dm. I d . Si~pier. Snnitd Vol. 20, N. 1 (1984) pp. 41-52

DRUGS ACTING AS MODULATORS OF NEUROCHEMICAL MECHANISMS OF GABAERGIC SYNAPTIC TRANSMISSION

[C. S. RAYEVSKY, G. I. KOVALEV, A. N. KHARLAMOV, A. V. PRIKHOZHAN and O. V. SHUMKOVA

Instilute of Pharmncology, Academy of Medica1 Sciences of the U S S R , M o s c w

Summary. - Some centra1 effects o/ a variety of GABA - related druqs (phenybut, pantogam, piracetarn, aminooxyacptic acid and sodium valproate) u)as studied on mice and rats. N o correlation was found between the total GABA content in the brain and the tranquillizing rffect displayed hy sodium valproate. Iuc3reased brain GABA level, protluced by AOAA, seem.s to be correla,ted with a genera1 " sedation " of animals, but not with a n "anxiolyt ic" effect. Valproate was shown to be the most efìment antaqonist o/ tl~ioserniearbazide and bicueulline convulsions. I t was o/ interest that vnl- proate toas demonstrated to impair the trace fixation of memory in rats (passive avoidanw techniqw). The lipophilic phenylderivative of GABA (phenybut) u a s shmun to produce n i th in mioromolar concentration rmqe a pronmunced increase in the efflux of [ i I f E GABA from superfused rat brain coviex synaptosomcs. Th i s effect i s probably due to the involvem.ent of an nnknmun mechanism., operating at the presynaptic terminals of gabaergic neurons.

Riassunto (Farmaci modulatori dei riieccanisnii iieurochimici della trasmissione sinaptica gabaer- gica). - Gli effetti centrali d i una serie d i farmaci yabaergici (fenibut, pantogam., piracetarn, .iialproato d i N a , acido aminossiacetico) sono stati studiati nel tnpo e nel ratto. f in d stato trovato u n rapporto tra i l rontennto i n GABA del cervello e l'effetto tranquillnnte del valproato, che invece d l'an,tagm,ista pitì eficace delle convulsioni da tiosemicarhazide e da bicucullina. L'aumento del contenuto in GABA cerebrale che s i

osserva dopo acido am,in,ossiacetico sembra collegnto con un effetto aedntivo piuttosto che con quello " ansio- litico ". Il valproato inoltre interferisce con l'appren- di,rnento d i un esercizio d i un evitam,ento passivo. I l fenibut (fenil-<lerivato lipofilo del GABA) produce a roncentrn,zioni micromolnri u n wu,rcato azlrnento della liberazione d i [?H]-GABA dai sinaptosomi 1l.i corteccia

cerebrale d i ratto superfusi; questo effetto *i dovuto ad un meccanismo ancora n,on chiarito, probabilm~nte dipendente da u n in@nza sui terminali presinaptici.

The involvement of y-,iniinot~utyrio iii:i~l ((li\ M) in the pathogenexis of ir vlrriaty i ~ f ni!iir«l~~~(i<~irl iiii~l pychiatric dinorilern, r7.q. , I ~ I I I I ~ , ~ I I ~ ~ , I I I I ' H ~ I I I I ~ I ~ , Parkinson'n ~linenne, convulxivo I i t ~ ~ r l r iind probahly schiao-nffcctive nt,aten l i a ~ I~cim recrnt.ly hypothesizcd [l-41.

A numher of drugs fiir whicli an interaction has been descrihed with gabaergic transmission can I>e considered as gabaergic driigs [.5, 61. Some of these compounds, such as haclofen, phenyl-GABA (phenybnt), progabide, pantogam, sodium valproate and piracetam appeared to be of a certain therapeutic value [2, 3, 61.

The aim of this paper is to test a numbcr of gaba- ergic compounds studying their influence on gaha- ergic synaptic transmission, mainly a t the presynaptic level.

The experiments werc carried oiit on l60 male Wistar rats weighing 180-200 g a d 180 male alhino mice weighing 18-22 g (nnimal farm " Stolbovaya ", 3foscow region). Drugs: phenybiit, pantogam, anino- oxyncetic acid ( A O A A ) , piracetani, sorlium valproate, hiciiciilline, picrotoxin, pentilentetraaol, obtained froni commercial sources. Corivulsions in albirio niice were elicited wit,h thioseniicarhazide (TSC, 18 mg/kg, S.O.) or bicuculline (ECC, 3 mg/kg. s.c.).

Rehavioral studies (conditioniiig, passive avuidance conflict situntion, etc.) were performed on Wistar

90.0

100.0

80.0

90.0

87.5

66.6 (n)

100.0

100.0

10. (i (a)

'I'he drugs were injected a t various interviils befare BCC (:i mg/kg 8.0.). (10 11 < 0.05.

Phenybnt (200 in&$, 30 inin after intraperitoneal iidministration, elevated the hrain GARA oontent without noticeal~le alterations in t,he activity of hoth GAD and GABA-transaminase (Fig. 1). Inter- cstingly, pantogam (SO0 ing/kg, i . ~ . ) , chemically

FTO. 1. - Effect of nome CABA annloguea nn tlie rnt brnin GABA conteiit, (:AD snd GABA - trnnsnniinaae activity. Oprn I>ms - (l.ZB.4 content,, nt,riped - <+Al), rrossorl - G4RA - 'l' ni;tivity. Ordinato = per cont rnodifiontioii~ af tlir hasail l , Besal v:has: GABA. - 2.22 pmole of CABA per g of hmin tisniie: OAD-34 + 3 (*mole GABA/lioi~r-'1100-' mg ii1' protlrili; l:.\Bi\-T 5!) 1.8.5 ~piiiolri HS.\;Ii<iiii. ~ ' / K ~ - ' or lwbiu t iwio * ,i < 1).05 (Stiirinnt t,-t.net,). ii.s. - ni,t aigiiifiriint.

C A O A A Pan. Pir. Vai. Va! BCC

Fio. 2. - Effect of gabnergie h ~ g s on " coiitiicl " belinvior in rats. Ordinate = number nf piinishod wnter4rinking; Ahscissa, = C: control; AOAA: aminooxyncetic neid (20mg/kp); Pari.: psntogam (500 mg/kg); Pir.: pirarctnm (1400 mg/kg); Vsl.: valproate (200 rng/lig); BCC: bicucitllino (9 tnglkg),

p < 0.06 (Studcnt's t-test).

t (s1 l st day 2 nd day 1 4 0 4

l 4 0 i C o n t r o l

160 j S c o p o l a m i n e 2.5 rng lkg

- Buffer A +-t K +30 m M .-. Ph 1 0 p M

FIG. 4. - A = Effects of Ei+ (30 mX) m d phenybut (10 VM) on []H]-GABA effliix from nynaptosonies; B = Conoentratioii- dependent effect of plicnybut on Kt-induoed eKlux oi [IHI-GABA. Eaoli d u e is tliu mcan 3, S.E.M. (6 eapriments).

l'li. - plienybut.

related t o ~antot~henic acid, increased GABA oontcnt, GAD and GARA-transan~inase activities. Piracetani a t the dose of 700 iiig/kg sliglitly decreascd tlie Iiraiii GABA content. with no significant changes in enzynie activities.

The presynaptic releasi. «f labelled GABA was studiod in a special set of experiments, uning super- fiision technique [IZ]. I t was fourid that phenyliut, within a wide ooncentration range (1-100 ILM) could increase both spontaneous and 30 pM KRC1 - evoked efflnx of trit,iniii froin synaptosoni~s preloaded d l i [)H]-GtlB-4 (lo-' M) (Fig. 4). This effect of pheiiyhut, a t the concent,rat,ioii of 10 pI!i i blocked by bicu- ciilliiie (10 pM) and I I ~ ~~icrotoxin (100 p51) a t con- ceiitrations mhich 77cr se do not affect GABA releane (Fig. 5 ) .

Drrigs which affect GARA-inediat,ed t~raiisiiiissioii are widely used in patieiits for tlin trcat,inciit o i a, vari+ of neiiropsyohopathological conditioiis. Tlicse compounds are also used for tlw ~t,ii<iy of i l w fiinii- tionalrole of GABAast1ieiii;~iii ii11iil~iLi~i.y t.n~iisiiiitt,ci, hoth in ceiitral ani1 pei.ililiiwl i i ( w ( ~ i i s sysli,iii 1.51.

Two lines of iiivestigations-lehavioral and iieurochemical-were undertaken in order to find out, whether or iiot t,liere is a correlation I~et~weeii these two aspects of gabaergic drug effect,~. Ko correlation was fouiid betweeri the anticonvulsant act,ivit,y of GABA aiialogues anci their al~ility t o elevate the t,otal GABA leve1 in the Iiraiii tissue. No drug-relnted changes in t,otal GABA content. in three braiii arcas wcre observed aft,rr miiuiii valproatc. This cari he explaincd Iiy the capaiit,y

FIG. 5. - Antn,ganiam hy piarot,oxin and hicucnlline of plienybut artion on ICi~-stirniilntod cffliis oi [?H]-GARA. Eacli rnliir is tlie nioeii + S.E.BI. (6 expt!riiiianla). P11 =

phenybrit,; BCC - hioucullino; Pt. = piorotoxin.

i d vdproate to accumulate GABA selcctively in i Iin fiinctionally important termina1 coinliartment cdf gabaergic neiirons [11]. Sirnilar considerat,ions iiiiglit be applietl t o tlie "anxiolyt~ic" cffect of wlproate and its influence on meniory proeesses. 'I'lin total increase of the brain GABA Ievel produced, I'or insta,nce, by AOAA a t the dose of 20mg/kg was followed hy a genera1 riepressant (sedative) i,lkct on miimal hehevior. In contrast t,o i t , the " tranrpdlizing " dose of valproat,e, 200 ing/kg, didn't ~xoduce any inhibitory effect on locomotor activity or latcncy of well-learned CAR in rats ['i]. Mean- while, AOAA heing a potent inhibitor of CABA t,ransaminase, elicited only a dight " :~nxiolyt,ic " rffect in rats exposed t o " conflict xituations ".

Phenybnt, used in patients no a sedative tranquilli- zer 1151, exerted a moderate increase in total brain UBBA cuntent, heing ineffective in the GABA uptake systein 111.

These fintlings may siiggest tlie exist,ence of GABA receptors of a special t,ype h a t e d on t,hc presynaptic terminals of tlie gabaergic neiirons, seilsitive to Iiiciicnlline aiid picrotrisin.

Stimnlation of thcse receptors hy phcnyhut wns dernonstrated in oiir conditions to cause an increase of ['H]-GABA efflux from hrain cortex synaptosomes. This type of autoreceptors iuight he implicated in the coiiiples regulatory iiiechaninm controlling presy- naptic release of GABA. Tliey differ fnnctionally from the GABA, sites [19], which seem to be hetero- rcceptom bccsiis~ t,heir fnnction is probably the inhihitory inodulation of the release of noii-aminoacid- ergic transmitters, cg., monoamines [l'i] and acet,yl- olioline. I t is known thnt phenyhut binds with the same t,ype of GABA receptor as baclofen 118, 191. Thus, phenybiit and prohably haclofen may interaet with GABA autoreceptors wliicli differ frani tliosc of GABA, type.

1. MELDRUM, B. S. 1978. Gamme-itniinobiityric aeid nnd tlio wnrcli fui. iiiw niktii:onviil*i~r~t drirgri. lxr»<.rl 2: :liM :Il#l.

2. EmRrcH, H. M., ZBRSSEN, D,, Klss~INa, W,, MOLLER, H, J . & \ V I N I I ~ I ~ I ' I I I I , i \ . I I I H I I . K l l ' ~ ~ l , ,>l' wo#li~~!bb v n l ~ w ~ ~ ~ ~ l "

inmia. Tlio GABA - Iiypotliesis uf nffective disorders. l / l V , 2 : l l i ) .

3. Emn, S. J. 1980. CABA nnd neiiropsychiatrio disor~lera. (!,<i,r<il. . l . .V~iir<il. Sri . 7: 2fi7 2511.

4. HARE, T. A. 1981. dlterntiona of centrnl GABAergic nct.ivity in nn,troli>gic i i ~ l I~ayi.iiiitl,i.ii. ilisiiriliii~: i~i,iiliiiilitm llirniigli msli.simmonts of CAB.4 and GAD activity in cprehronpiniil iiirid. M»l. ( ' P I I . Bior,lhr»~. 39: t>!J7-:Mi.$.

5. K n o ~ ~ s ~ i ~ n a ~ - I ~ n i ~ s ~ x , P. 1981. Gamma-aniinobutyrii aoid :~goniata. anti~gonista, ~ L I I I I ~~pt , i~l<c i n l ~ i l ~ i t ~ m I ) C Y ~ K I I i ~ t d tl1er~- peutio wperts. J . M&. C'h~m. 24: 1377-1383.

15. RAYSVSKY, I<. R. 1981. Nuilroc1ieniir:sl aspert,s of plinriiiarolagy of CABAergio subatanccs. Fu~<rl.»infid. TubiPoZ. (Mloscofo)

44.: 517-,528 (In Riianiati).

7. R ~ u w s x r . IC. S. k Kn~a~arnov, 4 . h'. 1983. C:ARAergic driig8: ~4feets un coiiditianiiip, nieriiory nnd learning. I'hrrr- mncol. I lps. Comm. 15: 86-90.

8. C!RAYYMAN, W,, H.msrx&, E. 9T PLWK, b1. 1955. Eino Metliode ziir qimntitntiven Bestimniimg dsr Aininos8iireiisiisnm- inonnotziing von Eiweiwliydralysatoii diirrh Iionihination von Eleit~roplioreno und Cliroiiintogrepliio. Hrip j>e -Sdw's 2. P/q&ol. Cl(wia. 229: 258.

9. Lows, J. I'., Ronr~s, E. k E Y ~ X A K , G. S. 1938. Tlio Hiioroinot,rir nini~siiroiiient i i l gliitaiiiio dcwrboxylaso nnd its Uistribiit,ion in hrain. J. .Vei,roi.liem. 3: 8-18.

10. Vnsri.'sv, V. \'W,, Sn~msKi, I. A. & R'ri,i>r.~irva, Z. l i . 18.70. Viirtlior iiivost,iptioii of t,lir p r q ~ r t i i ~ o or a(-;irrrinohi~ty- r;~tc-glilt:aninto-nniinotrr~nsferi~~c. I&khimi!,n 315: 656-561 (In R,~tmi;m).

11. l i o v n ~ i , : ~ , G. I., Pii~iuro~..~r~s, A. V. X: Ihrwsni., li. S. 1983. Prrsynaplic winliuia:i\t in tiir iiwrliiinisiii of plionybiit nction. Di!11. Ekkls,,. Hiol. J f d . 94: 69-61 (In R~imian).

12. It,~rr:nr, M., Ascr:r.mr, P. k I.svr, .T. 1972. A rimple iippnratiis for ntiidyiiig 1110 rrlc;is~ uf rii.iit~i,triitisiiiittera f rmi synnptuiiomes. Eilr . .I. Pbrii-mniul. 25: 411414.

13. Lownu, O. H., it0sir~nnriirr:ir. S. J., F.iim, -4. L. & IL~NLI.<LI,, 11. .T. I V f i I . 1'rolt;in L I W ~ I S ~ I . O I I I W ~ wit,lz tlie 1~'oliii phniiol rcagentL .l. 6Ui1. (lltim., 193: ?li5-Y7i1.

4 A I L A . . I : 1 . l I!cliillar comprtmenta of l:1\13.4 in hrnin siirl thcir reial,ian~liip lo niilicoiivii~sailt ihctivity. . lId, ('?Il. B im4~m. 39: :1111-:l:lO.

16. I<III\~NINI, I<. A. & LIP~S, 1. P. 1!l7(i. l'het~yhnt,, :b new t,ru~quillizw. l i l ~ i t u . f l u w ~ , 11,. 12: 122-127 [ lu ttwxi;~n].

17. I l i iwi . : l ; \ . i\. i:.. 1 1 i ~ , i , li. I!. k. I l ! i i t , ~ i t ~ . .t. 1.. I H S I . pH]-GAIM niid [3H]- l ,nr l r i fen aro lig:>rirls fnr tlir nani<. I>iriiitillinr-

i i i s i m i l i w x i l v i 1 8 1 ~ w l t t u u t l i ~ ~ t ~ ( ' W t i p w t p l i v n ~ w n l ~ ~ a ~ ~ c ~ . Hril. 7. l ' l ~ n ~ ~ m > l . 74: 22%223.

I :i 1 1 1 ; i i I ~ i i i i i i u I I i iIii.~r I i i , : i u l Iiwi,il Lliruiigli tlie Iiolr 1i.1'1 < ,p , i i l)! t l h , l t l , t , l ~ , , , ($1' I I I G , ~i~ilIot , inr and

, , l .< . l i l l l~ l l ( 'O l < , 1 1 1 1 . 1 1 <. l#: '< . . 111<.'1. I l l i l l ,<l l , i l \ '< ' l .c lP1>1.,!

r<~111xI~i~il '1 1iiiii.s > i d:iy S u i . l i ii i l ~ i ~ ! i i i l i l t l i i . aiiiiiials .;]io~taiii~i~iisi). ; i s i i r i i i ~ I I I h . l i i i i l l ~ i i i s i l i i , i i ~ v i l l i tlieii. ncck iniilrr tliv giiilliilitii, l i 1 1 1 < 1 i ~ \ i i i l i i i i i i o ~ q ~ ~ s i n ~ any resistanoi,.. TIIC~SC rills wi,rc, l k i l l i ~ l l,! r1~11icIly slicling down tlic 1iI;1ili. ii1'tIii, giiiIIii1 iiic,. N ~ i i \ . i , iiiiiiii;ils were left, in their hi,iii<.-ciigi. i'iir (i H iliiys. I . I i v i i I ; i l l d hy &illotine as were I i a , t i~ l l i i~~ l ~ ; ~ l ~ i l ~ i : ~ l ~ ~ ~ l rtit,s. Tlie foot shocl< stress consistcii nf i si4i.s r i l ' i~I~~i~tric;~l foot shoclcs delivered in iiidividual I ~ u x i x witli flocirs made of brass rods, 2 cui aliart,. Shocks wcro priiviilcil by a stiinulator wi-liioh clelivered a shock of 2inA every 320 insec witli 160 msec durat,ion. Rats were f o d ahooked continuoiisly for 3 min. All anirnals were killed betveen 10.00 and 12.00 h, the hrain rapidly renioved aud the ccrehral cortrx dissected.

I n order t o pe.rform [3H]-GABA binding, tlie fresli tifisue was homogonized in 10 vol of 0.32 sucrose. The P, pellet was dispersed in H,O iising a PT 10135 Polytron. Aftcr oentrifnga,tion the pellet was wasliecl oncc by resuspeii.;ion and eeiitrifugtioii i11 10 vol of 20 mM pot,assinm pliosphate huffer, p H T4 coii- taining 50 mM KC1 and frozen overnight. The frozen memlnanes were then thawed and exteiisi~~ely waihed in 20 inM potassinni pliosphate I)uffrr. 1iH 7.4, coii- taining 50 mM KC1. For 1,indjng as.%,y the t icsri~ was resuspended in the same pot,assiuiii phosphate huffer in tlie preseiice of d iaze~~an i (5 x 1O"M) and in the presence of 40-320 nM [?H]-GABA (spec. act. 35 Ci!iiimol. Aniersham) using 200-300 kg proteiri samples of hrain inenibranes in a t,ot,al incu- bation volume of 500 pl. -4fter 10 niin incubation the react,ioii was st~opperl by ncritrifugation a t 48,000 x g pe.r 10 min. The pellet \va.: gently aashed wit,li 4 nil H,O then dissolved in 3 in1 scintillat'ion fluid. Specific ['H]-GABA binding was calculated by suli- traoting the aniount of radioactivity l~ouiid in t,he prescnoe of L'H]-GABA alonc n-ith that Liouiid in t,ho

Table 1 shoms tliat cnrt,ical inciiiliranes froiii haud- ling-habituated rats have a liiglier iiunil~er of [3H] GABA hinding aites than do ineiiihinne~s froni naive rats. fhen foot shock was delivrred t,o bot,li iia,ive and handlirig-h,zbitiiate<t rats. t,his treatnient inark- edly deoreased [?H]-GAEA Iiiiiding in cortical niein- lirane preparatiun froin hsndling-hal~it,uat,ed rats 11ut failed to furtlicr decrease [3H]-GARA I~iiiding in tliose of naive ones. The foot shocl<-induceil drcreaie of 1%-GABA binding was excliisively duo t,o a de- orease in the density of GABA recognition sites. 111 fact, t,he total conceiitrati«ii of bincliiig sites in the cerehral cortex of liaiidling-hahituated rat,s and handling-ha,hituat,ed foot shocked ones was 6.02 i 0.60 and 3.61 5 0.36 pmoles, respectively.

On the other haiid, focrt shock rlecrcased t,he a p p rcnt affinit,y of GABA for it,s hindiiig sitrs (620 & 54 nM in lianclliiig-habituated and 291 3, 40 nM afker foot. shock).

In order tu Iietter clarify tlie act,ioii of stress oli GARA hinding s i t o , we stiidied wliethcr diazepani reversrd the decrease iii the density of G.kBA binding sites elicited by fuot shoclc. As sho\\?i iii Tahle 2 the additiori of diazepairi (5 x 10A M) t o the cortical menihrane preparat,ions froni Iiandling-liabituated foot shocked rat,s incroosed the low affinity QABA binding sites t u ap~iroxiniately tho leve1 found in handling-liabituated rats.

Moreover, tlie siiiiulta,~irous additi011 of Ru15-l788 ( 1 7 ) n specifie antagonist of lienzodiazepinc recept,ors' to iriemhrarics previously incnbated witli diazepan~, reverse,d tlie efiect o i tlie. M t e r hut failed to antagonize tlie effoct of fuc~t shock on GABA binding sites (resiilts iiot shown).

Tn,lili. 1. - ,T[/& «i foot shocli on the kinetic cuvrrpown/s of l'H]-GABA liirdis17 i 7 1 nuiw ( 1 7 d Jiic?idli~~y-I~abit~in(e$ rats. ~ ~ ~ ~ ~

---p

s r . i~111-<~4u.\

N d v e 1l:ilr Ii;iii<lliiic-lliilliluntod Rnt r

Hmhx ]<D Biiinr ED - ~

Control.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.50 j: 0 . 4 5 312135 5.84 i 0.66 (n) 6 2 6 i 6 2 ((1,)

Foot sliork . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.65 0.35 333&35 3.RZ -t 0 .42 ((il 357136 (71)

(n) p < 11.01 witli respcot t o naire rets. ( b ) p r; 0.01 a i t h ieqmct t,^ handlinp-lmhitu&t8d rats. E d i rnl iw is t l ip niem S.E. of 5 sepai.at,r experirnmt,u. 3 naive and 2 hnndliiig-hahituacL.d rats u-re used in eech

exprriiniwi. Bmas iii pol / i i iy pmtcin: lio in nX.

5. I , i . . A 4 . . S . . l . S I l , C , M. G., K ~ ~ , i'. C ! . ' & S . , l . I . 4 S ~ ~ P S R and 3-carl~olitioa ~WI.ULLHU t . 1 ~ < I w ~ i ( y o! IIW nlliiiil? OAUA binding sitea; ari ~f fcc t reverwd by dinicpani. i+& i i r s . 305: 13-18.

9. BRAESTRUP, C. & NIELSEX, M. 1981. 3H-propyl fi-carbolin~~3-ritrb~xyl~te 8ni a srlective radioligand for thr BZi benzo- diazopine rnroptor B U ~ C ~ ~ J S . J . ATeuroebem. 37: 333-:341.