- Email: [email protected]
Regional distribution of μ, δ and κ opioid receptors in human brains from controls and parkinsonian subjects
Brain Research, 414 (1987) 8-14
8
Elsevier BRE 12639
Regional distribution of 6 and opioid receptors in human brains from controls and parkinsonian subjects P. Delay-Goyet 1, J.-M. Zajac 1, F. Javoy-Agid 2, Y. Agid 2 and B.P. Roques 1 ID~partement de Chimie Organique, U.266 INS E RM, U.A. 498 C. N. R. S. , U. E. R. des Sciences Pharmaceutiq ues et B iologiques, Paris (France) and 2Laboratoire de MOdecine Exp~rimentale, U.289 INSERM, U.E.R. Piti~-Salp~tri~re, Paris (France)
(Accepted 4 November 1986) Key words." Opioid receptor; Selective ligand; di-Enkephalin analogue; Discriminative binding property;
Parkinson's disease; Human brain
The binding properties of/~ and 6 opioid receptors were investigated in several areas of human brain by using [3H]Tyr-D-Ala-Gly(Me)Phe-Gly-ol and [3H]Tyr-D-Thr-Gly-Phe-Leu-Thr as respective selective ligands, while the totality of opioid receptors was measured by using [3H]etorphine as a non-selective agonist. Receptor densities were highest in cerebral cortex, amygdala and striatum, and lowest in the substantia nigra (pars compacta). In the different brain areas of patients with Parkinson's disease, the density nd the proportion of the various opioid receptors were not significantly different from control subjects.
INTRODUCTION The existence of several classes of opioid receptors /~, 6 and K, differentially distributed in the rat brain, is now well documented (for review, see ref. 18). In human brain, these receptors have not yet been discriminated by direct binding studies 4,1°,12A3'15'16,2°. Biochemical and pharmacological studies have shown that 6 receptors are very likely involved in the physiological modulation of the nigrostriatal dopaminergic neurons in the rat and in the cat 5'19. Likewise, in the human brain, an anatomical relationship has been evidenced between dopaminergic neurons and enkephalinergic nerve terminals in the mesencephalon s. The major biochemical characteristic of Parkinson's disease is the marked degeneration of dopaminergic neurons of mesencephalic originl'll; Met- and Leu-enkephalin levels decrease in the pallidum and putamen, while only Met-enkephalin levels are affected in the substantia nigra and ventral tegmental area 26. These findings suggest that alterations
in enkephalinergic transmission are associated with a degeneration of dopaminergic pathways. However, contradictory data have been reported concerning the state of opioid receptor levels measured in brains of parkinsonian patients 14'22'23. The availability of tritiated probes with high selectivity and affinity for and 6 receptors, [3H]Tyr-D-Ala-Gly-(Me)Phe-Gly-ol ( D A G O ) 9 and [3H]Tyr-D-Thr-Gly-Phe-Leu-Thr (DTLET) 7'29 respectively, prompted us to investigate the regional distribution of kt, 6 and ~¢receptors in human brain and to re-examine to what extent opioid receptors are affected or regulated in Parkinson's disease. MATERIALS AND METHODS Chemicals
D A G O was prepared according to Handa et al. 9 and D T L E T as previously reported 29. [3H]DTLET (1.3 or 1.7 TBq/mmol) and [ 3 H ] D A G O (1.7 or 2.2 TBq/mmoi), obtained from their 2,5-dibromotyrosyl
Correspondence." B.P. Roques, D6partement de Chimie Organique, U.266 INSERM, U.A. 498 C.N.R.S., U.E.R. des Sciences Pharmaceutiques et Biologiques, 4 Avenue de l'Observatoire, 75006 Paris, France.
0006-8993/87/$03.50(~) 1987 Elsevier Science Publishers B.V. (Biomedical Division)
precursors, were from the CEA (Saclay, France). [3H]Ethylketocyclazocine (0.56 TBq/mmol) was purchased from New England Nuclear Corp., [3H]etorphine (1.2 TBq/mmol) from Amersham. Levorphanol tartrate was a gift from Hoffmann-La Roche.
Subjects In a first series of experiments, the characteristics of the opioid receptors were established in different brain regions from 19 control subjects, aged 65-90 years (mean 78.7 + 1.8 years). The post-mortem period varied from 2-27 h (mean 8.7 + 1.4). Ten brains were used for the study of the hypothalamus and substantia nigra and 9 others for the forebrain regions. In a second series of experiments, the comparative distribution of opioid binding sites in various areas of controls and parkinsonians was studied on brains from 37 control subjects (age 75.9 + 1.5 years) with no known neurological disease and from 32 patients with idiopathic Parkinson's disease (age 73.4 + 1.1 years). The time lapse between death and autopsy was 11.7 + 1.2 h for controls and 16.0 + 1.8 for parkinsonians. The immediate cause of death was sudden death, coma, cachexia, collapsus, asphyxia, bronchopneumonia, renal failure. The mean duration of Parkinson's disease was 11.8 + 1.5 years. Patients had received L-DOPA with a peripheral decarboxylase inhibitor alone or with an anticholinergic agent (1 patient). Three patients had only received anticholinergic agents. For 12 patients, drug treatment was continued up to the day of death and for the remainder, was discontinued at least 2 days (range 2 days to 4 years) prior to death. Doses of L-DOPA ranged from 375 to 1,000 mg/day. Criteria for parkinsonism was based on review of the clinical record, dopamine content of the caudate nucleus (less than 20% of the control values), and histopathological evidence of neuronal loss and presence of Lewy bodies in the substantia nigra. Within less than 2 h after autopsy the brains were stored at -70 °C. The forebrains and brainstems were cut rostrocaudally into 10-mm and 1-mm frontal sections respectively. The brain regions were dissected out from each tissue slice on a plate cooled at -15 °C. For each brain, equivalent regions from the sections were pooled, crushed on dry ice, and stored at -70 °C. An aliquot of each pool was weighed.
Preparation of membrane fractions and binding assays Membrane fractions were prepared as previously described 3°. Binding studies were carried out in 50 mM TrisHCI (pH 7.4). Each assay contained 0.3-1 mg of protein, the radioligand at the desired concentration and other additions in a final volume of 1 ml. All points were determined in triplicate and the non-specific binding was measured in the presence of 10 ~tM levorphanol. Following either a 40-min ([3H]DTLET) or a 30-min (other radioligands) incubation at 35 °C, the content of each tube was rapidly filtered over Whatman GF/B filters. The filters were washed twice with 5 ml of cold Tris-HCl buffer, dried and suspended in 5 ml of Beckman Ready Solv. EP cocktail for determination of bound radioactivity (counting efficiency --- 40%).
Data analysis The maximal number of binding sites (B~ax) and the dissociation constant (KD) for the radioligands were determined from least squares linear regression of Scatchard or Eadie-Hofstee analysis of saturation isotherms. When binding experiments were carried out with a single concentration of labelled ligand, the Bmax value was calculated from the concentration of bound radioligand (B) using the equation Bmax = B'(1 + KD/L), where KD is the dissociation constant and L the concentration of free ligand. Student's unpaired t-test was used for comparison between two means, with P < 0.05 considered significant. RESULTS
Binding properties of [3H]DA GO, [3HIDTLET and [3H]etorphine in different regions of human brains Equilibrium binding parameters obtained in various regions are presented in Table I. In all the brain regions studied, the specific binding of the 3 ligands was saturable and of high affinity. Eadie-Hofstee and Scatchard plots were linear and consistent with the hypothesis that each radioligand labelled a single class of binding sites. The mean (+ S.D.) K D values obtained for the different brain structures were 4.50 + 0.85 nM for [3H]DAGO, 1.51 + 0.19 nM for [3H]DTLET and 0.214 + 0.038 nM for [3H]etorphine, an opioid ligand
10 TABLE I Binding affinities and capacities of [3H] D A GO, [~H] D TL E T and [3H]etorphine in various human brain regions
The reported parameters + S.D. were obtained by linear regression analysis of Eadie-Hofstee representation isotherms. Experiments were performed using 8 concentrations between 0.5-12 nM, 0.1-5 nM and 0.1)5-0.60 nM of [3H]DAGO, [3H]DTLET and [3H]etorphine respectively; n = number of independent experiments (number of experiments performed with tissues pooled from different subjects). Brain structures
Nucleus caudatus Putamen Globus pallidus (external part) Thalamus Hypothalamus Amygdala Hippocampus Frontal lobe cortex (Brodmann area 9) Cingulate Cortex (Brodmann area 24) Substantia nigra (pars compacta)
[3H] D A G O
[3H] D TL E T
[3H] Etorphine
n
Ka (nM)
Bmax (femtomol/mg)
n
Ka (nM)
B~ax (femtomol/mg)
n
Ka (nM)
6 7 (1)
4.68 ___0.89 4.44 __+1.38
63 + 31 74 + 44
5 7 (1)
1.63 + 0.26 1.70+ 0.38
35 + 22 32 + 18
8 (1) 7 (1)
0.274 + 0.095 0.176+ 0.081
132 + 49 109 + 51
4 (4) 3 (1) 3 (2) 4 (4) 4 (3)
3.80 _+0.37 5.84 + 1.85 3.77 + 0.03 5.12+1.39 3.68 _+ 1.31
18 + 3 42 + 35 54 + 28 76+41 21 + 6
5 (4) 3 (1) 4 (3) 5(5) 4 (3)
1.52 + 0.34 1.67+0.56 1.36 + 0.24
11 + 5 <5 <5 31+7 13 _+4
5 (4) 3 (1) 3 (2) 4(4) 4 (3)
0.200 + 0.096 0.166 + 0.053 0.199 + 0.027 0.186+0.035 0.236 + 0.083
46 + 23 51 + 39 64 + 28 152+72 68 + 9
5 (3)
5.57 + 2.30
89 + 76
5 (3)
1.55 + 0.50
41 _+ 19
5 (3)
0.260 + 0.195
213 + 84
4 (1)
3.64 + 0.35
43 __+24
4 (1)
1.17 + 0.10
24 + 12
4 (1)
0.229 + 0.040
116 + 49
1 (1)
4.1
4(4)
-
<5
3(3)
0.267+0.097
21.7+1.2
9
which exhibits about the same affinity for the 3 opioid binding types 17. These values are close to those previously d e t e r m i n e d using rat brain m e m b r a n e s 29,3°. The affinities of [ 3 H ] D A G O and [3H]DTLET for the sites defined as/~ and 6, respectively, were similar in all brain regions. The standard deviations of the raw data (S.D. E r a d ) 31 were approximately 10% for each agonist saturation plot. These S.D. E r a d were smaller than the standard deviations (S.D.) of the means of Bmax values m e a s u r e d from different h u m a n brains. Thus, the standard deviation of the p a r a m eters r e p o r t e d in Table I most likely reflects individual variability in the population studied, since the differences in binding capacity are not correlated with age, p r e - m o r t e m conditions or p o s t - m o r t e m delay. [3H]Etorphine capacities were greater than the sum of [ 3 H ] D A G O and [3H]DTLET capacities (Table I). Due to the lack of highly selective r radioligands, sites were quantified as the difference between [3H]etorphine and the sum of [ 3 H ] D A G O and [3H]DTLET binding capacities. On the other hand, in the presence of 100 nM D A G O and 100 nM D T L E T , [3H]ethylketocyclazocine, a second putative ligand for ~ sites, binds to a residual high affinity site (nucleus caudatus : KD = 0.762 nM, Bmax = 49
B,nax (femtomol/mg)
femtomol/mg; putamen: K D = 0.728 nM, Bmax = 53 femtomol/mg; frontal cortex: K D = 1.12 nM, Bmax = 53 femtomol/mg). Thus, the remaining binding sites of [3H]etorphine, corresponding to non-/~, non-6 opioid sites, exhibit a high affinity for [3H]ethylketocyclazocine in agreement with their definition as ~csites. [OH]Ethylketocyclazocine eliciting about the same affinity f o r p , 6 and r receptors 6, the relative p r o p o r tions of these binding sites were also estimated in nucleus caudatus and frontal cortex by means of simple sequential inhibition, as described by Pfeiffer et al. 2°. Binding of [3H]ethylketocyclazocine (2 nM) in these areas was inhibited by D A G O in a complex m a n n e r which could be analyzed as a competition involving three different binding sites. The p r o p o r t i o n of [3H]ethylketocyclazocine binding displaced by D A G O with a low affinity (IC50 = 10,000 nM) was 30% in the nucleus caudatus and 50% in the frontal cortex. The proportions, calculated from the binding capacities of [3H]etorphine, are 26% and 39% respectively (Table I). These suggest either that [3H]etorphine and [3H]ethylketocyclazocine do not bind to the same r sites 2, or that they have different interactions with # and 6 sites 6.
11 T A B L E II
Comparison of the binding capacities (B,,ax) of [3H] DA GO, [3HIDTL ET and [3H]etorphine in various regions of brains from normal and parkinsonian patients Values are expressed as femtomol per m g of m e m b r a n e protein + S.D. T h e binding was performed with 2 n M [3H]DAGO, 1 n M [3H]DTLET, * 2 n M [3H]ethylketocyclazocine in presence of D A G O (100 nM) and D T L E T (100 nM) or 2 n M [3H]etorphine. Bmax of [3H]DAGO and [3H]DTLET were calculated from the equation: Bmax = B X (1 + Kd/L); n I and n 2 are the n u m b e r s of i n d e p e n d e n t normal or parkinsonian subjects, respectively; *, 1 pool; **, 2 pools of two samples each.
Brain region
n,
Nucleus caudatus Putamen Globus pallidus (external part) Amygdala Frontal cortex Substantia nigra (pars compacta)
n2
14 14 20 12 14 21"
[3H]DAGO
14 14 17 11 14 18"*
[3H]DTLET
[3H]Etorphine
Control
Parkinson
Control
Parkinson
Control
Parkinson
110 40 16 99 107 17
107 39 18 100 97 22
35 19 9.5 30 41 -
34 21 7.7 31 35 -
166 73 28 151 164 44 21
161 68 26 186 158 45 18
+ + + + + +
47 15 12 31 38 10
Distribution of opioid binding sites in various regions of parkinsonian brains The distribution of/~ and 6 binding sites was studied in regions innervated by the nigrostriatal or meso-
+ + + + + +
26 25 12 44 31 10
+ + + + +
19 9 6.4 18 15
+ + + + +
23 11 3.5 20 15
+ 36 + 32 + 27 + 50 + 53 + 20 +__6*
+ + + + + + +
38 30 27 62 50 16 9*
corticolimbic dopaminergic neurons, which are affected by Parkinson's disease (Table II). The use of a different sampling and the need, in the first series of experiments, to pool several different
1 6K 75
EB~O m +
¥¥ ++ 4-44-4-
,Q 50
0
25
0
+
~-
+ __44-4-
+ 44-
4-
_
4-44-4-
4-+ 4-4-
~ 4-4-
4-44-4-
44-:171
4-44-+ 4-+ 4-44-4++ 4-4-
++ 4-4++ 4-44-44-4++ 4-+~
+-0.1
4-44-4-
++ ++ ++ 4-4+4-I-+ ++ 4-4-
4-+1 ++l 4-+1 ++l 4-+1 ++. ++l
+4++ ++ ++ 4-+ ++ 4-+
+4-:1
++,
+4-
4-4-
++l 4- +1 4-+1 4- .~.i
+44-4-
++ +4-
++
4-+
-4444~+
+4-
4-44. +
++ 4-+
++ 4-4-
++--e o "~ 4- O •
4- + ~ J + 4- i i'li"l ~tael +4-n''' 4- 4- l * l i
4-+__ 4-4- •
++ 4-4-
4-+ ++~
++o
•
+ +....
++.
+ + o~-~
++o
o--
++o,o,o,.,
+-,-[
++ onon
++ ++
"44.4. 4-
"1" 444-
4444-
4- + I I " ~ 4- + l l l l i [ = 4elL[ 4- ++ il ll il li il
4'4" 44-
+ 44+
4"t" 44- 00
4- "~ .4. "l"
i
44- • 4- 00 4" •
0O • 00 •
ee 00
4- "l" 4- 4- •
:
•
O •
4- 44- 4-
4- "t" 4- 4- •
cpcpcp
cpcpcp
NUCLEUS CAUDATU
P U T ~ E N S
t
4444-
Olin ilDI JilL le~el iJlilllll
4- 4- J i l l 4- 4- l i I I i 4- 44- 4-
!ilk
cpcpcp GLOBUS PALL I DUB (EXTERNAL PART)
__
+ 444-
",1
Olel OIOI OIOI OIoIOIOI Illl~
+ 444-
4- + I I l I I .~. , . ~ l i l i l
4- 4- IOl llll l
+ + oriel
+ + 4- 4-
"4" + l l l l l 4- + i unon ....
4- 4- O I O I
4- 4-
.,.,
cpcpcp ~ Y G D A L A
--
4-4-
4-4-
t' •
"
++"" cpcpcp
FRONTAL CORTEX
+" cpcpcp
SUBSTANTIA S I O]WI% (PARS COMPACTA)
Fig. 1. Distribution of opioid binding sites in various brain areas. T h e p, 6 and r sites are expressed as percent of total b o u n d which was measured with 2 n M [3H]etorphine (Table II) (C, control; P, parkinsonian).
12 brains for saturation experiments, very likely account for the differences among control values reported in Tables I and II. Whatever the region sampled and the ligand used, large variations in the measurements are observed from one control to another. Nevertheless, the large number of independent measurements carried out (n > 11) permitted a suitable statistical comparison between controls and brain of parkinsonians (Table II). No significant changes in receptor densities (P < 5%) were found in the different regions examined, even in areas in which marked differences were observed, i.e. the amygdala [3H]etorphine: + 23%), the globus pallidus, external part, [3H]DTLET: -19%) and the substantia nigra, pars compacta, [3H]DAGO: +29%) (Table II). In amygdala, additional experiments (number of samples: controls 11, parkinsonians 13) confirmed the absence of significant variations of ~ sites binding using either [~H]etorphine (control: 228 + 74; Parkinson: 235 + 47 femtomol/mg), or [3H]ethylketocyclazocine in the presence of 100 nM D A G O and 100 nM DTLET (control: 95 + 23, Parkinson: 96 + 17 femtomol/mg). The moderate changes in receptor densities in the globus pailidus (external part) and substantia nigra (pars compacta) of brains of parkinsonians are most likely due to the inaccuracy related to a low number of binding sites in these areas (Table I). DISCUSSION The regional distribution of opioid sites in human brains (Table If, Fig. 1) is in good agreement with previous reports 12, in particular for the proportions of p and 6 sites observed by analysis of competition curves of non-selective ligands 2°. The large number of samples allowed a statistically safe comparison of the distribution of the various opioid receptors between brains from controls and parkinsonian patients. Owing to the fact that dissection and storage procedures do not affect opioid receptor binding capacities 25, the inaccuracy of the data is most likely attributable to individual variability. This is indicated by the fact that the ratios of p, 6 and sites in brains of controls or parkinsonian subjects varied to a lesser extent than the total capacity of binding sites. The absence of modification of either opioid receptors densities (Table II) or ratios of p, 6, ~: sites in
brains of parkinsonian subjects (Fig. 1), using selective p and 6 radioligands, is at variance with the previously reported reduction of 40-50% of [3H]naloxone sites in the caudate nucleus of brains of parkinsonian patients 22"23. However, using [3H]Met-enkephalinamide as ligand, an unmodified density of opioid receptors has also been observed in the nucleus caudatus, contrasting with a 42% decrease of the binding sites capacity in the substantia nigra 14. These discrepancies could be due to the differences in the radioligands used, or to the great variations associated with the use of human material from autopsy. Moreover, in the substantia nigra, where the levels of opioid binding sites are low, it is most likely that a marked (40%) diminution of the binding capacity must be reached in order to be statistically significant. On the other hand, an increase of [3H]Leu- and [3H]Met-enkephalin binding was reported in the caudate nucleus, putamen, nucleus accumbens, limbic cortex and hippocampus of parkinsonian subjects 23, suggesting a denervation supersensitivity of a population of postsynaptic opioid receptors. Using selective and metabolically resistant enkephalin analogues, we found no significant increase in the binding capacities (Table II), particularly in putamen, pallidum and substantia nigra where Met- and Leuenkephalin levels are decreased, indicating a change in peptide transmission 26. Similarly, no up-regulation of D 2 dopamine receptors was evidenced by positron emission tomography in parkinsonians27; however, the increase in dopamine receptor density in Parkinson's disease could be progressive and relative to the extent of the lesion 3. Using [3H]Leu-enkephalin or [3H]naloxone as ligands, a decrease in rat striatal opioid receptors has been observed after 6-OHDA-induced degeneration of the nigrostriatal dopaminergic pathway 21, while an increase of Da dopamine receptors has been shown 24. Using this animal model of the disease, an 80% decrease of tyrosine-hydroxylase activity in the striaturn was associated with a concomitant decrease of 30% of opioid binding sites capacity using either binding experiments on homogenates (unpublished observations) or autoradiography 28. Our data suggest that the neuronal localization of opioid receptors in striatum may be different in man and rat, or that as far as the degeneration process of
13 dopaminergic neurons is concerned, the neurochemical changes, subsequent to acute destruction of the nigrostriatal dopamine neurons in animal or to the chronic pathological process in Parkinson's disease, differ fundamentally. For example, natural and druginduced compensatory mechanisms for any possible opioid receptor decrease might develop in man after degeneration of the nigrostriatal dopaminergic pathway. It is likely that these features account for the discrepancies among data reported on brains of parkinsonians. It is noteworthy that a significant decrease of [3H]-naloxone binding, in the untreated parkinsonian caudate nucleus, has not been observed in patients treated with L - D O P A 23, suggesting that LD O P A therapy (the conventional treatment of Parkinson's disease) could stabilize the level of function-
REFERENCES 1 Agid, Y, Javoy-Agid, F. and Ruberg, M., The neurobiochemistry of Parkinson's disease. In C.D. Marsden and S, Fahn (Eds.), Movement Disorders-2, BIMR Neurology, Butterworths, 1986, in press. 2 Attali, B., Gouarderes, C., Mazarguil, H., Audigier, Y. and Cros, J. Evidence of multiple kappa binding sites by use of opioid peptides in the guinea pig lumbo-sacral cord, Neuropeptides, 3 (1982) 53-64. 3 Bokobza, B., Ruberg, M., Scanon, B., Javoy-Agid, F. and Agid, Y., [3H]Spiperone binding, dopamine and HVA concentrations in Parkinson's disease and supranuclear palsy, Eur. J. Pharmacol., 99 (1984) 167-175. 4 Bonnet, K.A., Groth, J., Gioannini, T., Cortes, M. and Simon, E.J., Opiate receptor heterogeneity in human brain regions, Brain Research, 221 (1981) 437-440. 5 Chesselet, M.-F., Cheramy, A., Reisine, T.D., Lubetzki, C., Glowinsky, J., Fournie-Zaluski, M.C. and Roques, B.P., Effects of various opiates including specific 6 and agonists on dopamine release from nigrostriatal dopaminergic neurons in vitro in the rat and in vivo in the cat, Life Sci., 31 (1982) 2291-2294. 6 Delay-Goyet, P., Roques, B.P. and Zajac, J.-M., Binding characteristics of non-selective opiates towards/~ and 6 receptor types, Life Sci., submitted. 7 Delay-Goyet, P., Zajac, J.-M., Rigaudy, P., Foucaud, B. and Roques, B.P., Comparative binding properties of linear and cyclic cS-selective enkephalin analogues: [3H]-[DThr2,LeuS]enkephalyl-Thr6 and [3H]-[D-P~n ,-7~nS]enkephalin, FEBS Lett. 183 (1985) 439-443. 8 Gaspar, P., Berger,B., Gay, M., Hamon, M., Cesselin, F., Vigny, A., Javoy-Agid, F. and Agid, Y., Tyrosine hydroxylase and methionine enkephalin in the human mesencephalon: immunohistochemical localization and relationships, J. Neurol. Sci., 58 (1983) 247-267. 9 Handa, B.K., Lane, A.C., Lord, J.A.H., Morgan, B.A., Rance, M.J. and Smith, C.F.C., Analogues of fl-LPH61_64 possessing selective agonist activity at/~-opiate receptors, Eur. J. Pharmacol., 70 (1981) 531-540.
al opioid receptors in the brain. However, the low number of L - D O P A untreated patients in our study does not allow this hypothesis to be confirmed. ACKNOWLEDGEMENTS Drs. Hauw, Lhermitte, Castaigne, Laplane (H6pital de la Salp~tri~re, Paris) and Drs. Beck, Bouchon, Moulias, Piette (H6pital Charles Foix, Ivry-surSeine) are gratefully acknowledged for providing human material. This work was supported by funds from the Centre National de la Recherche Scientifique, the Institu National de la Recherche M6dicale and the Universit6 Ren6 Descartes. We thank Dr. A. Beaumont for critical reading of the manuscript and A. Bouju for typing it.
10 Hiller, J.M., Pearson, J. and Simon, E.J., Distribution of stereospecific binding of the potent narcotic analgesic etorphine in the human brain: predominance in the limbic system, Res. Commun. Chem. Pathol. Pharmacol., 6 (1973) 1052-1062. 11 Hornykewicz, O., Dopamine ([3H]hydroxytyramine) and brain function, Pharmacol. Rev., 18 (1966) 925-964. 12 Itzhak, Y., Bonnet, K.A,, Groth, J., Hiller, J.M. and Simon, E.J., Multiple opiate binding sites in human brain regions: evidence for x and ty sites, Life Sci., 31 (1982) 1363-1366. 13 Kuhar, M.J., Pert, C.B. and Snyder, S.H., Regional distribution of opiate receptor binding in monkey and human brain, Nature (London), 245 (1973) 447-450. 14 Llorens-Cortes, C., Javoy-Agid, F., Agid, Y., Taquet, H. and Schwartz, J.-C., Enkephalinergic markers in substantia nigra and caudate nucleus from parkinsonian subjects, J. Neurochem., 3 (1984) 874-877. 15 Llorens, C., Malfroy, B., Schwartz, J.C., Gacel, G., Roques, B.P., Roy, J., Morgat, J.-L., Javoy-Agid, F. and Agid, Y., Enkephalin dipeptidyl carboxypeptidase (enkephalinase) activity: selective radioassay, properties and regional distribution in human brain, J. Neurochem., 39 (1982) 1081-1089. 16 Maurer, R., Cortes, R., Probst, A. and Palacios, J.M., Multiple opiate receptor in human brain: an autoradiographic investigation, Life Sci., 33 Suppl. I (1983) 231-234. 17 Gillan, M.G.C. and Kosterlitz, H.W., Spectrum of the p-, 6- and x-binding sites in homogenates of rat brain, Br. J. Pharmacol., 77 (1982) 461-469. 18 Paterson, S.J., Robson, L.E. and Kosterlitz, H.W., Opioid receptors. In S. Udenfriend and J. Meienhofer (Eds.), The Peptides: Analysis, Synthesis, Biology, Vol. 6, Academic, London, 1984, pp. 147-189. 19 Petit, F., Hamon, M., Fournie-Zaluski, M.-C., Roques, B.P. and Glowinsky, J., Further evidence for a role of 6opiate receptors in the presynaptic regulation of newly synthesized dopamine release, Eur. J. Pharmacol., 126 (1986) 1-9. 20 Pfeiffer, A., Pasi, A., Mehraein, P. and Herz, A., Opiate
14
21 22 23
24
25
26
receptor binding sites in human brain, Brain Research, 248 (1982) 87-96. Pollard, H., Llorens-Cortes, C. and Schwartz, J.-C., Enkephalin receptors on dopaminergic neurons in rat striatum, Nature (London), 268 (1977) 745-747. Reisine, T.D., Rossor, M., Spokes, E., lversen, L.L. and Yamamura, H.I., Alterations in brain opiate receptors in Parkinson's disease, Brain Research, 173 (1979) 378-382. Rinne, U.K., Rinne, J.K., Rinne, J.O., Laakso, K., Tenovuo, O., Lonnberg, P. and Koskinen, V., Brain enkephalin receptors in Parkinson's disease, J. Neural Transm., Suppl. 19 (1983) 163-171. Savasta, M., Dubois, A., Manier, M., Mouchet, P., Fourtier, O. and Scatton, B., Autoradiographie quantitative des sites r6cepteurs dopaminergiques D~ et D2 dans le striatum de rat: adaptation post-synaptique ~ la d6nervation nigrostri6e, Colloq. Natl. Neurosci., Bordeaux (1986). Schwartz, J.-C., Pollard, H., Martres, M.P., LlorensCortes, C., Javoy-Agid, F., Taquet, H., Agid, Y., Bouthenet, M.L. and Sales, N., Neurotransrnitter Markers in Human Area Postrema, Springer, Oxford, 1984. Taquet, H., Javoy-Agid, F., Hamon, M., Legrand, J.C., Agid, Y. and Cesselin, F., Parkinson's disease affects differently (MetS)-and (LeuS)-enkephalin in the human brain,
Brain Research, 280 (1983) 379-382. 27 Wagner, H.N., Wong, D.F., Dannals, R.F., Kuhar, M.J., Links, J,M., Frost, J.J., Folstein, M. and Folstein, S., Imaging Dopamine Receptors in the Human Brain, IVth World Congress of Biological Psychiatry, Philadelphia, 1985. 28 Waksman, G., Hamel, E., Delay-Goyet, P., Fournie-Zaluski, M.-C. and Roques, B.P., Radioautographic Visualization of Enkephalinase in Rat Brain with [SH]HACBOGly: Comparison with the Distribution of Opioid kt and 6 Receptors, International Narcotics Research Conference, North Falmouth, 1985. 29 Zajac, J.-M., Gacel, G., Petit, F., Dodey, P., Rossignol, P. and Roques, B.P., Deltakephalin, Tyr-D-Thr-Gly-PheLeu-Thr: a new highly potent and fully specific agonist for opiate 6-receptors, Biochem. Biophys. Res. Commun., 11 (1983) 390-397. 30 Zajac, J.-M. and Roques, B.P., Differences in binding properties of p and 6 opioid receptor subtypes from rat brain: kinetic analysis and effects of ions and nucleotides, J. Neurochem. , 44 (1985) 1605-1614. 31 Zivin, J.A, and Waud, D.R., How to analyze binding, enzyme and uptake data: the simplest case, a single phase, Life Sci., 30 (1982) 1407-1422.
8
Elsevier BRE 12639
Regional distribution of 6 and opioid receptors in human brains from controls and parkinsonian subjects P. Delay-Goyet 1, J.-M. Zajac 1, F. Javoy-Agid 2, Y. Agid 2 and B.P. Roques 1 ID~partement de Chimie Organique, U.266 INS E RM, U.A. 498 C. N. R. S. , U. E. R. des Sciences Pharmaceutiq ues et B iologiques, Paris (France) and 2Laboratoire de MOdecine Exp~rimentale, U.289 INSERM, U.E.R. Piti~-Salp~tri~re, Paris (France)
(Accepted 4 November 1986) Key words." Opioid receptor; Selective ligand; di-Enkephalin analogue; Discriminative binding property;
Parkinson's disease; Human brain
The binding properties of/~ and 6 opioid receptors were investigated in several areas of human brain by using [3H]Tyr-D-Ala-Gly(Me)Phe-Gly-ol and [3H]Tyr-D-Thr-Gly-Phe-Leu-Thr as respective selective ligands, while the totality of opioid receptors was measured by using [3H]etorphine as a non-selective agonist. Receptor densities were highest in cerebral cortex, amygdala and striatum, and lowest in the substantia nigra (pars compacta). In the different brain areas of patients with Parkinson's disease, the density nd the proportion of the various opioid receptors were not significantly different from control subjects.
INTRODUCTION The existence of several classes of opioid receptors /~, 6 and K, differentially distributed in the rat brain, is now well documented (for review, see ref. 18). In human brain, these receptors have not yet been discriminated by direct binding studies 4,1°,12A3'15'16,2°. Biochemical and pharmacological studies have shown that 6 receptors are very likely involved in the physiological modulation of the nigrostriatal dopaminergic neurons in the rat and in the cat 5'19. Likewise, in the human brain, an anatomical relationship has been evidenced between dopaminergic neurons and enkephalinergic nerve terminals in the mesencephalon s. The major biochemical characteristic of Parkinson's disease is the marked degeneration of dopaminergic neurons of mesencephalic originl'll; Met- and Leu-enkephalin levels decrease in the pallidum and putamen, while only Met-enkephalin levels are affected in the substantia nigra and ventral tegmental area 26. These findings suggest that alterations
in enkephalinergic transmission are associated with a degeneration of dopaminergic pathways. However, contradictory data have been reported concerning the state of opioid receptor levels measured in brains of parkinsonian patients 14'22'23. The availability of tritiated probes with high selectivity and affinity for and 6 receptors, [3H]Tyr-D-Ala-Gly-(Me)Phe-Gly-ol ( D A G O ) 9 and [3H]Tyr-D-Thr-Gly-Phe-Leu-Thr (DTLET) 7'29 respectively, prompted us to investigate the regional distribution of kt, 6 and ~¢receptors in human brain and to re-examine to what extent opioid receptors are affected or regulated in Parkinson's disease. MATERIALS AND METHODS Chemicals
D A G O was prepared according to Handa et al. 9 and D T L E T as previously reported 29. [3H]DTLET (1.3 or 1.7 TBq/mmol) and [ 3 H ] D A G O (1.7 or 2.2 TBq/mmoi), obtained from their 2,5-dibromotyrosyl
Correspondence." B.P. Roques, D6partement de Chimie Organique, U.266 INSERM, U.A. 498 C.N.R.S., U.E.R. des Sciences Pharmaceutiques et Biologiques, 4 Avenue de l'Observatoire, 75006 Paris, France.
0006-8993/87/$03.50(~) 1987 Elsevier Science Publishers B.V. (Biomedical Division)
precursors, were from the CEA (Saclay, France). [3H]Ethylketocyclazocine (0.56 TBq/mmol) was purchased from New England Nuclear Corp., [3H]etorphine (1.2 TBq/mmol) from Amersham. Levorphanol tartrate was a gift from Hoffmann-La Roche.
Subjects In a first series of experiments, the characteristics of the opioid receptors were established in different brain regions from 19 control subjects, aged 65-90 years (mean 78.7 + 1.8 years). The post-mortem period varied from 2-27 h (mean 8.7 + 1.4). Ten brains were used for the study of the hypothalamus and substantia nigra and 9 others for the forebrain regions. In a second series of experiments, the comparative distribution of opioid binding sites in various areas of controls and parkinsonians was studied on brains from 37 control subjects (age 75.9 + 1.5 years) with no known neurological disease and from 32 patients with idiopathic Parkinson's disease (age 73.4 + 1.1 years). The time lapse between death and autopsy was 11.7 + 1.2 h for controls and 16.0 + 1.8 for parkinsonians. The immediate cause of death was sudden death, coma, cachexia, collapsus, asphyxia, bronchopneumonia, renal failure. The mean duration of Parkinson's disease was 11.8 + 1.5 years. Patients had received L-DOPA with a peripheral decarboxylase inhibitor alone or with an anticholinergic agent (1 patient). Three patients had only received anticholinergic agents. For 12 patients, drug treatment was continued up to the day of death and for the remainder, was discontinued at least 2 days (range 2 days to 4 years) prior to death. Doses of L-DOPA ranged from 375 to 1,000 mg/day. Criteria for parkinsonism was based on review of the clinical record, dopamine content of the caudate nucleus (less than 20% of the control values), and histopathological evidence of neuronal loss and presence of Lewy bodies in the substantia nigra. Within less than 2 h after autopsy the brains were stored at -70 °C. The forebrains and brainstems were cut rostrocaudally into 10-mm and 1-mm frontal sections respectively. The brain regions were dissected out from each tissue slice on a plate cooled at -15 °C. For each brain, equivalent regions from the sections were pooled, crushed on dry ice, and stored at -70 °C. An aliquot of each pool was weighed.
Preparation of membrane fractions and binding assays Membrane fractions were prepared as previously described 3°. Binding studies were carried out in 50 mM TrisHCI (pH 7.4). Each assay contained 0.3-1 mg of protein, the radioligand at the desired concentration and other additions in a final volume of 1 ml. All points were determined in triplicate and the non-specific binding was measured in the presence of 10 ~tM levorphanol. Following either a 40-min ([3H]DTLET) or a 30-min (other radioligands) incubation at 35 °C, the content of each tube was rapidly filtered over Whatman GF/B filters. The filters were washed twice with 5 ml of cold Tris-HCl buffer, dried and suspended in 5 ml of Beckman Ready Solv. EP cocktail for determination of bound radioactivity (counting efficiency --- 40%).
Data analysis The maximal number of binding sites (B~ax) and the dissociation constant (KD) for the radioligands were determined from least squares linear regression of Scatchard or Eadie-Hofstee analysis of saturation isotherms. When binding experiments were carried out with a single concentration of labelled ligand, the Bmax value was calculated from the concentration of bound radioligand (B) using the equation Bmax = B'(1 + KD/L), where KD is the dissociation constant and L the concentration of free ligand. Student's unpaired t-test was used for comparison between two means, with P < 0.05 considered significant. RESULTS
Binding properties of [3H]DA GO, [3HIDTLET and [3H]etorphine in different regions of human brains Equilibrium binding parameters obtained in various regions are presented in Table I. In all the brain regions studied, the specific binding of the 3 ligands was saturable and of high affinity. Eadie-Hofstee and Scatchard plots were linear and consistent with the hypothesis that each radioligand labelled a single class of binding sites. The mean (+ S.D.) K D values obtained for the different brain structures were 4.50 + 0.85 nM for [3H]DAGO, 1.51 + 0.19 nM for [3H]DTLET and 0.214 + 0.038 nM for [3H]etorphine, an opioid ligand
10 TABLE I Binding affinities and capacities of [3H] D A GO, [~H] D TL E T and [3H]etorphine in various human brain regions
The reported parameters + S.D. were obtained by linear regression analysis of Eadie-Hofstee representation isotherms. Experiments were performed using 8 concentrations between 0.5-12 nM, 0.1-5 nM and 0.1)5-0.60 nM of [3H]DAGO, [3H]DTLET and [3H]etorphine respectively; n = number of independent experiments (number of experiments performed with tissues pooled from different subjects). Brain structures
Nucleus caudatus Putamen Globus pallidus (external part) Thalamus Hypothalamus Amygdala Hippocampus Frontal lobe cortex (Brodmann area 9) Cingulate Cortex (Brodmann area 24) Substantia nigra (pars compacta)
[3H] D A G O
[3H] D TL E T
[3H] Etorphine
n
Ka (nM)
Bmax (femtomol/mg)
n
Ka (nM)
B~ax (femtomol/mg)
n
Ka (nM)
6 7 (1)
4.68 ___0.89 4.44 __+1.38
63 + 31 74 + 44
5 7 (1)
1.63 + 0.26 1.70+ 0.38
35 + 22 32 + 18
8 (1) 7 (1)
0.274 + 0.095 0.176+ 0.081
132 + 49 109 + 51
4 (4) 3 (1) 3 (2) 4 (4) 4 (3)
3.80 _+0.37 5.84 + 1.85 3.77 + 0.03 5.12+1.39 3.68 _+ 1.31
18 + 3 42 + 35 54 + 28 76+41 21 + 6
5 (4) 3 (1) 4 (3) 5(5) 4 (3)
1.52 + 0.34 1.67+0.56 1.36 + 0.24
11 + 5 <5 <5 31+7 13 _+4
5 (4) 3 (1) 3 (2) 4(4) 4 (3)
0.200 + 0.096 0.166 + 0.053 0.199 + 0.027 0.186+0.035 0.236 + 0.083
46 + 23 51 + 39 64 + 28 152+72 68 + 9
5 (3)
5.57 + 2.30
89 + 76
5 (3)
1.55 + 0.50
41 _+ 19
5 (3)
0.260 + 0.195
213 + 84
4 (1)
3.64 + 0.35
43 __+24
4 (1)
1.17 + 0.10
24 + 12
4 (1)
0.229 + 0.040
116 + 49
1 (1)
4.1
4(4)
-
<5
3(3)
0.267+0.097
21.7+1.2
9
which exhibits about the same affinity for the 3 opioid binding types 17. These values are close to those previously d e t e r m i n e d using rat brain m e m b r a n e s 29,3°. The affinities of [ 3 H ] D A G O and [3H]DTLET for the sites defined as/~ and 6, respectively, were similar in all brain regions. The standard deviations of the raw data (S.D. E r a d ) 31 were approximately 10% for each agonist saturation plot. These S.D. E r a d were smaller than the standard deviations (S.D.) of the means of Bmax values m e a s u r e d from different h u m a n brains. Thus, the standard deviation of the p a r a m eters r e p o r t e d in Table I most likely reflects individual variability in the population studied, since the differences in binding capacity are not correlated with age, p r e - m o r t e m conditions or p o s t - m o r t e m delay. [3H]Etorphine capacities were greater than the sum of [ 3 H ] D A G O and [3H]DTLET capacities (Table I). Due to the lack of highly selective r radioligands, sites were quantified as the difference between [3H]etorphine and the sum of [ 3 H ] D A G O and [3H]DTLET binding capacities. On the other hand, in the presence of 100 nM D A G O and 100 nM D T L E T , [3H]ethylketocyclazocine, a second putative ligand for ~ sites, binds to a residual high affinity site (nucleus caudatus : KD = 0.762 nM, Bmax = 49
B,nax (femtomol/mg)
femtomol/mg; putamen: K D = 0.728 nM, Bmax = 53 femtomol/mg; frontal cortex: K D = 1.12 nM, Bmax = 53 femtomol/mg). Thus, the remaining binding sites of [3H]etorphine, corresponding to non-/~, non-6 opioid sites, exhibit a high affinity for [3H]ethylketocyclazocine in agreement with their definition as ~csites. [OH]Ethylketocyclazocine eliciting about the same affinity f o r p , 6 and r receptors 6, the relative p r o p o r tions of these binding sites were also estimated in nucleus caudatus and frontal cortex by means of simple sequential inhibition, as described by Pfeiffer et al. 2°. Binding of [3H]ethylketocyclazocine (2 nM) in these areas was inhibited by D A G O in a complex m a n n e r which could be analyzed as a competition involving three different binding sites. The p r o p o r t i o n of [3H]ethylketocyclazocine binding displaced by D A G O with a low affinity (IC50 = 10,000 nM) was 30% in the nucleus caudatus and 50% in the frontal cortex. The proportions, calculated from the binding capacities of [3H]etorphine, are 26% and 39% respectively (Table I). These suggest either that [3H]etorphine and [3H]ethylketocyclazocine do not bind to the same r sites 2, or that they have different interactions with # and 6 sites 6.
11 T A B L E II
Comparison of the binding capacities (B,,ax) of [3H] DA GO, [3HIDTL ET and [3H]etorphine in various regions of brains from normal and parkinsonian patients Values are expressed as femtomol per m g of m e m b r a n e protein + S.D. T h e binding was performed with 2 n M [3H]DAGO, 1 n M [3H]DTLET, * 2 n M [3H]ethylketocyclazocine in presence of D A G O (100 nM) and D T L E T (100 nM) or 2 n M [3H]etorphine. Bmax of [3H]DAGO and [3H]DTLET were calculated from the equation: Bmax = B X (1 + Kd/L); n I and n 2 are the n u m b e r s of i n d e p e n d e n t normal or parkinsonian subjects, respectively; *, 1 pool; **, 2 pools of two samples each.
Brain region
n,
Nucleus caudatus Putamen Globus pallidus (external part) Amygdala Frontal cortex Substantia nigra (pars compacta)
n2
14 14 20 12 14 21"
[3H]DAGO
14 14 17 11 14 18"*
[3H]DTLET
[3H]Etorphine
Control
Parkinson
Control
Parkinson
Control
Parkinson
110 40 16 99 107 17
107 39 18 100 97 22
35 19 9.5 30 41 -
34 21 7.7 31 35 -
166 73 28 151 164 44 21
161 68 26 186 158 45 18
+ + + + + +
47 15 12 31 38 10
Distribution of opioid binding sites in various regions of parkinsonian brains The distribution of/~ and 6 binding sites was studied in regions innervated by the nigrostriatal or meso-
+ + + + + +
26 25 12 44 31 10
+ + + + +
19 9 6.4 18 15
+ + + + +
23 11 3.5 20 15
+ 36 + 32 + 27 + 50 + 53 + 20 +__6*
+ + + + + + +
38 30 27 62 50 16 9*
corticolimbic dopaminergic neurons, which are affected by Parkinson's disease (Table II). The use of a different sampling and the need, in the first series of experiments, to pool several different
1 6K 75
EB~O m +
¥¥ ++ 4-44-4-
,Q 50
0
25
0
+
~-
+ __44-4-
+ 44-
4-
_
4-44-4-
4-+ 4-4-
~ 4-4-
4-44-4-
44-:171
4-44-+ 4-+ 4-44-4++ 4-4-
++ 4-4++ 4-44-44-4++ 4-+~
+-0.1
4-44-4-
++ ++ ++ 4-4+4-I-+ ++ 4-4-
4-+1 ++l 4-+1 ++l 4-+1 ++. ++l
+4++ ++ ++ 4-+ ++ 4-+
+4-:1
++,
+4-
4-4-
++l 4- +1 4-+1 4- .~.i
+44-4-
++ +4-
++
4-+
-4444~+
+4-
4-44. +
++ 4-+
++ 4-4-
++--e o "~ 4- O •
4- + ~ J + 4- i i'li"l ~tael +4-n''' 4- 4- l * l i
4-+__ 4-4- •
++ 4-4-
4-+ ++~
++o
•
+ +....
++.
+ + o~-~
++o
o--
++o,o,o,.,
+-,-[
++ onon
++ ++
"44.4. 4-
"1" 444-
4444-
4- + I I " ~ 4- + l l l l i [ = 4elL[ 4- ++ il ll il li il
4'4" 44-
+ 44+
4"t" 44- 00
4- "~ .4. "l"
i
44- • 4- 00 4" •
0O • 00 •
ee 00
4- "l" 4- 4- •
:
•
O •
4- 44- 4-
4- "t" 4- 4- •
cpcpcp
cpcpcp
NUCLEUS CAUDATU
P U T ~ E N S
t
4444-
Olin ilDI JilL le~el iJlilllll
4- 4- J i l l 4- 4- l i I I i 4- 44- 4-
!ilk
cpcpcp GLOBUS PALL I DUB (EXTERNAL PART)
__
+ 444-
",1
Olel OIOI OIOI OIoIOIOI Illl~
+ 444-
4- + I I l I I .~. , . ~ l i l i l
4- 4- IOl llll l
+ + oriel
+ + 4- 4-
"4" + l l l l l 4- + i unon ....
4- 4- O I O I
4- 4-
.,.,
cpcpcp ~ Y G D A L A
--
4-4-
4-4-
t' •
"
++"" cpcpcp
FRONTAL CORTEX
+" cpcpcp
SUBSTANTIA S I O]WI% (PARS COMPACTA)
Fig. 1. Distribution of opioid binding sites in various brain areas. T h e p, 6 and r sites are expressed as percent of total b o u n d which was measured with 2 n M [3H]etorphine (Table II) (C, control; P, parkinsonian).
12 brains for saturation experiments, very likely account for the differences among control values reported in Tables I and II. Whatever the region sampled and the ligand used, large variations in the measurements are observed from one control to another. Nevertheless, the large number of independent measurements carried out (n > 11) permitted a suitable statistical comparison between controls and brain of parkinsonians (Table II). No significant changes in receptor densities (P < 5%) were found in the different regions examined, even in areas in which marked differences were observed, i.e. the amygdala [3H]etorphine: + 23%), the globus pallidus, external part, [3H]DTLET: -19%) and the substantia nigra, pars compacta, [3H]DAGO: +29%) (Table II). In amygdala, additional experiments (number of samples: controls 11, parkinsonians 13) confirmed the absence of significant variations of ~ sites binding using either [~H]etorphine (control: 228 + 74; Parkinson: 235 + 47 femtomol/mg), or [3H]ethylketocyclazocine in the presence of 100 nM D A G O and 100 nM DTLET (control: 95 + 23, Parkinson: 96 + 17 femtomol/mg). The moderate changes in receptor densities in the globus pailidus (external part) and substantia nigra (pars compacta) of brains of parkinsonians are most likely due to the inaccuracy related to a low number of binding sites in these areas (Table I). DISCUSSION The regional distribution of opioid sites in human brains (Table If, Fig. 1) is in good agreement with previous reports 12, in particular for the proportions of p and 6 sites observed by analysis of competition curves of non-selective ligands 2°. The large number of samples allowed a statistically safe comparison of the distribution of the various opioid receptors between brains from controls and parkinsonian patients. Owing to the fact that dissection and storage procedures do not affect opioid receptor binding capacities 25, the inaccuracy of the data is most likely attributable to individual variability. This is indicated by the fact that the ratios of p, 6 and sites in brains of controls or parkinsonian subjects varied to a lesser extent than the total capacity of binding sites. The absence of modification of either opioid receptors densities (Table II) or ratios of p, 6, ~: sites in
brains of parkinsonian subjects (Fig. 1), using selective p and 6 radioligands, is at variance with the previously reported reduction of 40-50% of [3H]naloxone sites in the caudate nucleus of brains of parkinsonian patients 22"23. However, using [3H]Met-enkephalinamide as ligand, an unmodified density of opioid receptors has also been observed in the nucleus caudatus, contrasting with a 42% decrease of the binding sites capacity in the substantia nigra 14. These discrepancies could be due to the differences in the radioligands used, or to the great variations associated with the use of human material from autopsy. Moreover, in the substantia nigra, where the levels of opioid binding sites are low, it is most likely that a marked (40%) diminution of the binding capacity must be reached in order to be statistically significant. On the other hand, an increase of [3H]Leu- and [3H]Met-enkephalin binding was reported in the caudate nucleus, putamen, nucleus accumbens, limbic cortex and hippocampus of parkinsonian subjects 23, suggesting a denervation supersensitivity of a population of postsynaptic opioid receptors. Using selective and metabolically resistant enkephalin analogues, we found no significant increase in the binding capacities (Table II), particularly in putamen, pallidum and substantia nigra where Met- and Leuenkephalin levels are decreased, indicating a change in peptide transmission 26. Similarly, no up-regulation of D 2 dopamine receptors was evidenced by positron emission tomography in parkinsonians27; however, the increase in dopamine receptor density in Parkinson's disease could be progressive and relative to the extent of the lesion 3. Using [3H]Leu-enkephalin or [3H]naloxone as ligands, a decrease in rat striatal opioid receptors has been observed after 6-OHDA-induced degeneration of the nigrostriatal dopaminergic pathway 21, while an increase of Da dopamine receptors has been shown 24. Using this animal model of the disease, an 80% decrease of tyrosine-hydroxylase activity in the striaturn was associated with a concomitant decrease of 30% of opioid binding sites capacity using either binding experiments on homogenates (unpublished observations) or autoradiography 28. Our data suggest that the neuronal localization of opioid receptors in striatum may be different in man and rat, or that as far as the degeneration process of
13 dopaminergic neurons is concerned, the neurochemical changes, subsequent to acute destruction of the nigrostriatal dopamine neurons in animal or to the chronic pathological process in Parkinson's disease, differ fundamentally. For example, natural and druginduced compensatory mechanisms for any possible opioid receptor decrease might develop in man after degeneration of the nigrostriatal dopaminergic pathway. It is likely that these features account for the discrepancies among data reported on brains of parkinsonians. It is noteworthy that a significant decrease of [3H]-naloxone binding, in the untreated parkinsonian caudate nucleus, has not been observed in patients treated with L - D O P A 23, suggesting that LD O P A therapy (the conventional treatment of Parkinson's disease) could stabilize the level of function-
REFERENCES 1 Agid, Y, Javoy-Agid, F. and Ruberg, M., The neurobiochemistry of Parkinson's disease. In C.D. Marsden and S, Fahn (Eds.), Movement Disorders-2, BIMR Neurology, Butterworths, 1986, in press. 2 Attali, B., Gouarderes, C., Mazarguil, H., Audigier, Y. and Cros, J. Evidence of multiple kappa binding sites by use of opioid peptides in the guinea pig lumbo-sacral cord, Neuropeptides, 3 (1982) 53-64. 3 Bokobza, B., Ruberg, M., Scanon, B., Javoy-Agid, F. and Agid, Y., [3H]Spiperone binding, dopamine and HVA concentrations in Parkinson's disease and supranuclear palsy, Eur. J. Pharmacol., 99 (1984) 167-175. 4 Bonnet, K.A., Groth, J., Gioannini, T., Cortes, M. and Simon, E.J., Opiate receptor heterogeneity in human brain regions, Brain Research, 221 (1981) 437-440. 5 Chesselet, M.-F., Cheramy, A., Reisine, T.D., Lubetzki, C., Glowinsky, J., Fournie-Zaluski, M.C. and Roques, B.P., Effects of various opiates including specific 6 and agonists on dopamine release from nigrostriatal dopaminergic neurons in vitro in the rat and in vivo in the cat, Life Sci., 31 (1982) 2291-2294. 6 Delay-Goyet, P., Roques, B.P. and Zajac, J.-M., Binding characteristics of non-selective opiates towards/~ and 6 receptor types, Life Sci., submitted. 7 Delay-Goyet, P., Zajac, J.-M., Rigaudy, P., Foucaud, B. and Roques, B.P., Comparative binding properties of linear and cyclic cS-selective enkephalin analogues: [3H]-[DThr2,LeuS]enkephalyl-Thr6 and [3H]-[D-P~n ,-7~nS]enkephalin, FEBS Lett. 183 (1985) 439-443. 8 Gaspar, P., Berger,B., Gay, M., Hamon, M., Cesselin, F., Vigny, A., Javoy-Agid, F. and Agid, Y., Tyrosine hydroxylase and methionine enkephalin in the human mesencephalon: immunohistochemical localization and relationships, J. Neurol. Sci., 58 (1983) 247-267. 9 Handa, B.K., Lane, A.C., Lord, J.A.H., Morgan, B.A., Rance, M.J. and Smith, C.F.C., Analogues of fl-LPH61_64 possessing selective agonist activity at/~-opiate receptors, Eur. J. Pharmacol., 70 (1981) 531-540.
al opioid receptors in the brain. However, the low number of L - D O P A untreated patients in our study does not allow this hypothesis to be confirmed. ACKNOWLEDGEMENTS Drs. Hauw, Lhermitte, Castaigne, Laplane (H6pital de la Salp~tri~re, Paris) and Drs. Beck, Bouchon, Moulias, Piette (H6pital Charles Foix, Ivry-surSeine) are gratefully acknowledged for providing human material. This work was supported by funds from the Centre National de la Recherche Scientifique, the Institu National de la Recherche M6dicale and the Universit6 Ren6 Descartes. We thank Dr. A. Beaumont for critical reading of the manuscript and A. Bouju for typing it.
10 Hiller, J.M., Pearson, J. and Simon, E.J., Distribution of stereospecific binding of the potent narcotic analgesic etorphine in the human brain: predominance in the limbic system, Res. Commun. Chem. Pathol. Pharmacol., 6 (1973) 1052-1062. 11 Hornykewicz, O., Dopamine ([3H]hydroxytyramine) and brain function, Pharmacol. Rev., 18 (1966) 925-964. 12 Itzhak, Y., Bonnet, K.A,, Groth, J., Hiller, J.M. and Simon, E.J., Multiple opiate binding sites in human brain regions: evidence for x and ty sites, Life Sci., 31 (1982) 1363-1366. 13 Kuhar, M.J., Pert, C.B. and Snyder, S.H., Regional distribution of opiate receptor binding in monkey and human brain, Nature (London), 245 (1973) 447-450. 14 Llorens-Cortes, C., Javoy-Agid, F., Agid, Y., Taquet, H. and Schwartz, J.-C., Enkephalinergic markers in substantia nigra and caudate nucleus from parkinsonian subjects, J. Neurochem., 3 (1984) 874-877. 15 Llorens, C., Malfroy, B., Schwartz, J.C., Gacel, G., Roques, B.P., Roy, J., Morgat, J.-L., Javoy-Agid, F. and Agid, Y., Enkephalin dipeptidyl carboxypeptidase (enkephalinase) activity: selective radioassay, properties and regional distribution in human brain, J. Neurochem., 39 (1982) 1081-1089. 16 Maurer, R., Cortes, R., Probst, A. and Palacios, J.M., Multiple opiate receptor in human brain: an autoradiographic investigation, Life Sci., 33 Suppl. I (1983) 231-234. 17 Gillan, M.G.C. and Kosterlitz, H.W., Spectrum of the p-, 6- and x-binding sites in homogenates of rat brain, Br. J. Pharmacol., 77 (1982) 461-469. 18 Paterson, S.J., Robson, L.E. and Kosterlitz, H.W., Opioid receptors. In S. Udenfriend and J. Meienhofer (Eds.), The Peptides: Analysis, Synthesis, Biology, Vol. 6, Academic, London, 1984, pp. 147-189. 19 Petit, F., Hamon, M., Fournie-Zaluski, M.-C., Roques, B.P. and Glowinsky, J., Further evidence for a role of 6opiate receptors in the presynaptic regulation of newly synthesized dopamine release, Eur. J. Pharmacol., 126 (1986) 1-9. 20 Pfeiffer, A., Pasi, A., Mehraein, P. and Herz, A., Opiate
14
21 22 23
24
25
26
receptor binding sites in human brain, Brain Research, 248 (1982) 87-96. Pollard, H., Llorens-Cortes, C. and Schwartz, J.-C., Enkephalin receptors on dopaminergic neurons in rat striatum, Nature (London), 268 (1977) 745-747. Reisine, T.D., Rossor, M., Spokes, E., lversen, L.L. and Yamamura, H.I., Alterations in brain opiate receptors in Parkinson's disease, Brain Research, 173 (1979) 378-382. Rinne, U.K., Rinne, J.K., Rinne, J.O., Laakso, K., Tenovuo, O., Lonnberg, P. and Koskinen, V., Brain enkephalin receptors in Parkinson's disease, J. Neural Transm., Suppl. 19 (1983) 163-171. Savasta, M., Dubois, A., Manier, M., Mouchet, P., Fourtier, O. and Scatton, B., Autoradiographie quantitative des sites r6cepteurs dopaminergiques D~ et D2 dans le striatum de rat: adaptation post-synaptique ~ la d6nervation nigrostri6e, Colloq. Natl. Neurosci., Bordeaux (1986). Schwartz, J.-C., Pollard, H., Martres, M.P., LlorensCortes, C., Javoy-Agid, F., Taquet, H., Agid, Y., Bouthenet, M.L. and Sales, N., Neurotransrnitter Markers in Human Area Postrema, Springer, Oxford, 1984. Taquet, H., Javoy-Agid, F., Hamon, M., Legrand, J.C., Agid, Y. and Cesselin, F., Parkinson's disease affects differently (MetS)-and (LeuS)-enkephalin in the human brain,
Brain Research, 280 (1983) 379-382. 27 Wagner, H.N., Wong, D.F., Dannals, R.F., Kuhar, M.J., Links, J,M., Frost, J.J., Folstein, M. and Folstein, S., Imaging Dopamine Receptors in the Human Brain, IVth World Congress of Biological Psychiatry, Philadelphia, 1985. 28 Waksman, G., Hamel, E., Delay-Goyet, P., Fournie-Zaluski, M.-C. and Roques, B.P., Radioautographic Visualization of Enkephalinase in Rat Brain with [SH]HACBOGly: Comparison with the Distribution of Opioid kt and 6 Receptors, International Narcotics Research Conference, North Falmouth, 1985. 29 Zajac, J.-M., Gacel, G., Petit, F., Dodey, P., Rossignol, P. and Roques, B.P., Deltakephalin, Tyr-D-Thr-Gly-PheLeu-Thr: a new highly potent and fully specific agonist for opiate 6-receptors, Biochem. Biophys. Res. Commun., 11 (1983) 390-397. 30 Zajac, J.-M. and Roques, B.P., Differences in binding properties of p and 6 opioid receptor subtypes from rat brain: kinetic analysis and effects of ions and nucleotides, J. Neurochem. , 44 (1985) 1605-1614. 31 Zivin, J.A, and Waud, D.R., How to analyze binding, enzyme and uptake data: the simplest case, a single phase, Life Sci., 30 (1982) 1407-1422.