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Association of bromide with the synaptosomal fraction of mouse brain
~yü~Sc~e~c~r~Vt
1371-1380, 1970. Hrü'ain~ I' pp'
Pergamon Preae
A890CIATIOß OP SHOIIID~E "ITH T~ BYßAP'P0801UL 1rRACTIOß Oh 1DU8! eHAIß Zinya Enriya~a Division o! Rsurophar aoolop and ßeuzochsistry, Departcent of Psychiatry, State IIniversity of ßew Yori, Downstate Medical Ceatsr, Brooklyn, ßew York, II,S .A .
(Received 11 September 1970; in final form 19 October 1970)
IIBPITE o! well-known sedative and antiepileptic actions of bra~ide (Br ), the Bode of action of Hi is the central nervous gets (C1~) is still obscare. It has been deconstrated that Br diclnishes the sronoacine releases trac electrically sticnlated brain slice (1) and codifies the synaptic tranoission at choliaergic tercinals (4) . rapid clearance of Sr
Attar gstsic aüaistration of 8 : to rabbits,
irac the cerebro-spinal ilnid (C81~) was observed at low
concentration (possibly by native transport), while the toa~aulation of Br is the brain due to the establishssat of stasdr state squilibrint in brai~CBl~ blood ws touad when a high dose of Hi was adainistered (9),
It is inowd that
brain haswgenats binds Br (4), but no report is avai Lble to date ooncsraing the synaptosacal uptake of Hr which cat be one of the icportant factors is sincidating the action of Br in Q18 . The present investigation sits to detercine the nature of Br association with synaptosaces obtained trac souse brain hoswgenate. lfethods Sydaptosacai sad citochondrial fractions o! cows brain were prepared by density gradient csntriingation of crude citoohondrial (PS) preparations acoordiag to the cethods of dray and whittaker (3) .
Ths synaptosa~al (PZB)
layer was aspirated, dünted by slowly stirring in ice-cold distilled water until the sucrose oonceatration was approzicately 0.44 Y, and tidally sedLented by centritnging at 35,000 Z g, 90 cis, used directly .
1äs citoohondrial (P~C) pellet was
Preparation of subcellular particles was cads in epinoo Model
1371
HROA~E AND 3YNAPT080ME3
1372
L9-Bb H preparative ultracentrifuge.
Vol . 9, No. 23
The g lorces were alwlated using
average radii . IIaless otherwise sped üed, 1 tl o! synaptosastal suspensions containing 4 .~3 ag parti wlate protein/ al were used per tube for each asperiasnt . Aitsr iawbation the particle suspsnsioas were centrifuged at 0 90 aia and the supernate iaediatsly deanted .
C
at 38,100 % g for
The pellets were resuspended
into 1 ~1 0! ice-cold distilled water liter a areiul swabbing o! the interior snriace of the tubes . Yitochondriai tractions were handled esactiy the sue . Innlin-aetl~y- 38 was spioyed to obtain the estiaates of eztraparti wiats space in the several pellet swpeasions, and corrections were cade !or non-bound Br o! the pellst .
Iaulia is not bound or taten up by particles (8) . The radio-
activity o! the supernate and the resuspended pellst were aeasured by a liquid scintillation counting procedure using a tolusnr pPO-POpOP solution oontaiaing 1 wl o! hyaline .
The sodiv. salt of broaide was wed in all szperiawts and
was dster ined spectrophotaaetrialiy (7), in both supsrnate and rssuspended pellet, with tisane blancs and internal standards .
"hen sucrose solution was
used as a reaction =i:tore, the separation of Bi was achieved prior to assay by ion-ezchange ahrcaatographlc procedures acoording to De Oeiso et al (8) to avoid sucrose interference .
Beoovery o! Br after these separation procedures
was established by using a known Bt accordingly .
standard and correcting Br content
IInips otherwrise specified, the aee~aulation o! Br was ezprsssed
in~[,.eq/g protein .
protein was aeasured by the aethod o! Lowry st ai (9) . Rewlts and Mscsusioa
The results in TABI~ 1 show that the synaptosaaai fraction of souse brain binds Hr at OT C.
Hinding of Br
observed in Hinger s solution .
in escrow media was as low as 18 ~ of that
Addition of various ations invariably resulted
in the increase of Hs associated with the syaaptoscees .
Comparing the equiaolar
addition of 1faC1, ]fa1103 and lhI, the addition o! 1faC1 stisulated cost effectively the synaptoscaal binding o! Br ( ib .4 v. s . 8 .8 and B .OfLeq/g protein, respectively) . It is cost probable that iodide (I ) sad nitrate (1103 ) oaapets with Hr
for the
Vol. 9, No. 23
HROARDE AIE 3YNAPTOSOME3
1373
TABLE 1 Sifect o! several ions on the binding o! Br by syaaptosaaes Solution
Sr bound~teq/ag pmt)
Ringer's wlution 8ucross(250 a1Q Sucmse + llaCl(10 a1Q
Percent~~es ca~pa~s with the binding in the Ringer's wiutioa
19,9 (18 .7 - 20 .7) S,0 ( 9,8 -
100
9.S)
18
15,4 (14,9 - 1B .0)
80
Sucmse + !(allOa(10 flf)
8,6 ( 7,7 -
9 .8)
45
Sucmse + 1faI (10 a1n
8 .0 ( 7, 5 -
a. b)
49
Sucmse + ECl (10 ~
18, 4 (19.3 - 14.8)
89
Sucmse + YgCl~(10 ~
18 .0 (17.8 - 15 .8)
94
Sucmse + CaCl 9(10 alp
18 .9 (17,5 - 90,0)
98
All swpsnsions oontaiaad 90 aY glycylglycine (p8 7 . S) and 9rsghl of Br . Suspensions were kept for 30 ein at OTC. lfu'bers in parenthesis indicts supermental ranges obtained free three separate ezpsriaeata. unspsd i1c ionised ationic site%oa the syaaptosaaal asabrane, while chloride (C1 ) does not. that Ci
This phsnaasnoa shows good agtesent with the prsvions ündings
bas little ü any aswciation with synaptosaass obtained fana sows
brain hoaogenats under the physiologitral range of pe, although Î and l10~ are bound to the particles (10), The simulative effect of cationa for the binding of Hr suggests that phone nay be required to dminish ionic iateraotion of Br with the negatively charged surface of synaptosaaal a~abranes (11), Hr
binding of syaaptoscaes at ÔC as s function of p8 1s shown in Fig,1.
Acidifiption of the aediua resulted is the increase of Br binding.
This
suggests that sore atioaic ü:ed sites capable of the binding of Br
are
available on the synaptosaaal esbrans, and that fewer negative surface charges capable of inhibiting the approach of Br to the binding sites are present on the particles.
]fegativs surface charge of the synaptosoaes was found to be
caapletely abolished st p8 4,0 (11) .
By lowering ps, a smilar inarsase of Br
binding to liver aitochondria was previously reported (19) .
BRONZE AND 3YNAPTOrSOMEB
1374
Yol . 9, No. 23
ô â
P Q Y
Fig. 1 Eliect o! pH on the degree of Sr binding by syaaptosa~es . The results show are the average o! three separate szperi~ente. Inoabation o! synaptosa~es was perio:,ed !or 90 ein at OTC in the Ginger's solatioa containing 30 sd< tris-saleate bulfer over a range o! several pH values, and 4.5 - 5.0 ag/tial o! protein. At the end o! the incubation each segle wss receatriiuged (gee methods), and the pH of the supsrnate as ~easared to obtain the final pH o! the incubation ~izture. Ths ooncsntration o! Br added was 1 and 4~eq/al respectively u indicated. The rate o! Sr
binding as a !unction o! 8{ concentration in the reaction
~izture is shorn is Fig.l . Hoth at OTC gad 90sC, the rate o! H: binding was directly proportional to the Hr aeatal conditions a~ployed .
concentration o! the aedi~a under the ezpsri-
At SÔ C a slightly higher binding o! Bi than at
ÔC ws observed, bat this was not statistically significant.
Ser~a~ levels o!
Br (30 - d,! tleq/el) in broeide intazicatioa (13) are 8 - 10 bass higher than the H: added in this ezpsriasnt .
Bowsver within the sedative or antiepileptic
dose range, it slay well bs that the degree o! Br accu uiation in nervous cells is directly proportional to the Br concentration o! C8F or cerebral blood o! the surrounding tissues.
As shown in T11HIa S, Hr aacianlatioa by synaptosa~es
was not sigailioantiy a!lected b7 the addition o! glucose or ttstabolic inhibitors
HRO~E AND SYNAPTOBOMES
Vol. 9, No. 29
1375
3Ô C ÔC
ô
Rate o! bindiaH o! Br b7 s~naptos4es u a l~mation of Hr eoaosntratioa in the reaction ~iztnre . Ths particles were swpeaded is icr cold RinHer's solution containinH 30 sY trirnaleate (piH 7 .3) to Hire a protein conwntration o! X1 .3 - 5 .0 aH/L1 . Altar addition o! varions monta of H! , swpensionfs cers inanbated for ~0 min at O T C and 90 eC rsspectivsi~ . The values in the liHnrs are averaHes o! three separate s:psriaents . such as iodoacetate, DRp or onabain at either 0
C
and 90 e C inwbations .
Plavvate and lactate inhibited the Br acdaalation both at O vC and 90~C iaarbations, sugHesting that these organic anionic ca~poaads possibi~ oa~pete at the cationic site responsible for Hr bindiaH .
Hoth Hlnoose and p~ruvate are
substrates apabie o! sapportiaH ~aaiaobut~ric acid (OAHS) nptaks b7 tease brain synaptosates (8) .
The results suHgest that the Br acco alation b7
gnaptosc~es is lüsi~ to bs a tsperaturs or energy independent process . It is mown that liver titochondria binds Hi (1~ .
In this stnd~, Lhe
accosuLtion o! Hr b7 brain titochondria was also ezstined to sae the spsdücit~ of Br bindinH of synaptosates (TAHIa 3) .
Srotide eras bound
titochondria to a oonsiderabi~ lesser degree than b7 s~naptosotss .
M
brain
llitoohoadrisi
1378
BROb~E AI~ SYNAPT080ME3
Vol. 9, No. 23
~n~eLE s
Acaaulation o! Br b7 s~naptosaaes at O eC aad 90 eC= Inilneaces o! glnwse, p~rnvats, Lctats, and ~etabolic inhibitors. 8 bound ~prot)~S .D. Bnbstanoe tested O 90 ~ 90. 4 + 4.1 43.4 + 9.3 1Wne Iodoacetste ( 1 saI ) 19.8 t 9.0 (- 4) 91.8 + 9.4 (- e) 4, 4Dinitrophenol(Dl~ ( 0.4 elI ) 18.9+9.4 (-7) 19.8 t 4.0 (- 9) Ouabaia ( 0,9 ~ )
44.1+9.5 (- 5) 44.1 t 4.8 (+ 9)
19.0+4.0 (- 7) 18.8t9.4(-9)
49.0+1.9 (- 8) 41.4+4.4(-9)
18.5 t 1.8 (-19) e* 18.0 _+ 1.9 (-49)~ 18.1 + 1.9 (-41) ew
1ß .0 + 1.9 (-49) e 17 .8 _+ 1 .8 (-45) e 18.8 t 1.8 (-99) *
Olnoose ( 10 aa[ ) Oluoose(10 a1Q+Iodoacetate(1 n1Q Oluoose(10 w1AtD1~P(0.9 do p~ruvate ( 10 ~ ) p!=uvate(10 ~+D1~P(0 .4 ath Laotate ( 10 sY )
The results shown are the mean + B.D. obtained frame five separate szperiaents . e P(0.004, es PC0.05. lh~bers iâ parenthesis i~ioats percent changes oa~pared with the respective control values . The particles were suspended is ice-cold Ringer's solution containing 50 tY trirnaleate(pB 7 .9) to give a protein concentration o! 4.6 - 5 ag/al. Aitsr addition o! ~1esq/ai o! Barend test substances, the susp~sions were incubated !or 90 tin at 0'C aad 90eC respectiwl~ . pfavvate, lactate and iodoacetats were used as sodi~a salts and Ba+concentration in the reaction eiztnre ws tept constant b~ decreasing the pouat o! R1aC1 in Ringer's solution. TABLE 9 Acauaulation oi~Br bl gnaptoso~es aad brain a+itochondria Bfaaptosoaes Brain 1litochoadria
~Da^ot~t S.D. C C 19.4 + 9.5 94.3 + 3 .8 4.7 t 0.9
5.8 + 1.0
The results shown are the teen + S.D. obtained faros font separate azperiieats . ~periaeatal conditions were thi ssse as TABLE 9. oapadt~ to bind Sr wu appro:Latel~ 1/4 0! that was louad .in the sTaaptosawes . The inhibition o! Hr binding in sitoohondris b7 >~ , Î , p~ravats, aad lactate
Vol . 9, No. 23
BRONimE AND 3YNAPTOBOMEB
1377
was gwütativsi~ siailar to that reported !or cynaptosaasc (7äBIa 1 and 9) . These recuite indicate tint the aswdation o! Br with s~naptosaaet ie not a speaüia pheaaaenon in synaptosoaes or nerv endings, but ay occur in various snbcellular or cellular eocipart"eats of brain. TABIB 4 B!lect o! varions drugs on the binding of Br b7 synaptocaaes Bubctancs tected
Oongatration (a1Q
x
Chaa~e
picrotozin
1.0
+7
Btryclmiae
1.0
+ 4
Phsaobarbitai
1.0
- 4
Chlorpraaasine
1.0 0.1 0.01
-89 -30 -39
Acet~laholiae
1.0
+ S
s-H~draagtryptaaias
1.0
+ 7
Dlrlforepinephrias
1.0
+ 8
B:periaental conditions were the cane u TABTi 9, ezaept !or the tewpeatare of incabstion. All incnbatioac were carried out at OTC !or 90 ain. The rewits in TABIa 4 are the aeaa of three separate ezpsriaentc . 8tr~chnine sulfate, wdi~a phenobarbitai, chlorpiroaasine hydrochloride, aoet~laholine chloride, 3-hydrozytryptaaine ciwhnins sulfate, and Dlrnorepinephriae bitartrats were aced alter adjnstiag pR o! the drug wlation to 7 .0 . Bquiaolar ions, which were added with above amtioned drags, were adainistered to each control . The s!lsat o! various drugs is vitro oa the gnaptoca~al binding o! Br at OTC was shown in TABYB 4.
Central chaulants (piarotozin.aad stryc)mine),
phaoobarbital and putative transaitter substances (aaetylaholine, 5-~ydros, tryptsaine, norspinephriae and OABA) had ao sigaifiaat eilest oa the binding of Br by synaptosaaes at the ooncsntratioa of lÔ sl( or below.
Chlorproaa:iae(CPL)
was the only drug which showed a signiticaat inhibitory ailed on the syaaptocaaai binding o! Br aeiong the various substances tested .
CPZ at lÔ $, 10 4, cad 10 sY
ooaceatrations, diaiaished the Bi binding 82, SO cad 39
x
respectively .
HRON~E AI~ 3YNAPTOBOMEB
1378
Yol . 9, No. 23
It is well established t)rt CPL has rultipie elects on the reabrane psareability o! variow cells (14) .
The inhibitory elect o! CPfG on the pea~eability o!
ester (ib), H+a+ and L+(18), and acetate (17) ws reported in a variety o! cell r~branes.
It is probable that the inhibitory eliect o! CPL on the synaptoscui
binding o! Br is a relleotioa o! the general rsbrane eüscts o! this agent. Ibarapartiwlate apace in the pellet reasoned by im~lin-retbozr 9H wan not chaageE by the agent.
This suggests that the change o! oriti al wins o! the particles,
as observed in the inllueaa of CPL as the wires of erythrocyte (1B), ray not be the anse of the CPL e!leot on the Bfbinding o! syaaptosores . Oorrents The association o! Br with syaptoscaes iadiates that the synaptosaral r~brane contains es:posed, highly ionised cationic oonrtituents to which various anions ray bs board by ionic forces .
The synaptosaral and synaptic vesicle
r~branes also posses arposed and highir ionised polyanioaic groaps which are the site for binding sad retention o! various ationic substances each as potassia~ (19), aoetyicholine (90) and 3-hyd:orytryptarine (91) .
lrurtlur
erperirents wül be directed at characterising and ideatilying these anionic and cationic coastitusnts o! the syaaptosoral rsbraae which are apable o! binding various ionised substances . Although there is no doubt that the syaaptoscaes (prssynaptic endings) have a relatively high alüaity !or Br , the p~arraoologi a1 signiliante of this phenareoon in the Cl~ will require lurther investigation .
Binding o! bide by the synaptosaaes sad ritochondria o! rouse brain was studied upder a variety of ezperiremtal conditions .
The synaptosaral uptate o!
braride wan energy independent and was facilitated by the addition o! ations, While the presence o! other anions such as nitrate, iodide, pyauvate and lactate inhibited the association o! brcrids with synaptosares .
Acidili anon o! the
iawbatioa redis~ resulted is the increase o! brorids binding with synaptosaaes .
Vol . 9, No. 23
HROII~E AND SYNAPTOBOME3
1979
Brain sitoohoadria alw bound broside but such less extensively than synaptoscaea . The results are ooaristeat with the interpretation that the binding of brosids appears to resesble an anion ezohange process .
Oae of the attributes o!
the braside binding rite on the synaptoraaer is the prsseaoe of exposed, highlyionised cationic constitaants .
P~rthsx~ore, the results suggest that negative
surface charge of the synaptoroses say also bs a regniatory factor for the binding . It was found that asoag the various substances of neuropharaaoologigi interest tested only chlorprasasins inhibited the synaptosani binding of brallde . Admowled~sat The anchor is grateful to lliss ". t . Hoopea and Ilhr. 8. (nabs for stüllul technical sssistaace .
This woaic war supported is part by grants l~18883 and
III-18477 irca i
4, R.O, Hayden, H, Garoutte, J . lhgner,and R,H, Aird, Froc, Soc, Exp, Biol, ~7, 734 (1981) . S, E,(i, Gray, and V, P, hThi.ttater, J. Anat, (Load .) 98, 79 (198,
8 . !, Rnriyasa, H, "sinsteia, and E . Roberte, Brasa Res . 18, 479 (1989) . s 7, G. Hunter, Hioch~. J. 80, 981 (1935) . 8, R,C, Ds Gesso, A, Riasann III, sad 8 . Lindenbaa, J. Anal . Chs . 98, 7S 40 (1934), 9 . O.H . Lowry, hf .J, Rosebrough, A,L, Parr,and R, J . ltaadali, J . Mol . Chas 76S (1951) . 10, R . Ruriyasa, and E . Roberte, Abst . 4th Inc . Coag . on 1?haisaool .(Hasel, Jnly 1989) p . 395 (1989) .
isso
sao~E nrm sYxapTOSO~s
vo~.
9,
xo. as
11, J . Vos, t, lltri7saa, aad E, Boberts, Brain 8q . _, 994 (1988) . 19 . J .L, asnbie, ~r., Proc, eoc. Etp, Biel . Yed . ~S, 37b (1983) .
13 . D,J, Ilsller, Taas Yed. 84
4 , 79 (1986) .
14, P,B, Onth, and 1I,A, 8pirtes, Int. 8ee, YenroMoi . ~, 931 (1984) . ib . A,B. lre~aa, and Y,~. Bpirtes, Hioeh~ . Pha:aacol . i~, 19Sb (1983) . 18, J. ~risteasan, T,B,L. P~i, E . lbilq, aad A, ". "us, hed. Pt+oc. 17, 986 (19b8) . 17 . B,Y, ChaYoeels, and T,II, 8htnlaaa, Dhr, Biothia, Zh, ~ b8 (1989) . 18 . J. esn Stee~aiaei, ",t . Qiônmd, aad H,L . Hooi~, Biochea.Phasacol. 18, BS7 (1987) . 19 . H . "einstein, and t. Eari7saa, J, Yearochea, _, 493 (1970),
90, t. ihrijra, E . 8oberts, aad J . Yos, Brain 8es. 9, 931 (1988), 91, J,D, BoMason, J,H, Andersoa, and J .P. Qreen, J . Phasacol, a
~, !36 (198b) .
1371-1380, 1970. Hrü'ain~ I' pp'
Pergamon Preae
A890CIATIOß OP SHOIIID~E "ITH T~ BYßAP'P0801UL 1rRACTIOß Oh 1DU8! eHAIß Zinya Enriya~a Division o! Rsurophar aoolop and ßeuzochsistry, Departcent of Psychiatry, State IIniversity of ßew Yori, Downstate Medical Ceatsr, Brooklyn, ßew York, II,S .A .
(Received 11 September 1970; in final form 19 October 1970)
IIBPITE o! well-known sedative and antiepileptic actions of bra~ide (Br ), the Bode of action of Hi is the central nervous gets (C1~) is still obscare. It has been deconstrated that Br diclnishes the sronoacine releases trac electrically sticnlated brain slice (1) and codifies the synaptic tranoission at choliaergic tercinals (4) . rapid clearance of Sr
Attar gstsic aüaistration of 8 : to rabbits,
irac the cerebro-spinal ilnid (C81~) was observed at low
concentration (possibly by native transport), while the toa~aulation of Br is the brain due to the establishssat of stasdr state squilibrint in brai~CBl~ blood ws touad when a high dose of Hi was adainistered (9),
It is inowd that
brain haswgenats binds Br (4), but no report is avai Lble to date ooncsraing the synaptosacal uptake of Hr which cat be one of the icportant factors is sincidating the action of Br in Q18 . The present investigation sits to detercine the nature of Br association with synaptosaces obtained trac souse brain hoswgenate. lfethods Sydaptosacai sad citochondrial fractions o! cows brain were prepared by density gradient csntriingation of crude citoohondrial (PS) preparations acoordiag to the cethods of dray and whittaker (3) .
Ths synaptosa~al (PZB)
layer was aspirated, dünted by slowly stirring in ice-cold distilled water until the sucrose oonceatration was approzicately 0.44 Y, and tidally sedLented by centritnging at 35,000 Z g, 90 cis, used directly .
1äs citoohondrial (P~C) pellet was
Preparation of subcellular particles was cads in epinoo Model
1371
HROA~E AND 3YNAPT080ME3
1372
L9-Bb H preparative ultracentrifuge.
Vol . 9, No. 23
The g lorces were alwlated using
average radii . IIaless otherwise sped üed, 1 tl o! synaptosastal suspensions containing 4 .~3 ag parti wlate protein/ al were used per tube for each asperiasnt . Aitsr iawbation the particle suspsnsioas were centrifuged at 0 90 aia and the supernate iaediatsly deanted .
C
at 38,100 % g for
The pellets were resuspended
into 1 ~1 0! ice-cold distilled water liter a areiul swabbing o! the interior snriace of the tubes . Yitochondriai tractions were handled esactiy the sue . Innlin-aetl~y- 38 was spioyed to obtain the estiaates of eztraparti wiats space in the several pellet swpeasions, and corrections were cade !or non-bound Br o! the pellst .
Iaulia is not bound or taten up by particles (8) . The radio-
activity o! the supernate and the resuspended pellst were aeasured by a liquid scintillation counting procedure using a tolusnr pPO-POpOP solution oontaiaing 1 wl o! hyaline .
The sodiv. salt of broaide was wed in all szperiawts and
was dster ined spectrophotaaetrialiy (7), in both supsrnate and rssuspended pellet, with tisane blancs and internal standards .
"hen sucrose solution was
used as a reaction =i:tore, the separation of Bi was achieved prior to assay by ion-ezchange ahrcaatographlc procedures acoording to De Oeiso et al (8) to avoid sucrose interference .
Beoovery o! Br after these separation procedures
was established by using a known Bt accordingly .
standard and correcting Br content
IInips otherwrise specified, the aee~aulation o! Br was ezprsssed
in~[,.eq/g protein .
protein was aeasured by the aethod o! Lowry st ai (9) . Rewlts and Mscsusioa
The results in TABI~ 1 show that the synaptosaaai fraction of souse brain binds Hr at OT C.
Hinding of Br
observed in Hinger s solution .
in escrow media was as low as 18 ~ of that
Addition of various ations invariably resulted
in the increase of Hs associated with the syaaptoscees .
Comparing the equiaolar
addition of 1faC1, ]fa1103 and lhI, the addition o! 1faC1 stisulated cost effectively the synaptoscaal binding o! Br ( ib .4 v. s . 8 .8 and B .OfLeq/g protein, respectively) . It is cost probable that iodide (I ) sad nitrate (1103 ) oaapets with Hr
for the
Vol. 9, No. 23
HROARDE AIE 3YNAPTOSOME3
1373
TABLE 1 Sifect o! several ions on the binding o! Br by syaaptosaaes Solution
Sr bound~teq/ag pmt)
Ringer's wlution 8ucross(250 a1Q Sucmse + llaCl(10 a1Q
Percent~~es ca~pa~s with the binding in the Ringer's wiutioa
19,9 (18 .7 - 20 .7) S,0 ( 9,8 -
100
9.S)
18
15,4 (14,9 - 1B .0)
80
Sucmse + !(allOa(10 flf)
8,6 ( 7,7 -
9 .8)
45
Sucmse + 1faI (10 a1n
8 .0 ( 7, 5 -
a. b)
49
Sucmse + ECl (10 ~
18, 4 (19.3 - 14.8)
89
Sucmse + YgCl~(10 ~
18 .0 (17.8 - 15 .8)
94
Sucmse + CaCl 9(10 alp
18 .9 (17,5 - 90,0)
98
All swpsnsions oontaiaad 90 aY glycylglycine (p8 7 . S) and 9rsghl of Br . Suspensions were kept for 30 ein at OTC. lfu'bers in parenthesis indicts supermental ranges obtained free three separate ezpsriaeata. unspsd i1c ionised ationic site%oa the syaaptosaaal asabrane, while chloride (C1 ) does not. that Ci
This phsnaasnoa shows good agtesent with the prsvions ündings
bas little ü any aswciation with synaptosaass obtained fana sows
brain hoaogenats under the physiologitral range of pe, although Î and l10~ are bound to the particles (10), The simulative effect of cationa for the binding of Hr suggests that phone nay be required to dminish ionic iateraotion of Br with the negatively charged surface of synaptosaaal a~abranes (11), Hr
binding of syaaptoscaes at ÔC as s function of p8 1s shown in Fig,1.
Acidifiption of the aediua resulted is the increase of Br binding.
This
suggests that sore atioaic ü:ed sites capable of the binding of Br
are
available on the synaptosaaal esbrans, and that fewer negative surface charges capable of inhibiting the approach of Br to the binding sites are present on the particles.
]fegativs surface charge of the synaptosoaes was found to be
caapletely abolished st p8 4,0 (11) .
By lowering ps, a smilar inarsase of Br
binding to liver aitochondria was previously reported (19) .
BRONZE AND 3YNAPTOrSOMEB
1374
Yol . 9, No. 23
ô â
P Q Y
Fig. 1 Eliect o! pH on the degree of Sr binding by syaaptosa~es . The results show are the average o! three separate szperi~ente. Inoabation o! synaptosa~es was perio:,ed !or 90 ein at OTC in the Ginger's solatioa containing 30 sd< tris-saleate bulfer over a range o! several pH values, and 4.5 - 5.0 ag/tial o! protein. At the end o! the incubation each segle wss receatriiuged (gee methods), and the pH of the supsrnate as ~easared to obtain the final pH o! the incubation ~izture. Ths ooncsntration o! Br added was 1 and 4~eq/al respectively u indicated. The rate o! Sr
binding as a !unction o! 8{ concentration in the reaction
~izture is shorn is Fig.l . Hoth at OTC gad 90sC, the rate o! H: binding was directly proportional to the Hr aeatal conditions a~ployed .
concentration o! the aedi~a under the ezpsri-
At SÔ C a slightly higher binding o! Bi than at
ÔC ws observed, bat this was not statistically significant.
Ser~a~ levels o!
Br (30 - d,! tleq/el) in broeide intazicatioa (13) are 8 - 10 bass higher than the H: added in this ezpsriasnt .
Bowsver within the sedative or antiepileptic
dose range, it slay well bs that the degree o! Br accu uiation in nervous cells is directly proportional to the Br concentration o! C8F or cerebral blood o! the surrounding tissues.
As shown in T11HIa S, Hr aacianlatioa by synaptosa~es
was not sigailioantiy a!lected b7 the addition o! glucose or ttstabolic inhibitors
HRO~E AND SYNAPTOBOMES
Vol. 9, No. 29
1375
3Ô C ÔC
ô
Rate o! bindiaH o! Br b7 s~naptos4es u a l~mation of Hr eoaosntratioa in the reaction ~iztnre . Ths particles were swpeaded is icr cold RinHer's solution containinH 30 sY trirnaleate (piH 7 .3) to Hire a protein conwntration o! X1 .3 - 5 .0 aH/L1 . Altar addition o! varions monta of H! , swpensionfs cers inanbated for ~0 min at O T C and 90 eC rsspectivsi~ . The values in the liHnrs are averaHes o! three separate s:psriaents . such as iodoacetate, DRp or onabain at either 0
C
and 90 e C inwbations .
Plavvate and lactate inhibited the Br acdaalation both at O vC and 90~C iaarbations, sugHesting that these organic anionic ca~poaads possibi~ oa~pete at the cationic site responsible for Hr bindiaH .
Hoth Hlnoose and p~ruvate are
substrates apabie o! sapportiaH ~aaiaobut~ric acid (OAHS) nptaks b7 tease brain synaptosates (8) .
The results suHgest that the Br acco alation b7
gnaptosc~es is lüsi~ to bs a tsperaturs or energy independent process . It is mown that liver titochondria binds Hi (1~ .
In this stnd~, Lhe
accosuLtion o! Hr b7 brain titochondria was also ezstined to sae the spsdücit~ of Br bindinH of synaptosates (TAHIa 3) .
Srotide eras bound
titochondria to a oonsiderabi~ lesser degree than b7 s~naptosotss .
M
brain
llitoohoadrisi
1378
BROb~E AI~ SYNAPT080ME3
Vol. 9, No. 23
~n~eLE s
Acaaulation o! Br b7 s~naptosaaes at O eC aad 90 eC= Inilneaces o! glnwse, p~rnvats, Lctats, and ~etabolic inhibitors. 8 bound ~prot)~S .D. Bnbstanoe tested O 90 ~ 90. 4 + 4.1 43.4 + 9.3 1Wne Iodoacetste ( 1 saI ) 19.8 t 9.0 (- 4) 91.8 + 9.4 (- e) 4, 4Dinitrophenol(Dl~ ( 0.4 elI ) 18.9+9.4 (-7) 19.8 t 4.0 (- 9) Ouabaia ( 0,9 ~ )
44.1+9.5 (- 5) 44.1 t 4.8 (+ 9)
19.0+4.0 (- 7) 18.8t9.4(-9)
49.0+1.9 (- 8) 41.4+4.4(-9)
18.5 t 1.8 (-19) e* 18.0 _+ 1.9 (-49)~ 18.1 + 1.9 (-41) ew
1ß .0 + 1.9 (-49) e 17 .8 _+ 1 .8 (-45) e 18.8 t 1.8 (-99) *
Olnoose ( 10 aa[ ) Oluoose(10 a1Q+Iodoacetate(1 n1Q Oluoose(10 w1AtD1~P(0.9 do p~ruvate ( 10 ~ ) p!=uvate(10 ~+D1~P(0 .4 ath Laotate ( 10 sY )
The results shown are the mean + B.D. obtained frame five separate szperiaents . e P(0.004, es PC0.05. lh~bers iâ parenthesis i~ioats percent changes oa~pared with the respective control values . The particles were suspended is ice-cold Ringer's solution containing 50 tY trirnaleate(pB 7 .9) to give a protein concentration o! 4.6 - 5 ag/al. Aitsr addition o! ~1esq/ai o! Barend test substances, the susp~sions were incubated !or 90 tin at 0'C aad 90eC respectiwl~ . pfavvate, lactate and iodoacetats were used as sodi~a salts and Ba+concentration in the reaction eiztnre ws tept constant b~ decreasing the pouat o! R1aC1 in Ringer's solution. TABLE 9 Acauaulation oi~Br bl gnaptoso~es aad brain a+itochondria Bfaaptosoaes Brain 1litochoadria
~Da^ot~t S.D. C C 19.4 + 9.5 94.3 + 3 .8 4.7 t 0.9
5.8 + 1.0
The results shown are the teen + S.D. obtained faros font separate azperiieats . ~periaeatal conditions were thi ssse as TABLE 9. oapadt~ to bind Sr wu appro:Latel~ 1/4 0! that was louad .in the sTaaptosawes . The inhibition o! Hr binding in sitoohondris b7 >~ , Î , p~ravats, aad lactate
Vol . 9, No. 23
BRONimE AND 3YNAPTOBOMEB
1377
was gwütativsi~ siailar to that reported !or cynaptosaasc (7äBIa 1 and 9) . These recuite indicate tint the aswdation o! Br with s~naptosaaet ie not a speaüia pheaaaenon in synaptosoaes or nerv endings, but ay occur in various snbcellular or cellular eocipart"eats of brain. TABIB 4 B!lect o! varions drugs on the binding of Br b7 synaptocaaes Bubctancs tected
Oongatration (a1Q
x
Chaa~e
picrotozin
1.0
+7
Btryclmiae
1.0
+ 4
Phsaobarbitai
1.0
- 4
Chlorpraaasine
1.0 0.1 0.01
-89 -30 -39
Acet~laholiae
1.0
+ S
s-H~draagtryptaaias
1.0
+ 7
Dlrlforepinephrias
1.0
+ 8
B:periaental conditions were the cane u TABTi 9, ezaept !or the tewpeatare of incabstion. All incnbatioac were carried out at OTC !or 90 ain. The rewits in TABIa 4 are the aeaa of three separate ezpsriaentc . 8tr~chnine sulfate, wdi~a phenobarbitai, chlorpiroaasine hydrochloride, aoet~laholine chloride, 3-hydrozytryptaaine ciwhnins sulfate, and Dlrnorepinephriae bitartrats were aced alter adjnstiag pR o! the drug wlation to 7 .0 . Bquiaolar ions, which were added with above amtioned drags, were adainistered to each control . The s!lsat o! various drugs is vitro oa the gnaptoca~al binding o! Br at OTC was shown in TABYB 4.
Central chaulants (piarotozin.aad stryc)mine),
phaoobarbital and putative transaitter substances (aaetylaholine, 5-~ydros, tryptsaine, norspinephriae and OABA) had ao sigaifiaat eilest oa the binding of Br by synaptosaaes at the ooncsntratioa of lÔ sl( or below.
Chlorproaa:iae(CPL)
was the only drug which showed a signiticaat inhibitory ailed on the syaaptocaaai binding o! Br aeiong the various substances tested .
CPZ at lÔ $, 10 4, cad 10 sY
ooaceatrations, diaiaished the Bi binding 82, SO cad 39
x
respectively .
HRON~E AI~ 3YNAPTOBOMEB
1378
Yol . 9, No. 23
It is well established t)rt CPL has rultipie elects on the reabrane psareability o! variow cells (14) .
The inhibitory elect o! CPfG on the pea~eability o!
ester (ib), H+a+ and L+(18), and acetate (17) ws reported in a variety o! cell r~branes.
It is probable that the inhibitory eliect o! CPL on the synaptoscui
binding o! Br is a relleotioa o! the general rsbrane eüscts o! this agent. Ibarapartiwlate apace in the pellet reasoned by im~lin-retbozr 9H wan not chaageE by the agent.
This suggests that the change o! oriti al wins o! the particles,
as observed in the inllueaa of CPL as the wires of erythrocyte (1B), ray not be the anse of the CPL e!leot on the Bfbinding o! syaaptosores . Oorrents The association o! Br with syaptoscaes iadiates that the synaptosaral r~brane contains es:posed, highly ionised cationic oonrtituents to which various anions ray bs board by ionic forces .
The synaptosaral and synaptic vesicle
r~branes also posses arposed and highir ionised polyanioaic groaps which are the site for binding sad retention o! various ationic substances each as potassia~ (19), aoetyicholine (90) and 3-hyd:orytryptarine (91) .
lrurtlur
erperirents wül be directed at characterising and ideatilying these anionic and cationic coastitusnts o! the syaaptosoral rsbraae which are apable o! binding various ionised substances . Although there is no doubt that the syaaptoscaes (prssynaptic endings) have a relatively high alüaity !or Br , the p~arraoologi a1 signiliante of this phenareoon in the Cl~ will require lurther investigation .
Binding o! bide by the synaptosaaes sad ritochondria o! rouse brain was studied upder a variety of ezperiremtal conditions .
The synaptosaral uptate o!
braride wan energy independent and was facilitated by the addition o! ations, While the presence o! other anions such as nitrate, iodide, pyauvate and lactate inhibited the association o! brcrids with synaptosares .
Acidili anon o! the
iawbatioa redis~ resulted is the increase o! brorids binding with synaptosaaes .
Vol . 9, No. 23
HROII~E AND SYNAPTOBOME3
1979
Brain sitoohoadria alw bound broside but such less extensively than synaptoscaea . The results are ooaristeat with the interpretation that the binding of brosids appears to resesble an anion ezohange process .
Oae of the attributes o!
the braside binding rite on the synaptoraaer is the prsseaoe of exposed, highlyionised cationic constitaants .
P~rthsx~ore, the results suggest that negative
surface charge of the synaptoroses say also bs a regniatory factor for the binding . It was found that asoag the various substances of neuropharaaoologigi interest tested only chlorprasasins inhibited the synaptosani binding of brallde . Admowled~sat The anchor is grateful to lliss ". t . Hoopea and Ilhr. 8. (nabs for stüllul technical sssistaace .
This woaic war supported is part by grants l~18883 and
III-18477 irca i
4, R.O, Hayden, H, Garoutte, J . lhgner,and R,H, Aird, Froc, Soc, Exp, Biol, ~7, 734 (1981) . S, E,(i, Gray, and V, P, hThi.ttater, J. Anat, (Load .) 98, 79 (198,
8 . !, Rnriyasa, H, "sinsteia, and E . Roberte, Brasa Res . 18, 479 (1989) . s 7, G. Hunter, Hioch~. J. 80, 981 (1935) . 8, R,C, Ds Gesso, A, Riasann III, sad 8 . Lindenbaa, J. Anal . Chs . 98, 7S 40 (1934), 9 . O.H . Lowry, hf .J, Rosebrough, A,L, Parr,and R, J . ltaadali, J . Mol . Chas 76S (1951) . 10, R . Ruriyasa, and E . Roberte, Abst . 4th Inc . Coag . on 1?haisaool .(Hasel, Jnly 1989) p . 395 (1989) .
isso
sao~E nrm sYxapTOSO~s
vo~.
9,
xo. as
11, J . Vos, t, lltri7saa, aad E, Boberts, Brain 8q . _, 994 (1988) . 19 . J .L, asnbie, ~r., Proc, eoc. Etp, Biel . Yed . ~S, 37b (1983) .
13 . D,J, Ilsller, Taas Yed. 84
4 , 79 (1986) .
14, P,B, Onth, and 1I,A, 8pirtes, Int. 8ee, YenroMoi . ~, 931 (1984) . ib . A,B. lre~aa, and Y,~. Bpirtes, Hioeh~ . Pha:aacol . i~, 19Sb (1983) . 18, J. ~risteasan, T,B,L. P~i, E . lbilq, aad A, ". "us, hed. Pt+oc. 17, 986 (19b8) . 17 . B,Y, ChaYoeels, and T,II, 8htnlaaa, Dhr, Biothia, Zh, ~ b8 (1989) . 18 . J. esn Stee~aiaei, ",t . Qiônmd, aad H,L . Hooi~, Biochea.Phasacol. 18, BS7 (1987) . 19 . H . "einstein, and t. Eari7saa, J, Yearochea, _, 493 (1970),
90, t. ihrijra, E . 8oberts, aad J . Yos, Brain 8es. 9, 931 (1988), 91, J,D, BoMason, J,H, Andersoa, and J .P. Qreen, J . Phasacol, a
~, !36 (198b) .