~ne~nbrane hydrophobic binding site for tranquilizers. SEEMAN, k. et LEE, T. 1974. Enhanced binding of chlorprornazine to cholesterol- depleted synaptosome ...
Enhanced Binding of Chlorpromazine to Cholesterol-Depleted Synaptosome Fractions P. SEEMAN .AND T. LEE ~'/zurtn(r(.oIogy Dsparfrneni, &Inilrersib.yof Toronto. Toronfo,Ontrrrio .M5S IAH
Received August 29, 1973 SEEMAN,P., and LEE, T. 1974. Enhanced binding of chlorpromazine to cholesteroldepleted synaptosome fractions. Can. J. Physiol. Pharmacol. 52,522-525. Brain synaptosome fractions were treated by Murphy's method in order to deplete the menabrane cholesterol. The binding of "3-chlorpromazine to these treated membranes was meas~rred. It was found that the mennbranelbuffer partition eoefl~cierlt of the drug was threefold greater in the treated synaptosome-rich fractions compared with the untreated fractions. The results suggest that cholesterol may not be a part of the ~ n e ~ n b r a nhydrophobic e binding site for tranquilizers. SEEMAN,k. et LEE, T. 1974. Enhanced binding of chlorprornazine to cholesteroldepleted synaptosome fractions. Can. J . Physiol. Pharmacol. 52,522-525. kes fractions riches en synaptosomes du cerveau ont kt6 traitees & I'aide de la rnCthode de Murphy afin de les dCpleter en cholestCrol membranaire. Nous avcms CtudiC la iixation de "'S-chlorpromazine par ces membranes. Comparativemeut aux fractions non traitCcs, le cocfHiciemt dc partition enlrc les membranes et la solution tampon est trois fois plus GBcv6 d a m %ecas des fractions traitkes. Ces rksultats sugg&rcn& que le ckolestCrol ne ferait pas partie des sites hydrophobes qui lient la chlorpromazine. [Traduit par le journal]
relate with the octanol/water partition coeficients (Sceman 1572; Seernan ct al. 19748~). Thc major tranquilizers, such as chlorThe extremely high number of these hydropromazinc, have many effects on biological membranes (Seeman 1972). but the mem- phobic sites (80 I~mol/rngof dry membrane, brane sites to which the drugs bind have not which virtually amorlnts to a monolayer around yet been identified. Current views suggest that the nlembrane; see Kwarnt and Seernan 1969a; chlorprornazinc may hind to the following: Sesman and Weinstein 1966) does not indicate ( i ) doparnine receptor sites on membranes of whcther the sites are protein or- lipid, since dopaminei-gic synapses (Kebabian ef al. 1972; both protein (Krieglstein and Kuschinsky Horn anti Snyder 1971; Andhn et al. 1970; 1969: Jiinchen et crl. 1969; Krieglstein et a/. Hornykiewicz 1966; Yexk 1972; Kehr et al. 1972) and lipid (Walzcr p t aE. 8 968) avidly 1972) ; ( i i ) nonspecific ( i . ~ . non-receplor) absor1-r thcse hydrophobic phenothiaaines. Rathcr than measure the binding of chlorhydrophc~bic sites in the rneml-prane, these promazinc to nlernbranc proteins that have k i n g either membrane lipid or- membrane been extracted and denatured ( c f . Metcalfe et protein (Kwant and Seernan 19690, 1969b: al. 1 9 6 8 ) , wc decided to measure the amount Seeman 4972; Kwant and Sceman 4971; Holn~esand Piette 1970; Balzar et al. 1968) ; of chlorprebrnazinc binding to membranes from and (iii) anionic sites in the membrane at which some lipid (cholesterol) was removed which competitive binding occurs between without using any protein-denaturing organic chlorpromazine and Caw ((Secman ct al. solarents (Gralnam and Green 8 969). 1974a, 1974b; Kwant and Seeman 1969b). Methods The evidence for hydrophobic handing sites $reparadion of ~ y n a p d u s f ~ r ) z c ~ - Fracrions ~ic?? is (a) that the enthalpy, as well as the entropy, ?ynaptosc~me-rick fractions were prepared from of chlorprctmazine binding become more negaguinea pig brain using minor modifications of thc tive at higher temperature (Kwant and Seeman procedure of Gray and Whittaker (1962) (cf. See196%); and ( b ) that the nerve-blocking ps- man ct al. 1972). fllbino guinea pigs (400-500 g ) dcncies of the phenothiazlne tranquilizers cor- were anesthetizcd with sodium pentobarbital (45
523
SEEMAN AND 1,EE: CHLORPROMAZINE BINDING IN SYNAPTOSBMES
TABLE 1. 35S-ch10rproma~ineadsorption to synaptosom~es Nor~nalsynaptosomes
Cholesterol-depleted synaptosomes
Cmernbrane Ctota~
Gree
(pmolll)
(pnlol/l)
(mmol/kg membrane)
Cmernbrane
normal
6;otal
Cfree
(ctmol/l)
(pmol/l)
(mmol/kg membrane)
pdeglcted* pdepleted
pnorma~
*Mean = 3.12 k 0.82. N/-{E: C represents the drug concentrntion in the various states. P,,,,m,Irepresents the partition coefficiealt for normal synaptosomes ( = - 6',:,,,for the cholesterol-depleted synnptosomes 4 C , , e m b r s n e / C r r e e for depleted syiiaptosomes).
bronr/Crrec). Pdeplrled represents the partition coefficient
mgikg intraperitoneal) and sacrificed by cardiac puncture. The brains were immediately removed, rinsed in 154 mM NaC1 ( 4 "), and honaogenized in 10 vcslurnes of 0.32 134 sucrose ( 4 " ) in a glass homogenizer with a Teflon piston (type A; clearance 8.005 in. (8.127 mm); A. H. Thomas, Philadelphia, Pa.). The homogenate was centrifuged at 1000 X g for 10 min at 4", the supernatant of which was recentrifuged at I7 000 X g for 55 nmin nt 4 " (Sorvrall RC-2B refrigerated centrifuge). This final pellet was layered onto a sucrose discontintlous density gradient of 0.32 r%f, 0.8 M. and 1.2 M sucrose and centrifuged at 25 $08 r.p.m. for 2 h at 4" in the SW41 swinging bucket tit;uniiarn rotor of the Bcclinlan L-2-65B ultracentrifuge (Beckman Instr~~ments,PaIo Alto, Calif .) . The synaptosome-rich band layering at the 0.8 hf - 1.2 144 sucrose interface, was diluted with an ecpaal volume of 0.32 A4 sucrose and recentrifuged at 37 000 X g for 60 rnin at 4"; the pellet was washed 3 times by resuspension in 10 miM trishydroxymethylaminomethane-HC1 buffer (pH 7.4) and recentrifugation, and was finally restaspended in this buffer at a final concentration of 0.5 g dry membranes per 100 ml buffer. The synaptosome-rich pellet had more than 60% synaptosorne profiles on thi~n-sectionelectron microscopy. Clzoicstc)rol Depletion o f Syrzayts,so~~~c-Rich Fraction The method of Murphy (1962a, B962b) was used to deplete the synaptosome membranes of unesterified cholssterol. The process is reversible (Bruckdorfer tJt al. 1968) and removes the majority of the membrane's tanesteriffed cholesterol without changing the amount sf the other major csalstituents of the membrane (Murphy 19620, 1962b; Bruckdorfer ct ni. 1948: Bruckdorfer and Green 1967: Basford c f ai. 1964; Bell and Schwartz 1971; but see Deuticke and Zoll~ler ( B 972) about the possibility of digerent cholesterol "pools"). Essentially, the procedaire was to deplete the membrane cholesterol by incubation overnight with cholesterol-depleted plasma lipoproteins as follows. Fresh plasma was obtained from blood withdrawn from the guinea pig aorta. The plasmla was divided into two portions. One portion ("pre-
-
incubated sernm"; Murphy 1962er) was incubated for 16-18 h in a water bath at 37 "C in order tts esterify the non-esterified choIcstero1 (Murphy 1862a), and then heated at 55 " C for 18 min to inactivate the esterase; the other portion was kept at 4 " C until later use, The cfepletion was done by mixing 1 volume (0.1-0.2 n21) of the synaptosorne-rich fraction with 8 volumes of preincrlbated serum, and incubating ;at 37 'C for a further 16-18 Bn to allow desorption of the mlembr-ane cholesterol and adsorption to the plasma lipoprotein. As a control, the fresh untreated plasma, which had been stored at 4 "@, was also incubated with an aliquot of the synaptosomerich fraction. The fracf ions were centrifuged and washed befare testing for drug binding. Hirlding o f C/~lor.pronzazfneto Chol~stcrol-Deplcted~d .Synuptosofnc Fractions The binding of chlorpromlazine to the synaptosornerich fraction was done by adding a 0.2 1111 aliquot of cl?lorpromazine solution, containing 75 nCi of "JSchlorpromazine (.4mersham/Searle, Chicago, f 11.1, to 0.5 ml of the synaptosome-rich s ~ ~ s p e n s i oall n ~ in 10 rnM Tris-HCI buffer, pH 7.4. After 15 wain at room temperature (22 " C ) the synaptosomes were centrifuged, and the supernatants were rno~aitoredfor unboalnd ""IS-chlorpromazine using liquid scintillation spectrometry; complete details are available elsewhere for both the radioactive monitoring procedure (Kwant and Seeman 196%) and the method for calculating the amount of drug adsorbed to the membrane (Seeman et 01. 1972). Membrane protein was measured by Lswry9s method (Lc~wry et al. 1951 ).
Results The results shown in Table 1 indicate that the chlsrprsmazine membrane/buffer partition coefficients for the treated synaptosorne fractions were generally about threefold higher than those for the untreated sgmaptosome fractions. Table 2 shows that the chlsrpromazine binding was relatively more enhanced in
524
C A N . 9. PMYSIOI,. PHARMACCIL. V O L . 52, I974
adsorption to synaptosomes from difrerent regions of the brain TABLE 2. 35S-chlorpr~ma~ine --
Normnal synaptosomes
--
-
-- - - - -- - -
.
--
-
-.-.
Cholestercdl-depleted synaptosornes
Cmembrane Gota1
(ptnol/I) Hypothalamras Hippocampus Caudate Whole brain -
G,,,
(mmol/kg ( pmoll/B) membrane) Pnorm,,
8 -02 6.16 7.75 7.56
9.79 9.79 9.79 9.79 --
I .31 1.31 1.73 2.38 --
163 212 223 3 15
cholesterol-dcpletcd synaptosomes from the ~ ~ ~ P O C ~ I P S P( UP,,,.,, S I ,,,,,,,jP,Io ,.,,, = 6.77, where P ,,,.,,,,.,,.,, is the partition coeflicient of tlme depletcd syrraptosomes and P,,,,.,,,,2,, is that for the the nornaal synaptosomes); this finding was xac~tstatistically significant, howe\~cr,since only two experiments werc done here, and since Trahle I ia~dicatesthat one of the values for the brain P,,,,,,,,,,,,,iiH",l,)l 1,1:11 was 7.73.
since there arc only 27 tetrodotoxin sites per square micrometer of rnenabrane area (Co9qukoun and Ritchie 1972) whcrerts the density of membrane cholesterol cites is about 1 000 000 times greater than this (sec references in Seeman 2 972; Seeman ee mi. 1973). This work was srapported by the Ontario Mesmtnl Health Foundatioxm (grant 274) and the McdicaI KesearcTa Co~incilof Canada (grant bZT-295 1 ) . . % N D ~ .NN., E . , R I J - ~ C H ES R . C;., , CORROI)B. ti., FUXE, K.,
'Thc n-e~srltsindicatc that synaptosome-rich and B_!NGF.BQS-I.EDT, 8!. 1940. Keceplor activity and turnover s f dopamine an(%noradrenalil~eafter ncurofractions, tseatcd to reduce membrane chc~lesIcptics. E inhibitors rescrpinc, chlc~rprorn;tr.,ine and preny lnsnine (Ashworth :and Green 19663). Tt is also ~ ~ C P W I ? L to the lipids of the rnernbranes of the st~rcoplasrnic t1113t the rncnabrane actions of elcctroskabilizing reticulum. Na~enyn-SClimiedebergsArch. Pharmakol. Exp. Pathoi. 260,456-473. drugs (with tht? exception of tctrc~dotoxin)are ~ , and GRL~:N. C. 1964. t:xw r y similar. qa~aIitatively and quantitativelyg BASFORD,J. M., G ~ . o v t i%., change of cholesterol betweer1 hrrrasan bct;ifor the erythrocyte membrane arnd tlmc neurolilpcrproteins and erqithrocytei;. Biochirn. Biopiiiys. Henma (Secn~an 1972; Roth and Seeman Acta, 84, 764-766. BEI.!.,1;. P., and SCHWARI-z, C. J. 1971. Exch:ansgeal9iliiy 1971). s f clmolesterol between swine serun-i Ijpop~'oteirms;:rmct The enhanced chlurpromazine binding by erythrocytes, ill 13itr.o. Biochim. Biophys. Act;r. 231, the chc~lestc'ro1-depleted synaptosomes s~aggests 553-557. that cholesterol is not an important hydro- BRLICKUORFEK, K . R.. G K A F I A M J. , M . , ;inel C i ~ t ~(1. t~, 1968. 'The ir~corporation of steroid snolecrsles into phobic birding sitc for tranquilizers. The hylecithin sols, beta-lipoproteins and cellular memdrophobic moieties of thc membrane proteins branes. Enr. J . Biochem. 4,512-518. or the residual ~ncmbrrxne lipids (primarily R R U C K D ~ R ~K.E R , ~and v GREEN,C. 1967. 'l'he exchange phospholipids) would, therefore, be the sites of unesterified cholesterol between human Lowdensity lipoproteins and rat erythrocyte "ghosts". of binding. Tlnc enhancetf binding of chlorRischem. J . 104,270-274. promazinc in these cholesterol-dcpletsd synC
