Studies on Cyclophilin-D and NAADP on Ca
2+-mediated events
PhD thesis
Miklós Mándi, MD
Semmelweis University
Neurosciences (‘János Szentágothai’) PhD School Functional Neurosciences Program
Supervisors:
Official reviewers:
Chairman of committee:
Members of committee:
Veronika Ádám, MD, DSc
Balázs Sümegi, MD, DSc Gábor Bánhegyi, MD, DSc Erzsébet Ligeti, MD, DSc
Zsolt Liposits, MD, DSc Csaba Sőti, MD, PhD
Budapest, 2011
Introduction:
Cal cium is on e of t h e most im po rt ant and versatil e si gn all in g agent t hat det ermin es a v id e variet y o f intra- and in tercellu lar p ro cess es. The p att erns o f ch an ges i n th e con cent rati on o f calci u m are ti ghtl y regu l at ed b y several ex tracell ular fact ors , as well as v ario us int racell ul ar s econd m es sen ger pat hwa ys . Comp artm ent aliz ation an d si gnal -in du ced rel eas e of cal ci um from intracel lul ar o rgan el les (e. g. endo pl asmi c/s arcopl asmi c reti culum, l ys os omes, mitochon dria, Gol gi memb ran e, nu cl eu s) or th e ex tracellul ar space fo rms t he basi s of calci um si gnal trans du ctio n. Nicotini c aci d-ad eni ne di nu cleotid e phos ph at e (NAADP ) is rapi dl y em ergin g as an i ntracellul ar s econ d m ess en ger medi ati n g cal cium rel eas e from m ainl y th e aci di c calci um stores (e. g.
l ys os omes, an d d eriv ativ es: mi croso mes ). Th e NAADP-ind uced C a2 +- mobiliz atio n is usu al l y li mit ed in sp ace and tim e, acti n g as a tri gger th at m a y be p rop agat ed and gen eraliz ed b y C a2 +-indu ced C a2 +-rel eas e (C ICR) vi a r yano din e recepto rs and in osito l-1,4 ,5 -t risp hos ph at e (In sP3) recepto rs (e. g.
ooc yt e-ferti lizati on ) or serv es as a local effecto r of e. g. insuli n-secret ion from p an creat ic β-cells, or n eu rite ou t gro wth an d n eu ro tran sm itter s ecreti on.
Mito cho nd ria pl a y a more comp lex rol e in intracell ular cal cium hom eost asi s, acti n g as a ‘calci um sin k’ and v ice v ers a, ch an ges of m at rix calci um co ncent ratio n d et ermi nes s ev eral mito cho nd ri al fun ct ions, ex p and in g from regul ati on of k e y st eps of th e Sz ent g yö rg yi -Kreb s c ycl e to mit ochon dri al perm eabilit y trans it ion (mPT). R ev ers ible or i rreversi bl e p ermeabi lit y transiti on occurs in resp ons e t o di fferent int ra- an d ex tracell ul ar si gnal transd uction p ath ways o r m et ab oli c ch an ges con vergin g to enh an ced ROS - pro du ctio n or hi ghl y el ev at ed m at rix calci um con cent rati on o r hi gh free phos ph at e (Pi) con centratio n. C ycl op hilin -D (C yp D) i s an im p ort ant , cal ci um- sen sitiv e regul ato r o f th e p ermeab ilit y t ransi tion po re’s op en in g, p ro moti n g perm eabilit y t ransiti on
Objectives:
#1 The primar y aim of t he p res ent t hesis was to cl arif y th e mo dul ato r y rol e of c ycl oph ilin -D in t he Ca2 +-ind uced permeabilit y t ransi tion and it ’s rel atio n to th e bi oen ergeti c stat e o f mi to cho nd ri a. W e firs t v erifi ed that su bst rat e- starv ed mito ch ond ria are ind eed m ore s us ceptibl e t o Ca2 +-i ndu ced perm eabilit y tran siti on and s ho wed th at gen eti c d el eti on o f C yp D provid es similar p rot ection t o th at o f c yclo spo rin-A i n wild -t yp e, subst rat e-s tarv ed mitochon dria. Also , we p erform ed p aral lel recordin gs o f swellin g and C a2 + upt ak e int o mit o cho ndri a in t he p resence o f R u36 0 o r an u nco upl er. Th es e results l ed to t he quest ion wh eth er high Ca2 + acts o n the su rface o f mitochon dria o r is ent erin g mit ochon dria b y a RuRed/Ru 3 60-i nd ep en dent pat hwa y. To sh ed l ight amon g t h ese p ossibi liti es, we ch allen ged in sit u mitochon dria wi th Ru360 o r R uth eni u m Red, and record ed Ca2 + u pt ak e cap abiliti es . Nex t, we cl ari fi ed th e co ntri butio n o f t h e di fferent compl ex es o f the el ectron t rans po rt ch ain to C a2 +-i nd uced swelli n g b y u s in g in hibit ors o f Compl ex I, III and IV. Fin all y, we d emonst rated t he div ers e effect o f t he abl atio n of c ycl op hil in-D o n t he s wel lin g of in sit u mit ochon dri a of n eu ron s and ast ro c yt es. M oreov er, we sho wed th at th e del eti on of cycl ophil in -D not onl y d el a yed init i al swelli n g o f n eu ron al mit ochon dria ind uced b y ex citot ox icit y (glut amat e-gl ycin e), but it also rend ers n eu ro ns less su scept ibl e to d el a yed cal cium d eregul atio n.
#2 On th e o th er hand, i n regard to c yt oso li c C a2 +-si gn alizati on, the m ai n obj ecti v e of ou r wo rk was to est ablis h NAADP as a di stin ct pat hwa y for C a2 + rel eas e i n h ep ato c yt e mi croso mes. Fi rst l y, we d efi ned th e activ el y lo ad ed micros omal C a2 +sto re t o b e th apsi gargi n -, but not b afilom yc in A1-sensiti ve.
In th e n ex t step, we verifi ed th at NAADP was in deed acti ve in m obilizi n g Ca2 + from rat liv er micros omes and we com pared t h e C a2 + rel easin g po tenti al of NAADP t o tho se of InsP3 and cADPR . Als o, we dem onst rat ed th e l ack o f cross -d es ens itizat ion between NAADP, cADPR and In sP3 as an evid en ce fo r sep arate recepto rs for each C a2 +-mo bili zin g seco nd mess en gers. Fo cu sin g o n the pro p erti es of t h e NAADP -d ep en dent Ca2 + releas e, we p ro vid ed an NAADP
dos e-depend en ce curve, as well as pH- and pC a-depend en ce cu rv es fo r th e NAADP -in du ced C a2 + effl ux from liv er micros omes. W e verifi ed fo r th e first time in vert eb rate t issu es t h e u niqu e i nactiv ati on pattern of th e NAADP - medi at ed Ca2 + rel eas e, wh ere ‘p er s e’ no n-C a2 +-mo bilizin g d oses o f NAADP are abl e to abolis h the effect of a satu ratin g q u antu m of NAADP.
Fu rth ermo re, we in cub at ed p re-lo ad ed micros om es with thapsi gargin and bafilom yci n A1 to s uppo rt t he noti on th at th e o ne-p ool m od el is ap pli cable fo r th e C a2 + rel eas e from th e micros omal C a2 + stores. Fin all y, b y dem onst ratin g th e p harm acolo gical p ro p ert ies o f th e NAADP-ind uced Ca2 + efflux , we provi ded stro n g ev id en ce fo r NAADP b ein g an ind ep end ent medi ato r from In sP3 and cADPR in liv er micros omes.
Methods:
Isol atio n of br ain mi tochon dria fro m WT and cyp D K O mice - C57Bl/ 6 WT and KO fo r c yc lophili n-D litt ermate mice were a k ind gi ft from Drs . Nik a Dani al an d Ann a Schi nzel , fro m Howard Hu gh es Medi cal In stitu te and Dana-Farber Cancer Insti tut e, Harv ard M edi cal Scho ol. Mice were cro ss- bread fo r 8 gen erati ons prio r to harv est in g brai n tissu es from WT and KO age-mat ch ed an imals fo r th e pu rp os e of mitochon drial i sol ati on and cult uri n g of n eu ron s and astro c yt es. No n-s yn apti c brain mit ochon dri a from adul t mal e WT an d KO fo r C ypD mi ce (aged 87 -1 15 d a ys ) were is olat ed o n a Percoll gradi ent as d es crib ed previ ousl y (Sim s et al., 199 0) with min or mo difi catio ns det ail ed in (Chi nop o ulos C. et al. , 20 03 ). All anim al p ro cedu res were carri ed out accordin g to t he l ocal anim al care an d u se comm ittee (Eg yet em i Áll atkís erl eti Bizot ts ág) guid elin es .
Prepar atio n of rat li ver micros omes
Li v er mi cros omes were p rep ared as d es crib ed p revio usl y (Fl eishn er an d Krau s-Fri edm ann , 19 86). Bri efl y, Sp ragu e-Dawl e y rat liv er was hom o genis ed in an ice-cold m ediu m of 0.3 2 M sucros e, 20 mM MOPS-bu ffer (pH 7 .2 ), 0.5 mM EGTA also cont ainin g 1 mM d ithioth reito le (DTT) and 0 .2 mM phen ylm eth yl s ulfon yl fl uo rid e (PMS F) as prot ease in hibit ors and centrifu ged at 2 000x g fo r 1 5 minut es at 4 °C . The su pern at ant was cent ri fu ged at
15,0 00x g fo r 45 mi nut es, an d th e resul tin g s up ernatant was collect ed and fu rth er cent rifu ged at 100 ,00 0x g fo r 9 0 minut es. Fin all y the p ell et was resus pend ed in a sol ution cont ain in g 0. 3 2 M su cros e, 20 mM MOPS (p H=7.2 ), 1 mM DTT and 0. 2 mM PMSF. P rot ein con cent ratio n was set for ∼2 0 m g/ml whi ch was m easu red b y t he Lo wr y ass a y u sin g bo vin e s erum al bumi ne as stan dard . The s am pl es were froz en in li quid n itro gen and st ored at -80 °C until requ ired .
Mito ch ond rial memb ran e p ot entia l (∆Ψm) deter mi natio n
∆Ψm was est imated usin g flu ores cen ce quenchin g o f th e cationi c d ye safrani ne O, b ecau se o f its accum ul ation ins id e en ergiz ed mit ochon dri a (Akerm an et al., 1 976 ). Mit ochon dria (1 m g) were add ed to 2 ml o f an incub ation m edi um con tai nin g 1 20 mM KCl, 20 mM Hep es (acid ), 1 0 mM pot assiu m pho sph at e, 1 mM M gC l2, 0 .005 mM EGTA, 5 mM potas sium gl ut amate, 5 mM p otas sium m al at e, 0 . 001 mM c yclo spo ri ne A, 0. 05 mM AP5A, 0 .5 m g/ml BS A an d 5 µ M s afranin e O (pH 6 .8 o r p H 7. 8). Flu ores cen ce was reco rd ed in a Hit achi F-4 500 spectrofl uo rim et er (Hit achi Hi gh Tech nolo gi es, Maid enhead , UK) at a 5 Hz acqui sitio n rat e, us in g 495 and 585 nm ex cit atio n and emiss ion wav el en gt hs, resp ectiv el y. Ex perim ent s were perform ed at 37 °C . To con vert s afrani n e O fl uo rescen ce int o millivol ts (mV), a volt age– fl uo res cence calib ratio n cu rve was con structed . To this end , safrani ne O fl uo rescen ce was reco rd ed i n the p res en ce of 2 nM valinom ycin and st ep wis e in creasin g [K+] (in th e 0 .2–1 20 mM ran ge), which all owed calculation o f ∆Ψm b y th e Nernst eq uat io n, ass umin g a m at rix [K+] of 1 20 m M (Akerm an et al ., 197 6).
Ca2 + up ta ke of isol at ed mit ochon dria
Mito cho nd rial-d epen den t remo v al o f m edium C a2 + was foll o wed usi n g the imp ermeant h ex apot assiu m s alt of th e fluo res cent d ye C alcium Green 5 N (CaGreen ) (Mol ecul ar Pro bes, Po rtl and , OR, USA). C aGr (500 nM) was ad ded to a 2 ml medium cont ainin g mit ochon d ria (0.1 25 m g/ml ) and 1 20 mM KCl , 10 mM Tris, 5 mM KH2P O4, 1 mM M gCl2, p H 7 .6. Sub st rat es were add ed wh ere i ndi cated. Al l ex perim ent s were performed at 37 ° C. Fluo res cence
intensit y was m easu red in a Hit achi F-4 500 fluo res cen ce sp ectroph otom et er (To k yo , J ap an ) usin g 517 nm ex ci tati on and 5 35 nm emis sion wavel en gth s.
Measu rement of mito cho ndri al s well ing
Swell in g o f isol at ed mitochon dri a was ass ess ed b y m easu rin g li ght scatt er at 52 0 nm i n a GBC UV/V IS 9 20 s pect rop hot ometer. Mito cho nd ri a were add ed at a fi nal con cent ratio n o f 0. 125 m g/ml to 2 ml of m edi um con tai nin g 1 20 m M KCl, 10 mM Tris, 5 mM KH2PO4, 1 m M M gCl2, pH 7.6 . Subst rat es were add ed wh ere ind icat ed . At th e end of each ex perim ent , th e non -s electi ve po re-formi n g p ept id e al amethi ci n (40 µ g) was add ed as a calib rat ion st and ard to caus e m ax imal swelli n g. All ex perim ents were perform ed at 3 7 °C .
Acti ve l oadi ng of mi cro somes with Ca2 + and C a2 + r el eas e ass ay
Ca2 + upt ak e an d rel ease were m easu red usin g 4 5Ca2 + isot op e to d et ect Ca2 + mov ements . Th e micro som es were d iluted in a s oluti on of 15 0 mM KCl , 20 mM M OPS (p H 7 .2), 0 .5 mM M gCl2, 10 µ M C a2 +. In each ex p erim en t, 20 - 40 nC i 4 5CaCl2 was us ed per as sa y p oi nt. Th e C a2 + up take was start ed b y injecti n g 1 mM of ATP in t he sol utio n at ro om t emp erat ure. Ca2 + releas e was preform ed b y add in g 10 0 µ M EGTA i n th e p res en ce o r ab s en ce o f t he C a2 + rel easi n g agents (1 0 µ M InsP3, 10 µ M cADPR and 1 0 µ M NAADP ). Th e
4 5Ca2 + rem ai nin g in the v esi cl es was det ermin ed b y filt ratio n o f 0.5 ml micros omes t hrou gh a nit ro cellul os e Mi llipo re filt er (HAW P, 0. 45 µm p ore size) und er v acuum . The filt ers were wash ed with 5 ml o f quench sol utio n (15 0 mM KCl, 20 m M MOPS (p H 7.2 ), 10 mM M gCl2 an d 1 mM LaCl3) to lower t he rate o f unsp ecifi call y b ou nd radi oacti vit y. The radio activit y ret ai ned o n t he fil ter was m easu red b y st andard s cintil lat ion counti n g.
Passi ve l oadi ng of micro so mes a nd C a2 + rel eas e
Li v er mi croso mes were p assi v el y lo ad ed with 5mM 4 5CaCl2 (20-40 nCi per ass a y p oint ) b y incub ati on fo r at l east 5 hou rs in an ice-col d med ium con tai nin g 1 50 mM KCl, 20 mM MOP S (pH 7. 2), 4 5Ca2 + and 5 mM Ca2 +. Passi ve lo ad ed v esi cl es were dilut ed 1 0-fold int o a Ca2 + rel easin g m ediu m con tai nin g 150 m M KCl, 2 0 mM MOPS (pH 7 .2 ) and 5 00 µ M of EGTA to
adj ust pCa to 6 at room t em peratu re an d C a2 + rel easin g agonists . Th e C a2 + rel eas e was sto pp ed b y 5 -fol d dil utio n wit h th e s am e quench so lutio n des crib ed abov e, th en th e s amp les were filtrat ed t hrou gh Mill ipore filt ers an d wash ed b y 5 ml o f q uen ch s oluti on. Th e ret ai ned radi oacti vit y was measu red b y st an d ard s cinti llat ion co untin g.
Results and discusion:
#1
Compl ex co ntrib utio n of Cycl ophil in -D i n br ain -sp ecifi c mit och ond rial per meabi lity tr ansiti on in du ced by Ca2 +Fi rstl y, we ch all en g ed is ol at ed b rain m itochon dri a b y C aC l2 in th e pres en ce an d abs en ce o f glut am at e and mal at e, and reco rd ed li ght s catt eri n g spect rop hot ometri cal l y at 520 nm. W e us ed a th ree-p uls e C aCl2 prot ocol for this, and all s ubs eq u ent sim ilar ex perim ents: 20 µ M C aCl2 was gi ven at 1 00 sec, follo wed b y 200 µ M CaCl2 at 3 00 s ec and again at 50 0 s ec. Th e addi tion of 20 µ M C aCl2 to subs trat e-sup pl em ent ed or su bst rate-st arved b rain mitochon dria of wil d t yp e mi ce did n o t caus e a decreas e in li ght scatt er;
inst ead, a cess ation in th e b as elin e decreas e i n li ght s catter was obs erv ed.
However, th e su bs equent 20 0 µ M CaCl2 pulse in du ced a l arge d ecreas e in li ght s cat ter in subst rat e-st arv ed, but not sub strat e-s upp lem en ted mitochon dria. Th e n ex t 200 µ M CaCl2 p ulse gi v en at 5 00 sec did not i ndu ce an y furth er chan ges i n li ght s catt er for th e s ubst rat e-s tarv ed mitochon dria, b ut it cau sed a m ino r ch an ge in sub strat e-su ppl ement ed m ito cho ndri a. The effect of th e fi rst 20 0 µ M CaCl2 puls e was cycl ospo ri n A-sensiti ve, h owev er, th e secon d addi tion o f 200 µ M CaCl2 ov errod e t he p rot ectiv e effect o f C ys A.
Subs equ ent ex p erim ents b en efited from the availabilit y o f c yclo phil in-D kno ck -ou t mice. Ou r res ults o btain ed from s ubst rat e-s tarv ed mito cho nd ri a from C yp D-KO mi ce were strikin gl y si milar to t hos e o bt ai ned from C ys A- treat ed WT mi ce. The p resen ce o f s u bstrat es , h owev er, did n ot provid e add ition al prot ect ion in th e C yp D-KO mi tochon dri a. Th es e results s ho w th at hi gh -C a2 + lo ads can indu ce PTP i n th e absence of sub strat es . In o ur hands , abs en ce of sub strat es prev en ted is ol at ed mito ch ond ri a from b uildi n g a
memb ran e pot enti al of hi gher th an -10 mV, a con ditio n wh ere mito ch on dri al Ca2 + upt ak e is u nfav orabl e. Ind eed, recordin gs of ex tramito ch ond rial Ca2 + b y Cal cium Geeen 5 N reveal ed t hat i n th e abs en ce of s ubst rat es, mit o cho ndria were un abl e to perform Ca2 + s equ est rat ion, yet ex hibi ted large ch an ges in li ght s catt er. Elect ron mi cros cop y im agi n g of mito ch ond ri a th at ex hibit ed large ch an ges in li gh t scatt er con fi rm ed t hat this was du e t o s wel lin g.
To ad dress th e sit e o f action o f C a2 + on t he li ght s catter, we pre-t reated mitochon dria wit h th e C a2 + un ipo rt er inh i bito r, R u36 0 at a co ncent ratio n th at was foun d to prev en t the upt ak e o f ex tramitochon drial C a2 + and sho wed t hat WT mitoch ond ri a sti ll ex hibit ed hi gh-C a2 +-ind uced ch an ges in ligh t scatt er in the pres ence of Ru3 60. Th e l ack of effect o f R u36 0 was als o ob serv ed in th e pres en ce of C ys A or wh en th e effect of C a2 + was comp ared in W T v ers us C yp D-KO mi to cho ndri a. Th e pres en ce o f th e u ncou pler comp let el y dep ol arizi n g mito ch ond ria failed to affo rd ex tra p rot ection agai nst hi gh-C a2 + indu ced s wellin g. Furth ermo re, th e p resen ce o f th e un co u pler n egat ed th e prot ectiv e effect s of subst rat es i n WT mitochon dri a. Th e fail ure of Ru 360 t o prot ect against t he Ca2 +-in du ced large ch an ges in l i ght s catter coul d b e ex plai ned b y as su min g th at C a2 + act ed o n th e ex t ram it och ond ri al si de.
Surp risi n gl y, isol at ed mitocho ndria l oad ed wi th Fu ra 2 an d imaged u nd er wid e-fi eld epi flu orescen ce mi cros cop y showed ro bust i ncreas es in m at rix - ent rapp ed Fu ra 2 flu ores cence wh en p erfused wit h a buffer co ntai nin g 0.1 m M CaCl2. This effect ex hibited onl y a p arti al s ensi tivit y to Ru 3 60 (10 µ M ) and ruth en ium red (10 µ M), arguin g agai nst the assum ptio n th at Ca2 + was actin g ex clus iv el y o n an ex tramit ochon dri al si te wh en indu ci n g chan ges in li ght scatt er.
Nex t, we p ret reat ed mito ch ond ri a wi th compl ex I (rot eno ne o r pieri cidi n A), comp lex III (m yx o thi azo l or sti gmat elli n), and co mpl ex IV (KC N) in hibit ors to add ress th e cont ribu t ion of res pi rat or y ch ain comp on ents to th e protecti ve effect o f sub strat es on C a2 +-in du ced ch an ges in li ght s catt er.
The em ergi n g p ict ure b as ed on o ur resu lts is t hat ro tenon e or p ieri cidi n A affo rd ed p rot ection from C a2 +-i ndu ced ch an ges in li ght scatt er, irresp ectiv e o f the p res en ce o r abs cen ce o f su bst rat es , whil e m yx o thi azol, sti gmatell in and KCN n ot onl y fali ed to co nfer p rot ecti on, th e y als o negated the p ro tecti ve effect of s ubst rat es. Hi gh co ncent ratio ns of m yx othi azol an d sti gm at ellin (10
µ M and 2 µ M , resp ectiv el y) th at als o bl ock compl ex I b indi n g t o a different site, fail ed to affo rd prot ectio n, as op pos ed to ro tenon e an d p iericidin A.
To add ress th e rol e of C yp D in t he op eni n g o f b rai n-sp eci fic P TP in situ, s wellin g o f C yp D-d eficient v ers us wil d t yp e mi to cho nd ri a wit hin neu ro ns or ast ro c yt es of th e s ame cu lture were comp ared duri n g Ca2 + overlo ad, i ndu ced b y add ition o f cal cim ycin (1 µ M 4 Br-A2 318 7) and visu alized b y wid e-field epifluo res cence imagi n g of mito chon dri all y t arget ed DsR ed2 . Neu ron s and ast ro c yt es were dist in guish ed b y thei r di fferent mitochon drial morp holo g y and mi to ch ond rial swell in g was mo nito red b y calculation o f chan ges in m ean mito cho ndri al di am et ers usi n g th e thinn es s rat io t echniq u e. Th e ons et o f swelli n g was defin ed b y th e su d den decreas e in the forem enti on ed th inn ess ratio . Both W T and c yp D-KO mit o cho nd ri a withi n neu ro ns swell ed and fragm ent ed in res po nse to C a2 + overlo ad induced b y t he add ition o f cal cim yc in wi thin 60 0-800 sec. Wh en NaC N was co -appli ed wit h calci m ycin in a glu cose-free media i n th e pres en ce o f 2 mM 2-d eox ygl u cos e, swellin g o f mit ocho ndri a was almo st im medi at e in bot h wild t yp e and C yp D- KO n euro ns. Ho wever, C a2 + ov erlo ad of u ncoupl ed mito chond ri a (b y co - app licati on o f 1 µ M SF 68 47 ) tri ggered s wel lin g at a si gni ficantl y earli er tim e in C yp D-KO t h an i n wi ld t yp e neuron s. In cont rast t o t h e neurons, C a2 + overlo ad o f un cou p led mito ch ond ri a t riggered swell in g at a si gnificant l y earli er tim e in wild t yp e th an in C yp D-KO ast ro c yt es.
Fin all y, in o rd er t o ex pos e neuron al in s itu mito cho nd ria t o hi gh -C a2 + ch all en ge b y an alt ernativ e mech ani sm, we ex po sed neuron s to ex cito tox ic lev els o f gl ut amate and gl ycin e, i n th e absence o f M g2 +. Glu tam at e ex pos ure tri ggered a bip hasic mitochon drial s wel li n g respo ns e com pri s ed of an i niti al, 1s t an d a well s ep arat ed, d ela yed 2n d drop . Th e 1s t d rop o f thin ness ratio inv ari abl y coi nci ded with th e ini tial [Ca2 +]i-respo ns e to glut amat e and th e 2n d dro p t o th e s econd ar y, i rrev ersibl e ri se of [Ca2 +]i, t ermed del a yed cal ciu m deregul ati on, DC D. In cultu res prep ared from C yp D-KO mi ce the 1s t ph as e of mitochon drial swelli n g was det ect ed o nl y i n 60 % o f th e neu ron s and o nl y 4 0
% o f C yp D-KO n eu ron s ex hibit ed th e s econd ar y swelli n g o f mit ochon dria duri n g DC D. In add ition , the in iti al swelli n g of mi tochon dri a was si gni fi cantl y d ela yed in C yp D-KO n eu ro ns comp ared to wil d t yp e, whil e t he
time o f ons et o f th e second ar y mito cho ndri al s wellin g was not stati sti call y different .
#2
Ca2 + r el eas e t rigg er ed by NAADP in h ep ato cyt e mi cro so mesHepat ic micro som al vesi cl es rapid l y s eq u est ered 4 5Ca2 + in th e pres en ce of ATP , with an upt ake o f 4.0 ± 0.2 nm ol/ mg p rot ei n (n =13 ). The max imum o f Ca2 + upt ak e was fou nd wit hin 5-10 and about 9 0% of th e sp ecifi cal l y ret ai ned micros omal C a2 + was rapidl y rel eased b y ion om ycin (5 µ M). Th e Ca2 + accumul atio n o f mi cros omes was n earl y abol ish ed b y 1 µ M thap si gargin (a sel ectiv e in hibit or o f SERC A), whi le 1 µ M bafilo m ycin A1 (an es tablish ed blo ck er o f th e V-t yp e ATP -as e) do es n ot affect su bst anti all y the C a2 + uptake mech anism s of li ver micros omes. In th e li ght o f these result s, it is the SER CA that rep res ents th e m ain mech anism resp o nsibl e fo r t he act iv e loadin g o f li ver micros omes.
NAADP (10 µ M ), InsP3 (1 0 µ M ) and cADPR (1 0 µ M ) i nd uced a fast Ca2 + effl ux , whi ch differed si gni fi cantl y from cont rol mi crosom es (C ICR ).
Aft er 5 s econ ds o f Ca2 + rel ease, th e tot al amou nt o f C a2 + effl ux eli cit ed b y C ICR was 0. 165 ± 0.06 n mol/m g protein, In sP3 releas ed 0 .7 ± 0.0 9 nmol Ca2 +/m g p ro tei n, wh ile cADPR eli cit ed 0.82 1 ± 0.1 nmol C a2 +/m g p ro tei n.
Und er th e s ame co nditio ns, NAADP rel eased 0 .42 ± 0.0 8 nm ol C a2 +/m g prot ei n. Thu s NAADP is a p otent, but so mewhat l ess effectiv e C a2 + rel easin g mess en ger th an cADPR an d InsP3 in l iv er h ep ato c yt e mi cro somes. NAADP indu ced C a2 + rel eas e in rat liv er mi cro somes in a do se-d epen dent m an ner, with a hal f-m ax imal co ncent ratio n (EC5 0) o f 0 .93 ± 0 .1 µ M . Nex t, we t est ed subs eq uent C a2 + rel ease from acti vel y l oad ed liv er mi croso mes b y cADPR, In sP3 and NAADP i n th e p res ence o f an ATP -regeneratin g s yst em. NAADP man aged to eli cit m ax imal C a2 + efflux wh en appli ed aft er cADPR and In sP3 hav e al read y been p robed. Thu s cross -d es ensi tizati on t o InsP3 and cADPR b y NAADP di d not o ccur. This res ult fu rth er supp orts th e view t hat NAADP acts upon a Ca2 + rel eas e mech anism disti nct from th at o f In sP3 an d cADPR from rat li ver micro som es . When tested fo r s el f-desensitiz atio n, fi rst injecti on of a subth resh old con cen tration o f NAADP (0.1 µ M) t o micros o mes at the th ird
minut e du rin g activ e lo adin g did n ot resul t in sub stanti al Ca2 + releas e b y itsel f, h owev er aft er 2 minut es o f in cub atio n, 10 µ M NAADP rel eas ed on l y 0.14 ± 0.0 4 nm ol Ca2 +/mg p ro tei n comp ared to 0 .39 ± 0. 04 n mol/m g p ro tei n Ca2 + rel eas ed from non p re-i ncub at ed micro som es. Thus , NAADP ma y fun cti on as its o wn speci fi c antagon ist with an IC5 0 of 30 n M. Th e cu rv es o f dos e depend en c y an d residu al C a2 + effl ux after p re-in cub at ion wit h var yi n g con cent ratio ns of NAADP fo rm a U-sh ape as NAADP d es ens itize i ts recept ors with an IC5 0 that i s one order o f mag nitud e lo wer th an the EC5 0. Wh en activ el y lo ad ed m icros omes were in cu bat ed fo r at least 5 min utes with bafilom yci n A1 (1 µ M), we foun d th at b oth NAADP (1 0 µ M ) an d cADPR (10 µ M) eli cit ed an enti rel y no rmal Ca2 + rel eas e res pon se. On th e oth er hand, pre- treatm ent with th ap si gargin (1 µ M) redu ced th e am ount o f Ca2 + efflux eli cit ed b y NAADP, simil arl y to t hat o f cADPR. Bas ed on our res ults the Ca2 + releas e from mi crosom es can be descri bed wit h a on e-po ol mo del, b ein g a mix tu re o f Ca2 + sto res deriv in g from bot h l ys os o mes an d t he ER, filled m ainl y b y SERCA an d con tai ni n g recept ors for In sP3, cADPR and NAADP.
We sh ow th at th e pC a respo ns e cu rves o f t he In sP3 and cADPR app eared to b e b ell -sh ap ed with an op timal pC a at 7 an d 6 resp ectiv el y, howev er, t he NAADP-i ndu ced C a2 + releas e was fai rl y i n dep en d ent o f t he ex trav esi cul ar Ca2 + con cent ratio n. Als o, we fo und th at th e NAADP -in du ced Ca2 + releas e in hepatoc yt e mi cros om es was not affected b y ch an gin g t he p H of th e in cub ati on bu ffer from 6 .4 to 7. 8, h owev er, the respo ns e to cADPR was mostl y depend ent o n pH, sho win g an optim al p H o f 7. 2. NAADP- an d cADPR-tri ggered C a2 + rel ease from li ver mi crosom es was differen tial l y affected b y p H, prov idin g furt her evi d en ce th at t h es e ago nist s si gnal th ro u gh fun cti on all y disti nct pat hwa ys . Fin all y, h ep arin (10 0 µ g/ ml, a well - est abli sh ed i nhi bito r of th e InsP3R-s) in hibit ed t h e C a2 + rel ease elici ted b y In sP3 an d did not alt er th e effect o f cADPR and NAADP. Th e R yR antagon ists (r yan odi n e [5 µ M] and ruth eni um red [5 µ M]), bl ocked th e cADPR-ind uced Ca2 + efflux , leavin g that of In sP3 an d NAADP un alt ered . The L-t yp e C a2 + recept or bl ockers , v erap ami l (10 0 µ M) and di lti azem (100 µ M) abolis hed speci fi cal l y, b ut onl y p arti all y t he C a2 + rel easin g effect o f NAADP h avi n g minim al effect on t h e C a2 + rel eas e b y In sP3 and cADPR . To sum u p, n eit her hep arin , n or r yan od i ne and rut henium red were abl e t o bl ock sub stanti all y th e
NAADP -in du ced C a2 + releas e, wh ile v erap amil an d dil tiaz em were effectiv e inhibit ors of NAADP recepto rs st ren gt h eni n g th e noti on th at th e NAADP medi at ed Ca2 + rel eas e i n h ep at oc yt e micros omes is in deed disti nct and ind ep end ent fro m th at of In sP3 and cADPR.
List of publications:
Rel ated t o th e pr es en t th esis
Mán di M. , Tóth B. , Timár G y. an d Bak J . (200 6): C a2 + rel ease t ri ggered b y NAADP in hepato c yt e mi cros omes.
Bio chem . J . 3 95: 233 -23 8.
[ IF: 4. 100 (200 6)]
Mán di M . & Bak J . (20 08 ): Ni cotin ic acid ad en in e n ucl eo ti de dipo hos ph at e (NAADP) and Ca2 + mobiliz atio n.
J . Recep t. Si gn al. Trans du ct. Res. 23 (3 ), 163 -1 84.
[ IF: 1. 540 (200 8)]
Chino poul os, C ., Vajda, S., Cs an ád y, L. , M ándi , M. , M áth é, K., and Ád ám - Vizi, V. (2 009 ): A n ovel kin eti c as sa y o f mito cho nd ri al ATP -ADP ex chan ge rat e medi ated b y t he ANT.
Bio ph ys .J . 9 6, 249 0-2504 . [ IF: 4. 683 (200 9)]
Vaj da, S., Mándi, M ., Konrád, C. , Kis s, G., Amb rus , A., Ád ám-Vizi , V. , and Chino poul os, C . (2 0 09): A re-ev alu atio n of th e rol e o f mat ri x acidi fi cation i n uncoup ler-ind uced C a2 + rel eas e from mit o cho nd ri a. FEBS J . 2 7 6, 2 713 -2 724 . [ IF: 3. 139 (200 9)]
Chino poul os, C ., Gerencsér, AA., M ándi , M., Mát h é, K., Tö rőcsik , B. , Dó czi, J ., Tu riák, L., Ki ss, G., Kon rád, Cs ., Vajd a, Sz. , Vereczk i, V., Oh , RJ . and Ád ám-Vizi, V. (2 01 0): Fo rward op erati on o f ad enin e n u cleotid e translo cas e duri n g F0 F1 -ATPas e rev ers al: crit ical ro le o f m at rix sub strat e-level phos pho r yl ati on. FASEB J . 24 (7), 2 405 -2416 .
[ IF: 7. 049 (200 9)]
Dó czi, J ., Tu riák, L., Vaj da Sz., M ándi, M., Tö rőcsi k, B., Geren cs ér, A.A., Kiss , G. , Ko nrád , Cs ., Ád ám -Vizi, V., an d Chino pou los, C . (2011 ): Co mpl ex con tri buti on o f C yc lophili n-D to Ca2 +-indu ced p ermeabilit y t rans ition in brain mito ch ond ri a, with rel atio n to th e b ioenergeti c s tat e.
J . Bio l. C hem . 2 86 (8), 63 45 -63 53.
[ IF: 5. 52 (2 009 )]
Not r elat ed to th e p r es ent th esis
Kon rád, C s.; Kiss, G.; Törőcsik, B.; Lá bár, J .; Gerencsér, A.A.; M án di, M .;
Ád ám-Vizi, V. , and Chino poul os, C. (2011 ): A di stin ct s equ en ce i n th e ad eni ne nu cl eoti de transl ocas e from Art emi a fran ci scan a emb r yo s is ass oci at ed with ins ensitivi t y to bon gk rekat e and at yp i cal effects of adenin e nucleotid es on Ca2 + upt ak e and s equ est ration. FEBS J . 278 (5 ), 822 -8 36.
[ IF: 3. 139 (200 9)]
