Neuropharmacology Vol. 29, No. 5, pp. 445452, Printed in Great Britain. All rights reserved
002X-3908/90 $3.043 + 0.00
1990
Copyright 0 1990Pergamon Press plc
MODULATION OF MU, DELTA AND KAPPA OPIOID RECEPTORS IN RAT BRAIN BY METAL IONS AND HISTIDINE G. A. TEIWANI* and S. H. HANISSIAN~ Department of Pharmacology, College of Medicine, The Ohio State University, Columbus, Ohio 43210-1239, U.S.A. (Accepted 20 October 1989)
Summary-The effect of zinc (Zn*+) and several other trace elements was studied on the binding of the opioid receptor agonists [‘HI DAGO (pyr-o-Ala-Gly-Methyl-Phe-Glyol]-enkephaliny, rH] DSTLE ([Tyr-o-Ser-Gly-Phe-Leu-Thr]-enkephalin) and [3H]EKC (ethylketocyclazocine), which are specific for the mu, delta and kappa opioid receptors, respectively, in the cerebral cortex of the rat. Physiological concentrations of zinc were inhibitory to mu receptor binding, whereas the delta and kappa receptors were relatively insensitive to this inhibition. Scatchard analysis, using these opioid agonists, revealed curvilinear plots; concentrations of zinc equal to or less than the IC, (the concentration of cation which caused 50% inhibition of the binding of opioid ligand to its receptor), increased the KD (the dissociation constant) of all three subtypes of receptor, with no effect on the B,,,,, (the maximum number of binding sites) and abolished the high affinity sites of the delta and kappa receptors. Copper, cadmium and mercury also inhibited the binding of these ligands to their receptors. Histidine was most effective in preventing the inhibitory effects of zinc and copper, whereas it was less effective on cadmium and without any effect on the inhibition caused by mercury. Magnesium and manganese were stimulatory to opioid receptor binding, whereas cobalt and nickel had dual (stimulatory and inhibitory) effects. Non-inhibitory concentrations of zinc significantly decreased the stimulatory effects of magnesium and manganese on the mu and delta receptors, suggesting that part of the effect of zinc was through prevention of the actions of stimulatory cations. The reducing reagent dithiothreitol partially protected against inhibition by zinc and the oxidizing reagent dithiobisnitrobenzoic acid notentiated the inhibitorv effects of zinc on the binding of 13H1DSTLE and [‘HI DAGO. These results suggest that physiological concentrations of free zinc can inhibit mu but not d lta and kappa receptors. The lower susceptibility of delta and kappa receptors to inhibition by zinc may be, due to the presence of fewer sulthydryl-groups in these receptors, or due to their inaccessibility to binding by zinc. Relative physiological concentrations of free zinc and histidine may govern the extent of binding of the mu ligands to their receptors. Key words-opioid
receptors, DAGO, DSTLE, EKC, zinc, histidine
Several lines of evidence suggest that essential trace elements, including zinc, are involved in neurotransmitter homeostasis in addition to having effects on the steady-state levels of neurotransmitters. A number of reports have indicated modulation of opioid receptors by divalent cations (Pasternak, Snowman and Snyder, 1975; Simantov, Snowman and Snyder, 1976; Zajac and Roques, 1985; Paterson, Robson and Kosterlitz, 1986). Recently, it was observed that zinc ions inhibited the binding of [3H] naloxone to the cortex, striatum, midbrain and hippocampus of the rat in vitro and this inhibition by zinc was abolished by histidine (Hanissian and Tejwani, 1988). Stengaard-Pedersen, Fredens and Larson (1981) have suggested that there might be a relationship between zinc and the enkephalins and
*To whom all correspondence should be addressed. tPresent address: Division of Cell Biology, Burroughs Wellcome Co., 3030 Cornwallis Road, Research Triangle Park, North Carolina 27709, U.S.A. 445
that zinc ions may be physiologically important modulators of the function of opioid receptors in the hippocampal mossy fiber system. Furthermore, Stengaard-Pedersen (1982) has shown that zinc ions have the ability to inhibit the stereospecific binding of [3H] enkephalinamide to opioid receptors, possibly due to a zinc-thiol interaction. In addition, Baraldi, Caselgrandi and Santi (1984) have reported that zinc, in vitro, inhibits the binding of [‘HI naloxone to membranes from the brain of the rat. In view of these reports, the present study was conducted to explore the role of zinc ions, which are found in large concentrations in the brain (Donaldson, St. Pierre, Minnich and Barbeau, 1973) and are known to oxidize sulfhydryl groups and histidine, which is an endogenous metal chelator and an essential part of the opioid receptor (Roy and Ng, 1982), in modulating the binding of the different subtypes of opioid receptors in the cortex of the rat. In addition, the effect of other trace elements on these receptors were compared with that of zinc, to examine any similarities and differences in their mode of inter-
446
G. A. TEIWANI and
action with opioid receptors. A preliminary report on this work has been presented (Tejwani and Hanissian, 1988). METHODS
Reagents
The radioactive opioid ligands, used in these studies, were the following: [3H] DAGO (specific activity, 30.3 Ci/mmol); [3H] DSTLE (specific activity, 32.1 Ci/mmol); and [3H] ethylketocyclazocine (specific activity, 28.9 Ci/mmol), all obtained from NEN Research Products (Boston, Massachusetts). The purity of all the radioactive materials was > 98%. The highly purified metals zinc (ZnCl,) copper (CuCI,) cadmium (CdCl,) magnesium (MgCI,) and mercury (HgCl,), as well as naloxone, histidine, histamine, imidazoleacetic acid and citrate were all received from the Sigma Chemical Company (St Louis, Missouri). Manganese (MnCl,) and cobalt (CoCI,) were obtained from Mallinckrodt (Paris, Kentucky). Nickel (NiCl,) was a product of EM Science (Cherry Hill, New Jersey). The liquid scintillation cocktail used was Scinti Verse E, obtained from Fisher (Cincinnati, Ohio). Levorphanol was obtained from Hoffmann-LaRoche, Inc. (Nutley, New Jersey). Both DAGO and DADLE were supplied by Bachem (Torrance, California). Concentrations of protein in samples of brain were determined using the bicinchoninic acid method, with bovine serum albumin as standard (Smith, Krohn, Hermanson, Mallia, Gartner, Provenzano, Fujimoto, Goeke, Olson and Klenk, 1985). Animals
Male SpragueDawley rats (25&3OOg) were obtained from Zivic Miller Laboratories, Inc. (Allison Park, Pennsylvania). Before being sacrificed, the animals were maintained on normal laboratory rat chow for at least 3 days, as described before (Vaswani, Tejwani and Mousa, 1983). Preparation of membranes from brain
Preparations of membranes from the cerebral cortex of adult male Sprague-Dawley rats (250-300 g), sacrificed by decapitation, were prepared according to the method of Wood, 1986, with slight modifications. Briefly, the brains were dissected and their cerebral cortices pooled and homogenized in 20 mM Hepes buffer, pH 7.5 (100 mg wet weight/ml) and centrifuged at 49,000 g for 15 min at 4°C. The supernatant was discarded and the pellet homogenized in its original volume of buffer. This step was repeated and the homogenized pellet was incubated at 37°C for 40 min to allow any endogenous opioid peptides to dissociate from their receptors and be degraded by proteolytic enzymes. The suspension was finaliy centrifuged at 49,000g for 15 min at 4°C and the pellet homogenized in 22 ml of buffer/250 mg original tissue wet weight. This homogenate represented
S. H. HANISSIAN
the working membrane Tejwani, 1988).
preparation
(Hanissian
and
Opioid receptor binding assays
Assays, using membrane suspensions (1 ml, consisting of about 1 mg protein) from the cerebral cortex of the rat, described above were performed in duplicates. The ligand [3H] DAGO (1 nM), [3H] DSTLE (1 nM) or [3H] EKC (2 nM) was incubated with various concentrations of cations and/or 1 mM histidine for 1 hr at 25°C which was sufficient for all the ligands to reach equilibrium. The suspensions were then rapidly filtered on Whatman GF/B filters, using a Brandel cell harvester to trap the labelled membranes and followed by 3 x 3 ml washes with ice-cold Hepes buffer (20 mM, pH 7.5). The filters were then placed in vials, containing liquid scintillation cocktail and the radioactivity counted using a Beckman liquid scintillation counter. Specific binding of the radioactive ligands to the opioid receptors was determined as the difference between the amount bound in the absence and in the presence of 1 PM levorphanol (Hanissian and Tejwani, 1988) (specific binding = total binding - non-specific binding). When [3H] EKC was used, DAGO and DADLE (100 nM each) were included in the assay mixture to suppress the binding of this ligand to mu and delta receptors (Goldstein, 1987). For saturation studies, [3H] DAGO and [3H] DSTLE were used over a concentration range of &20 nM and [“HI EKC over a range of O-10 nM. All incubations were carried out for 1 hr at 25°C. There are two widely used specific ligands to study the binding of delta opioid agonists to their receptors. Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE) is a selective delta receptor ligand (Mosberg, Hurst, Hruby, Galligan, Burks, Gee and Yamamura, 1982). However, consistent results were not obtained using this compound, especially in presence of zinc; it was thought that this was because zinc may react with the dithioether group of the ligand and prevent it from binding to delta receptors (Mosberg, Omnaas and Goldstein, 1987). Instead, another delta receptor selective ligand Tyr-p-Ser-Gly-Phe-Leu-Threnkephalin (DSTLE or DSLET) (David Moisand, Meunier, Morgat, Gacel and Roques, 1982) was used. The ligand DSTLE has 20-fold greater affinity for delta receptors than it has for mu receptors and more than IOOO-foldhigher affinity for delta receptors than for kappa receptors (James and Goldstein, 1984). Scatchard analysis
The radioactive opioid ligands were used over the same concentration range as that described for the saturation experiments. Binding was carried out using 1 ml of the working membrane suspensions in the presence and in the absence of 30 p M ZnC1, and/or 1 mM histidine. When using [3H] DSTLE, 100pM ZnC1, was included in the assay mixture and 150 PM
447
Effect of zinc and histidine on brain opioid receptors 0.12,,
,
I
0.100,
I
I
Fig 1. The inhbibition of the binding of [3H] EKC to opioid receptors in the cortex of the rat by ZnCI, in the presence (0) and absence (0) of 1 mM histidine. The values shown are the mean of 4-6 experiments. Inter-experimental variations in the values were less than 10%.
of ZnCl, was included when [3H] EKC was the ligand. The tubes were incubated for 1 hr at 25°C. Binding was terminated by rapid filtration as described above. Specific binding was determined as the difference between the amount of radioactivity bound, in the absence and in the presence of 1 PM levorphanol. In addition, DAGO and DADLE (100 nM each) were included in the assay mixture when [‘HI EKC was used. The I& values of the cations were determined on the IBM PC computer, using the GraphPad program and non-linear regression. Equilibrium binding parameters, such as Ko and B micx,were determined using the limiting slopes technique, followed by curve peeling by the Rosenthal method (Rodbard, Munson and Thakur, 1980).
0
50
100150200250 Bound (fmollmg
0
50
100
150
200
protein)
Fig. 2. Scatchard analysis of the binding of [‘HI DAGO to opioid receptors in the cortex of the rat. A. Control, K,, = 0.40 + 0.08, B,,, = 30 k 10.0, KD2= 1.94 + 0.20, = 110 f 14.0. B. In the presence of I mM histidine, B Kyi 0.35 k 0.025, B,,,, = 30 + 10.0, KD2= 3.70 f 0.75, B ,,,_Z = 140 f 2.50. C. In the presence of 30pM ZnCl,, KD2= 4.35 + 0.23, K,, = 2.0 k 0.20, B,,,, = 30 + 9.50, B mr2 = 150 f 12.50. D. In the presence of 30 p M ZnCl, and KD, = 1.40 k 0.13, B,,, = 30 + 5.0, 1mM histidine, KD2 = 3.40 + 0.33, BmXZ = 135 f 7.50. The values for the K,‘s and B,,‘s reported are the mean + SD from 4 to 6 assays; K,, and K,, are expressed in nM; B,,,, and Bmlz are expressed in fmol/mg protein.
RESULTS 1. Efsect of zinc on the binding of [‘H] DAGO, [‘H] DSTLE and [‘H] EKC to opioid receptors in the cerebral cortex of the rat in the presence and in the absence of histidine
It was observed that the mu opioid receptors in the cerebral cortex of the rat were inhibited by zinc ions to a greater extent than the kappa or the delta receptors. The I&, of zinc for the binding of [3H] DAGO, [3H] EKC, and [3H] DSTLE to the mu, kappa and delta receptors was 37, 150 and 550 PM, Table
respectively. An example of inhibition of opioid receptor binding by zinc is shown in Fig. 1. Histidine (1 mM) increased the I& for zinc by 6.8, 4.2 and 1.1 fold, respectively (Table 1). Scatchard analysis, using these opioid agonists, revealed curvilinear plots (Figs 24); zinc increased the KD of all three subtypes of receptor with no effect on their B,,,. In addition, zinc abolished the high affinity sites of the delta and kappa receptors. A summary of the equilibrium binding parameters, derived from these scatchard plots, is shown in Table 2. Histidine alone had no
1.Effect of divalent cations on the binding of [‘HI DAGO and [‘H] DSTLE to opioid receptors in the cortex of the rat
[‘HI DAGO
Cation Zn’+ :;:: HE?+
-Histidine 37 + 7.0 20*f 2.5 23 1.0 9f
1.0
+Histidine 250 f 25.0* (6.8) 130 25fO.l*(l.3) + 12.5*(5.7) 7 f 0.6 (0.8)
[‘HI DSTLE -Histidine 550 + 45 58 f 2.5 33 2.4 14 + 2.5
+ Histidine 620*70(1.1) 130 85 + * 5’ IO’(2.2) (2.6) 12+ 1.5(0.9)
IC, = The concentration of the cation which caused 50% inhibition of [‘HI Ligand binding. The values reported are the mean f SD from 6 to 8 experiments. Histidine = I mM. Statistical analysis was performed using Student’s f-test; *P < 0.05 vs control. Numbers in parentheses represent the increase in the IC,, of the cations, in the presence of I mM histidine.
448
G. A. TEJWANIand S. H. HANWIAN
. L i0
D
0.025 0.020 0.015
.
0.010 0.005
50
100
150
0
50
100
150
0
50
100
150
0.000
Bound (fmollmg proteln)
Fig. 3 Scatchard analysis of the binding of [‘HI DSTLE to opioid receptors in the cortex of the rat. A. Control, K,, = 0.09 * 0.05, B,,, = 20 k 4.10, KD2= 0.82 + 0.20, Bmax2 = 80 k 8.20. B. In the presence of 1 mM histidine, KD2= 1.80 k 0.30, Bmar2 = 135 + 4.10. C. In the presence of 100pM ZnCl,, KD2= 3.10* 0.25, Bmar2= 132 f 11.10. D. In the presence of 100pM ZnCl, and 1 mM histidine, KD2= 2.80 f 0.60, Bmar2 = 150 + 2.10. The values for the K,‘s and B,,,,,' s reported are the mean f SD from 4 to 6 assays; K,,and KD2are expressed in nM; B,,,,,, and Bmar2 are expressed in fmol/mg protein.
I’Hl Ligands
used
[‘HI DSTLE,
parameters derived from the Scatchard and [‘HI EKC in the cortex of the rat
DAGO
0
50
DSTLE
plots of [‘HI EKC
I. Control 0.40 +_0.08 30 + 10.0 I .94 * 0.20 110+ 14.0
K”, kI,XI K”2 Bmar* 2. Histidine K”, B null 42 Bd 3. Zinc K”, f&X, K”2
&X2 4. Histidine
KM B rndX1
KD2 RWdXZ
0.09 20 0.82 80
f f f +
0.05 4.10 0.20 8.20
0.35 30 3.70 140
k + * +
0.025 10.0 0.75 2.50
1.80 f 0.30*,*’ 135 + 4.10*
2.0 30 4.35 150
* + f f
0.20’ 9.50 0.23’ 12.50
3.10 + 0.25’ 132 f 11.10’
+ zinc 1.4oio.13* 30 * 5.0 3.40 f 0.33* 135 + 7.50
l
100
150 I IO
Fig. 4. Scatchard analysis of the binding of [3H] EKC to opioid receptors in the cortex of the rat. A. Control, K,,= 1.43 + 0.10, Bmax, = 25 k 5.10, KD2= 5.0 f 0.09, Bmar2 = 145 & 2.40. B. In the presence of 1 mM histidine, K,,= 1.41 + 0.05, Bmx,= 30 + 8.0, KD2= 5.60 f 0.17, Bmax2 = 155 k4.10. C. In the presence of 150pM ZnCl,, K,,= 8.50 + 0.80, Bmarz = 148 f 12.0. D. In the presence of 150 PM ZnCl, and 1 mM histidine, KD2= 5.10 + 0.05, Bmr2= 150+ 1.25. The values for the K,'sand B,,'s reported are the mean + SD from 4 to 6 assays; KD,and KD2 are expressed in nM; B,,, and Bmar2 are expressed in fmol/mg protein.
Table 2. Summary of equilibrium binding DAGO,
’
-
-
2.80 f 0.60’ 150 f 2.10’
1.43 25 5.0 145
f f + +
0.10 5.10 0.09 2.40
1.41 30 5.60 155
f 0.05 f 8.0 f 0.17” 54.10
8.50 f 0.80* I48 + 12.0
5.10+0.05** 150; 1.25
The values reported were obtained from 4 to 6 experiments. The Kol and KD2 are in nM; B,,,, and B,,,, are expressed in fmol/mg protein. Histidine = I mM; zinc = 30 FM with DAGO, IOOpM with DSTLE and l50pM with EKC. Statistical analysis was performed using Student’s r-test. *P < 0.05 vs control; l*f < 0.05 vs zinc. The data shown in this table were derived by dissecting curvilinear Scatchard plots shown in Figs 2-4 as indicated in Methods section.
449
Effect of zinc and histidine on brain opioid receptors
effect on the &, and B_ of the mu and kappa receptors for [3H] DAGO and [3H] EKC, respectively, whereas it abolished the high affinity site of the delta receptors and increased both the Ko and B,,,,, of its low affinity site (Table 2). The presence of histidine in the assay mixture did not alter the effect of zinc on the mu and delta receptors. However, histidine completely prevented the effect of zinc on the low affinity site of the kappa receptors, but not on its high affinity site (Table 2). 2. .!Zf)^ect of tlarious trace elements on binding to mu and delta opioid receptors in the cerebral cortex of the rat
The binding of [‘H] DAGO and [3H] DSTLE to opioid receptors in the cerebral cortex of the rat was inhibited by Hg’+, Cd’+ and Cu2+, although to different extents. The IC,,s of these cations for the binding of [3H] DAGO are presented in Table 1, which shows that their order of inhibition was Hg2+ > Cd2+, Cu2+ > Zn2+. Histidine prevented the inhibitory effect on the mu receptors and increased the I&, in the following order: Zn2+ > Cu2+ > Cd2+ > Hg2+ (Table 1). The inhibitory effects of these cations on the binding of [‘HI DSTLE was as follows: Hg2+ > Cu2+ > Cd’+ > Zn2+. Histidine raised the IC, in the order: Cu2+ > Cd2+ > Zn2+ > Hg2+ (Table 1). The divalent cations Mg2+ and Mn2+ were found to be stimulatory to binding to mu and delta receptors Magnesium ions stimulated the binding of [3H] DAGO in the cortex of the rat with an EC& of about 100 p M; this effect was completely prevented when a concentration of zinc as small as 30pM was included in the assay mixture, with increasing concentrations of Mg2+ (Fig. 5). This effect was also seen when [3H] naloxone was used as the ligand (Hanissian and Tejwani, 1988). Thus, it appears that Zn2+ may be acting as an allosteric inhibitor of binding to mu receptors, since it was capable of overcoming the effect of millimolar concentrations of Mg2+. Magnesium also stimulated the binding of
$300 g 0 ::
0
0
’
’
10-5
“““”
’
“““”
10-4
’ 10-3
t’ll’l” 10-2
MgC12 CM]
Fig. 6. The effect of MgCl, on the binding of [jH] DSTLE to opioid receptors in the cortex of the rat in the presence (0) and absence (a) of 100 PM ZnCl,. The values shown are the mean of 46 experiments. Inter-experimental variations in the values were less than 10%.
[3H] DSTLE, but the EC,, was larger (500 PM). The inclusion of 100 p M zinc in the assay mixture by itself had no effect on the binding of [3H] DSTLE, but decreased the extent of stimulation by Mg2+ by 33% (Fig. 6). These results again point to important structural and regulatory differences between the mu and delta opioid receptors and show that the delta receptors were much less susceptible to inhibition by zinc and did not appear to be under tight regulation by zinc as the mu receptors. Manganese was also stimulatory to opioid receptor binding. The ECSo for mu receptors was about 300 PM and for delta receptors about 100 PM. The extent of stimulation of these receptors by Mn2+ was about the same (50%). The presence of zinc completely prevented the stimulatory effects of Mn2+ on both the mu and delta receptors (data not shown). The divalent cations Co’+ and Ni2+ had dual effects, first stimulating the binding of [3H] DAGO, then inhibiting it at greater concentrations. The stimulation achieved by Co2+ was at a concentration between 5 and 50/1M, whereas that by Ni2+ was between 50 and 500 /IM. Histidine decreased the effect of Ni2+ and completely prevented the stimulatory effect of Co2+ but not its inhibitory effects (data not shown). 3. Effect of suljhydryl reagents on the inhibition of opioid receptor binding by zinc
Fig. 5. The effect of MgCI, on the binding of 13H1DAGO to opioid receptors in the Eortex of the rat in ihe-presence (0) and absence (0) of 30 uM ZnCl,. The values shown are the. mean of 4-6 k@erimeks. Inter-experimental variations in the values were less than 10%.
The reducing reagent dithiothreitol (DTT) was capable of preventing the effects of zinc on mu and delta receptors, significantly (Figs 7A and 8A). The oxidizing reagent dithiobisnitrobenzoic acid (DTNB) inhibited mu and delta receptor binding in a dosedependent manner and up to about 75% at a concentration of 2 mM. In the presence of both DTNB and zinc, the inhibition was more extensive than that observed in presence of DTNB or zinc alone (Figs 7B and 8B). This suggests that part of the inhibitory effect of zinc was by oxidation of SH-groups on the
G. A. TEJWANI and S. H. HANISSIAN
(B) DTNB
B
v)
C Zn
1CQpM -*a
1mM
1OmM
-tZn
-rzn
1OOpM
1mM
2mM
-+i!n
-+Zn
-+Zn
Fig. 7. The effect of (A) dithiothreitol (DTT) and (B) dithiobisnitrobenzoic acid (DTNB) on the binding of [‘HI DAGO to opioid receptors in the cortex of the rat in the presence and absence of ZnCl,. C = Control; Zn = in the presence of 30 FM ZnCl,; (-) = without ZnCl,; (+Zn) = in the presence of 30 PM ZnCl, and DTT/or
DTNB. See the Methods section for experimental details. mu receptors which could be overcome by DTT and potentiated in the presence of DTNB. The effect of zinc on the delta receptors is believed to be nonphysiological, since very large concentrations (> 500 PM) of zinc were needed to achieve any inhibition of delta receptor binding. DISCUSSION
A paucity of information exists on the effect of essential trace elements and particularly zinc, on various subtypes of opioid receptors in brain. The total concentration of Zn*+ in the cortex of the rat is about 190pM (Donaldson et al., 1973) and that of histidine is about 45 PM (Taylor and Snyder, 1972). It has been reported that about 20% of zinc in the brain is either free or bound to ligands of molecular weight less than 10,000 Daltons (Crawford and Harris, 1984). The remaining 80% of zinc is
(A)
(B) OTT
1OOpM 1mM -+Zn -+zn
DTNB
1OmM -*Zn
1CQghl 1mM -+zn -+zn
2mM -+zn
Fig. 8. The effect of (A) dithiothreitol (DTT), and (B) dithiobisnitrobenzoic acid (DTNB) on the binding of the [)H] DSTLE to opioid receptors in the cortex of the rat in the presence and absence of ZnCl,. C = Control; Zn = in the presence of SOOpM ZnCl,; (-) = without ZnCl,; (+Zn) = in the presence of 500 PM ZnCl, and DTT/or DTNB. See the Methods section for experimental details.
apparently chelated or structurally bound to larger molecules. The present study showed that, at physiological concentrations of zinc, the mu opioid receptors were under inhibition by zinc, whereas the delta and kappa receptors were not. Histidine was capable of preventing the effect of zinc on the mu and kappa receptors but not on the delta receptors. Thus, if zinc was exerting its effects by inhibiting essential sulfhydryl-groups on the opioid receptors, as reported (Stengaard-Pedersen, 1982; Mazullo and Hine, 1980), the present study showed that the mu, delta and kappa receptors were sensitive to regulation by zinc to different extents, suggesting the differential presence of essential sulfhydryl-groups on these receptors and accessibility to zinc ions and hence structural differences among these subtypes of opioid receptor. To date, few reports have examined the effect of zinc on opioid receptors. Stengaard-Pedersen (1982) was the first to show that zinc inhibited the binding of [3H] enkephalinamide to opioid receptors in the brain of the rat and increased the K, while decreasing the B,, of these receptors at large concentrations of zinc. Baraldi et al. (1984) then showed that zinc decreased the affinity of opioid receptors in the brain of the rat for binding of naloxone, without any effect on the number of binding sites. In agreement with this study it was shown that physiological concentrations of zinc (30 PM) increased the KD of opioid receptors in the cortex and midbrain of the rat for [3H] naloxone, without changing the number of binding sites and this zinc effect was prevented by histidine (Hanissian and Tejwani, 1988). Similar results are now reported using the mu receptor against DAGO. Zinc inhibited the binding of [‘HI DAGO in the cortex of the rat. Histidine by itself had no effect on mu receptor binding and was not effective in preventing the zinc-induced increase in the K, of mu receptors for [3H] DAGO (Fig. 2). However, histidine increased the I& of zinc significantly, suggesting that it was acting mainly as a metal chelator (Table 1). Histidine also increased the IC,, of zinc for kappa receptors but by itself had no effect on the equilibrium binding parameters of kappa receptors, using [3H] EKC as ligand (Table 2). Histidine prevented the zinc-induced increase in the KD of kappa receptors. However, inhibition by zinc of kappa receptors was observed only when using large concentrations of this ion, hence its effects did not seem to be physiological (Fig. 1). Other metals, for example copper, cadmium and mercury inhibited the binding of opioid agonists to the mu receptors much more than to delta receptors (Table 1). In addition, copper, cadmium and mercury were found to be more potent inhibitors of opioid receptor binding than was zinc (Table 1); histidine was most effective in preventing the inhibitory effects of zinc and copper and had no effect on the inhibition caused by mercury. Even though histidine has a high affinity for mercury (Klotz, 1954), it is not certain
Effect of zinc and histidine on brain opioid receptors
why it was not effective in preventing the inhibitory effects of this metal ion on the opioid receptors. It is possible that mercury binds very strongly to membrane proteins and hence histidine is not able to complex with it. Magnesium and manganese ions were stimulatory to the binding of DAGO and DSTLE; the inclusion of sub-I& concentrations of zinc prevented the stimulatory effects on the mu and delta receptors, significantly (Figs 5 and 6). These findings again suggest that zinc was acting by another mechanism, besides oxidizing SH-groups, since physiological concentrations of zinc had no effect on delta receptor binding but prevented the effects of stimulatory ions such as Mg*+ and Mn’+, possibly by competing with them for the divalent cation binding sites on the receptors and preventing their binding. These results are in agreement with those of Paterson et al. (1986) who reported that Mn’+ and Mg*+ were stimulatory to the binding of naloxone, DAGO, DPDPE and DADLE at concentrations up to 1 mM but were inhibitory to the binding of dynorphin. Histidine residues have been shown to be essential for opioid receptor activity, for when inactivated or altered, the receptors lose their activity (Roy et al., 1982). The present study showed that histidine was an important modulator of opioid receptor activity in the brain and that by complexing the cations Zn2+, Cu*+ and Cd*+ it prevented their inhibitory effect (Table 1) and that histidine by itself had direct effects on the delta receptors (Table 2). Opioid receptors have been reported to have essential sulfbydryl groups which must be in the reduced form for the receptors to be active (Mazullo and Hine, 1980; Simon and Groth, 1975). Since divalent metal ions, such as copper and zinc, are also oxidizing agents, they could inhibit the binding of opioid ligands by oxidizing the essential thiol groups on the opioid receptors and decreasing their affinity for the hgands. Histidine is capable of preventing their effects by chelating them (Hanissian and Tejwani, 1988). The reducing reagent DTT had no effect on mu and delta receptor binding by itself but increased the binding in the presence of zinc by about 50% (Fig. 7A) and 71% (Fig. 8A), respectively. Dithiothreitol could be preventing the effect of zinc on the mu receptors by keeping the sulfhydryl-groups in the reduced form and possibly by chelating zinc. Stengaard-Pedersen (1982) observed that among several reducing reagents, only DTT was capable of partially preventing the inhibitory effects of zinc on the binding of [3H] enkephahnamide. In the present study, these findings were confirmed and extended using the mu receptor agonist DAGO and the delta agonist DSTLE. In addition, it was observed that histidine was much more effective in preventing the inhibitory effects of zinc on opioid receptors than any other amino acid or reducing agent used so far (Hanissian and Tejwani, 1988; Stengaard-Pedersen, 1982).
451
The oxidizing reagent DTNB decreased the binding of DAGO and DSTLE by itself and, in the presence of sub-I& concentrations of zinc, its inhibitory effects were potentiated (Figs 7B and 8B). Thus, these results support previous findings (Hanissian and Tejwani, 1988) that the inhibitory effects of zinc were not only due to the oxidation of the sulfbydryl-groups on the opioid receptors, since its effects were not completely reversed by reducing reagents and were potentiated by the oxidizing reagent DTNB. This also supports the previous hypothesis that the inhibitory effects of zinc were at least partially due to prevention of the effects of stimulatory ions, such as magnesium and manganese. Thus, the present study revealed that essential trace elements are important modulators of opioid receptor activity in the brain and possibly in the periphery and that their deficiency or excess will certainly have important effects on the opioid system. In addition, these findings show that there are important structural differences among the different subtypes of opioid receptors and that not all of them may have sulfbydryl-groups, essential for binding or that they are inaccessible to zinc. In addition, zinc ions prevented the stimulatory effects of Mg*+ and Mn*+, as well as those of Co’+ and Ni*+, by preventing their action on the divalent cation sites of the opioid receptors, suggesting that zinc has a dual action, first oxidizing sulthydryl-groups essential for binding on the mu receptors and by allosterically preventing the effect of stimulatory ions. Lastly, it was shown that histidine is an important endogenous chelator of divalent cations in the brain and also has modulatory effects on the opioid receptors, either directly, by increasing the KD and B,,, of the low affinity sites of delta receptors, or indirectly by complexing these ions and preventing their effects on the opioid receptors. Acknowledgement-We
thank the American Health Assistance Foundation for partially supporting this study. REFERENCES
Baraldi M., Caselgrandi E. and Santi M. (1984) Reduction of withdrawal symptoms in morphine-dependent rats by zinc: Behavioral and biochemical studies. Neurosci. Left. s18: s371. Crawford I. L. and Harris N. F. (1984) Distribution and accumulation of zinc in whole brain and subcellular fractions of hippocampal homogenates. In: The Neurobiology of Zinc, part A, pp. 157-171. Alan R. Liss, New York. David M., Moisand C., Meunier J.-C., Morgat J.-C., Gacel G. and Roaues B. P. (1982) l”HlTvr-D-Ser-Glv-Phe-LeuThr: A specific probe for the delta-opiate recepior subtype in brain membranes. Eur. J. Pharmac. 78: 385-387. Donaldson J., St. Pierre T., Minnich J. L. and Barbeau A. (1973) Determination of Na+, K+, Mg*+, CU*+, Zn*+ in rat brain regions. Can. J. Biochem. 51: 87-92. Goldstein A. (1987) Binding selectivity profiles for ligands of receptor - multiple ._. - types: - ._. Focus ._^ on opioid receptors. ‘l’rends Pharmac. Sci. 8: 456-459.
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Hanissian S. H. and Tejwani G. A. (1988) Histidine abolishes the inhibition by zinc of naloxone binding to opioid receptors in rat brain. Neuropharmacology 27: 1145-l 149. James I. F. and Goldstein A. (1984) Site-directed alkylation of multiple opioid receptors. I. Binding selectivity. Molec. Pharmac.
25: 331-342.
Klotz I. M. (1954) Thermodynamic and molecular properties of some metal-protein complexes. In: The Mechanism of EnzymeAction (McElroy W. D. and Glass B., Eds), pp. 257-285. The Johns Hopkins Press, Baltimore. Mazullo G. and Hine B. (1980) Opiate receptor function may be modulated through an oxidation-reduction mechanism. Science 20s: 1171-1173. Mosberg H. I., Hurst R., Hruby V. G., Galligan J. J., Burks T. F., Gee K. and Yamamura H. I. (1982) [o-Pen2, L-Cys’]-enkephalinamide and [D-Pen’, o-Cys’]-enkephalinamide, conformationally constrained cyclic enkephalinamide analogs with delta receptor specificity. Biochem. biophys. Res. Commun. 106: 506512. Mosberg H. I., Omnaas J. R. and Goldstein A. (1987) Structural requirements for delta opioid receptor binding. Molec. Pharmac. 31: 599602.
Pastemak G. W., Snowman A. M. and Snyder S. H. (1975) Selective enhancement of [3H]-opiate agonist binding by divalent cations. Molec. Pharmac. 11: 735-744. Paterson S. J., Robson L. E. and Kosterlitz H. W. (1986) Control by cations of opioid binding in guinea pig brain membranes. Proc. natn. Acad. Sci. U.S.A. 83: 62166220.
Rodbard D., Munson P. J. and Thakur A. K. (1980) Quantitative characterization of hormone receptors. Cancer 46: 2907-2918. Rov B. P. and Ne A. Y. (1982) Chemical modification of opiate recept&s with ‘ethoxyformic anhydride and photooxidation: Evidence for essential histidine residues. Biochem. biophys. Res. Commun. 109: 518-526.
Simantov R., Snowman A. M. and Snyder S. H. (1976) Temperature and ionic influences on opiate receptor binding. Molec. Pharmac. 12: 977-986. Simon E. J. and Groth J. (1975) Kinetics of opiate receptor inactivation by sulfbydryl reagents: Evidence for conformational change in presence of sodium ions. Proc. natn. Acad. Sci. U.S.A. 72: 24042407.
Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fjuimoto E. K., Goeke N. M., Olson B. J. and Klenk D. C. (1985) Measurement of protein using bicinchoninic acid. Analyf. Biochem.
150: 76-85.
Stengaard-Pedersen K. (1982) Inhibition of enkephalin binding to opiate receptors by zinc ions: Possible physiological importance in the brain. Acta pharmac. toxic. JO: 213-220.
Stengaard-Pedersen K., Fredens K. and Larson L. I. (1981) Enkephalin and zinc in the hippocampal mossy fiber system. Brain Res. 212: 230-233. Taylor K. M. and Snyder S. H. (1972) Isotopic microassay of histamine, histidine, hitidine decarboxylase and histamine methyltransferase in brain tissue. J. Neurochem. 19: 1343-1358. Tejwani G. A. and Hanissian S. H. (1988) Regulation of delta opioid receptor binding by divalent cations. PASEB J. 2: 4528.
Vaswani K. K., Tejwani G. A. and Mousa S. (1983) Stress induced differential intake of various diets and water by rats: The role of opiate system. Life Sci. 32: 198331996. Wood P. L. (1986) Multiple opioid receptors in the central nervous system. In: Neuromethods, Vol. 4, Receptor Binding (Boulton A. A., Baker G. and Hrdina P. D.. Eds). pp. 329-363. The Humana Press, Clifton. Zaiac J.-M. and Roaues B. P. 11985) Differences in binding properties of mu’ and delta opioid receptor subtypes from rat brain: Kinetic analysis and effects of ions and nucleotides. J. Neurochem. 44: 160551614.
002X-3908/90 $3.043 + 0.00
1990
Copyright 0 1990Pergamon Press plc
MODULATION OF MU, DELTA AND KAPPA OPIOID RECEPTORS IN RAT BRAIN BY METAL IONS AND HISTIDINE G. A. TEIWANI* and S. H. HANISSIAN~ Department of Pharmacology, College of Medicine, The Ohio State University, Columbus, Ohio 43210-1239, U.S.A. (Accepted 20 October 1989)
Summary-The effect of zinc (Zn*+) and several other trace elements was studied on the binding of the opioid receptor agonists [‘HI DAGO (pyr-o-Ala-Gly-Methyl-Phe-Glyol]-enkephaliny, rH] DSTLE ([Tyr-o-Ser-Gly-Phe-Leu-Thr]-enkephalin) and [3H]EKC (ethylketocyclazocine), which are specific for the mu, delta and kappa opioid receptors, respectively, in the cerebral cortex of the rat. Physiological concentrations of zinc were inhibitory to mu receptor binding, whereas the delta and kappa receptors were relatively insensitive to this inhibition. Scatchard analysis, using these opioid agonists, revealed curvilinear plots; concentrations of zinc equal to or less than the IC, (the concentration of cation which caused 50% inhibition of the binding of opioid ligand to its receptor), increased the KD (the dissociation constant) of all three subtypes of receptor, with no effect on the B,,,,, (the maximum number of binding sites) and abolished the high affinity sites of the delta and kappa receptors. Copper, cadmium and mercury also inhibited the binding of these ligands to their receptors. Histidine was most effective in preventing the inhibitory effects of zinc and copper, whereas it was less effective on cadmium and without any effect on the inhibition caused by mercury. Magnesium and manganese were stimulatory to opioid receptor binding, whereas cobalt and nickel had dual (stimulatory and inhibitory) effects. Non-inhibitory concentrations of zinc significantly decreased the stimulatory effects of magnesium and manganese on the mu and delta receptors, suggesting that part of the effect of zinc was through prevention of the actions of stimulatory cations. The reducing reagent dithiothreitol partially protected against inhibition by zinc and the oxidizing reagent dithiobisnitrobenzoic acid notentiated the inhibitorv effects of zinc on the binding of 13H1DSTLE and [‘HI DAGO. These results suggest that physiological concentrations of free zinc can inhibit mu but not d lta and kappa receptors. The lower susceptibility of delta and kappa receptors to inhibition by zinc may be, due to the presence of fewer sulthydryl-groups in these receptors, or due to their inaccessibility to binding by zinc. Relative physiological concentrations of free zinc and histidine may govern the extent of binding of the mu ligands to their receptors. Key words-opioid
receptors, DAGO, DSTLE, EKC, zinc, histidine
Several lines of evidence suggest that essential trace elements, including zinc, are involved in neurotransmitter homeostasis in addition to having effects on the steady-state levels of neurotransmitters. A number of reports have indicated modulation of opioid receptors by divalent cations (Pasternak, Snowman and Snyder, 1975; Simantov, Snowman and Snyder, 1976; Zajac and Roques, 1985; Paterson, Robson and Kosterlitz, 1986). Recently, it was observed that zinc ions inhibited the binding of [3H] naloxone to the cortex, striatum, midbrain and hippocampus of the rat in vitro and this inhibition by zinc was abolished by histidine (Hanissian and Tejwani, 1988). Stengaard-Pedersen, Fredens and Larson (1981) have suggested that there might be a relationship between zinc and the enkephalins and
*To whom all correspondence should be addressed. tPresent address: Division of Cell Biology, Burroughs Wellcome Co., 3030 Cornwallis Road, Research Triangle Park, North Carolina 27709, U.S.A. 445
that zinc ions may be physiologically important modulators of the function of opioid receptors in the hippocampal mossy fiber system. Furthermore, Stengaard-Pedersen (1982) has shown that zinc ions have the ability to inhibit the stereospecific binding of [3H] enkephalinamide to opioid receptors, possibly due to a zinc-thiol interaction. In addition, Baraldi, Caselgrandi and Santi (1984) have reported that zinc, in vitro, inhibits the binding of [‘HI naloxone to membranes from the brain of the rat. In view of these reports, the present study was conducted to explore the role of zinc ions, which are found in large concentrations in the brain (Donaldson, St. Pierre, Minnich and Barbeau, 1973) and are known to oxidize sulfhydryl groups and histidine, which is an endogenous metal chelator and an essential part of the opioid receptor (Roy and Ng, 1982), in modulating the binding of the different subtypes of opioid receptors in the cortex of the rat. In addition, the effect of other trace elements on these receptors were compared with that of zinc, to examine any similarities and differences in their mode of inter-
446
G. A. TEIWANI and
action with opioid receptors. A preliminary report on this work has been presented (Tejwani and Hanissian, 1988). METHODS
Reagents
The radioactive opioid ligands, used in these studies, were the following: [3H] DAGO (specific activity, 30.3 Ci/mmol); [3H] DSTLE (specific activity, 32.1 Ci/mmol); and [3H] ethylketocyclazocine (specific activity, 28.9 Ci/mmol), all obtained from NEN Research Products (Boston, Massachusetts). The purity of all the radioactive materials was > 98%. The highly purified metals zinc (ZnCl,) copper (CuCI,) cadmium (CdCl,) magnesium (MgCI,) and mercury (HgCl,), as well as naloxone, histidine, histamine, imidazoleacetic acid and citrate were all received from the Sigma Chemical Company (St Louis, Missouri). Manganese (MnCl,) and cobalt (CoCI,) were obtained from Mallinckrodt (Paris, Kentucky). Nickel (NiCl,) was a product of EM Science (Cherry Hill, New Jersey). The liquid scintillation cocktail used was Scinti Verse E, obtained from Fisher (Cincinnati, Ohio). Levorphanol was obtained from Hoffmann-LaRoche, Inc. (Nutley, New Jersey). Both DAGO and DADLE were supplied by Bachem (Torrance, California). Concentrations of protein in samples of brain were determined using the bicinchoninic acid method, with bovine serum albumin as standard (Smith, Krohn, Hermanson, Mallia, Gartner, Provenzano, Fujimoto, Goeke, Olson and Klenk, 1985). Animals
Male SpragueDawley rats (25&3OOg) were obtained from Zivic Miller Laboratories, Inc. (Allison Park, Pennsylvania). Before being sacrificed, the animals were maintained on normal laboratory rat chow for at least 3 days, as described before (Vaswani, Tejwani and Mousa, 1983). Preparation of membranes from brain
Preparations of membranes from the cerebral cortex of adult male Sprague-Dawley rats (250-300 g), sacrificed by decapitation, were prepared according to the method of Wood, 1986, with slight modifications. Briefly, the brains were dissected and their cerebral cortices pooled and homogenized in 20 mM Hepes buffer, pH 7.5 (100 mg wet weight/ml) and centrifuged at 49,000 g for 15 min at 4°C. The supernatant was discarded and the pellet homogenized in its original volume of buffer. This step was repeated and the homogenized pellet was incubated at 37°C for 40 min to allow any endogenous opioid peptides to dissociate from their receptors and be degraded by proteolytic enzymes. The suspension was finaliy centrifuged at 49,000g for 15 min at 4°C and the pellet homogenized in 22 ml of buffer/250 mg original tissue wet weight. This homogenate represented
S. H. HANISSIAN
the working membrane Tejwani, 1988).
preparation
(Hanissian
and
Opioid receptor binding assays
Assays, using membrane suspensions (1 ml, consisting of about 1 mg protein) from the cerebral cortex of the rat, described above were performed in duplicates. The ligand [3H] DAGO (1 nM), [3H] DSTLE (1 nM) or [3H] EKC (2 nM) was incubated with various concentrations of cations and/or 1 mM histidine for 1 hr at 25°C which was sufficient for all the ligands to reach equilibrium. The suspensions were then rapidly filtered on Whatman GF/B filters, using a Brandel cell harvester to trap the labelled membranes and followed by 3 x 3 ml washes with ice-cold Hepes buffer (20 mM, pH 7.5). The filters were then placed in vials, containing liquid scintillation cocktail and the radioactivity counted using a Beckman liquid scintillation counter. Specific binding of the radioactive ligands to the opioid receptors was determined as the difference between the amount bound in the absence and in the presence of 1 PM levorphanol (Hanissian and Tejwani, 1988) (specific binding = total binding - non-specific binding). When [3H] EKC was used, DAGO and DADLE (100 nM each) were included in the assay mixture to suppress the binding of this ligand to mu and delta receptors (Goldstein, 1987). For saturation studies, [3H] DAGO and [3H] DSTLE were used over a concentration range of &20 nM and [“HI EKC over a range of O-10 nM. All incubations were carried out for 1 hr at 25°C. There are two widely used specific ligands to study the binding of delta opioid agonists to their receptors. Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE) is a selective delta receptor ligand (Mosberg, Hurst, Hruby, Galligan, Burks, Gee and Yamamura, 1982). However, consistent results were not obtained using this compound, especially in presence of zinc; it was thought that this was because zinc may react with the dithioether group of the ligand and prevent it from binding to delta receptors (Mosberg, Omnaas and Goldstein, 1987). Instead, another delta receptor selective ligand Tyr-p-Ser-Gly-Phe-Leu-Threnkephalin (DSTLE or DSLET) (David Moisand, Meunier, Morgat, Gacel and Roques, 1982) was used. The ligand DSTLE has 20-fold greater affinity for delta receptors than it has for mu receptors and more than IOOO-foldhigher affinity for delta receptors than for kappa receptors (James and Goldstein, 1984). Scatchard analysis
The radioactive opioid ligands were used over the same concentration range as that described for the saturation experiments. Binding was carried out using 1 ml of the working membrane suspensions in the presence and in the absence of 30 p M ZnC1, and/or 1 mM histidine. When using [3H] DSTLE, 100pM ZnC1, was included in the assay mixture and 150 PM
447
Effect of zinc and histidine on brain opioid receptors 0.12,,
,
I
0.100,
I
I
Fig 1. The inhbibition of the binding of [3H] EKC to opioid receptors in the cortex of the rat by ZnCI, in the presence (0) and absence (0) of 1 mM histidine. The values shown are the mean of 4-6 experiments. Inter-experimental variations in the values were less than 10%.
of ZnCl, was included when [3H] EKC was the ligand. The tubes were incubated for 1 hr at 25°C. Binding was terminated by rapid filtration as described above. Specific binding was determined as the difference between the amount of radioactivity bound, in the absence and in the presence of 1 PM levorphanol. In addition, DAGO and DADLE (100 nM each) were included in the assay mixture when [‘HI EKC was used. The I& values of the cations were determined on the IBM PC computer, using the GraphPad program and non-linear regression. Equilibrium binding parameters, such as Ko and B micx,were determined using the limiting slopes technique, followed by curve peeling by the Rosenthal method (Rodbard, Munson and Thakur, 1980).
0
50
100150200250 Bound (fmollmg
0
50
100
150
200
protein)
Fig. 2. Scatchard analysis of the binding of [‘HI DAGO to opioid receptors in the cortex of the rat. A. Control, K,, = 0.40 + 0.08, B,,, = 30 k 10.0, KD2= 1.94 + 0.20, = 110 f 14.0. B. In the presence of I mM histidine, B Kyi 0.35 k 0.025, B,,,, = 30 + 10.0, KD2= 3.70 f 0.75, B ,,,_Z = 140 f 2.50. C. In the presence of 30pM ZnCl,, KD2= 4.35 + 0.23, K,, = 2.0 k 0.20, B,,,, = 30 + 9.50, B mr2 = 150 f 12.50. D. In the presence of 30 p M ZnCl, and KD, = 1.40 k 0.13, B,,, = 30 + 5.0, 1mM histidine, KD2 = 3.40 + 0.33, BmXZ = 135 f 7.50. The values for the K,‘s and B,,‘s reported are the mean + SD from 4 to 6 assays; K,, and K,, are expressed in nM; B,,,, and Bmlz are expressed in fmol/mg protein.
RESULTS 1. Efsect of zinc on the binding of [‘H] DAGO, [‘H] DSTLE and [‘H] EKC to opioid receptors in the cerebral cortex of the rat in the presence and in the absence of histidine
It was observed that the mu opioid receptors in the cerebral cortex of the rat were inhibited by zinc ions to a greater extent than the kappa or the delta receptors. The I&, of zinc for the binding of [3H] DAGO, [3H] EKC, and [3H] DSTLE to the mu, kappa and delta receptors was 37, 150 and 550 PM, Table
respectively. An example of inhibition of opioid receptor binding by zinc is shown in Fig. 1. Histidine (1 mM) increased the I& for zinc by 6.8, 4.2 and 1.1 fold, respectively (Table 1). Scatchard analysis, using these opioid agonists, revealed curvilinear plots (Figs 24); zinc increased the KD of all three subtypes of receptor with no effect on their B,,,. In addition, zinc abolished the high affinity sites of the delta and kappa receptors. A summary of the equilibrium binding parameters, derived from these scatchard plots, is shown in Table 2. Histidine alone had no
1.Effect of divalent cations on the binding of [‘HI DAGO and [‘H] DSTLE to opioid receptors in the cortex of the rat
[‘HI DAGO
Cation Zn’+ :;:: HE?+
-Histidine 37 + 7.0 20*f 2.5 23 1.0 9f
1.0
+Histidine 250 f 25.0* (6.8) 130 25fO.l*(l.3) + 12.5*(5.7) 7 f 0.6 (0.8)
[‘HI DSTLE -Histidine 550 + 45 58 f 2.5 33 2.4 14 + 2.5
+ Histidine 620*70(1.1) 130 85 + * 5’ IO’(2.2) (2.6) 12+ 1.5(0.9)
IC, = The concentration of the cation which caused 50% inhibition of [‘HI Ligand binding. The values reported are the mean f SD from 6 to 8 experiments. Histidine = I mM. Statistical analysis was performed using Student’s f-test; *P < 0.05 vs control. Numbers in parentheses represent the increase in the IC,, of the cations, in the presence of I mM histidine.
448
G. A. TEJWANIand S. H. HANWIAN
. L i0
D
0.025 0.020 0.015
.
0.010 0.005
50
100
150
0
50
100
150
0
50
100
150
0.000
Bound (fmollmg proteln)
Fig. 3 Scatchard analysis of the binding of [‘HI DSTLE to opioid receptors in the cortex of the rat. A. Control, K,, = 0.09 * 0.05, B,,, = 20 k 4.10, KD2= 0.82 + 0.20, Bmax2 = 80 k 8.20. B. In the presence of 1 mM histidine, KD2= 1.80 k 0.30, Bmar2 = 135 + 4.10. C. In the presence of 100pM ZnCl,, KD2= 3.10* 0.25, Bmar2= 132 f 11.10. D. In the presence of 100pM ZnCl, and 1 mM histidine, KD2= 2.80 f 0.60, Bmar2 = 150 + 2.10. The values for the K,‘s and B,,,,,' s reported are the mean f SD from 4 to 6 assays; K,,and KD2are expressed in nM; B,,,,,, and Bmar2 are expressed in fmol/mg protein.
I’Hl Ligands
used
[‘HI DSTLE,
parameters derived from the Scatchard and [‘HI EKC in the cortex of the rat
DAGO
0
50
DSTLE
plots of [‘HI EKC
I. Control 0.40 +_0.08 30 + 10.0 I .94 * 0.20 110+ 14.0
K”, kI,XI K”2 Bmar* 2. Histidine K”, B null 42 Bd 3. Zinc K”, f&X, K”2
&X2 4. Histidine
KM B rndX1
KD2 RWdXZ
0.09 20 0.82 80
f f f +
0.05 4.10 0.20 8.20
0.35 30 3.70 140
k + * +
0.025 10.0 0.75 2.50
1.80 f 0.30*,*’ 135 + 4.10*
2.0 30 4.35 150
* + f f
0.20’ 9.50 0.23’ 12.50
3.10 + 0.25’ 132 f 11.10’
+ zinc 1.4oio.13* 30 * 5.0 3.40 f 0.33* 135 + 7.50
l
100
150 I IO
Fig. 4. Scatchard analysis of the binding of [3H] EKC to opioid receptors in the cortex of the rat. A. Control, K,,= 1.43 + 0.10, Bmax, = 25 k 5.10, KD2= 5.0 f 0.09, Bmar2 = 145 & 2.40. B. In the presence of 1 mM histidine, K,,= 1.41 + 0.05, Bmx,= 30 + 8.0, KD2= 5.60 f 0.17, Bmax2 = 155 k4.10. C. In the presence of 150pM ZnCl,, K,,= 8.50 + 0.80, Bmarz = 148 f 12.0. D. In the presence of 150 PM ZnCl, and 1 mM histidine, KD2= 5.10 + 0.05, Bmr2= 150+ 1.25. The values for the K,'sand B,,'s reported are the mean + SD from 4 to 6 assays; KD,and KD2 are expressed in nM; B,,, and Bmar2 are expressed in fmol/mg protein.
Table 2. Summary of equilibrium binding DAGO,
’
-
-
2.80 f 0.60’ 150 f 2.10’
1.43 25 5.0 145
f f + +
0.10 5.10 0.09 2.40
1.41 30 5.60 155
f 0.05 f 8.0 f 0.17” 54.10
8.50 f 0.80* I48 + 12.0
5.10+0.05** 150; 1.25
The values reported were obtained from 4 to 6 experiments. The Kol and KD2 are in nM; B,,,, and B,,,, are expressed in fmol/mg protein. Histidine = I mM; zinc = 30 FM with DAGO, IOOpM with DSTLE and l50pM with EKC. Statistical analysis was performed using Student’s r-test. *P < 0.05 vs control; l*f < 0.05 vs zinc. The data shown in this table were derived by dissecting curvilinear Scatchard plots shown in Figs 2-4 as indicated in Methods section.
449
Effect of zinc and histidine on brain opioid receptors
effect on the &, and B_ of the mu and kappa receptors for [3H] DAGO and [3H] EKC, respectively, whereas it abolished the high affinity site of the delta receptors and increased both the Ko and B,,,,, of its low affinity site (Table 2). The presence of histidine in the assay mixture did not alter the effect of zinc on the mu and delta receptors. However, histidine completely prevented the effect of zinc on the low affinity site of the kappa receptors, but not on its high affinity site (Table 2). 2. .!Zf)^ect of tlarious trace elements on binding to mu and delta opioid receptors in the cerebral cortex of the rat
The binding of [‘H] DAGO and [3H] DSTLE to opioid receptors in the cerebral cortex of the rat was inhibited by Hg’+, Cd’+ and Cu2+, although to different extents. The IC,,s of these cations for the binding of [3H] DAGO are presented in Table 1, which shows that their order of inhibition was Hg2+ > Cd2+, Cu2+ > Zn2+. Histidine prevented the inhibitory effect on the mu receptors and increased the I&, in the following order: Zn2+ > Cu2+ > Cd2+ > Hg2+ (Table 1). The inhibitory effects of these cations on the binding of [‘HI DSTLE was as follows: Hg2+ > Cu2+ > Cd’+ > Zn2+. Histidine raised the IC, in the order: Cu2+ > Cd2+ > Zn2+ > Hg2+ (Table 1). The divalent cations Mg2+ and Mn2+ were found to be stimulatory to binding to mu and delta receptors Magnesium ions stimulated the binding of [3H] DAGO in the cortex of the rat with an EC& of about 100 p M; this effect was completely prevented when a concentration of zinc as small as 30pM was included in the assay mixture, with increasing concentrations of Mg2+ (Fig. 5). This effect was also seen when [3H] naloxone was used as the ligand (Hanissian and Tejwani, 1988). Thus, it appears that Zn2+ may be acting as an allosteric inhibitor of binding to mu receptors, since it was capable of overcoming the effect of millimolar concentrations of Mg2+. Magnesium also stimulated the binding of
$300 g 0 ::
0
0
’
’
10-5
“““”
’
“““”
10-4
’ 10-3
t’ll’l” 10-2
MgC12 CM]
Fig. 6. The effect of MgCl, on the binding of [jH] DSTLE to opioid receptors in the cortex of the rat in the presence (0) and absence (a) of 100 PM ZnCl,. The values shown are the mean of 46 experiments. Inter-experimental variations in the values were less than 10%.
[3H] DSTLE, but the EC,, was larger (500 PM). The inclusion of 100 p M zinc in the assay mixture by itself had no effect on the binding of [3H] DSTLE, but decreased the extent of stimulation by Mg2+ by 33% (Fig. 6). These results again point to important structural and regulatory differences between the mu and delta opioid receptors and show that the delta receptors were much less susceptible to inhibition by zinc and did not appear to be under tight regulation by zinc as the mu receptors. Manganese was also stimulatory to opioid receptor binding. The ECSo for mu receptors was about 300 PM and for delta receptors about 100 PM. The extent of stimulation of these receptors by Mn2+ was about the same (50%). The presence of zinc completely prevented the stimulatory effects of Mn2+ on both the mu and delta receptors (data not shown). The divalent cations Co’+ and Ni2+ had dual effects, first stimulating the binding of [3H] DAGO, then inhibiting it at greater concentrations. The stimulation achieved by Co2+ was at a concentration between 5 and 50/1M, whereas that by Ni2+ was between 50 and 500 /IM. Histidine decreased the effect of Ni2+ and completely prevented the stimulatory effect of Co2+ but not its inhibitory effects (data not shown). 3. Effect of suljhydryl reagents on the inhibition of opioid receptor binding by zinc
Fig. 5. The effect of MgCI, on the binding of 13H1DAGO to opioid receptors in the Eortex of the rat in ihe-presence (0) and absence (0) of 30 uM ZnCl,. The values shown are the. mean of 4-6 k@erimeks. Inter-experimental variations in the values were less than 10%.
The reducing reagent dithiothreitol (DTT) was capable of preventing the effects of zinc on mu and delta receptors, significantly (Figs 7A and 8A). The oxidizing reagent dithiobisnitrobenzoic acid (DTNB) inhibited mu and delta receptor binding in a dosedependent manner and up to about 75% at a concentration of 2 mM. In the presence of both DTNB and zinc, the inhibition was more extensive than that observed in presence of DTNB or zinc alone (Figs 7B and 8B). This suggests that part of the inhibitory effect of zinc was by oxidation of SH-groups on the
G. A. TEJWANI and S. H. HANISSIAN
(B) DTNB
B
v)
C Zn
1CQpM -*a
1mM
1OmM
-tZn
-rzn
1OOpM
1mM
2mM
-+i!n
-+Zn
-+Zn
Fig. 7. The effect of (A) dithiothreitol (DTT) and (B) dithiobisnitrobenzoic acid (DTNB) on the binding of [‘HI DAGO to opioid receptors in the cortex of the rat in the presence and absence of ZnCl,. C = Control; Zn = in the presence of 30 FM ZnCl,; (-) = without ZnCl,; (+Zn) = in the presence of 30 PM ZnCl, and DTT/or
DTNB. See the Methods section for experimental details. mu receptors which could be overcome by DTT and potentiated in the presence of DTNB. The effect of zinc on the delta receptors is believed to be nonphysiological, since very large concentrations (> 500 PM) of zinc were needed to achieve any inhibition of delta receptor binding. DISCUSSION
A paucity of information exists on the effect of essential trace elements and particularly zinc, on various subtypes of opioid receptors in brain. The total concentration of Zn*+ in the cortex of the rat is about 190pM (Donaldson et al., 1973) and that of histidine is about 45 PM (Taylor and Snyder, 1972). It has been reported that about 20% of zinc in the brain is either free or bound to ligands of molecular weight less than 10,000 Daltons (Crawford and Harris, 1984). The remaining 80% of zinc is
(A)
(B) OTT
1OOpM 1mM -+Zn -+zn
DTNB
1OmM -*Zn
1CQghl 1mM -+zn -+zn
2mM -+zn
Fig. 8. The effect of (A) dithiothreitol (DTT), and (B) dithiobisnitrobenzoic acid (DTNB) on the binding of the [)H] DSTLE to opioid receptors in the cortex of the rat in the presence and absence of ZnCl,. C = Control; Zn = in the presence of SOOpM ZnCl,; (-) = without ZnCl,; (+Zn) = in the presence of 500 PM ZnCl, and DTT/or DTNB. See the Methods section for experimental details.
apparently chelated or structurally bound to larger molecules. The present study showed that, at physiological concentrations of zinc, the mu opioid receptors were under inhibition by zinc, whereas the delta and kappa receptors were not. Histidine was capable of preventing the effect of zinc on the mu and kappa receptors but not on the delta receptors. Thus, if zinc was exerting its effects by inhibiting essential sulfhydryl-groups on the opioid receptors, as reported (Stengaard-Pedersen, 1982; Mazullo and Hine, 1980), the present study showed that the mu, delta and kappa receptors were sensitive to regulation by zinc to different extents, suggesting the differential presence of essential sulfhydryl-groups on these receptors and accessibility to zinc ions and hence structural differences among these subtypes of opioid receptor. To date, few reports have examined the effect of zinc on opioid receptors. Stengaard-Pedersen (1982) was the first to show that zinc inhibited the binding of [3H] enkephalinamide to opioid receptors in the brain of the rat and increased the K, while decreasing the B,, of these receptors at large concentrations of zinc. Baraldi et al. (1984) then showed that zinc decreased the affinity of opioid receptors in the brain of the rat for binding of naloxone, without any effect on the number of binding sites. In agreement with this study it was shown that physiological concentrations of zinc (30 PM) increased the KD of opioid receptors in the cortex and midbrain of the rat for [3H] naloxone, without changing the number of binding sites and this zinc effect was prevented by histidine (Hanissian and Tejwani, 1988). Similar results are now reported using the mu receptor against DAGO. Zinc inhibited the binding of [‘HI DAGO in the cortex of the rat. Histidine by itself had no effect on mu receptor binding and was not effective in preventing the zinc-induced increase in the K, of mu receptors for [3H] DAGO (Fig. 2). However, histidine increased the I& of zinc significantly, suggesting that it was acting mainly as a metal chelator (Table 1). Histidine also increased the IC,, of zinc for kappa receptors but by itself had no effect on the equilibrium binding parameters of kappa receptors, using [3H] EKC as ligand (Table 2). Histidine prevented the zinc-induced increase in the KD of kappa receptors. However, inhibition by zinc of kappa receptors was observed only when using large concentrations of this ion, hence its effects did not seem to be physiological (Fig. 1). Other metals, for example copper, cadmium and mercury inhibited the binding of opioid agonists to the mu receptors much more than to delta receptors (Table 1). In addition, copper, cadmium and mercury were found to be more potent inhibitors of opioid receptor binding than was zinc (Table 1); histidine was most effective in preventing the inhibitory effects of zinc and copper and had no effect on the inhibition caused by mercury. Even though histidine has a high affinity for mercury (Klotz, 1954), it is not certain
Effect of zinc and histidine on brain opioid receptors
why it was not effective in preventing the inhibitory effects of this metal ion on the opioid receptors. It is possible that mercury binds very strongly to membrane proteins and hence histidine is not able to complex with it. Magnesium and manganese ions were stimulatory to the binding of DAGO and DSTLE; the inclusion of sub-I& concentrations of zinc prevented the stimulatory effects on the mu and delta receptors, significantly (Figs 5 and 6). These findings again suggest that zinc was acting by another mechanism, besides oxidizing SH-groups, since physiological concentrations of zinc had no effect on delta receptor binding but prevented the effects of stimulatory ions such as Mg*+ and Mn’+, possibly by competing with them for the divalent cation binding sites on the receptors and preventing their binding. These results are in agreement with those of Paterson et al. (1986) who reported that Mn’+ and Mg*+ were stimulatory to the binding of naloxone, DAGO, DPDPE and DADLE at concentrations up to 1 mM but were inhibitory to the binding of dynorphin. Histidine residues have been shown to be essential for opioid receptor activity, for when inactivated or altered, the receptors lose their activity (Roy et al., 1982). The present study showed that histidine was an important modulator of opioid receptor activity in the brain and that by complexing the cations Zn2+, Cu*+ and Cd*+ it prevented their inhibitory effect (Table 1) and that histidine by itself had direct effects on the delta receptors (Table 2). Opioid receptors have been reported to have essential sulfbydryl groups which must be in the reduced form for the receptors to be active (Mazullo and Hine, 1980; Simon and Groth, 1975). Since divalent metal ions, such as copper and zinc, are also oxidizing agents, they could inhibit the binding of opioid ligands by oxidizing the essential thiol groups on the opioid receptors and decreasing their affinity for the hgands. Histidine is capable of preventing their effects by chelating them (Hanissian and Tejwani, 1988). The reducing reagent DTT had no effect on mu and delta receptor binding by itself but increased the binding in the presence of zinc by about 50% (Fig. 7A) and 71% (Fig. 8A), respectively. Dithiothreitol could be preventing the effect of zinc on the mu receptors by keeping the sulfhydryl-groups in the reduced form and possibly by chelating zinc. Stengaard-Pedersen (1982) observed that among several reducing reagents, only DTT was capable of partially preventing the inhibitory effects of zinc on the binding of [3H] enkephahnamide. In the present study, these findings were confirmed and extended using the mu receptor agonist DAGO and the delta agonist DSTLE. In addition, it was observed that histidine was much more effective in preventing the inhibitory effects of zinc on opioid receptors than any other amino acid or reducing agent used so far (Hanissian and Tejwani, 1988; Stengaard-Pedersen, 1982).
451
The oxidizing reagent DTNB decreased the binding of DAGO and DSTLE by itself and, in the presence of sub-I& concentrations of zinc, its inhibitory effects were potentiated (Figs 7B and 8B). Thus, these results support previous findings (Hanissian and Tejwani, 1988) that the inhibitory effects of zinc were not only due to the oxidation of the sulfbydryl-groups on the opioid receptors, since its effects were not completely reversed by reducing reagents and were potentiated by the oxidizing reagent DTNB. This also supports the previous hypothesis that the inhibitory effects of zinc were at least partially due to prevention of the effects of stimulatory ions, such as magnesium and manganese. Thus, the present study revealed that essential trace elements are important modulators of opioid receptor activity in the brain and possibly in the periphery and that their deficiency or excess will certainly have important effects on the opioid system. In addition, these findings show that there are important structural differences among the different subtypes of opioid receptors and that not all of them may have sulfbydryl-groups, essential for binding or that they are inaccessible to zinc. In addition, zinc ions prevented the stimulatory effects of Mg*+ and Mn*+, as well as those of Co’+ and Ni*+, by preventing their action on the divalent cation sites of the opioid receptors, suggesting that zinc has a dual action, first oxidizing sulthydryl-groups essential for binding on the mu receptors and by allosterically preventing the effect of stimulatory ions. Lastly, it was shown that histidine is an important endogenous chelator of divalent cations in the brain and also has modulatory effects on the opioid receptors, either directly, by increasing the KD and B,,, of the low affinity sites of delta receptors, or indirectly by complexing these ions and preventing their effects on the opioid receptors. Acknowledgement-We
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