), bu only in a mino dec ease in [Ca2+],, o a ound 70 )&I in AP cells (Fig. 5D). The small CaZ+ esponse o TRH obse ed in AP cells canno be a ibu ed o inhibi ion o Ca2+ elease by cy osolic Ca2+, as ound o HeLa cells [2,14], because loading he cells wi h BAPTA did no modi y he e ec o TRH on [Ca2+],, (da a no shown). In con as o his s iking di e ence in he [Ca2+],, esponses, measu emen s o [Caz+], in GH, (Fig. 5E) and AP cells (Fig. 5F) loaded wi h Fu a- and s imula ed wi h TRH showed e y simila esponses. In bo h cases, mos o he indi idual cells (50- 750/0) esponded o TRH wi h a as and ansien [Ca2+], 0 Ha cou B ace & Co. L d 1998 Vi al ans e o ER- a ge ed aequo in 93 A 700 600 500 400 300 200 100 0 His Ca B 700 600 3 500 b 400 +” 300 ( I 0 200 2 100 0 Ca2+ Ca Ca 2 Fig. 6 E ec s o his amine and ca eine on [Ca*+],,in pHSVe AEQ- in ec ed bo ine ch oma in cells. Cells we e in ec ed wi h 3.6 x lo4 i plml24h p io o measu emen s. (A) 10 FM his amine (His) o 10 mM ca eine (Ca ) was added when indica ed. (B) Two pulses o 10 mM ca eine we e added as indica ed. The o al luminescence coun s pe co e slip we e 1.05 2 0.16 x lo8 cps (n = 14). O he de ails as in Figu e 3. These expe imen s a e ep esen a i e o a leas 3 simila ones. inc ease. The a e age inc ease o [Ca2+], a he peak was abou 600-800 no in bo h cell ypes. One o he main ad an ages o he HSV amplicon sys em is he abili y o in ec non-di iding cells such as neu ons. This allows using he pHSVe AEQ i us o s udy he dynamics o neu onal Caz+ s o es. In his epo , we ha e employed wo di e en cell ypes: ch oma m and ce ebella g anule cells. Ad enal medulla ch oma in cells can be ega ded as a highly specialized o m o pos - ganglionic sympa he ic neu ons. They de i e emb yo- logically om he neu al c es , a e elec ically exci able and elease esicle-con ained subs ances in esponse o speci ic s imuli. They a e equen ly u ilized in s udies on CICR mechanisms and he ela ionship be ween InsP,- and yanodine-sensi i e s o es. He e, we ha e used his amine o ac i a e he InsP,-sensi i e Ca*+ elease mechanism and ca eine o ac i a e he CICR mechanism p esen in hese cells 1301. Figu e 6 shows he esul s ob ained in pHSVe AEQ in ec ed bo ine ch oma in cells. The addi ion o 1 mM Ca2+ o Ca2+-deple ed ch oma in cells induced an ele a ion o he [Ca2+le up o a s eady- A Ca2+ 600 GllJ B 600 I 500 Y 400 jg 300 + c l 200 z 100 0 Ca2+ 2 min Fig. 7 E ec s o glu ama e (Glu) and high K+ on [Ca2+le in pHSVe AEQ-in ec ed ce ebella a anule cells. Cells we e in ec ed wi h 7.2 x 1 O4 i plml 24 h p io o-measu emen s. 100 PM glu ama e (A) and 50 mM K+ (B) we e added as indica ed. The o al luminescence coun s pe co e slip we e 0.19 0.07 x 1 O6 cps (n = 4). O he de ails as in Figu e 3. These expe imen s a e ep esen a i e o a leas 3 simila ones. s a e o 650 100 mM (n = 13) wi hin 4-5 min (Fig. 6A). Subsequen exposu e o 10 @VI his amine igge ed a apid dec ease o [Ca2+le, o abou 60% o he ini ial le el. A e washing ou he agonis , he ER eloaded wi h Ca*+ up o he same s eady-s a e, and he cells esponded o a la e applica ion o 10 mM ca eine wi h a second d op in [Ca2+],,. A simila pa e n was ob ained, bo h in ime cou se and ampli ude, when he cells we e challenged wi h wo consecu i e pulses o 10 mM ca eine (Fig. 6B). These da a indica e ha wo di e en s imuli, ac ing ei he ia InsP, ecep o s o ia yanodine ecep o s, a e able o elease compa able amoun s o Ca*+ om he ER in ch oma m cells. Finally, a p ima y pos mi o ic neu onal cul u e was used. We deli e ed he a ge ed aequo in in o a ce ebella g anule cells by in ec ing hem wi h he pHSVe AEQ i us. Figu e 7 illus a es a ep esen a i e example in which g anule cells we e s imula ed ei he wi h glu ama e o wi h 50 mM K+. The s eady-s a e le el o [Caz+], was 300 + 60 I.~M (n = 3), somewha lowe han in he o he cell ypes s udied. Addi ion o 100 PM 0 Ha cou B ace & Co. L d 1998 Cell Calcium (1998) 24(2), 87-96 94 M T Alonso, M J Ba e o, E Ca nice o, M Mon e o, J Ga cia-Sancho, J Al a ez glu ama e (Fig. 7A) elici ed an inc ease in [Ca2+], ollowed by a slow decline accele a ed by emo al o he agonis . Me abo opic glu ama e ecep o agonis s like quisquala e ( 1 ClM) and ia% 1 aminocyclopen ane- 1 S,3Rdica boxylic acid, (E dns-ACPD, 100 @VI) did no induce any signi ican change in he [Ca2+], le el (da a no shown). This esul is consis en wi h he lack o e ec o hese agonis s on [Ca2+], in single cell imaging expe imen s in ou g anule cell cul u es (da a no shown). Finally, a 2 min depola iza ion pulse wi h 50 mM K+ (Fig. 7B) also induced an inc ease in he [Caz+],, signal e y simila o ha e oked by glu ama e. DISCUSSION Conside able in e es is p esen ly ocused on he dynamics o [Ca2+],, due o i s ole as he main ese oi o Ca2+, able o be apidly eleased in o he cy osol upon cell s imula ion. Howe e , he es ima es o he s eady-s a e [Ca2+],, epo ed in he li e a u e ha e been con lic ing, p obably because o he di e en me hodologies used [31]. Many o he measu emen s we e unde aken wi h low-a ini y luo escen p obes, which ha e impo an limi a ions such as non-speci ic localiza ion h oughou he cell and in e e ence wi h Mg2+. In ac , due o he need o elimina e he s ong dye signal a ising om he cy osol, mos o hese s udies ha e been pe o med in pe meabilized, quenched o pa ched cells 132,331. Se e al o hese p oblems can be o e come using ER- a ge ed pho op o eins. They a e speci ically localized in he ER, hus allowing selec i e moni o ing o lumenal [Ca2+],, changes in in ac cells. Fu he mo e, aequo in does no display Mg2+-sensi i e luminescence and i has a g ea e dynamic ange han luo escen dyes. Howe e , he i s a emp s o measu e [Ca2+],, using ER- a ge ed aequo in u ned ou o be pa icula ly di icul because o he apid aequo in consump ion esul ing om he high [Caz+] p esen in his compa men . Thus, no only he wild ype [ 10,l 11, bu also he low-a ini y e sion o aequo in a e endowed wi h a Ca2+ a&-&y oo high o eliably measu e [Ca2+],, [ 1,131. We ha e ecen ly sol ed his p oblem by using coelen e azine n, a chemically modi ied o m o he p os he ic g oup ha educes he a e o luminescence, hus allowing measu emen s o s eady-s a e [Caz+],, a ound 500 @VI in HeLa cells [2,14]. These esul s we e in close ag eemen wi h da a ecen ly epo ed, also in HeLa cells, using an ER- a ge ed chime a dubbed ‘cameleon’ (34). The au ho s ound ha 90% o he cell popula ion had [Ca”‘], a s eady s a e a ound 400 @VI [35]. The s eady-s a e [Caz+],, es ima ed he e in he di e en cell ypes s udied we e also in he same ange, om 300 l. ~ in g anule cells o 800 l&I in GH, cells. Up o now, s udies o he dynamics o [Ca2+],, using ER- a ge ed cons uc s (aequo in- o cameleon-based) ha e Cell Calcium (1998) 24(2), 87-96 been mos ly pe o med in adhe en cell lines (pa icula ly HeLa cells) and e y limi ed in o ma ion is a ailable o p ima y adul cells. The eason is ha non-di iding cells a e e ac o y o he mo e common me hods o in oduc ion o DNA like Ca2+ phospha e. He pes i al ec o s a e a powe ul ool o gene ans e in o pos mi o ic cells and, mo e speci ically, in o adul neu ons. Mo eo e , HSV-1 can e icien ly in ec a wide a ie y o es ablished lines o di e se o igin, hus o e coming many o he limi a ions concomi an o DNA ans ec ion o he selec ion o a s able ans ec ed clone. Gi en he physiological ele ance o he Ca2+ s o es in p ima y exci able cells, we decided o exploi he wide hos ange o he HSV-1 sys em o deli e he ER- a ge ed aequo in o a numbe o di e en cell ypes. Thus, we ha e gene a ed a eplica ion de icien he pes simplex ype 1 i us con aining he ER- a ge ed low a ini y aequo in gene @HSVe AEQ. Vi al in ec ion o bo h p ima y pos mi o ic cells and es ablished cell lines led o he co ec exp ession o he chime ic aequo in selec i ely in o he lumen o he ER as de e mined by immunos aining and by unc ional [Ca2+],, s udies. The ansduced chime ic ER- a ge ed aequo in displayed Ca2+-sensi i e luminescence and could be easily used o moni o [Ca2+],, changes elici ed by InsP,-gene a ing agonis s, ca eine and ac i a ion o ol age-ga ed o ecep o -ope a ed Ca2+ channels in a a ie y o cells. The da a p esen ed he e a e he i s epo o di ec measu emen s o he dynamics o [Ca2+],, in pi ui a y cells (bo h GH, and AP cells), PC12, ch oma in and ce ebella g anule cells. The use o an adeno i us ec o o a ge wild ype aequo in o he ER has been ecen ly epo ed 1361. Howe e , al hough he au ho s showed exp ession o he apop o ein in he ER o he in ec ed cells, no measu emen s o [Ca2+],, we e p o ided. As we ha e al eady discussed, wild ype aequo in econs i u ed wi h na i e coelen e azine canno p o ide eliable measu emen s o [Ca2+],, due o he apid consump ion o he p obe [ 131. Rega ding he physiological da a epo ed in his s udy, i was su p ising o ind ha TRH p oduced such a small e ec in AP cells compa ed wi h ha ob ained in GH, cells (compa e Fig. 5D and C), e en hough he agonis is able o induce a compa able [Ca2+], inc emen in bo h cell ypes (compa e Fig. 5E and F and see [ 17,251). Appa en ly, a ela i ely modes mean dec ease o abou 70 @I in [Ca2+le is enough o p oduce a [Ca2+lc peak o 700 nM in 50-75~0 o he AP cells analyzed by single cell imaging. This dissocia ion be ween he dec ease in [Ca2+],, and he [Ca2+], peak is in ag eemen wi h ou p e ious esul s in his amine-s imula ed HeLa cells, whe e he changes in [Ca2+],, and [Ca”], we e di ec ly compa ed [2,14]. Those esul s we e in e p e ed in e ms o a s ong and apid inhibi ion o he InsP,-ga ed 0 Ha cou i B ace & Co. L d 1998 Vi al ans e o E/?- a ge ed aequo in 95 channels by mic odomains o high [Ca’+], in he icini y o he channels. Howe e , expe imen s designed o es his hypo hesis by loading he AP cells wi h BAPTA p io o TRH s imula ion showed no accele a ion o he [Caz+],, d op (da a no shown), sugges ing ha he mechanism may be mo e complex in his case. Zn ac , he magni ude o he esponse o [Ca*+],, o an InsP,-p oducing agonis appea s o be qui e a iable depending on ei he he cell o he agonis used. Thus, his amine in ch oma im cells o o he InsP,-p oducing s imuli like UTP o b adykinin in PC12 cells did induce a apid, la ge Ca*+ elease om he ER (see Figs 6A & 4A). In any case, one impo an conclusion om hese esul s is ha he e is no always a di ec co ela ion be ween he heigh o he [Ca’+], peak measu ed wi h luo escence indica o s and he amoun o Ca eleased om he ER. Fo his eason, he con en o he s o es canno be es ima ed accu a ely om he heigh o he [Ca2+], peak induced by a Ca2+- eleasing agonis and di ec measu emen o [Ca2+], becomes essen ial. Gi en he na u al p opensi y o he pes i us o in ec pos mi o ic neu ons, an immedia e applica ion o his echnique is o s udy neu onal Ca2+ s o es. I has become clea in ecen yea s ha in acellula Ca2+-s o age si es can play a c ucial ole in egula ing complex Ca2+ signals and neu onal esponses such as exci abili y, synap ic plas ici y, elease o neu o ansmi e s and pe haps neu onal dea h [371. I is well known ha s imula ion o glu ama e ecep o s in neu onal cells elici s an inc ease o he [Ca2+lc due o Ca2+ en y h ough bo h he NMDA ‘channel complex and o he ol age-ga ed Ca2+ channels [38]. Ou p esen esul s in ce ebella g anule cells (see Fig. 7A) show ha s imula ion wi h glu ama e induces a apid inc ease in [Ca2+le , which can be explained om he s imula ion o he sa co- and endoplasmic e iculum Ca2+-ATPases (SERCA) by he inc ease in [Ca2+], due o ac i a ed Ca2+ en y Al hough ecen esul s sugges ha he iono opic ecep o - media ed [Ca2+], signals migh also in ol e elease o Ca2+ om in acellula s o es by CICR 1391, ou da a do no suppo his hypo hesis in ce ebella g anule cells. On he o he hand, addi ion o quisquala e, o he mo e selec i e me abo opic glu ama e ecep o agonis uns-ACPD, o he neu onal cul u e did no p oduce any esponse ei he in [Ca’+],, o in [Ca2+],, sugges ing ha ou cul u es o g anule cells lack me abo opic glu ama e ecep o s. S udies in a numbe o neu onal p epa a ions ha e indica ed ha depola izing s imuli can ac i a e a calcium- induced-calcium- eleased (CICR) mechanism [39]. Howe e , he in ol emen o CICR in he gene a ion o [Caz+], peaks has been di licul o es ima e due o he pa allel ac i a ion o plasma memb ane Ca2+ channels and he subsequen massi e Ca2+ en y. Specula ions on he ole o CICR ha e been qui e a iable depending on he me hodology used o p oduce depola iza ion, leading o con lic ing esul s in he li e a u e. In ou expe imen al condi ions we did no obse e any sign o ac i a ion o CICR upon a 2 min pulse wi h high K+ medium in GH,, AP cells o g anule neu ons (see Figs 5A,B & 7B). Ins ead, high K+ depola iza ion always elici ed a signi ican inc ease in [Ca2+],, in all cells es ed. Again, he mos p obable explana ion o his e ec is he s imula ion o he SERCA by he inc ease in [Ca2+],. Ano he ool o en used o examine he ole o CICR is he abili y o ca eine o elici elease o in acellula Ca2+. Ou measu emen s o [Ca2+],, in ad enal ch oma m cells (Fig. 6) con i m ha ca eine mobilizes Ca2+ om he in acellula s o es in hese cells, a esul consis en wi h ea lie epo s using luo ime ic echniques o measu e [Ca”‘], [30,40]. In conclusion, he da a p esen ed he e demons a e ha he HSV based echnology is an e icien me hod o deli e he ER- a ge ed aequo in o a numbe o mam- malian cells, bo h es ablished lines and pos mi o ic p ima y cells. Fu u e de elopmen s may include p o- ducing new HSV ec o s ca ying aequo in chime as a ge ed o o he compa men s (e.g. nucleus, mi o- chond ia) o exp essing o he a ge ed indica o genes such as he ecen ly epo ed cameleons [34] ha allow pe o ming single-cell imaging expe imen s. This app oach will acili a e he s udy o key physiological issues such as he ole o neu onal calcium s o es o he beha io o unc ionally dis inc Ca2+ pools wi hin he ER. ACKNOWLEDGEMENTS We a e indeb ed o D F. Lim o helping us o es ablish he HSV media ed gene ans e echnique in ou labo a o y and o his e e p esen ad ise on he amplicon sys em. We hank P. Ala &n o help wi h he imaging expe imen s and bo h P. Ala con and D A. Sanchez o gene ously p o iding AP cells. We acknowledge D P. Michelena and D A. Ga cia o ch oma Bm cells. We also hank J. Fe nandez o echnical assis ance. We a e g a e ul o S. Calleja o assis ance wi h con ocal mic oscopy We specially hank D T. Schimmang o his help ul commen s on he manusc ip . This esea ch was suppo ed by g an s om Spanish Go e nmen Agencies o Heal h Resea ch FIS 96/1443 (MTA), FIS 96/0456 (JA) and DGICYT PB92/0268 (JGS) and ellowships o MJB and EC. REFERENCES 1, Mon e o M., B ini M., Ma saul R. e al. Moni o ing dynamic changes in ee Ca2+ concen a ion in he endoplasmic e iculum o in ac cells. EMBOJ 1995; 14: 5467-5475. 2. Mon e o M., Ba e o MJ., Al a ez J. [Ca*+] mic odomains con ol agonis -induced Ca2+ elease in in ac cells. FASEB / 1997; 11: 881-886. 3. Boo man M.D., Be idge MJ. The elemen al p inciples o calcium signaling. Cell 1995; 83: 675-678. 0 Ha cou B ace & Co. L d 1998 Cell Calcium (1998) 24(2), 87-96