Proc. Natl. Acad. Sci. USA Vol. 76, No. 11, pp. 5455-5459, November 1979
Biochemistry
Properties and localization of f-endorphin receptor in rat brain (opiate receptor/regional distribution/cation effect/enzymatic treatment/relative potency)
PING-YEE LAW, HORACE H. LOH, AND CHOH HAO LI* Langley Porter Neuropsychiatric Institute, Department of Pharmacology and *Hormone Research Laboratory, University of California, San Francisco, California 94143
Contributed by Choh Hao Li, July 6, 1979
Stereospecific high-affinity binding sites for brain homogenate. Scatchard analysis of the binding data revealed binding sites with Kd values of 0.81 and 6.8 nM and density of 120 and 240 fmol/mg of protein. Distribution of {h13H1endorphin binding in various brain regions parallels that of opiate receptor:striatum > thalamus > amyg ala > hypothalamus, septum > cortex > midbrain, brainstem. Similar to their effect on 3H-labeled agonist binding, Na+ and other monovalent cations, GTP, trypsin, chymotrypsin, phospholipase A2, and N-ethylmaleimide all inhibited the specific binding of In contrast to their action on alkaloid and fth[3HJendorphin. enkephalin binding, Ca2+, Mg2+, and Mn2+ also inhibited 4h4[3Hlendorphin binding. These data suggest a difference between Ph-endorphin and alkaloid/enkephalin binding sites. ABSTRACT
#h-[3HJendorphin could be demonstrated in the P2 pellet of rat
Since the discovery of its opioid activities, the pharmacological properties of fl-endorphin have been well characterized. It is accepted that the potent analgesic activity of ,B-endorphin (1, 2) results from the interaction of the peptide with the opiate receptors present in the brain. However, with the exception of some displacement studies (3, 4), the nature of ,B-endorphin binding to the putative opiate receptor has not yet been elucidated because of a lack of availability of radioactive f3-endorphin. This was resolved by the successful tritiation and iodination of the peptide reported by Houghten and Li (5) and Hazum et al. (6), respectively. In both cases the biological activities of f3-endorphin were retained. Furthermore, using the iodinated peptide, Hazum et al. (6) found some dissimilarities between alkaloid or enkephalin and the f3-endorphin binding to the receptor. In order to investigate the hypothesis of heterogeneous opiate receptor, interaction between f3-endorphin and the opiate receptor was carried out by using 3-[3HTyr27]endorphin. In a preliminary note (7), we described the ability of the radioactive peptide to interact with the crude synaptosomal fraction of rat brain. We report here the characterization of this interaction.
MATERIALS AND METHODS [Met]- and [Leu]enkephalin were purchased from Biochem Laboratory (Marina del Reyes, CA). f-Lipotropin (f3-LPH) was isolated from sheep pituitary (8). f3-[3H]Endorphin and 3-endorphin and other related peptides were synthesized as described (5, 9). Morphine was from Mallinckrodt; levorphanol and dextrorphan were from Hoffmann-LaRoche; naloxone was from Endo Laboratory (Garden City, NJ); trypsin (bovine pancreas) type III, chymotrypsin (bovine pancreas), phospholipase A2 (bee venom), o-phenanthroline, N-ethylmaleimide, bovine serum albumin fraction V, and Hepes were from Sigma; choline chloride was supplied by Baker. 3h-[3H]Endorphin binding was measured as reported (7). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Crude synaptosomal preparations, P2 pellets (22,000 X g for 20 min), were obtained from the excised brain (minus cerebellum) of male adult Sprague-Dawley rats (200-240 g) as described (7). The final lysed pellets were resuspended (1.5 g/40 ml) in 25 mM Hepes/0.1% albumin, pH 7.7 (buffer A). These membrane preparations were kept at -65°C until used. Specific binding of 43h-[3H]endorphin was measured at 23-250C for 1 hr in 1.0 ml of buffer A containing 2 mM 1,10-o-phenanthroline (buffer B). Membrane preparation (0.5 ml; 0.4-0.6 mg of protein) was added to the reaction vessel (1.5-ml polypropylene centrifuge tube) 5 min prior to the initiation of the binding assays. Assays were started by the addition of appropriate aliquots of 13h-[3H]endorphin. The tubes were then capped and vortexed immediately. After 1 hr of incubation, the reaction mixtures were removed with a pasteur pipette and applied to Whatman G glass-fiber GF-C filter discs (2.4 cm) prewetted with washing buffer [buffer A plus 0.01% Triton X-100 plus 100 mM choline chloride at pH 7.7 (buffer C)]. The filters were then washed three times with 5 ml of buffer C. Radioactivity on each filter was determined by liquid scintillation counting in a Beckman LS150 after the filters were allowed to stand in 8.0 ml of Scintiverse (Fisher) overnight. Specific binding of 3h-[3H]endorphin was defined as the difference in the average radioactivity bound to sets of triplicate samples in the presence of 0.1 ,uM dextrorphan or 10,M levorphanol plus 0.1 MuM dextrorphan. RESULTS Affinity of 4h[3H]Endorphin Binding. In an earlier study (7), we demonstrated that, by washing the filters with buffer C, adsorption of flh-[3H]endorphin by the glass fiber filters was greatly diminished. The adsorption by the filters was decreased from 20-30% of the total applied radioactivity when washed with buffer A to 1% when washed with buffer C. It was also demonstrated by washing with buffer C that the high-affinity binding of 3H-labeled [D-Ala2, D-Leu5]enkephalin was not altered. Thus, by using such a washing procedure with the rapid filtration method, we were able to demonstrate a proteindependent and temperature-dependent high-affinity association of the 13h-[3H]endorphin with the brain membrane (7). Moreover, this high-affinity binding can be demonstrated to be stereospecific (7). The stereoactive isomer levorphanol or (-)naloxone was at least 1000-fold more potent in displacing 3h-[3H]endorphin binding than was the inactive isomer dextrorphan or (+)naloxone, respectively. When the binding of this radioactive ligand was determined at various concentrations and the binding data were analyzed by the method of Scatchard (10), a two-component or single site with negative cooperativity typical of the opiate binding was observed (Fig. 1). The Kd values (with 95% confidence limits) determined from linear regression analysis of the two binding Abbreviation: LPH, lipotropin.
5455
Biochemistry:
5456
Law et al.
Proc.
7.Or
6.0[ Kd = 0.81 nM 5.0O 0 0
x 4.0 a)
%0.
Ic
3.0 0
0
m
2.0
0
°0
1.0
I-
50
I- \ 100
Kd = 6.8 nM 0
\
200 250 150 Bound, fmol/mg FIG. 1. Scatchard analysis of specific (3h-[3H]endorphin binding to P2 membranes prepared from whole rat brain minus cerebellum. -
---
sites were 0.81 nM (0.80-0.81 nM) and 6.8 nM (6.6-7.0 nM), respectively. The number of sites at the saturation level of binding were determined to be 0.12 and 0.24 pmol mg of protein for the high- and low-affinity binding sites, respectively. The number of binding sites thus obtained corresponds excellently with the number of sites for [3H]etorphine (0.15-0.2
pmol/mg of protein) as reported by Simon et al. (11). Although the Kd of the low-affinity binding site is significantly different from that obtained with 3H-labeled alkaloid or enkephalin (20-30 nM) (12, 13), the similarity in the values suggests that flh-endorphin is interacting with the previously identified opiate receptor.
ph[3H1Endorphin Binding at Various Brain Regions. If 1B4-[3H]endorphin is indeed associated with the opiate re-
the
ceptor previously characterized with radioactive alkaloid, the
distribution of the 4h-[3H]endorphin binding activities should coincide with the distribution of the putative opiate receptor. When the brains of 14 rats were dissected into various regions and the crude synaptosomal membranes were prepared from
Natl. Acad. Sci.
USA
76 (1979)
the pooled tissues, the binding of f3h-[3H]endorphin to the membranes from different regions varied significantly. As in the case of 3H-labeled alkaloid binding (14), the 13h-[3H]endorphin binding was concentrated at the thalamus, hypothalamus, and midbrain region (Table 1). No specific binding was observed in the cerebellum. Scatchard analysis revealed that two-component binding was present in all brain areas tested, except in midbrain (the concentration of 13h-[3H]endorphin used was probably not high enough to permit detection of the second binding site in the midbrain). When the Kd values of endorphin binding sites were determined by linear regression analysis of the Scatchard plots, the affinities of various brain regions for ,B-endorphin differed significantly. Affinity for 13h-[3H]endorphin was highest in the septal region and lowest in the thalamus. The distribution of 13h-[3H]endorphin binding sites did not coincide with the level of jB-endorphin in various brain areas as determined by radioimmunoassay (15). Although f3-endorphin was not detected in the striatum or cortical area (15), a large number of ,3h-[3H]endorphin binding sites were found in these two areas. In these two areas, f3h-[3Hjendorphin conceivably could be interacting with the opiate receptor that has enkephalin as its natural ligand. It appears that 13h-[3Hlendorphin also has affinity for the "enkephalin receptor" as proposed by Hazum et al. (6). Inhibition of ,[h43HJEndorphin Binding by Various Agents. It has been reported that opiate agonist binding to the brain membrane can be attenuated by prior treatment of the membranes with degradative enzymes (17). Both trypsin and chymotrypsin were potent inhibitors of 134-[3H]endorphin binding (Fig. 2). Phospholipase A2 in the presence of 5 mM CaCl2 exhibited a concentration-dependent inhibition of fth-[3H]endorphin binding. This action of phospholipase was abolished by the addition of 2 mM ethylene glycol bis(,B-aminoethylether)-N,N,N',N'-tetraacetic acid to the incubation mixtures.
Similar to 3H-labeled agonist binding (18, 19), fOh-[3H]endorphin binding to the brain membrane was also inhibited by N-ethylmaleimide. When the membranes were incubated with 0.5 mM N-ethylmaleimide for different durations, a timedependent inhibition of 13h-[3H]endorphin binding could be observed (data not shown). The presence of 100 mM Na+ in the
Table 1. Regional distribution of f3-endorphin and enkephalin and of 1h-[3H]endorphin binding activities
Ilh-[3H]Endorphin binding Brain region
Hypothalamus
Enkephalin* 120 ± 7
f3-Endorphin* 490 + 30
Thalamus
36 ± 5
329 ± 19
Septum
85 ± 7
234 ± 34
Midbrain
32 + 1
207 ± 15
sites, fmol/mg
Kd, nM 6.6
0.30
(0.26-0.36)
(4.7-11.1)
0.68 (0.63-0.75) 0.19 (0.13-0.36)
12.6 (12.5-12.8) 3.6 (3.3-4.0)
111.4
386.9
150.6
591.3
138.5
393.4 185.5
10.3
(9.9-10.8) Brainstem
Striatum
30 + 4 112 + 11
179 ± 5
None
0.46
4.4
(0.41-0.54)
(3.5-5.1)
0.48
(0.477-0.48) Cortex
15 ± 2
Cerebellum
5± 1
Amygdala
55
8.2
71.5
164.0
270.1
690.0
110.1
256.1
(5.4-16.4)
None
0.36
3.2
(2.7-3.8)
None
(0.34-0.37) ND
-
0.51
10.4
ND 144.7
559.4
(9.5-11.6) (0.48-0.54) ND = not detectable. The values in parentheses represent the 95% confidence limits. * The mean (±SEM) levels of enkephalin (as enkephalin units/g of tissue) and fl-endorphin (as ng/g of tissue) are from Rossier et al. (15) and Simantov et al. (16).
Biochemistry:
Proc. Natl. Acad. Sci. USA 76 (1979)
Law et al.
5457
lOOr-
1 00r
0
80k c 0
80
*- 60
C
*c 40
2 60
.1E0
0
c
20k_
0 ._l
-D 40 l_ C
gg/ml Chymotrypsin
20
10Or
80[C
0
,FJ
60k-
-c ._
DI
40[20k100 30 10 Phospholipase A2, ng/ml
FIG. 2. Effect of various concentrations of trypsin, chymotrypsin, and phospholipase A2 on /,31-[3H]endorphin binding. Membranes devoid of buffer B were resuspended in 10 mM Hepes buffer at pH 7.5. Treatments with enzymes were carried out at room temperature for 30 min in a final volume of 5 ml. The actions of trypsin and chymotrypsin were terminated by addition of 0.25 ml of 40 mM 1,10-ophenanthroline; the action of phospholipase A2 was terminated by 0.25 ml of 100 mM ethylene glycol bis(f3-aminoethyl ether)-NNN',N'-tetraacetic acid. The 22,000 X g (20 min) pellets were resuspended in buffer B and specific binding of A3h-PH]endorphin (1.2 nM) was then determined. Incubations of the membranes with various concentrations of phospholipase A2 were carried out in the presence of 5 mM CaCl2 (3) or 2 mM ethylene glycol bis(f3-aminoethyl ether)N,N,N',N'-tetraacetic acid (-).
modulated by the presence of guanine nucleotide (20,21). The presence of GTP, at concentrations as low as 1 MM, inhibited the specific binding of 13h-[3Hlendorphin (Table 2). In this respect, 3B4-[3H]endorphin binding is more sensitive to GTP than is 3H-labeled alkaloid or [3H]enkephalin binding. [3H]Enkephalin binding to the membrane of NG108-15 cells (21) and to the rat brain (20) was slightly inhibited by 1 gM GTP. Effects of Cations on fth[3HJEndorphin Binding. The opiate agonist binding to brain membrane has been reported to be inhibited greatly by the presence of Na+ (22).
3h-[3H]Endorphin binding to the brain membranes was similarly affected by Na+ (Fig. 3). As in the case of [3H]dihydromorphine binding (22), 1h-[3H]endorphin binding to the membrane was almost completely abolished at 100 mM Na+. This inhibitory action was not limited to Na+; 10 mM LiCl, 100 [CaX2 80
1X
None Cations (10 mM): Na+ K+ Li+ Cs+ NH4+ GTP: 1 yM
56.7
100
28.6 60.3 34.4 41.5 35.3
50.4 106.4 60.7 73.2 62.2
26.8 27.6 12.8 15.5
47.3 48.7 22.6 27.3
% control
1o-
1X
2X
5X
10-4
100 [Mg2+] 80I__ 60_40 20-
1X 10-6
100
5X
X 2X 1X 2X 1X
i0
10-4
n-
80-
60-
The specific binding of /lh-[3H]endorphin (1.2 nM) to 0.6 mg of P2 lysate was determined in the presence and absence of the monovalent cations at 10 mM. All incubations were carried out at room temper-
ature (250C) for 1 hr.
2X
1X
-
fmol/mg
1 mM
2X
106
c
Agent
100,OM
li-
40
Table 2. Effect of monovalent cations and GTP on 13hH]endorphin specific binding
Specific binding,
r
60
incubation mixture did not attenuate the inhibition as in the case of antagonist binding (19). Thus, the response of 13h-[3H]endorphin binding to N-ethylmaleimide treatment is characteristic of an opiate agonist. Recent studies have indicated that the opiate binding can be
10 AM
100
50
Na+, mM flh-[3H]endorphin (1.2 nM) binding of specific Inhibition FIG. 3. by various concentrations of NaCl.
40
20
FIG. 4. Inhibition of specific 13h-[3H]endorphin (1.2 nM) binding by different concentrations of CaCl2, MgCl2, and MnCl2 in the incubation mixture.
Proc. Natl. Acad Sci. USA 76 (1979) 54585 Biochemistry: Law et al.
CSC1, and NH4Cl all had similar inhibitory effects (Table 2) but 10 mM KCI did not. The action of these cations on 3h-[3H]endorphin binding is similar to this action on [3H]dihydromorphine binding (22). 13h-[3H]Endorphin binding to the membranes was inhibited by the divalent cations Ca 2, Mg+2, and Mn+2 at 10 ,uM (Fig. 4). In the case of Mn 2, inhibition of binding was observed at 1 ,uM. The inhibitory activity of these cations on flh-[3H]endorphin binding was in direct contrast to that observed with [3H]dihydromorphine binding (23), cation concentrations >100 mM being required. Moreover, Pasternak et al. (23) reported that the inhibitory effect of 100 mM Na+ on 3H-labeled agonist binding could be reversed by the presence of 1 mM Mn+2. This was not the case with h-[3H]endorphin; in fact, 1 mM Mn+2 abolished the 3h-[3H]endorphin binding completely. In this respect the binding of 13h-[3H]endorphin to the opiate receptor is different from that of other radioactive opioid ligands. Displacement of PBO3H]Endorphin by Various Alkaloids and Peptides. 3h-[3H]Endorphin bound to the brain membrane could be displaced by various alkaloids and peptides (Fig. 5). When the concentration (1C50 value) of each of these compounds required to displace 50% of f3-[3H]-endorphin specifically bound was compared to that of fl-endorphin, the order of relative potencies among these compounds was different depending on the tritiated ligand used in the binding assay (Table 3). Enkephalin was more potent than f3-endorphin in displacing [3H]enkephalin and alkaloids such as diprenorphine and levorphanol were more potent than f3-endorphin in displacing [3H]dihydromorphine. However, f3-endorphin had the greatest potency in displacing h-[3H]endorphin. The order of potencies of various alkaloids in displacing fh-[3H]endorphin was similar to the order in displacing 3H-labeled alkaloid or
enkephalin. There appeared to be a secondary recognition site other than the enkephalin pentapeptide sequence for the 3h-[3H]endorphin binding. This was concluded from the observations that 100
9 --
80 60
Met']Enkephalin
20 :t
o;
.0
10-10
.c ._
10-9
10-7 Peptide, M
10-8
106
I{
1 0-5
E E 0
1100
0-R
Diprenorphine
80
60
A
2° |
Levorphanol-/ A
40-
1Naloxane N~aloxane Morphine
/ 0
/
/
20 0
n
~O-.O
0
lo-1°
10-9
lo-,A
10-7
10-6
/3-Endorphin a-Endorphin [Met5]Enkephalin [Leu5]Enkephalin
1.0 0.17 6.6
1.0 0.03 0.32
1 0.021 0.019
3.1
0.17
0.022
f3-LPH f3-LPH (66-91)
0.006 0.002 2.0 4.30 0.60 Cyclazocine Diprenorphine 1.09 4.30 0.52 0.07 1.30 0.023 Morphine 0.25 0.54 0.016 (-)Naloxone
Biochemistry
Properties and localization of f-endorphin receptor in rat brain (opiate receptor/regional distribution/cation effect/enzymatic treatment/relative potency)
PING-YEE LAW, HORACE H. LOH, AND CHOH HAO LI* Langley Porter Neuropsychiatric Institute, Department of Pharmacology and *Hormone Research Laboratory, University of California, San Francisco, California 94143
Contributed by Choh Hao Li, July 6, 1979
Stereospecific high-affinity binding sites for brain homogenate. Scatchard analysis of the binding data revealed binding sites with Kd values of 0.81 and 6.8 nM and density of 120 and 240 fmol/mg of protein. Distribution of {h13H1endorphin binding in various brain regions parallels that of opiate receptor:striatum > thalamus > amyg ala > hypothalamus, septum > cortex > midbrain, brainstem. Similar to their effect on 3H-labeled agonist binding, Na+ and other monovalent cations, GTP, trypsin, chymotrypsin, phospholipase A2, and N-ethylmaleimide all inhibited the specific binding of In contrast to their action on alkaloid and fth[3HJendorphin. enkephalin binding, Ca2+, Mg2+, and Mn2+ also inhibited 4h4[3Hlendorphin binding. These data suggest a difference between Ph-endorphin and alkaloid/enkephalin binding sites. ABSTRACT
#h-[3HJendorphin could be demonstrated in the P2 pellet of rat
Since the discovery of its opioid activities, the pharmacological properties of fl-endorphin have been well characterized. It is accepted that the potent analgesic activity of ,B-endorphin (1, 2) results from the interaction of the peptide with the opiate receptors present in the brain. However, with the exception of some displacement studies (3, 4), the nature of ,B-endorphin binding to the putative opiate receptor has not yet been elucidated because of a lack of availability of radioactive f3-endorphin. This was resolved by the successful tritiation and iodination of the peptide reported by Houghten and Li (5) and Hazum et al. (6), respectively. In both cases the biological activities of f3-endorphin were retained. Furthermore, using the iodinated peptide, Hazum et al. (6) found some dissimilarities between alkaloid or enkephalin and the f3-endorphin binding to the receptor. In order to investigate the hypothesis of heterogeneous opiate receptor, interaction between f3-endorphin and the opiate receptor was carried out by using 3-[3HTyr27]endorphin. In a preliminary note (7), we described the ability of the radioactive peptide to interact with the crude synaptosomal fraction of rat brain. We report here the characterization of this interaction.
MATERIALS AND METHODS [Met]- and [Leu]enkephalin were purchased from Biochem Laboratory (Marina del Reyes, CA). f-Lipotropin (f3-LPH) was isolated from sheep pituitary (8). f3-[3H]Endorphin and 3-endorphin and other related peptides were synthesized as described (5, 9). Morphine was from Mallinckrodt; levorphanol and dextrorphan were from Hoffmann-LaRoche; naloxone was from Endo Laboratory (Garden City, NJ); trypsin (bovine pancreas) type III, chymotrypsin (bovine pancreas), phospholipase A2 (bee venom), o-phenanthroline, N-ethylmaleimide, bovine serum albumin fraction V, and Hepes were from Sigma; choline chloride was supplied by Baker. 3h-[3H]Endorphin binding was measured as reported (7). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Crude synaptosomal preparations, P2 pellets (22,000 X g for 20 min), were obtained from the excised brain (minus cerebellum) of male adult Sprague-Dawley rats (200-240 g) as described (7). The final lysed pellets were resuspended (1.5 g/40 ml) in 25 mM Hepes/0.1% albumin, pH 7.7 (buffer A). These membrane preparations were kept at -65°C until used. Specific binding of 43h-[3H]endorphin was measured at 23-250C for 1 hr in 1.0 ml of buffer A containing 2 mM 1,10-o-phenanthroline (buffer B). Membrane preparation (0.5 ml; 0.4-0.6 mg of protein) was added to the reaction vessel (1.5-ml polypropylene centrifuge tube) 5 min prior to the initiation of the binding assays. Assays were started by the addition of appropriate aliquots of 13h-[3H]endorphin. The tubes were then capped and vortexed immediately. After 1 hr of incubation, the reaction mixtures were removed with a pasteur pipette and applied to Whatman G glass-fiber GF-C filter discs (2.4 cm) prewetted with washing buffer [buffer A plus 0.01% Triton X-100 plus 100 mM choline chloride at pH 7.7 (buffer C)]. The filters were then washed three times with 5 ml of buffer C. Radioactivity on each filter was determined by liquid scintillation counting in a Beckman LS150 after the filters were allowed to stand in 8.0 ml of Scintiverse (Fisher) overnight. Specific binding of 3h-[3H]endorphin was defined as the difference in the average radioactivity bound to sets of triplicate samples in the presence of 0.1 ,uM dextrorphan or 10,M levorphanol plus 0.1 MuM dextrorphan. RESULTS Affinity of 4h[3H]Endorphin Binding. In an earlier study (7), we demonstrated that, by washing the filters with buffer C, adsorption of flh-[3H]endorphin by the glass fiber filters was greatly diminished. The adsorption by the filters was decreased from 20-30% of the total applied radioactivity when washed with buffer A to 1% when washed with buffer C. It was also demonstrated by washing with buffer C that the high-affinity binding of 3H-labeled [D-Ala2, D-Leu5]enkephalin was not altered. Thus, by using such a washing procedure with the rapid filtration method, we were able to demonstrate a proteindependent and temperature-dependent high-affinity association of the 13h-[3H]endorphin with the brain membrane (7). Moreover, this high-affinity binding can be demonstrated to be stereospecific (7). The stereoactive isomer levorphanol or (-)naloxone was at least 1000-fold more potent in displacing 3h-[3H]endorphin binding than was the inactive isomer dextrorphan or (+)naloxone, respectively. When the binding of this radioactive ligand was determined at various concentrations and the binding data were analyzed by the method of Scatchard (10), a two-component or single site with negative cooperativity typical of the opiate binding was observed (Fig. 1). The Kd values (with 95% confidence limits) determined from linear regression analysis of the two binding Abbreviation: LPH, lipotropin.
5455
Biochemistry:
5456
Law et al.
Proc.
7.Or
6.0[ Kd = 0.81 nM 5.0O 0 0
x 4.0 a)
%0.
Ic
3.0 0
0
m
2.0
0
°0
1.0
I-
50
I- \ 100
Kd = 6.8 nM 0
\
200 250 150 Bound, fmol/mg FIG. 1. Scatchard analysis of specific (3h-[3H]endorphin binding to P2 membranes prepared from whole rat brain minus cerebellum. -
---
sites were 0.81 nM (0.80-0.81 nM) and 6.8 nM (6.6-7.0 nM), respectively. The number of sites at the saturation level of binding were determined to be 0.12 and 0.24 pmol mg of protein for the high- and low-affinity binding sites, respectively. The number of binding sites thus obtained corresponds excellently with the number of sites for [3H]etorphine (0.15-0.2
pmol/mg of protein) as reported by Simon et al. (11). Although the Kd of the low-affinity binding site is significantly different from that obtained with 3H-labeled alkaloid or enkephalin (20-30 nM) (12, 13), the similarity in the values suggests that flh-endorphin is interacting with the previously identified opiate receptor.
ph[3H1Endorphin Binding at Various Brain Regions. If 1B4-[3H]endorphin is indeed associated with the opiate re-
the
ceptor previously characterized with radioactive alkaloid, the
distribution of the 4h-[3H]endorphin binding activities should coincide with the distribution of the putative opiate receptor. When the brains of 14 rats were dissected into various regions and the crude synaptosomal membranes were prepared from
Natl. Acad. Sci.
USA
76 (1979)
the pooled tissues, the binding of f3h-[3H]endorphin to the membranes from different regions varied significantly. As in the case of 3H-labeled alkaloid binding (14), the 13h-[3H]endorphin binding was concentrated at the thalamus, hypothalamus, and midbrain region (Table 1). No specific binding was observed in the cerebellum. Scatchard analysis revealed that two-component binding was present in all brain areas tested, except in midbrain (the concentration of 13h-[3H]endorphin used was probably not high enough to permit detection of the second binding site in the midbrain). When the Kd values of endorphin binding sites were determined by linear regression analysis of the Scatchard plots, the affinities of various brain regions for ,B-endorphin differed significantly. Affinity for 13h-[3H]endorphin was highest in the septal region and lowest in the thalamus. The distribution of 13h-[3H]endorphin binding sites did not coincide with the level of jB-endorphin in various brain areas as determined by radioimmunoassay (15). Although f3-endorphin was not detected in the striatum or cortical area (15), a large number of ,3h-[3H]endorphin binding sites were found in these two areas. In these two areas, f3h-[3Hjendorphin conceivably could be interacting with the opiate receptor that has enkephalin as its natural ligand. It appears that 13h-[3Hlendorphin also has affinity for the "enkephalin receptor" as proposed by Hazum et al. (6). Inhibition of ,[h43HJEndorphin Binding by Various Agents. It has been reported that opiate agonist binding to the brain membrane can be attenuated by prior treatment of the membranes with degradative enzymes (17). Both trypsin and chymotrypsin were potent inhibitors of 134-[3H]endorphin binding (Fig. 2). Phospholipase A2 in the presence of 5 mM CaCl2 exhibited a concentration-dependent inhibition of fth-[3H]endorphin binding. This action of phospholipase was abolished by the addition of 2 mM ethylene glycol bis(,B-aminoethylether)-N,N,N',N'-tetraacetic acid to the incubation mixtures.
Similar to 3H-labeled agonist binding (18, 19), fOh-[3H]endorphin binding to the brain membrane was also inhibited by N-ethylmaleimide. When the membranes were incubated with 0.5 mM N-ethylmaleimide for different durations, a timedependent inhibition of 13h-[3H]endorphin binding could be observed (data not shown). The presence of 100 mM Na+ in the
Table 1. Regional distribution of f3-endorphin and enkephalin and of 1h-[3H]endorphin binding activities
Ilh-[3H]Endorphin binding Brain region
Hypothalamus
Enkephalin* 120 ± 7
f3-Endorphin* 490 + 30
Thalamus
36 ± 5
329 ± 19
Septum
85 ± 7
234 ± 34
Midbrain
32 + 1
207 ± 15
sites, fmol/mg
Kd, nM 6.6
0.30
(0.26-0.36)
(4.7-11.1)
0.68 (0.63-0.75) 0.19 (0.13-0.36)
12.6 (12.5-12.8) 3.6 (3.3-4.0)
111.4
386.9
150.6
591.3
138.5
393.4 185.5
10.3
(9.9-10.8) Brainstem
Striatum
30 + 4 112 + 11
179 ± 5
None
0.46
4.4
(0.41-0.54)
(3.5-5.1)
0.48
(0.477-0.48) Cortex
15 ± 2
Cerebellum
5± 1
Amygdala
55
8.2
71.5
164.0
270.1
690.0
110.1
256.1
(5.4-16.4)
None
0.36
3.2
(2.7-3.8)
None
(0.34-0.37) ND
-
0.51
10.4
ND 144.7
559.4
(9.5-11.6) (0.48-0.54) ND = not detectable. The values in parentheses represent the 95% confidence limits. * The mean (±SEM) levels of enkephalin (as enkephalin units/g of tissue) and fl-endorphin (as ng/g of tissue) are from Rossier et al. (15) and Simantov et al. (16).
Biochemistry:
Proc. Natl. Acad. Sci. USA 76 (1979)
Law et al.
5457
lOOr-
1 00r
0
80k c 0
80
*- 60
C
*c 40
2 60
.1E0
0
c
20k_
0 ._l
-D 40 l_ C
gg/ml Chymotrypsin
20
10Or
80[C
0
,FJ
60k-
-c ._
DI
40[20k100 30 10 Phospholipase A2, ng/ml
FIG. 2. Effect of various concentrations of trypsin, chymotrypsin, and phospholipase A2 on /,31-[3H]endorphin binding. Membranes devoid of buffer B were resuspended in 10 mM Hepes buffer at pH 7.5. Treatments with enzymes were carried out at room temperature for 30 min in a final volume of 5 ml. The actions of trypsin and chymotrypsin were terminated by addition of 0.25 ml of 40 mM 1,10-ophenanthroline; the action of phospholipase A2 was terminated by 0.25 ml of 100 mM ethylene glycol bis(f3-aminoethyl ether)-NNN',N'-tetraacetic acid. The 22,000 X g (20 min) pellets were resuspended in buffer B and specific binding of A3h-PH]endorphin (1.2 nM) was then determined. Incubations of the membranes with various concentrations of phospholipase A2 were carried out in the presence of 5 mM CaCl2 (3) or 2 mM ethylene glycol bis(f3-aminoethyl ether)N,N,N',N'-tetraacetic acid (-).
modulated by the presence of guanine nucleotide (20,21). The presence of GTP, at concentrations as low as 1 MM, inhibited the specific binding of 13h-[3Hlendorphin (Table 2). In this respect, 3B4-[3H]endorphin binding is more sensitive to GTP than is 3H-labeled alkaloid or [3H]enkephalin binding. [3H]Enkephalin binding to the membrane of NG108-15 cells (21) and to the rat brain (20) was slightly inhibited by 1 gM GTP. Effects of Cations on fth[3HJEndorphin Binding. The opiate agonist binding to brain membrane has been reported to be inhibited greatly by the presence of Na+ (22).
3h-[3H]Endorphin binding to the brain membranes was similarly affected by Na+ (Fig. 3). As in the case of [3H]dihydromorphine binding (22), 1h-[3H]endorphin binding to the membrane was almost completely abolished at 100 mM Na+. This inhibitory action was not limited to Na+; 10 mM LiCl, 100 [CaX2 80
1X
None Cations (10 mM): Na+ K+ Li+ Cs+ NH4+ GTP: 1 yM
56.7
100
28.6 60.3 34.4 41.5 35.3
50.4 106.4 60.7 73.2 62.2
26.8 27.6 12.8 15.5
47.3 48.7 22.6 27.3
% control
1o-
1X
2X
5X
10-4
100 [Mg2+] 80I__ 60_40 20-
1X 10-6
100
5X
X 2X 1X 2X 1X
i0
10-4
n-
80-
60-
The specific binding of /lh-[3H]endorphin (1.2 nM) to 0.6 mg of P2 lysate was determined in the presence and absence of the monovalent cations at 10 mM. All incubations were carried out at room temper-
ature (250C) for 1 hr.
2X
1X
-
fmol/mg
1 mM
2X
106
c
Agent
100,OM
li-
40
Table 2. Effect of monovalent cations and GTP on 13hH]endorphin specific binding
Specific binding,
r
60
incubation mixture did not attenuate the inhibition as in the case of antagonist binding (19). Thus, the response of 13h-[3H]endorphin binding to N-ethylmaleimide treatment is characteristic of an opiate agonist. Recent studies have indicated that the opiate binding can be
10 AM
100
50
Na+, mM flh-[3H]endorphin (1.2 nM) binding of specific Inhibition FIG. 3. by various concentrations of NaCl.
40
20
FIG. 4. Inhibition of specific 13h-[3H]endorphin (1.2 nM) binding by different concentrations of CaCl2, MgCl2, and MnCl2 in the incubation mixture.
Proc. Natl. Acad Sci. USA 76 (1979) 54585 Biochemistry: Law et al.
CSC1, and NH4Cl all had similar inhibitory effects (Table 2) but 10 mM KCI did not. The action of these cations on 3h-[3H]endorphin binding is similar to this action on [3H]dihydromorphine binding (22). 13h-[3H]Endorphin binding to the membranes was inhibited by the divalent cations Ca 2, Mg+2, and Mn+2 at 10 ,uM (Fig. 4). In the case of Mn 2, inhibition of binding was observed at 1 ,uM. The inhibitory activity of these cations on flh-[3H]endorphin binding was in direct contrast to that observed with [3H]dihydromorphine binding (23), cation concentrations >100 mM being required. Moreover, Pasternak et al. (23) reported that the inhibitory effect of 100 mM Na+ on 3H-labeled agonist binding could be reversed by the presence of 1 mM Mn+2. This was not the case with h-[3H]endorphin; in fact, 1 mM Mn+2 abolished the 3h-[3H]endorphin binding completely. In this respect the binding of 13h-[3H]endorphin to the opiate receptor is different from that of other radioactive opioid ligands. Displacement of PBO3H]Endorphin by Various Alkaloids and Peptides. 3h-[3H]Endorphin bound to the brain membrane could be displaced by various alkaloids and peptides (Fig. 5). When the concentration (1C50 value) of each of these compounds required to displace 50% of f3-[3H]-endorphin specifically bound was compared to that of fl-endorphin, the order of relative potencies among these compounds was different depending on the tritiated ligand used in the binding assay (Table 3). Enkephalin was more potent than f3-endorphin in displacing [3H]enkephalin and alkaloids such as diprenorphine and levorphanol were more potent than f3-endorphin in displacing [3H]dihydromorphine. However, f3-endorphin had the greatest potency in displacing h-[3H]endorphin. The order of potencies of various alkaloids in displacing fh-[3H]endorphin was similar to the order in displacing 3H-labeled alkaloid or
enkephalin. There appeared to be a secondary recognition site other than the enkephalin pentapeptide sequence for the 3h-[3H]endorphin binding. This was concluded from the observations that 100
9 --
80 60
Met']Enkephalin
20 :t
o;
.0
10-10
.c ._
10-9
10-7 Peptide, M
10-8
106
I{
1 0-5
E E 0
1100
0-R
Diprenorphine
80
60
A
2° |
Levorphanol-/ A
40-
1Naloxane N~aloxane Morphine
/ 0
/
/
20 0
n
~O-.O
0
lo-1°
10-9
lo-,A
10-7
10-6
/3-Endorphin a-Endorphin [Met5]Enkephalin [Leu5]Enkephalin
1.0 0.17 6.6
1.0 0.03 0.32
1 0.021 0.019
3.1
0.17
0.022
f3-LPH f3-LPH (66-91)
0.006 0.002 2.0 4.30 0.60 Cyclazocine Diprenorphine 1.09 4.30 0.52 0.07 1.30 0.023 Morphine 0.25 0.54 0.016 (-)Naloxone
