MEMBRANE-RELATED EFFECTS OF ATYPICAL ANTIDEPRESSANTS

I , y o t i . i i i iiindiiic~s it,h rtxctivity. (!mrplin~ nf 1 ip i11I -

l , c r i l ~rit , l i acleiiylati~ ry<:lase stri~iigly I I I S ~ > ~ ~ ~ < I S nii t,lii: fliiidity of li11id 11ilayi.i..

l3iit,li c:l~ssi:il trir:yclir a,nd at,yliic;~l AJ)'s, iri ailditioii t.i> t.livii. uwll rl~icrimciitcrl cficct,~ o11 MA iipt;Lkr, int,crai;l. ilir~.atl,y vitli a iiuiiil~cr ol' c c i i t d lieurotransiiiitlcr rcccriti~rs [5j. Hiiwcver, thcre is iio evidenoe of a corrclation 11ct~eeii tliis e f i c t ani1 t,lii: clinica1 effic,acy. Sonie at,ypical AU's do not afict tlie uptakc of noradreii;tlii~c (NA) aiiii scrotoiiiii (5HT) aiid liavc iio afinity to iiiiiprsiiiine (11121) i>r desiiiethyliiiiilirii~iiiiie (DMI) hiiiding sitw, Iiowcver thcy niodify t,lie transfer <if NA sigiial LGJ.

Tlie rolc of n~cnil~raric ~ ~ l i o ~ ~ ~ l i u l i l ~ i r l iiietliylat~iiiii i i i

the transfer of tlic receptor-rncdiated biologica1 iiifcir- niation has Lccii rcoeiitly deiiioiistri~tcil ['i]. Iieceiit result,n sliow t,h;it phuslilioli1~id iiietliylatiuii iri m t cortical ineiiibraiies is affected by acute adiiiiiiistratiiiii of DMI 181. This troatiiieut pruiliiccs clianges in t,lir fliiidit,y characteristics of tlir iriciiihraiic aiid tlir sigiial traiisier hetwoon NA aiid its rec,c~itors cali IIC modified. It is also posiiblc that AD'S inay act Iiy a corninon, as yet unkiiown, iiiechaiiisiii of iict,ioii. UTe proposed tha t some fuiidainental, perliaps. iiie~iil~rai~c-relaterl eficcts of N ' s could Iead to t;iniiiltn.iieuus altrratioiis iii ticveral iieurot~ransiiiittcr systciiis. l'ri!liiiiirinry findiiigs liavc becn lirevioiisly ]1r'!"""t<1 [Y-l l].

Tlio a,iiii of this study wan te> iiivestigst,e tlie rciii- tioiislii~is betweeii t,he affinity for memhraiio lipiils of severa1 AD's aud their effeot oli iicurotransiiiit~teus uptake.

Tho 31I-iiiii1~raiiiino biiidiiig studics were carricd out aocording tu Raisriiaii rd nl. L13].

Tlie intcractioii of AU's with ii~odcl lipirl iiiciiil~ia- ncs was studied using tlie fluorcsoeiit probu l-aiiiliii- oiiaphtalin-8-siilphonattc (ANS) wliich biiids to t>lic polar liead groups regio11 of lipid bilaycr [13J. Mudcl li$ bilayer liposornes werc prepared acourdiiig to Batzri et ul. [14]. Fluorenccuce of t,he probe was initiatcd a t ), = 360 niii aud registered a t ). = 480 niii (spectrofluoriinetcr " Optoii ", BltD).

The influeiice of d r u p oii thr. uptnkc uf iicuro- transniit,ters was estimated oli crude synapt«suiiial preparation using radioisotopc tccliiiiqucs [15, I G J . Kinet,ic studies were oarried out in the samr ooiiditioiis, but incubatioii l a skd 3 iiiin and probes were rapirlly filtered on Milliporc, filters (0.45 p ) .

Synaptosomal inonibranes were obt,ained ;1s dc- scribed b y Christianscn [l?']. Relative hylropho- bicity of drug? was estimat,ed accoi.diiig t o their

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M - I ' IIUU~Ol~O

Iiisn = ooiiceiit,rnt,ion of drug tlint inliibit,~ 50'%, of t l i ~ qxxiifii: Liiiiding of 'H-iniilrnmine.

Sludy o/ wldive / ~ l r i ~ ~ I ì i l / y o A1)'s. - 111 ordor t« firid out wlict~liei. tlrr iiiterxctiiui of driipi with yhospliolipiil iiieiulirancs i ~ , or is iiot alilx

Ittiiprsmitir . . . . . . . . . . . . . . . lJl:l7 Il. 1 Li5 1 1 . 5

ll3ll . . . . . . . . . . . . . . . . . . . . . . i l . llli 6.7 sii i l . 5

~'liimiprnminc . . . . . . . . . . . . . . iI.%l4 1 8 . 8 X? Ii.iC5 I:i:fwalirio. . . . . . . . . . . . . . . . . . i l 279 li.? E 0.25 I'irlin<lol . . . . . . . . . . . . . . . . . . . O 1141 4 . 6 11.5 11.375

~p

I<u = liindiiig roriiit,nrit. K = niiiiil>rr of Iiiiidiug s i tw I; = cl r~nges of the surfiiit: oIe~tri<: cll iwg~ of t h ~ ~nt.~nImm~~.

tliiir siiiiple triiiisitiiiii f rmi wntrr to i~r,n;iiiic sril\~i~rits. tlie rch~tive Iiyilrcil~liobicit~ of tliese i lr i ip was iimlictl (Talilc 3). A corrclatiiiii wns foiiiiil ( r = 0 X) Iictwecri Iii l i p (rchitive l i y i l r l i l ~ i c i t y ) mi l K1, (hiiuling coiistaiit \ d i iiieiiiliraiic lipids). Riit iio currelatiim wati found Iirtwern tlie p r t i t io i i cuelli- cients ani1 the niinilicr uf I~iiiiliiig sites (also K,N) ii,ii<l the irifliience un the siirface elcctric cliarge of tlie iiieiiiliranc. Therehre, iiitrr;ictiori of clrugs with iiienilirnne lipii1 Iiilnycr iliffers frnin their siiiiple traiisitii~ii froni watcr tu orgniiic soiveiit.

,Vezwotrrmsrnitte.r uytaI<e iii vitro. - It W ~ S ri!l~urt~!~l that thc activity of Ne, K-ATPase, a iiieiii1ir;riir-- I>«uiiil enzyiiie systeiii connecteil with the riiccliiiiiisiii uf iieurotransniitter trnnsport, cari he iiioduh~teil I I ~ chniigcs i i f the st,ate of iiieiiil~rane lipirls 1,201. 'l'ho elyect of AD'S ilrugs oii t,hc accnniulntiuii of iiviiro- trantiiiiitters itiid i~enr~nc t ive :~iniiio acids 11y ci.liiIi. synaptonoiiial preparation froiii rnt hraiii is F ~ I J M ~ I

in Tuble 4. Atypicnl m ' s were foun~l ti, hc Iiw active in agreeiiiciit with previuuu data j5J.

A Iiiglily sigiiitic;i,iit, corr~~l;rtiii~i w n s liiiiii<l I i i~t~rwii t,hc inHueiice r ~ f d i . i i ~ s ~ ~ i i tlie siirft~cu dcctr i~: ~:liiii.:'~' of tlie n ieni l~r~i~ie lipid l~ilayor ;imI thrir i~iliil~itioii of MA ii~itake ('l';~l>li: 5 ) . Tlicri:i'iirr, ;icciiiiiiiliitiiiii of AD'S cntions in tlii! liliiil Ilihyvr oI' i i i ~ i i i . i ~ i i ~ i l

iiieiiibr;~ni!, t l ~ t iiiiliici:~ L i i i I I i i i i * l ~ , i , t i i i

clinrge i ~ f tlie iiii:iiilii.iriii! siii~lii<:i*. I I I I L V Iii! i . i . I i i l i~1

t o chimgcr i i i ;utivit,y iif t l i i ! ) l i \ li~iiinl~airt. rydvi i i~ . 'l'hc kiiictic ~~iitilysis ~ I I ~ N I * ~ ~ Ilin1 M ~ I I I I ~ 4 4 ' I h , 1 1 1 i p

stbi~licil wi,ri! iiiiii~:~iiiilsil i l iw iiiliiliilisr* # i l 3ll4l' i i101aiLv

( i 2 ) 'I'lii, i l i i l i r s l i t i i v I l i i i l . I l i i ~ u u < I I . I I U N i l i o t i r i 1

t i I ilio Ii.tiii;<i~iiili~ru i.iii.rit-r. I i i i l I C I I I I V ~

i i i l i i l i i t . i i i . i i i . i i i i i i l iilil~iki- Imwvww ~ i ~ l t i i ~ i ~ l l r . . i ~ l ~ ~ i i + t ( i i i ~ I I i i i ~wolwrtiw "1' i i i i ~ ~ i i l i r i i ~ i v l i l i i i l f i .

p-- P- - -- -~ ~~ ~- - ~~ ~

~ - - ~

- - P

~ ~ - - ~ - - - -P~~ ~

. ~ . - - <r,~lllli,l.lllilll l",,', ,,t I. ,111 I111I.1

1, Il 1; P---- -P

ill C \ UI.I I \ 'Il 5ll'r 811 <;.\IL\ ~ ~ l ' ~ c l ~ # ~ : ~ $ ~ s , ~ ~ ~ P - - ~~~

~p

1 r 1 1 rhn,r r r / r , i%sh o/ s / r .~ssr~ l II,J/~M</S. ~-

T st;itr: i ~ i syiiapt~iisii i i i ; i l iiic.iiilii.;~iirs «f strcssi.il

a ~ i i r i i ~ ~ ~ l s ~ I i i k r s 111ar1~dIy f'rorii c,oiit,rd rat,s (T:i1~11! 7). T l ~ e i i i i i ~ u i i t of ANS--liri,lir: iii syiialitusoiii;d riiciiilii.a,-

i i w i i i c r r s ~ c i l . Tlir I i im l i ng <:riiirt.aiil. of t l i r . priilii~ is

I i ipl icr i11 st~resscil n i i i i i i i ~ l s wl i i le t l w i i i i i i i l ~e r c i i

I i i r idir ig s i t r s in lowcr. ( 'haractcrist iw nf' ANS I i i i i r l i r ig

t u " r i u i t r d " iiiciiibi.tiiic,- i1 l i ta i i i i 4 f ro l l i c i r i i t r d r;ils

coi i ic idc \\%li data i$ ut l i c r nut l iors I l 7 J Af l i i i i t y iif i i io i i r ia i~ i i i ics cnrricrs iii i i i c~~ i l i i . s i i i~s o i

s t rcssr~l a i i i i i i d s i i i c r w w < l ( T d i l c H) t i i i ~ l t l i e i ~ i ~ i i i l ~ i ~ r

riC ca r r i rw diil i i u t cli;i~igi:. T l i c nct,ivat,ioii of tliv i i ]~ t ; ikv s y s t ~ ~ r n i i ~ y 1 m ~ I ~ ; c x ~II;III~~~s of I I A t , r ~ n s -

ii i issii i i i. Nii, JC--4Tl';isv ;int,ivity r1ccrrai;cil i n st~rcss<,il

;ri i i i i ia~ls (So l i l r !l), iriirl alsii t,lie d i o priit,eii i: lipicl

I\'onidnni:iliiii, . . . . . . . . . . . . . . . . . . . . . . . . . . 11.17 $ il.4S - 0.7 + 0 . 2 5 --(1.8 _L i i . I ~ ( i r ) -11.s <:o. l8

Uripiuiiiiiii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.7 $ I l . X t --O.:: +IL45 -11.5 +( ! .4 t i - O , ! l ~ l l , l l s [ l ~ j

BHT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . l l ! 1 . 4 -1.5 l : - 0 . 9 L 0.0s ( h ) CL4B.Z . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1 ,i ii.4!1 -11.4 *O.*:$ -0.S L 0,2!!(uj - l . o * O.~l~i1~2(1~1

l~liit,:itii;itio . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.33 j 11.41 0 . 4 - 11.!1 i l ( ) - I I S l lllli (rij ~~ ~ - - - ~ - P ~ - ~ - -- ~- ~ ~~ . ~ .--p- ~

p~ --p--- - . -p

-~ P~

-p- -

~ ~ ' ~ ~ I I I ~ P s il, tllble m: rnrrclatinii codiii:ii:iits (r i m) I letiwrii r l i n rwt~r is l i i~s r i l inli~l.;i~vlin>i "t' n~iliililiiossaiils jyibli l i l~>namw (Iiorizoiits~lly) mid bheii. iniliieiiio oii iiphki? ot' iii>iiri>fi.iitirinittni.r (v«i.tirally).

( ( 1 ) = p .,, 0.05; ( h ) p , 0 1 ; n = 3.

Table fi. - Charactekstics of anlidepressnnts interactimb icith synapfoaomal mewhranes nbtained frnm rnl brain

Imipramine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.687 2.42 -

Deriprsniine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.57 2 .37 O.!l

Clomipramine .................................................................... 0.13 2 . 2 O . M

Pirseidal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.16 1.38 0 .75

-- - -~ .-

KB = binding constant; N = number of binding aites; f, - chsnge of t,hs surface uleotric charge of the membrane.

.-

x, (M-') N cnr) KBN KDIM) ~ n i n ~

Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 . l X 1 W 1.7 x 10-3 18.7 6.1 :C IOY 4 . :1 x 1 0 - 6

Ko - binding mmtant; N = number of binding sites; Ko = diswcirition coiistmt of ANA-membrane oomplex; FmOl = molar fluorescenes. All data ani expreasd per mg of protein. Protein content WlL8 evaluated by the mothod of Lawry e1 al. (1951).

Table 8. - Chamcteristics o/ ?beurotransvbitters upluke .qptemn « / M chrowic Stress. -

D O N T R O L R T R B R 8

N I U H O T K A N S H I T T B H

KdM) V-IM) K m I W V-IM)

......................................... Norsdrtinaline 6.8 x 10-7 L .U x 10-12 9 . 7 x 10-7 L .4 x 10-12

............................................. Dopamine I#. L X 10.8 7 . 4 x J ,i. I x I O 0 7 . l X I O 13

................................................ 5 H T . . 0.6 x 10-M 4 . 1 x 1 lW l : x l :I.<! v 10 11

................................................ GABA 0 . 4 x 1u6 4.2 i: 10-ti 2.7 x io n .i.n .< I O 11

.- -~ p - . .~~ . . . . . A

KM = Xiohaelia canatant; V,, = number of carrieni.

Table R. - Aclimity of Na, K-ATPasc alte? chronP'c stress. . -- ---- -- - - -- -

N&. K-ATPss* nctivity. mcM Pilnill iiI piotein p r 1 hour

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Synsptosamal membrane8 fmm 1st brain 12.6 8.9

A'iiiiiwriiis 1iiir.s or i~viil<.iiw niigpst t,li;ct iiiciiil,r;~iir~ lil~irls ~il;iy a iIyii;tiiiic rolv i i i thr; ligt111~1-r~:cr~~t~~~r iiitcrncliiiii 1211. TIN: 11ri1;ir 1i i~; icI g.uiilis 111' pli<isplii~- lipirls scrvc as rcrogiiiti<iii sitcs 10r varioiis ligaiiils aiid r~!giilato recqit:r Iiiii<Linl: activity. Hiitli tlio iiiiiiil~cr iif l~iiidiiig sitcs anrl c~iiililiiig 111' agoiiist- rrciqkur coiiililox wit,li sccoiirl iiicssimgci. is sri1ist;iii- tially iiiodifieil I I ~ tlic stnt,c of' iiiciiil>iaiii: lil~irls, Tlic act,irit,y of N;i, Ii74TI'iis~ syst,riii, i.clatcd ti, t.raiisiiiitt,cr iiptake iiiochaiiisiiis. dclieiiils grrntly ori t ~ I i s i c a l st,at.c of riiriiilir;inc liliiils 120].

Oiir eslicriiiieiits sliii~vcil tliiit AD'S witli iliffiwiit, cliciiiic;il cli:mactcristics nin Iri~iiiiil iii i.lie pl;ir Iicnil groiqi rcfiiuii of iiiriiilirniir (Talile 2 ) . Tliis is iii iiwonl- arice ait,li rlat,a I,y ot,licr aiitliim r~l~taiiicil wit,li spiri-lalieled prol~cs 1221. Tlieir clet,,z siiggrst t,liiil, AD'S art. l o c i ~ t d in lirilai. Iicad groii1is regiini o i incnibrane li~iirls anrl iicnnilept~ics penct,i.ate rlrc~ier iiito lilrid Iiilayci aiirl a,re l i ~ c a t < ~ l i i i t'lie rrgioii rif Iiydrophobic chniiis of Iipirl iiioleculrs. Correlat~iiiii annlyfiis sliouwl tliat bindiiig of AWs wit,h iiirii1i.l iiieiiihranes diffcrs frorii their siiiil~le traiisitiriii frriiii wa.t,er tu organic solvent, t,li«ugli tlicir liinrling ciin- stants arc deterrriiiieil liy t,hc rehtive liyr1ropliul~icit.y of roinl~ouii(1s. Oiir exl~criiiient,s sliowrrl (Salilc 1) tliat n.t,ypical AD'S are 3-4 iirdrrs o r iiiag~iiturl(~ less potent in tho rlisplaccnieiit of lal~elcd iniipraiiiiiio froin it,s liinding sit,es t l ~ a u trir,ylic a i i t i i I rp~.~.ssa~i t~~. Tliis rcsults in iii accordniicc nit,li ot,her data prcvioiilsy reported [IS]. Rowevcr tlie atypical AD'S iiiliiliit, iiciirotransiiiittc?rs iipta,kc. A liiglily significant corre- Iation I~ct~rveeii the inflir~mcr of >W's on thc iii.fa,cc rlectrir oharge of tlie ineml)rane lipirl I~ilayrr and tlieir inliiliition of ncurotraiisiiiitters uptakc siiggests t.li;~t, t,hr accuiiiiilation of AD'S ca,t,ions iii tlie lipid l~ilayer of presynapt)ic nienilrraiies iiiduues chaiiges in tlie clcot,ric chargc of thc mcinbraiie surface wliicli niay Le partly r e l a t d t,u tlie cliaiigos in thc inriiii~. aininc transport systmi. A correlation u w alni foiitid bctwoen tlie iiifluciicc of drugs on tlie surfacc electric chargc of synapt,osomal rneniliraii~:~ and their influence on monoaniines uptake. Tliis suggcsts tliat our liyl~ot.liesis o i tlie possible act,ion uf All's a t tlie iiieiiil~raiic lcvcl sliiruld hc takeii i i i tu consi- deratioii. Thc noncoiiipct~it~ivc iiiliil>itioii oF 5HT uptake niiggests t,liat t'liese . m ' s do iiot sct directly on tlie act,ive sit,e of the carrier, but inliibit uptake

i ~ ~ i l i r i c t l , y l l 1 1 1 1 i 1 i g I ~ r r r t i s i i r

iiiei~iI)rii~~e l i~~ir ls , 1 1 1 1 i i e ~ r o l i ~ i ~ i i c ~ l h t , a coiirci.iiiiig thc nic-

cliaiiisiiis iif actiiiii « i AI)'.: ara ril~taiiir.il iisiiig nii.iii- Iiraiirs of "iiuriiinl" aiiiiiials. Iiiit i t is \wll kiio\vii tliat AU's liroiliice tlieir tlici;il~<&c niitiilrlir(wivr v f f r ~ t uiily oii ilclircsst~rl li;it,ients, Iiiit iiot oli licnltliv 11111teers. Aftcr chrimii. strr:ss \W ol~serverl iii niiiiiials a ilcpresrioii- lilw stiit,,., Rioclicnii~uI ~t~rii l i i~s d i w w l tlint prot,ciii/liliiil rat,ii~ iii syiial~ti~si~iiial niriii- Iiranw of st,rt.sscd niiiiiials cliiiiigc~l iii c i ~ ~ n ~ ~ a r i s o ~ ~ t,o iiieiiil~raiies obt,aincrl fiiiiii coiitrul aniiiinls, 1,rcaiisr. of a rkcrcase in proteiii coiicciitration ci~iitent. WI! ~ i r ~ ~ l ~ r m " c l t,liat tliis is coiiii~ctrd wit,li tlir Iack nf si~inc pcri~rlirral prut,eiiis, due t,o t,lic st,ress, wliicli inny affcct iueinliraiie traiispurt syst,eiiis ai111 eiizyiiit: :tctivit,irs. Clianges iii niciiiliraiic mrface wcre tlit. c;iun: of different cliaraeterist~icii of ANS Iiiniliiig t,ri syiinl~i.os<iiiia,l ineiiiliraiics froiii " dq~ressed " aiiiiiials. Iricrease o" tlie aiiiouiit of fliiore.:ocneo niay 11e ~clst~crl t,o t,lic Iiiglier hyrlrol~lioliicity of inciriliranc suriacc d i i ~ tu t110 clecrcascil iiuiril~cr id ~~criplieral prot,eins. Niiiiiliei. of ANS liinding sitcs a lw drcreaserl, ilceaiisc this ~lrtillc biiids t,ii iiliids and als« tri protciiis. Kiiictic stiiilics showerl t,liat affiiiity of c:irriers f i r iieiirotransinittt.rs iii syiiaptr~siinics of del~ressrd rats inc,reases. Iiiit niiinlicr of carriers remains oi~iistniit~. Tliis nixy Iic t,he reasoii of tlie ~1t.ficit of t,lic iiioiio- aniiiicrgic t~rarisiiiission, ai111 tlic iiiliihit~ion of upt,akr liy AD'S inny " noriiialize " t,lie situatioii.

Al1 topt l icr , our data shriw t,hat niimliraiie- relat,cil processes in iioiiiial animals differ froni t,hiisc in <lopresseil oncs. Tliereforc i t in vrry iniport,ant evalriiite thc ii~iiroclieinical anrl hcliavioral par;irne- teix of aiitirlc1iressaiit~s ori aiiin~als witli a clepressive- like stai,c.

Takiiig iiito accoiiiit t,liu rolr: of spcifio iiieinliraiir tsrgrts fin ant~idel~ressaiits \ve waiit t o st,ress tlic sigiiificaiice of " specifio " cffccts of iim-spooifir iiiaiiiliraiie-rt!latr*<l act,iori of thwe driigs. Sincc tlir csscaile of cyclic AMP-iiiediated evriits fuiict~ioii as a Iiiglily effeet,ive aiiiplification iiiccl~nriisiii [23]' siiwll chaiiges iii tlie stat,c of inciiibi~i~iic lipirls cali ~iiarkeilly cliange thc traiisfer of NA inlnit sigiial. Tlic ineinliiaiie-related cffect of AD'S is a nori- qiccific one. It can operat,e only a t tlie first ste11 of tlic Iirocess of desensitization of fi-a,drenergic sciisit~ivr adeiiylsto cyclase, < . e . , ali alteration in coupling. It is therehre iiryort,ant that pliospliolipid uiet,liylation iii ciirt,ical incinbranes is affect,ed Iiy ~diiiinistration of UMI [8]. Pliosl~lioliliid iiietliylat~iun regulates the fluidit,y of' nieiiil~rane lipids, and this niay infliience t,hc transfer of recept,or-mediat,ed signal ttu th r transducer systeni. m ' s chaiige the