European Journal of Pharmacology, 50 (1978) 283--284

283

© Elsevier/North-Holland Biomedical Press

Rapid c o m m u n i c a t i o n TWO BINDING SITES FOR 3 H-SPIROPERIDOL ON RAT STRIATAL MEMBRANES

M. BRILEY and S.Z. LANGER Synth~labo, L.E.R.S., Department of Biology, Synaptic Receptor Group, 58, rue de la Glaciate, 75013 Paris, France

Received 25 May 1978, accepted 9 June 1978 The butyropherone neuroleptic, 3H-spiroperidol (3H-spiperone) was used by Fields et al. (1977) to label dopaminergic receptors in the central nervous system. These authors have shown that in the striatum the ligand binds with a single affinity constant. Leysen et al. (1978a), however, found a single affinity constant but a biphasic dissociation of 3 H-spiroperidol from rat striatal membranes. Furthermore, Howlett and Nahorski (1978) found a suggestion of a low affinity binding site for 3 H-spiroperidol and 3 H-haloperidol in various regions of rat central nervous system. We demonstrate here that there are clearly two specific binding sites for 3H-spiroperidol on rat striatal membranes. Rat striata were homogenised (Virtis 45 homogeniser) in 50 volumes of ice-cold buffer (50 mM Tris/HC1, pH 7.4, 120 mM NaC1, 5 mM KC1), washed by centrifugation at 30,000 × g and finally resuspended at 10 mg original wet weight/ml in the same buffer. This membrane suspension was incubated at 37°C for 5 min with 10 ~M pargyline and 0.1% ascorbic acid. Binding was determined by incubating 2.2 ml of membrane suspension with varying concentrations of 3 H-spiroperidol (NEN chemicals GmbH, 26.4 C i / m m o l e ) a t 37°C for 15 min. Two 1 ml samples from each tube were filtered under vacuum through Whatman GF/B filters and washed with two 10 ml rinses of ice-cold buffer. The filters

were dried at 110°C for 20 min and counted in toluene, PPO (5 g/l), P O P O P (0.1 g/l), scintillation liquid. Specific binding was defined as that displaced by 10 ~ M haloperidol and represented 85% of the total binding at 0.25 n M 3 H~spiroperidol. Scatchard plots of the binding of 3Hspiroperidol (0.05 nM--20 nM) to rat striatal. membranes (fig.l) repeatedly showed two components with dissociation constants differing by a factor of approximately 8, Kdl = 0.17 ± 0.04 nM (mean ± S.E.M., n = 9), Kd2 ffi 1.32 ± 0.25 riM. The higher affinity component comprised 41.4 ± 4.3% of the total specific binding which was 290 + 20 fmoles/mg protein. These values were not altered if the membranes were stored frozen (--80°C) for up to 6 weeks. The addition of CaC12 (10 raM) to the incubation medium, as frequently used by other groups, had no effect on the above results. Both binding sites were displaced by neuroleptics such as haloperidol (Ki's 0.09 nM and 0.95 nM) and by dopamine receptor agonists such as dopamine (Ki's 3.6 /~M and 30 ~M) and apomorphine (Ki's 0.95 /~M and 5.88 ~M). Both sites were stereospecific, butaclamol (+) (Ki's 0.16 nM and 1.65 nM) being approximately 1,000fold more potent than butaclamol (--) at displacing 3H~spiroperidol from each site. These results demonstrate the importance of studying the binding of both the

284

0.6

BOUND FREE 0.4

0.2

,\ I I I I

0.1

0.2 BOUND

~t

0.3

Fig.1. A typical Scatchard plot of specific s H-spiroperidol binding to rat striatal membranes. Specific binding was determined as described in the text. The axes are expressed as bound/free (pmoles 3 H-spiroperidol bound/mg protein/pmoles s H-spiroperidol/ml) and bound (pmoles s H-spiroperidol bound/mg protein). Each line was fitted by linear regression. The affinity constants, calculated from the inverse of the slope of each line were Kd~ = 0.24 + 0.03 nM and Kd~ = 1.11 + 0.09 nM. The estimates of maximum binding, calculated from the intercepts on the x-axis were Bmax I ffi 135 ± 11 fmolcs/mg protein; Bmaxtotal = 290 + 29 fmoles/mg protein. Maximum low affinity binding (Bmax 2 ) calculated from Bmaxtota 1 -- Bmax~ = 155 + 18 fmolas/mg protein. The figure shows a typical experiment with at least 9 observations.

radioactive ligand and the displacing drugs over a wide range of concentrations. M a n y studies have used only a few points over a narrow range of concentrations around the affinity constant and have therefore failed to observe the existence of a second binding site.

Clarification o f t h e p h y s i o l o g i c a l significance o f t h e t w o t y p e s o f n e u r o l e p t i c binding sites m a y c o n t r i b u t e significantly t o o u r k n o w l e d g e o f c e n t r a l d o p a m i n e r g i c transm i s s i o n a n d t h e role o f n e u r o l e p t i c s in t h e treatment of schizophrenia. The following f a c t o r s m i g h t b e involved in o u r observations: (a) t h e sites m a y be l o c a t e d pre- a n d p o s t s y n a p t i c a l l y , t h e p r e s y n a p t i c sites b e i n g situated on nerve terminals emanating from e i t h e r t h e s u b s t a n t i a nigra o r t h e c o r t e x o r b o t h ; (b) it has b e e n suggested t h a t t h e r e are d o p a m i n e r e c e p t o r s o n glial cells in t h e s t r i a t u m a n d t h a t these m a y d i f f e r f r o m t h e neuronal postsynaptic dopamine receptors ( H e n n et al., 1 9 7 7 ) ; (c) t h e r e is r e c e n t evidence that neuroleptics bind not only to d o p a m i n e r g i c r e c e p t o r s b u t also t o a s e r o t o n ergic r e c e p t o r in t h e s t r i a t u m a n d f r o n t a l c o r t e x ( L e y s e n et al., 1 9 7 8 b ) . In t h e light o f t h e e x i s t e n c e o f t w o binding sites f o r n e u r o l e p t i c s in t h e s t r i a t u m , the effects of denervation, supersensitivity a n d s u b s e n s i t i v i t y o n 3 H - s p i r o p e r i d o l binding r e q u i r e d e t a i l e d r e i n v e s t i g a t i o n .

References

Fields, J.Z., T.D. Reisine and H.I. Yamura, 1977, Biochemical demonstration of dopaminergic receptors in rat and human brain using (s H)-spiroperidol, Brain Res. 136, 578. Howlett, D.R. and S.R. Nahorski, 1978, A comparative study of (SH)-haloperidol and (SH)-spiroperidol binding to receptors on rat cerebral membranes, FEBS Letters 87,152. Leysen, J.E., W. Gommeren and P.M. Laduron, 1978a, Spiperone: a ligand of choice for neuroleptic receptors. 1. Kinetics and characteristics of in vitro binding, Biochem. Pharmaeol. 27,307. Leysen, J.E., C.J.E. Niemegeers, J.P. Tollenaere and P.M. Laduron, 1978b, Serotonergic component of neuroleptic receptors, Nature 272, 168. Henn, F.A., D.J. Anderson and A. Sellstr6m, 1977, Possible relationship between glial cells, dopamine and the effects of antipsyehotic drugs, Nature 266,637.