Effects of lesions on 3H-apomorphine and 3H-spiperone stereospecific binding sites in subcellular fractions of rat striatum

Neurmhemlstri lnternatumal, Vol 3, Nos 3,'4, pp 219 228 1981 Printed m Great Britain

0197 0186,81/030219 10.$0200,'0 © 1981 Pergamon Press Ltd

EFFECTS OF LESIONS ON 3H-APOMORPHINE AND 3H-SPIPERONE STEREOSPECIFIC BINDING SITES IN SUBCELLULAR FRACTIONS OF RAT STRIATUM J. E. LEYSEN, W GOMMERENand P VAN GOMPEL Department of Biochemical Pharmacology, Janssen Pharmaceutlca Research Laboratories, B-2340 Beerse, Belgium

(Received 9 March 1981, accepted 11 May 1981) Abstraet--3H-splperone and 3H-apomorphine showed saturable stereospecific binding to membranes of both a mlcrosomal and a mltochondrlal fraction of rat strlatum. For each hgand, the Ko-values were similar in both subcellular fractions, but the mlcrosomal fraction contained a 2 times higher binding site density than the mitochondrial fraction In both subcellular preparations the number of aH-splperone stereospeclfiC binding sites was about two times higher than the number of 3H-apomorphlne stereospecific binding sites Unilateral strlatal kamlc acid lesions caused a reduction of 67°0 of 3H-splperone and of 71~o of aH-apomorphme stereospecific binding in the mlcrosomal fraction In the mltochondnal fraction the reduction in stereospecific binding sites was only 33°0 and 40°~ for the 3H-hgands respectively Scatchard plots of 3H-splperone stereospeclfiC binding were linear in the two subcellular fractions of both lesioned and control strlata, and binding affinities were similar throughout Scatchard plots of 3H-apomorphine stereospeclfiC binding appeared to be non-linear in conditions where non-specific binding exceeded stereospeclfiC binding i e in the mltochondrlal fractions and in the mlcrosomal fraction of the lesioned strlata To allow clear analysis of the data, original experimental measurements of total binding and non-specific binding are reported The impact of variations in blank values on the final outcome of the analysis of specific binding is discussed Unilateral cortical ablation caused a 22~o reduction of both 3H-splperone and SH-apomorphlne stereospeclfiC binding in the mlcrosomal fraction, but did not affect the binding m the mltochondrlal fraction SH-splperone and 3H-apomorphme stereospecific binding sites appear to occur concomitantly and to be affected in parallel by the lesions Binding sites recovered in the striatal mlcrosomal and mltochondrlal fraction show the same binding characteristics but are apparently of different cellular origin Dopamme receptors on lntrastrlatal neurones and cortical strlatal afferents are mostly recovered in the microsomal fraction, and are likely to be localized on plasma membranes or associated with small subcellular transport particles The origin of dopamine receptors recovered in the mltochondnal fraction is unknown The presently reported findings corroborate the hypothesis that 3H-splperone and 3H-apomorphine label the same unitary dopamine receptor complex, but probably interact with different sub-unit sites of the receptor

D o p a m m e receptors have been labelled m vttro in erglc nerve terminals has been rejected In contrast to membrane preparations of dopaminergic brain areas the findings of Nagy et al. (1978) several other with 3H-dopamine antagonists (mostly butyropheresearchers found no destruction of strlatal nones), and 3H-dopamine agonists (mostly catechol 3H-dopamlne agonlst or 3H-dopamIne antagonist derivatives) (see references in Creese and Slbley, 1979; binding sites after successful 6-hydroxydopamlne Leysen, 1981) The bone of contention in the lesions of nlgro-strlatal pathways (Creese and Snyder, 1979; Leysen, 1979, Fuxe, Hall and Kohler, 1979, dopamlne receptor study has been whether the antaGoldsteln, Lew, Asano and Veta, 1980). On the other gonists and the agonlsts bind to multiple sites (Creese, hand, cortical ablations revealed that part of the striaBurt and Snyder, 1975; Tlteler, Welnrich, Sinclair and tal dopaminergic receptors labelled with 3H-antagoSeeman, 1978), whether sub-unit sites on a unitary nists occurred on corticostrlatal afferents (Schwarcz, receptor complex are involved (Leysen 1979), or Creese, Coyle and Snyder, 1978). Also stnatal kalnlC whether the data can be reconciled with distinct (post and/or presynaptlc) receptors localized on different acid lesions, destroying cell bodies in the vicinity of membranes (Nagy, Lee, Seeman and Flblger, 1978). the kalnlC acid injection site (Schwarcz and Coyle, 1977) caused a marked reduction of both 3H-antagoThe early hypothesis that dopamlne agonlsts prenists and 3H-catechol-hke agonlst labelled sites ferentially labelled autoreceptors on strtatal dopamln219

220

I E L]'~,,rN, W G'OMMfR!N and P VaN GoMPII

(Fields, Relslne and Yamamura, 1978, Schwarcz et al, 1978, Schwarcz, Fuxe, Hokfelt, Terenlus and Goldstem, 1980, Leysen, 1979, Creese, Usdm and Snyder, 1979, Davis, Woodruff, Poat and F r e e d m a n 1980), suggesting that the binding sites are locahzed on stnatal lnterneurones However, no agreement was reached whether antagonist and agonlst b m d l n g sites were reduced to the same extent. Surprisingly, in contrast to the generally made observation, Welnrlch and Seeman (1980) did not observe a reduction m 3H-apom o r p h m e (agonlst) binding after strmtal kalnlC acid lesions Investigations of the subcellular locahzatlon of 3H-splperone (antagonists) a n d 3H-apomorphlne binding sites in rat strtatum showed that about 50% of the sttes of b o t h hgands occurred in the mlcrosomal and 25°0 in the heavy mltochondrlal fraction However, the heawer subcellular fraction containing the binding sites appeared not to be synaptosomes from dopamlnerglc neurones (Leysen, 1979). Recently Davts et al (1980) showed that high affinity 3 H - A D T N b m d l n g sites on mlcrosomal membranes were more affected by kalnlC acid lesion than the binding s~tes in a m e m b r a n e fraction containing synaptosomes The functionality a n d cellular o n g m of the dopamlnerglc receptors on the different subcellular fractions is still u n k n o w n In th~s study our hypothes~s of a concomitant occurrence of 3H-antagonist and 3H-agonlst stereospecific binding sites on a unitary d o p a m i n e receptor (Leysen, 1979) was further investigated The objective of our study was to find out whether dopamlnerglc receptors, recovered m the two different subcellular fracnons, correspond to dopammerglc receptors on different neurones The assumption was made that the receptors, locahzed on nerve terminals of cortlcostrlatal afferents, would probably be found m the mltochondrml fraction Therefore we examined the influence of strlatal kamlc acid lesions and cortical ablations on 3H-apomorphlne and 3H-splperone binding, in m e m b r a n e s of mlcrosomal and mltochond n a l fracnons of rat strlatum In recent papers we analyzed the problem of the inconsistency of the 3H-dopamine agomst binding, 3H-antagonist binding is more uniform. The major handicap of studies with the catechol-hke agonlsts has proven to be the various types of high non-specific binding predominating a low specific binding (Leysen. 1980, Leysen and G o m m e r e n , 1981) The use of different assay condlttons a n d different ways for assessment of non-specific b m d m g m the m a n y tzrewous studies has rendered comparison of the data very dtfficult In this study 3H-splperone and 3 H - a p o m o r p h m e bmd-

mg were measured in cond~hons which are prone to afford optimal assessment of stereospeclfiC binding (Leysen and G o m m e r e n , 1981) Moreover, ~pecml attention ts prod to reporting raw experimental measurements to allow direct e v a l u a h o n of the data The importance of variations m non-specific binding, m the final outcome of the analysis of specific binding, wdl be discussed

EXPERIMENTAL PROCEDURES Neuronal lestons

Female Wlstar rats (150g) were anaesthetized with 2 5 mg/kg (1 p ) alfentand, a short acting narcotic analgesic (Nlemegeers and Janssen, 1981), kamlc acid (2 ttg/0 5,1 solunon m sahne buffered at pH 5-6) was rejected over a 5-mln period into the right stnatum (coordinates A = 8 0, V = + 1 4, L = 3 0 according to Komg and Khppel, 1963) using a micro syringe mounted on a David Kopf stereotaxic apparatus Three groups of 40 rats were treated on &fferent days The animals were housed m groups of ten Up to 4 days after the operation the ammals received Bactocdhne (27 mg/kg i m/day) and 7 ml Ringer soluhon was administered subcutaneously twice daffy At day 21 after the operation the animals were sacrificed by decapitation, brains removed from the skull, and stnata rapidly &ssected For cortical ablation the right front side of the skull was removed and the right cortex was sucked off from the prefrontal cortex to the bregma Bleedmg was controlled by gently pressing oxidized cellulose on the wound After the operation ammals were kept m mdwldual cages for 5 days [a sulficlently long period to cause reducnon of strmtal 3H-halopendol binding [Schwarcz et al, 1978]) and were then sacrificed The controlateral s~de was used as control m both experiments Earher stu&es on total membrane fracnons had shown no &fference m 3H-hgand between controlateral sides of lesioned rats, of sham operated rats and of nawe rats (Leysen, 1979) Tt~sue preparatton

Stnata were rapidly &ssected after decap~tatlon, stnata from the control or lesmned side were respectively pooled to form 6 groups of 11-13 kalmc acid lesioned rats, and 3 groups of 5 cortically ablated rats The fresh nssue was homogemzed m 30 volumes 0 25 M ice-cold sucrose using an Elvejhem Potter homogemzer and ahquots were stored at -40°C for determmanon of enzymanc activittes, proteins and endogenous dopamme (storage m perchlonc acid) The homogenate was centrifuged at 745 g x 10 mln to remove the cell debris and the nuclear fraction The pellet was rehomogemzed m 15 volumes 0.25 M sucrose and recentrffuged Pooled supernatants were centrifuged at 30,000g x 10mln to obtain the heavy and light mitochondrml (M + L) fraction The pellet was washed by resuspensmn m 15 volumes 025 M sucrose followed by recentnfugatlon The pooled supernatants were further centrifuged at 251,800 g × 45 mm to yield the m~crosomal {P) fraction Finally, the (M + L)

3H-apomorphme and 3H-splperone binding sites and P-fractmn pellets were washed first in 40volumes Tns-HCI buffer 15 mM, pH 74, EDTA 1 mM, ascorblc acid 001%, mcubatmn at 37°C for 5mln and centrffugatmn and then m 40 volumes Tns-HCI buffer 50 raM, pH 7 7 The final pellets were suspended m 20 volumes of the latter buffer

Receptor bmdm# assays For 3H-apomorphme binding, part of the tissue suspenstuns was 5-fold dduted with buffer to a final concentratmn of 10rag ortgmal wet weight t~ssue per ml Tns-HC1 15 mM, pH 7 5, EDTA 1 mM, ascorblc acid 0 01~o (TnsEDTA buffer) To 1 ml samples of that suspensmn ahquots of 50 #1 solvent or (+)-butaclamol and 50 #1 3H-apomorphine spec. act 30 Cl/mmole were added and mcubatmns were run for 30mm at 25°C 3H-Apomorphme concentratmns ranged from 0 2 to 6 nM. For SH-splperone bradmg the remaining part of the tissue suspensmns was dduted 40-fold with buffer to a final concentration of 1 25 mg original wet weight per ml Tns-HCI 50 mM, pH 7.6, NaC1 120 mM, KCI 5 mM, CaCI2 2 mM, MgCI2 1 mM, pargyhne 1/~M, ascorblc aod 0.1°,o (Tns-SALT buffer) To 5 ml samples of the tissue suspensmn ahquots of 250/~1 solvent or (+}-butaclamol and 250/~1 3H-splperone spec act 25 5Cl/mmole were added, lncubatmns were run for 15 mm at 37°C 3H-Splperone concentratmns ranged from 0 02 to 0 8 nM For the determlnatmn of non-specific binding ( + )-butaclamol was added m 1000-fold excess over the labelled llgands Membrane bound radioactlwty was harvested by rapid vacuum filtration through Whatman GF/B glass fibre filters using a filtratmn manifold. Falters were rinsed three times with 5 ml Ice-cold Tns-HCI buffer Radmactwlty on the filters was counted in an Intertechmque hqmd scmtdlatmn spectrometer eqmpped with a built-in computer to calculate DPM-values according to experimental cahbratlon quench-curves B~ochemwal measurements Proteins were assayed according to Lowry, Rosebrough, Farr and Randall (1951) Chohne acetyl transferase (CAT) was measured according to Fonnum (1969) using ahquots of the total homogenate containing 05 mg original wet weight tissue Glutamlc acid decarboxylase (GAD) was assayed in condmons as described by Chalmers, McGeer, Wlckson and McGeer (1970) using ahquots of the total homogenate contalnlng 067mg tissue. 14C-CO2 gas hberated from the 14C-L-glutamlc acid (spec act 50 mCl/mmol) substrate was adsorbed m a mixture of ethanolamme 2-methoxyethanol (2 1), contained m a test tube connected hermetically to the mcubatmn tube Endogenous dopamme was measured by the enzymatic method (Coyle and Henry, 1973) using partmlly purified catechol-O-methyl transferase and 3H-S-adenosyl methmnine (spec act 10.5 Ci/mmol) as methyl donor. Labelled substances were purchased from New England Nuclear (Germany), (+)-butaclamol was kindly donated by Ayerst Laboratories Drugs were &ssolved and stored m pure ethanol and prmr to the assay dduted m 10~ ethanol m water RESULTS 3H-Spiperone and 3H-apomorphine concentratmn binding curves were investigated in membranes from

221

a mlcrosomal and a mitochondnal fraction of strlata from naive rats. The assay con&tions (see Methods) used for the 3H-spiperone and 3H-apomorphine binding, respectively, were previously found to be optimal. Figure l(a and b) shows that linear Scatchard plots were obtained for 3H-spiperone stereospecific binding (0.03~3.8 nM) and for 3H-apomorphine (0.2-6nM). The binding affinities of the ligands were virtually similar m both subcellular fractions, i.e. for 3H-spiperone between 0.05 and 0.08 nM, and for 3H-apomorphlne between 0.7 and 0.9 nM. However, the maximal number of binding sites per gram wet weight of tissue was nearly two times higher In the microsomal fraction (21 fmol/mg tissue for 3H-splperone, 12 fmol/mg tissue for 3H-apomorphme) than in the mltochondrlal fraction (13.7 fmol/mg tissue for aH-spiperone, 6 fmoi/mg tissue for 3H-apomorphine). It was noted that m both subcellular fractions SH-spiperone labelled about twice as much sites as 3H-apomorphme. It ~s worth mentioning that, for 3H-apomorphine, binding variability between assays performed in different sessions at the same day was very low, but it was high between assays performed with 2 month intervals

Effects of unilateral kainic acid leszons in the striatum Twenty-one days after unilateral injection of kalnic acid into the striatum of rats, 3H-spiperone and SH-apomorphlne binding curves were measured in the mlcrosomal and the mltochondrial fractions of striata from the lesioned and the control side. Measurements on 6 groups of animals were performed independently from each other on 3 consecutive days using for each group separately prepared tissue fractions and ligand dilutions. For each tissue preparation 3H-spiperone and SH-apomorphine binding were measured in parallel. Means of ligand concentration, of total binding and of nonspecific binding are summarized in Tables 1 and 2. This detailed information on raw experimental measurements allows direct evaluation of the level of binding, the percentage of bound versus totally added hgand and the ratio between specific and non-specific binding in each of the tissue preparations. For 3H-spiperone, the nonspecific binding constituted only a minor portion ( < 2 5 ~ ) of the total binding and the non-specific binding did not vary in the various tissue fractions. Thus variations in binding in the different tissues were entirely due to variations in stereospecific binding. For 3H-apomorphlne, the non-specific binding was much more pronounced and constituted In certain tissue preparations a substantial portion (up to 78~) of the total binding. Non-specific 3H-apomorphine

~33

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I l','q',J, W GOMMIRt'~ arid P V a \ (5()MP]I

OMAL FRACTION

~=~°=~°~'2~2°g,g:. ,,=,

,,=, N 0 0 10 20 3H SPIPERONE STEREOSPECIFIC BINDING (fmoles ml -!

o~- +~; "T

ICROSOMAL FRACT)ON

00s MITOCHONDRIAL

-

~,

0.

.

.

. 50 100 3H APOMORPHINE STEREOSPECIFIC BINDING (fmoles rnl -))

Fig 1 Scatchard plots of stereospeclfiC binding to washed membranes from a rat strlatal mlcrosomal and mitochondrlal fractmn of (a) 3H-splperone (0 02-0 8 nM) using an assay volume of 5 5 ml containing 6 25 mg wet weight tissue in Trls-SALT buffer. 37°C Mlcrosomal fraction K o = 0 0 5 3 + 0 0 0 5 n M . Bm,~=21 + 2 f m o l / m g tissue Mltochondrial fraction Ko = 0078 _+ 0007 nM. Bmax = 13 7 + 0 2 fmol/mg tissue (bl 3H-apomorphme (0 2-6 nM) using an assay volume of 1 1 ml containing 10mg wet wmght tissue in Trls-EDTA buffer. 25'-C Microsomal fraction Ko = 0 9 _ 0 1 n M . Bm~ = 120_+ 1 2 fmol/mg tissue Mltochondnal fractmn K D = 0 7 _+ 0 3 nM. Bma~ = 6 4 +__3 3 fmol/mg tissue Stereospeclfic binding was taken as the difference between binding in the absence and the presence of 1000-fold excess (+)-butaclamol The free hgand concentrations were calculated as the difference between the concentrations of totally added and totally bound hgand Points are the means of 8 (mlcrosomal) and 5 (mltochondrlal) experiments K o and Bm~-values are the means of the values derived from regression lines calculated by the method of least squares for each of the experiments

binding was higher )n the mltochondrlal than in the mlcrosomal fraction, but in both subcellular fractions somewhat lower in the tissue from the lesioned than from the control side It is clear that in such cases the derived ~tereospeclfiC binding of 3H-apomorphlne wa~ less well defined Scatchard plots of stereospeclfiC 3H-spjperone binding in the mlcrosomal fractions {Fig 2a) and in the mitochondrlal fractions (Fig 2b) obtained from strlata at the control and lesioned side respectively, were linear with v]rtually similar slopes Krrvalues were a b o u t 0 07 nM, corresponding to the Ko-values of 3H-sp]perone measured m the strlatal subcellular fractions from naive rats The n u m b e r of binding sites in the microsomal 121 5 f m o l / m g tissue) and mltochondrlal fractions (14 2 fmol/mg tissue) from strlata at the control side were similar to the n u m b e r of sites m corresponding tissue fractions of naive rats In the microsomal fractions from the lesioned striata a reduction of 67°., m the maximal n u m b e r of receptor sites per mg tissue was measured In mitochondrial fractions from lesioned s t n a t a the reduction in Bmax was less p r o n o u n c e d and a m o u n t e d only to 33°. In Table 1 the percentages of reduction in stereospec]fic 3H-sp]perone binding observed at the various concentrations of the hgand are indicated All values are s|mdar to the reduction in binding calculated from Bmj~-~alues The Scatchard plots of stereospeclfiC 3H-apomorphme binding m lesioned and control s m a t a are shown m Figs 3(a) and (b) Using a mlcrosomal fraction from the control side, the plot was linear between 0 2 and 3 n M . and the point measured at 6 n M deviated from the line The Ko- and Bmax-values were slightly higher than those for the mlcrosomal fraction from s t n a t a of nmve rats Scatchard plots of measurements m the mlcrosomal fraction at the lesioned side and m the mltochondrml fractions at b o t h the control and the lesioned side appeared to be non-linear However. the data m Table 2 show that in the tissue preparatlons yielding curved plots, the non-specific binding constitutes 50-70°0 of the total binding In such cases slight variations of non-specific b m d l n g or systematic errors in the determination of the non-specific binding can considerably influence the values of the stereospeclfiC b l n d m g Therefore. the shape of the Scatchard plots could be due to an artefact (see discuss]on) In order to compare variations in the n u m b e r of 3 H - a p o m o r p h m e stereospeclfiC binding sites m lesioned and control strlata, the mean value of the reduction m binding at the various hgand concentrations was considered This a m o u n t e d to 71°,, in the mlcrosomal fraction and to 40°. m the mitochondrlal

3H-apomorphme and 3H-splperone binding sites

223

(a)

03

~o2

CONTROL

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°1 LESIONED

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~ 0 5 10 15 20 25 3H SPIPERONESTEREOSPEClFICBINDING(f moles rat'1 ) IN THE MICROSOMALFRACTION (b)

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+

z~ol

't

/

0

0

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10

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3H SPIPERONESTEREOSPECIFICBINDING( f moles rag-l} IN THE MITOCHONDRIALFRACTION

Fig. 2. Scatchard plots of stereospeclfic 3H-splperone binding (0025-0 3 nM) to membranes of (a) a mlcrosomal and (b) a mltochondrlal fraction from rat stnata at the control (C) and the lesioned side (L), 21 days after unilateral mtrastnatal rejection of kamm acid Assay condmons were as mdmated m legend to Fig l(a). Unprocessed experimental data are in Table 1 Mmrosomal fraction C Ko = 0074 + 0005 nM, Bmax = 21 5 + 0.9 fmol/mg tissue, L' Ko = 0063 + 0.005 nM, Bm~ = 7.2 _ 0 8 fmol/mg tissue. Mltochondrlal frachon C" Ko = 0 076 ___ 0.005 nM, Bmax = 142 ___+0 4 fmol/mg tissue, L K o = 0070 _ 0.005 nM, Bm~ = 95 + 0.9 fmol/mg tissue. Points are mean values _ SEM of five independent experiments. K o and Bmax-values are the means of the values derived from regression hnes calculated by the method of least squares for each of the experiments.

__4

J E LI-'fSEN W GOMMER[N and P VaN (J()MP, I

Table 1 ~H-Splperone binding (pmoles:ml + SEM, n = 5)* to membranes of la) a mlcrosomal and (b) a mnot, hondnal fracnon of rat strm)a 21 days after unilateral strlatal rejections of katmc acid 3H-Splperone* concentranon+ pmoles/ml

Control sldc Non-speclficall? Total b bound bound

Les~oned side Non-specifically Totally bound bound

(at

00256 0 0504 o 100 0 150 0 197 0295

+ 00005 _+ 0 0004 ± 0001 -+ 0002 _+ 0001 _+ 0004

00058 _+ 00002 000054 _+ 000005 00027 _+ 0000l 000046 _+ 000005 0 0093 _+ 0 0004 0 00074 ± 0 00005 0 0039 + 0 0002 0 00059 ± 0 0t)003 00144_+00005 00013+00001 00059_+00003 00011 ± 00001 00172 -+ 00009 00015 -+ 00001 00074 _+ 00003 00015 _+ 00001 00194 _+ 00008 00021 _4- 00001 00081 -+ 00004 00019 ± 00001 00224 + 00007 00033 _+ 00001 00095 _+ 00004 00030 + 00001

(b)

0 0256 0 0504 0 100 0 150 0 197 0295

± 0 0005 -+ 0 0004 -+ 0 001 ± 0002 _+ 0 001 -+ 0004

0 0042 0 0064 0 0099 00122 0 0137 00156

_+ 0 0002 0 00054 _+ 0 00005 -+ 0 0002 0 00078 -+ 0 00006 -+ 0 0002 0 0010 _+ 0 0001 -+ 00003 00016 -+ 00001 _+ 0 0003 0 0022 -+ 0 0001 -+ 00004 00032 -+ 00001

0 0032 0 0048 0 0073 00090 0.0098 00177

_+ 0 0002 0 00053 _+ 0 00005 + 0 0003 0 00069 + 0 00005 _+ 0 0004 0 0012 + 0 0001 -+ 00004 00016 _+ 00001 -+ 0 0004 0 0021 + 0 0001 _+ 00006 00031 _+ 00001

Reducm>n m stereo speofic binding C,,) ~7 (~1 64 (~q 64 66 mean 62 _+! 28 27 ~2 30 33 31 mean 30 +_1

* Assays were performed with a volume of 5 5 ml containing 1 14 mg tlssue/ml f Concentratmns were exper,mentally determined for each dilution by counting the radloacnvlty

fractxon It can be seen m Table 2 that the reduction in b i n d i n g calculated at each o f the h g a n d c o n c e n trations varied at r a n d o m a r o u n d the m e a n value a n d that there is no systematic shift from the low t o w a r d s the high h g a n d c o n c e n t r a n o n s . Hence, the p e r c e n t a g e o f r e d u c t i o n in the n u m b e r of sites in lesioned strlata

was virtually similar w h e n assayed with 3 H - s p l p e r o n e a n d 3 H - a p o m o r p h l n e , a n d with b o t h h g a n d s the reduction m b i n d i n g sites in the m l c r o s o m a l fraction nearly d o u b l e d the reduction in b i n d i n g sites in the m l t o c h o n d r l a l fraction D a t a on p r o t e i n content, content o f e n d o g e n o u s d o p a m l n e , C A T activity a n d 0 05-

0 08 ~

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100 3H APOMORPHINE STEREO6PECIFIC BINDING ( f moles mi - ] ) iN THE MICROSOMAL FRACTION S0

0

o

2'o

~o

6'o

3H APOMORPHINE STEREOSPECIFIC BINDING (f moles ml - l ) IN THE MITOCHONDRIAL FRACTION

(a) (b) Fig 3 Scatchard plots of stereospeclfiC 3H-apomorphlne binding (02-6 nM) to membranes as in legend to Fig. 2 Assay conditions were as m legend to Fig. 1(b). Unprocessed experimental data are in Table 2 Mlcrosomal fraction C' KD = 23 + 0.5nM, Bmax = 167 _+ 1.8fmol/mg tissue Points are mean values __ SEM of six independent experiments Ko and B,,=x-values are the means of the values derived from regressmn hnes calculated by the method of least squares for each of the experiments at 3H-apomorphine concentrations between 0.2 and 3 nM

aH-apomorphine and 3H-splperone binding sites

225

Table 2 3H-Apomorphlne blndmg (pmoles/ml _+ SEM, n = 6* to membranes of (a) a mlcrosomal and (b) a mltochondrxal fraction of rat stnata 21 days after unilateral strlatal rejection of kamlc acid 3H-apomorphme concentrat~ont pmoles/ml

Control side Non-specifically Totally bound bound

Lesioned side Non-specifically Totally bound bound

Reduction in stereospecific binding (°/o) 67 73 72 75 70 72 mean 71 _+1 49 25 52 39 28 44 mean 40 + 5

(a)

0198_+0.002 0394_+0002 0795 _+ 0.004 1 50 _+ 0.01 304 -+ 002 613-+005

0017_+0.001 0.031 _+0.001 0056 _+ 0002 0089 _+ 0.003 0.141 _+ 0004 0219_+0007

00051 _+00002 00090_+0.0003 00177 4- 0.0007 0030 _+ 0.001 0059 4- 0003 0100_+0004

00079_+00003 00140_+0.0004 00241 _+ 0.0006 0040 _+ 0001 0069 -+ 0003 0113_+0003

00040_+00002 00081 _+00005 00135 -+ 00008 0025 4- 0001 0044 -+ 0003 0079_+0002

(b)

0.198 4- 0002 0394 -+ 0.002 0795 -+ 0004 l 50 -+ 0.0l 304-+002 6.13_+0.05

00139 _+ 00007 00233 4- 00005 0042 4- 000l 0070 _+ 0002 0.119_+0.005 022_+001

00065 _+ 00003 00114 -+ 00004 0.022 -+ 0001 0039 4- 0002 0083_+0003 0156_+0007

0.0010 4- 0.0005 0018 4- 0.001 0030 -4- 0001 0056 4- 0002 0097_+0004 0.169_+0007

0.0062 -+ 00004 0.009 4- 0001 0021 -+ 0.001 0037 -+ 0001 0.071 _+0.003 0.133_+0005

* Assays were performed with a volume of 1.1 ml containing 9.09 mg tlssue/ml "?Concentrauons were experimentally determined for each ddutlon by counting the ra&oactlwty

G A D activity in lesioned and control straata are summarized in Table 3(a). It Is noted that the protein content m the mlcrosomal fraction was reduced by 40~o at the lesioned side, but was virtually similar at b o t h sides in the m l t o c h o n d n a l fraction The d o p a m m e content was not significantly altered, the CAT activity was reduced by 43~o in the total h o m o genate of lesioned s t n a t a and the G A D activity was reduced by 489~,. It was noted that the d o p a m l n e content and the CAT activity measured in the contralateral side was slmdar to measurements m animals not-operated on, a n d other investigators & d not find

a difference between naive controls and contralateral sides, 1 year after kalmc acid lesions (Schwarcz et al., 1980)

Effects of umlateral cortical ablations D a t a on 3H-spiperone and SH-apomorphlne binding in subcellular fractions of s t n a t a from rats 5 days after unilateral cortical ablations are shown in Table 4. Measurements were performed at a single ligand concentration. Cortical ablation caused a significant reduction of 2 2 ~ in stereospeclfiC binding of b o t h llgands in the mlcrosomal fraction. In the mitochon-

Table 3. Biochemical measurements m strlata of rats with (a) unilateral kamxc acid lesions (n = 6) and (b) cortical ablation (n = 3) Protein mg/g Mlcrosomal Mltochondrlal fraction fraction (a) Control side Lesioned side Reduction Not operated controls (b) Control side Lesioned side Reduction Not operated* control

Dopamlne content /~g/g

CAT pmoles/mg mm

GAD pmoles/mg mln

20.2 + 0 8 12 4- 1 40%

40.0 4- 0.8 36 _+ I l 0~o

9.7 4- 1 4 84 _+ 07 13~o

156 _+ 3 67 _+ 4 57%

187 _+ 14 117 + 13 38~o

155 + 0 6 11 0 + 0 4 29%

33 + l 28 + 1 15%

10 3 + 1 0 66 + 0.4 6.4 + 0.2 3~o

164 + 5 90 + 2 89 + 3 1~o

168 + 8 165 + 6 2%

78

92

205

* Duplicate measurements performed along with the determination m cortical ablated rats

226

1 F kl'(st_\ W GOMMIRt', and P VA'~ GOMPt) Table 4 Binding (pmoles/ml + SEM, n = 31 of -~H-splperone at 025 nM and -~H-apomorphlne tit ~ nM w strlatal subcetlular fraction of rats 5 days after umlateral cortical ablations Mlcrosomal fraction Control side Lesioned side

(al ~H-Sptperone Total binding Non-spemfic banding Stereospeclfic binding

00209 + 00006 00055 _+ 00005 00154 _+ 00007

Reductmn (b) 3H-Apomorphme Total binding Non-speofic binding Stereosplofic binding Reduction

0.017 + 0001 00052 4- 0001 00118 _+ 00006

Mltochondrlal fraction Control side Lesioned side 0012 _ 0001 00043 _+ 00004 00076 _+ 00004

22",,, P < 0 05* 0151 _+0006 0 083 _+ 0 003 0 068 _+ 0 003

0132_+0006 0 079 4- 0 003 0 053 _+ 0 002 22"., P < 005*

0.0123 + 00006 00047 _+ 00004 00076 _+ 00001 I",, P > 0 5

0127_+0006 0 094 _+ 0 003 0 033 _+ 0 002

0129_+0003 0 099 + 0 001 0 030 _+ 0 002 9",, P > 05

* P-values Student's t-test indicating of &fference betvmen values at lesmned and control side

drlal fraction there was no significant change in binding with either hgand StereospeclfiC binding at the contralateral side was a httle lower than the stereospecific binding at the contralateral side in the kalnlC acid lesioned rats (see Tables 1, 2 and 4). The differences appeared primarily to be due to a higher nonspecific binding in the cortically ablated rats Table 3(b) show that the protein content in the strlatal mlcrosomal fractions was reduced by 29°o and in the mltochondrlal fractions reduced by 15°o at the lesioned side. The striatal d o p a m l n e content, CAT activity a n d G A D activity were not affected by the lesion. D o p a m l n e content and CAT activity were similar In the contralateral control sides and in the simultaneously measured strlata of a non-operated rat. The strlatal G A D activity in the cortically ablated rats was somewhat lower than in the naive rat. Some lnterday variability between control measurements in nmve rats was noted Therefore comparison was made with naive controls measured at the same day as the operated rats

DISCUSSION Binding experiments in the mlcrosomal and mitochondrial fractions of strlata of naive rats revealed that the KD-values of SH-splperone and 3H-apomorphlne, respectively, are the same in the two subcellular fractmns and also similar to the KD-values previously measured in a total particulate preparation of rat strlatum (Leysen and G o m m e r e n , 1981). In b o t h subcellular fractions SH-splperone labelled a b o u t twice the n u m b e r of stereospeclfiC sites as SH-apomorphine, which is in agreement with the ratio of 3H-spiperone a n d a H - a p o m o r p h i n e sites found in the

total particulate fraction. That indicates that both subcellular fractions contain a d o p a m l n e receptor with similar binding characteristics. However, the receptor density is nearly two times higher in the mlcrosomal fraction than in the mltochondrlal fraction KalnlC acid lesions of the strlatum caused a much higher reduction in the n u m b e r of stereospecific binding sites m the strlatal mlcrosomal fraction than in the mltochondrlal fraction. Binding experiments with 3H-splperone, in the subcellular fractions of lesioned and control strlata, revealed straight lines, with similar slopes m all tissue preparations, hence binding affinities were not changed in the lesioned tissue and were similar to those found in naive rats Binding experiments with 3H-apomorphlne revealed apparently non-linear Scatchard plots m the mlcrosomal fractions of the lesioned strlata and m the mltochondrlal fractions of b o t h the lesioned and the control striata However, in the latter case straight lines could be drawn, when the first a n d the last points on the curve were neglected This rendered e v a l u a n o n of the effect of the leslonlng difficult according to the hnes whmh could be drawn subjectively through the points, different speculations are possible However, when considering the original experimental measurements on total and non-specific binding, it is apparent that non-linear Scatchard plots were observed m tissue preparations, where stereospeclfiC binding was inferior to a high non-specific binding This SltUanon can easily give rise to artefacts, which are not necessarily ruled out in independently repeated experiments Indeed, previous investigations have shown that nonspecific 3H-apomorphxne binding is very complex and that specific binding is difficult to assess correctly

3H-apomorphme and 3H-splperone bmding sites The use of (+)-butaclamol for defining non-specific binding appears to be the most appropriate way. However, when the non-specific binding is much higher than the specific binding, the shghtest systematic error in the definition of the non-specific binding will seriously affect the derived specific binding. It is clear that in such situations it is very difficult to derive unbmsed conclusions from Scatchard plots The most detached way to evaluate the effect of the kalnlC acid lesion on the number of 3H-apomorphlne binding sites, seemed to be a comparison of the reduction in stereospeclfic binding at the various 3H-apomorphine concentrations. The percentage of reduction at the different concentrations varied at random around the mean value, therefore demonstrating the vahdlty of the evaluation procedure It appeared that stereospecific 3H-apomorphlne bmdlng was two times more reduced in the mlcrosomal than in the mltochondrlal fraction, and in both subcellular fractions the percentage in reduction of 3H-apomorphlne binding was similar to the percentage in reduction of 3H-splperone binding. The adequacy of the kalnlC acid lesions was demonstrated by the reduction in activity of the enzymes (CAT and GAD) which are contained in neurones with striatal cell bodies. The fact that the endogenous dopamlne content was not significantly changed m the lesioned striata demonstrated that the dopamlnergic strlatal afferents remained intact It cannot readily be explained why the protein content is substantially reduced in the mlcrosomal fraction at the lesioned slde, and is virtually unaffected in the mltochondrlal fraction. Because of this unidentified reduction in proteins it has seemed more appropriate to express alterations in binding and enzyme activities per original wet weight of tissue. Unilateral cortical ablations caused a s~gnificant and similar reduction of both 3H-splperone and 3H-apomorphlne binding in the mlcrosomal fraction, but the binding in the mitochondrlal fraction was not affected The fact that the dopamlne content and CAT activity were s~mllar in strlata at the lesioned side and the contralateral control side, as well as in strlata of a naive rat, indicated that dopamlnerglc neurones and strlatal chohnerglc lnterneurones remained intact Also GAD activity was s~mllar in the lesioned and the controlateral side but the activity was somewhat less than m a naive a n l m a l ~ o r e controls are necessary to find out the significance of this observation In general, our data corroborate the hypothesis that part of the striatal dopamlnergxc receptors are localized on cortico-striatal afferents (Schwarcz et al, 1980). For technical reasons, binding experiments m cortical ablated rats were only performed at a single

227

aH-ligand concentration, and hence extenswe conclusions cannot be drawn. It is important, however, that the findings m the cortical ablated rats gwe support to conclusions which can be derived from findings in kalmc acid lesioned rats. 3H-Spiperone and 3H-apomorphine binding seem to be affected in a parallel way and receptors in the mlcrosomal and the mltochondnal fractions are differently influenced. Also the binding measurements in the controlateral sides are in accordance with observations in naive rats and in controlateral sides of kalnlC acid lesioned rats: binding In the microsomal fraction is two times higher than in the mltochondrlal fraction and in each tissue preparation the splperone binding is the double of the apomorphlne binding The presently reported data have further shown that apparently inevitable methodological problems are a major drawback in the evaluation of the localization and regulation of dopamine agonxst binding sites compared to the dopamine antagonist sites A description of the original results seemed to be the only way to allow an objective analysis and to render comparison with future studies possible According to the analysis--which was thought to be the most appropriate---further evidence was gained that both 3H-splperone and 3H-apomorphlne stereospeclfiC binding sites occur concomitantly and are affected in a parallel way. In different subcellular fractions and after different neuronal lesions the number of splperone to apomorphlne sites were consistently proportlonate to a factor of about 2. These observations substantiate the hypothesis that both splperone and apomorphlne label the same unitary receptor, although they probably interact with different subunits, occurring in a ratio of 2:1 on the receptor macromolecular complex (Leysen, 1979) Further, the presently described investigations were undertaken to obtain information about the neuronal origin and function of dopamxnerglc receptors in the mltochondrlal fraction. The fact that the receptors on both subcellular particles are differentially influenced by neuronal lesions, demonstrates that they are receptors of different origin and that the d~fferent subcellular distribution is not an artefact of the preparation procedure. It has appeared that receptors occurring in the m~crosomal fraction are more readily destroyed by both kalnlC acid lesions and cortical ablation. So the currently discussed dopamlne receptors which are localized on lntra-strlatal neurones and on corticostrmtal afferents are both recovered In the m~crosomal fraction This suggests that they are localized on plasma membranes. The possibility also exists that part of the receptors recovered in this fraction are

228

J E L~sv~. W GOMMERENand P VAN GOMPFL

locahzed on small intracellular particles which are transported towards the sites where they must be incorporated in the plasma membrane (Laduron, 1980) The idea that receptors recovered in the heawer subcellular fraction would be associated wtth nerve terminals of cortlco-strlatal afferents was rejected The presently available data do not allow further speculation on the origin of the receptors in the heawer subcellular fraction However. evidence has now been obtained that they are associated with other cells than the receptors in the mlcrosomal fraction This ind~cites that further elaboration of the problem using different techniques and cell markers is necessary Acknowledgement--Part of th~s work was supported by a grant from I W O N L We thank Dr A Wauquier for his advice on the les~onmg procedures and we are indebted to W Mehs for his skillful assistance m performing the lesions Sincere thanks to Dr Jan Tollenaere and David Ashton for their help m preparing the manuscript

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