Regional interactions of opioid peptides at μ and δ sites in rat brain

Peptides. Vol. 4, pp. 853-858, 1983. ~'~Ankho InternationalInc. Printed in the U.S.A.

Regional Interactions of Opioid Peptides at/x and (3 Sites in Rat Brain W I L L I A M A. H E W L E T T A N D J A C K D. B A R C H A S

N a n c y Pritzker Laboratm:v o f Behavioral N e u r o c h e m i s t o ' D e p a r t m e n t o f Psychiato, and Behavioral Sciences, Stanford Unit'ersity School o f Medicine Stanfi~rd, CA 94305 R e c e i v e d 29 July 1983 HEWLETT, W. A. AND J. D. BARCHAS. Regional interactions of opioid peptides at IX and 6 sites in rat brain. PEPTIDES 4(6) 853-858, 1983.--Opiate binding sites in five brain regions were labeled with the tx and ~ markers, :~Hmorphine and aH-[D-Ala2,D-leuS]enkephalin, respectively. The highest densities of both 3H-morphine and 3H-DADLE labeled sites are found in striatum and frontal cortex. Hypothalamus and midbrain contain predominantly :~H-morphine labeled sites. The selectivity of the opioid peptides [D-Ala2,D-leu~]enkephalin,/3-endorphin and dynorphin(1-13) for the two opiate sites was investigated by comparing the potency of these unlabeled compounds against the Ix and ~ markers in different brain regions. This determination has the effect of controlling for the breakdown of peptides within each region. While the enkephalin analogue shows a preference for the ~ binding site and/3-endorphin is more nearly equipotent towards the two binding sites, dynorphin(1-13) shows a high affinity and selective preference for the/~ binding site over the 6 site. The potency of the opioid peptides in displacing the Ix and 6 markers varies from region to region according to the relative densities of the two opiate binding site populations. Multiple opiate receptors Rat brain regions Dynorphin selectivity fl-Endorphin selectivity

all-morphine binding

3H-DADLE binding

preferentially label the IXand (3 opiate receptor populations in

THE complexity of endogenous opioids is evidenced by the existence of several biochemicaily and antomically distinct opioid peptide systems, as well as multiple opiate receptors, At least three distinct classes of opioids have been described: enkephalins,/3-endorphin and related peptides, and the more recently discovered dynorphins and neoendorphins. At least five different classes of CNS opiate receptors have been proposed. Of these, the so called Ix and 8 and K opiate receptors have been the best characterized by pharmacological studies [l l, 23, 24], competitive binding assays [5, 18, 21, 23], selective protection experiments [31,34] and autoradiography [14]. The 8 receptor is pharamcologically defined as the receptor with high affinity for enkephalin but low affinity for morphine. The Ix receptor has been defined as the predominate site of action of morphine. As yet, no endogenous ligand for the Ix receptor has been found. Finally, the K receptor has a low affinity for morphine and DADLE but binds the benzomorphan, ethylketocyclazocine (EKC), and has a high affinity for the opioid peptides dynorphin and a-neo-endorphin [7, 9, 19, 36]. In light of the high number of/x and (3 sites in rat brain and the possibility that a released peptide might interact at any available receptor, we have examined the effects of three classes of peptides at these two sites. Thus, in the this paper, we examine the relative potency of the three opioids, [DAla2,D-leu'~]enkephalin (DADLE), dynorphin(1-13) and fl-endorphin at all-morphine and 3H-DADLE labeled sites in rat brain regions to determine the selectivity of these opioids for these sites. The binding paradigm employed here resembles that of Chang and Cuatrecasas [5] who have shown that low concentrations of labeled dihydromorphine and DADLE

a noncooperative manner. This group and others [10,33] have examined the regional binding of tx and 6 labels in brain regions using a competitive binding assay. We have employed a similar paradigm and generally confirmed and extended their earlier findings using the radioligands 3Hmorphine and 3H-[D-Ala2,D-leuS]enkephalin. A preliminary report of this work has been presented elsewhere [15]. METHOD [1-3H]Morphine (22 Ci/mMol) and D-Ala2-[tyrosyl-3,5-aH] enkephalin (5-D-leucine) (25 Ci/mMol) were purchased from Amersham Corp. Dynorphin(1-13) was purchased from Peninsula Laboratories (San Carlos, CA). [D-Ala2,D-leu5] enkephalin was purchased from Biosearch (San Rafael, CA). Male Sprague-Dawley rats, 160-200 g, were decapitated, their brains quickly removed and the cerebella were discarded. In experiments with brain parts, the individual regions were dissected according to a modification of the procedure by Holman et al. [17]. Each brain was placed on an ice cold petri dish and washed periodically throughout dissection with ice cold buffer. Hypothalamus including the preoptic area was separated from surrounding olfactory structures as closely as possible along natural lines of demarcation extending rostrally to the diagonal tract of Broca. A horizontal cut approximately 2 mm in depth corresponding to an extension of ventral midbrain separated hypothalamus from thalamus. Hippocampi were removed as a whole. Striata were dissected from the cortices bilaterally, using the corpus callosum as the external demarcation. Frontal cortex

853

854

H E W L E T T AND BARCHAS

was isolated by coronal slice anterior to the septal area at approximately 3 mm from the frontal poles. Thalamus and the remaining cortical areas were separated from midbrain by cuts on the lateral and rostral margins of the superior colliculi. A coronal cut caudal to the inferior colliculus but rostral to the obex separated medulla-pons from our midbrain dissection. Tissue from septum, thalamus, posterior cortex, and medulla-ports was discarded. Brain regions were weighed and then homogenized by Brinkman Polytron in cold 50 mM TRIS.HCI (Sigma) pH 7.4 at 50 mg/ml for 40 sec. Each homogenate was preincubated at 37°C for 40 rain to allow for dissociation of endogenous ligand, centrifuged at 30,000 g for 20 rain, resuspended in buffer at 50 mg/ml, incubated for 40 rain at 25°C, then placed on ice. Membrane preparation of 0.3/.d was incubated with 1 nM concentration o f a tritiated ligand and 3-7 concentrations of unlabeled ligand (0 nM, 0.2 nM, 1 nM, 5 nM, 25 nM, 125 nM, 1/zM) at 4°C to minimize the potential breakdown of the peptide ligands by the membrane preparation [21,26] for a period of 2 hr in a total volume of 500 p,l. Polypropylene incubation tubes were precoated with buffer containing 2.0 g percent crystallized bovine serum albumin (Schwarz/Mann) and 0.1 g percent polylysine hydrobromide Type V (Sigma), to minimize the loss of dynorphin(l-13) due to adhesion to the wall of the plastic tube. After incubation the homogenate was then diluted with 4.5 ml cold buffer and rapidly filtered under vacuum through Whatman GF/B filters. Incubation tubes and filters were then rinsed with 4.5 ml cold buffer. Radioactivity on filters was assessed in scintillation cocktail using an external standard quench correction to compute DPM. In all experiments specific binding for a given :~H ligand was defined as total binding minus binding in the presence of 1 /zM concentration of the corresponding unlabeled ligand. Each estimate of specific displacement represented the average of 2-4 replicates within a given experiment. Each experiment compared the displacement of the two :3Hligands by at least two of the unlabeled ligands in the same regional preparations under identical conditions. In a given experiment 4-5 regions were tested simultaneously. IC:,, values were determined by plots of percent control specific binding versus log concentration of cold ligand. Mean IC~o values and standard errors were computed using log transformed IC:,o values from different experiments. RESULTS To determine whether dynorphin(1-13) and ]3-endorphin were more like morphine or more like DADLE, displacement curves of morphine and D A D L E were carried out against 1 nM concentrations of 3H-DADLE and 3Hmorphine. Our results in whole brain confirmed that at these concentrations the radio-ligands are primarily labeling distinct populations of opiate binding sites as indicated by the diminished ability of the unlabeled ligands to displace the opposite labeled ligand (see Tables 2a and 2b). Thus, in whole brain unlabeled D A D L E is twenty times more potent in displacing 3H-DADLE than is unlabeled morphine. Similarly, unlabeled morphine is five times more potent in displacing 3H-morphine than is unlabeled D A D L E . This finding is in agreement with several other groups who have determined that/~ and 8 binding in whole brain is stereospecific, saturable, and non-cooperative [5, 8, 20, 21, 23]. We then examined the binding characteristics of each ligand in different brain regions. The apparent affinity constants (Ka) of the two opiates for their respective high and

low affinity sites were 2.47 nM and 243 nM for morphine and 1.85 nM and 13.4 nM for D A D L E as determined by a nonlinear statistical fit [25] of the binding data based on the mass action law: K,,

-

[R,I*IL,] [131

for both morphine and DADLE, assuming a two receptor non-cooperative model in striatum the region with the highest concentration o f b o t h sites (IK,~]-dissociation constant: [Rr]--ffee concentration of receptor: [L,.]--ffee concentration of ligand; [B]--concentration of receptor-ligand complex). Table 1, which shows the binding of 3H-morphine and aH-DADLE in brain regions, generally confirms the work of Chang e t a / . ]41 showing that striatum and cortex contain the highest concentration of both :~H-DADLE binding sites and :~H-morphine binding sites and that hypothalamus and midbrain (while having less of each binding site) contain a higher proportion of ~H-morphine binding sites (Table 1). Having established a differential regional distribution of the binding sites for the two opiate labels, we carried out displacement curves of unlabeled DADLE, morphine, /3-endorphin, and dynorphin(1-13) against the two labeled ligands in each brain region. The log average potencies of these unlabeled ligands against :~H-DADLE and :~Hmorphine over" all experiments in the different brain regions are given in Tables 2a and b. The most striking differences can be seen when comparing morphine's profile in striatum and hypothalamus. In striatum, morphine displaces :~Hmorphine with moderate affinity (IC~o-5 nM), yet is uniquely inactive against :~H-DADLE (IC~,,=242 nM), thus exhibiting almost a 50-fold difference in potency. In hypothalamus, the morphine IC~ against :~H-morphine is not different than in striatum (6 riM), but morphine's ability to displace :~H-DADLE is much improved (16.2 riM), showing less than a 3-fold difference in relative potency between these regions. Thus the selectivity of morphine observed in striatum (and to some extent in whole brain) disappears in hypothalamus. DADLE is also more discriminating in striatum (IC.-,o against :~H-DADLE=3.7 nM against :~Hmorphine=21 riM) than in hypothalamus (1C~,,'s 9.2 nM vs. 30 nM), but the shift is not as dramatic. Using the same analysis, the cortex resembles the striatum, and the midbrain resembles the hypothalamus, while hippocampus lies in between. Thus the regional differences seen with analysis based on binding densities (c.f., Table 1 and [4] can be demonstrated more dramatically with a cross-competition analysis shown here. When one looks at the ability of dynorphin(1-13) to displace ~H-opiates in brain regions, one sees a pattern which resembles that of morphine. This means both high potency in competing with morphine, and a similar pattern of selectivity within and across regions. Against :~H-morphine, dynorphin( l - l 3) is most potent in striatum and frontal cortex and less potent in hypothalamus and midbrain. Dynorphin(1-13) is morepotent than morphine itself in displacing :~H-morphine in every region. Dynorphin(l-13) shows a moderate potency (lC:,0 10.4 nM) in displacing 3H-DADLE from ~ sites in striatum but is almost four times more potent than DADLE itself in displacing the tritiated enkephalin analogue from sites in hypothalamus. In fact, dynorphin(1-13) shows greater potency against :~H-DADLE in this region than D A D L E shows in displacing itself in any region (Table 2). /3-Endorphin, on the other hand. shows uniformly high a

/~ A N D 6 B I N D I N G

OF OPIOID PEPTIDES

855

TABLE 1 SPECIFIC BINDING OF :~H-DADLE AND 3H-MORPHINE IN BRAIN REGIONS AND WHOLE BRAIN (fmol/mg wet wt) ~H-DADLE

Striatum Frontal Cortex Hippocampus Midbrain Hypothalamus Whole Brain

:~H-Morphine

Mean

+SE

(N)

Mean

_+SE

(N)

Binding Ratio*

4.4 3.3 1.7 0.97 0.87 1.7

0.1 0.2 0.1 0.05 0.06 0.1

(13) (8) ( 11 ) (13) (12) (6)

3.8 3.0 2.4 2.1 1.7 2.1

0.2 0.3 0.1 0. I 0.1 0.2

(10) (7) (8) (10) (9) (7)

0.86 0.91 1.4 2.2 1.9 1.2

Values represent the average specific binding in fmols/mg region (wet wt) when 1 nM concentration of 3H-DADLE or :*H-morphine is incubated with 30 mg/ml concentration of membrane preparation at 4°C. Specific binding for a given 3H ligand is defined as total binding minus binding in the presence of 1 txM concentration of the corresponding unlabeled ligand. Nonspecific binding was constant over brain regions for each :*H-ligand and equal to 1.3 fmol/mg region for 3H-morphine and 0.7 fmol/mg region for '~H-DADLE. Numbers in parentheses indicate the number of experiments for each region. *Binding ratio represents the ratio of specific binding of 3H-morphine to that :~H-DADLE in each region.

TABLE

2

POTENCIES (nM) OF DADLE, MORPHINE, DYNORPHIN(I-13), AND fi-ENDORPHIN AGAINST :'H-DADLE AND 3H-MORPHINE IN BRAIN REGIONS AND WHOLE BRAIN DADLE IC~,,

(N)

Morphine SEM*

IC:,.

(N)

Dynorphin SEM*

fi-Endorphin

IC~,,

(N)

SEM*

IC:,,,

(N)

SEM*

10.4 7.5 4.3 2.5 5.0 8.6

(4) (3) (4) (5) (4) (3)

0.056 0.009 0.030 0.048 0.074 0.058

2.9 2.3 1.4 2.5 2.8 2.9

(4) (3) (5) (3) (5) (4)

0,088 0.119 0. 110 0.096 0.101 0.106

1.3 1.1 1.0 2.4 2.3 1.3

(4) (3) (4) (4) (4) (3)

0.022 0.097 0.086 0.078 0.029 0.102

4.8 3.6 3.3 4.7 4.5 3.5

(5) (3) (5) (5) (4) (2)

0.055 0.053 0.102 0.100 0.048 0.316

(a) :~H-DADLE Striatum Frontal Cortex Hippocampus Hypothalamus Midbrain Whole Brain

3.7 4.9 5.2 9.2 12.8 5.4

(5) (3) (4) (4) (6) (3)

0.052 0.064 0.086 0.026 0.054 0.119

121.0 77.0 18.0 8.1 19.6 59.0

Striatum Frontal Cortex Hippocampus Hypothalamus Midbrain Whole Brain

21.0 23.0 19.0 30.0 34.0 27.0

(5) (2) (4) (3) (5) (3)

0.062 0.123 0.109 0.115 0.051 0.098

2.5 2.6 2.2 3.0 3.3 2.9

(4) (3) (5) (5) (6) (3)

0.046 0.131 0.108 0.101 0.065 0.127

(b) :~H-Morphine (5) (3) (4) (4) (5) (4)

0.088 0.033 0.108 0.029 0.072 0.079

A 1 nM concentration of :~H-DADLE or ZH-morphine was incubated with 3-7 concentrations of cold ligand and 30 mg/ml membrane preparation for 2 hours at 4°C. IC~0 values are determined by plots of percent control specific binding versus log concentration o f cold ligand. Specific binding for a given 3H-ligand is defined as total binding minus binding in the presence of I ~M concentration o f the corresponding unlabeled ligand. IC~,, values given represent the log average of the IC:,~ values of (N) experiments and are expressed in nM units of concentration. *Standard errors are expressed in log units.

856

H E W L E T T AND BARCHAS

potency against 3H-DADLE in every region. It is particulary potent against 3H-DADLE in hippocampus, fi-Endorphin is less potent than either dynorphin(l-13) or morphine in displacing '~H-morphine in every region, however, it is significantly more potent than D A D L E in displacing ~H-morphine. /3-Endorphin shows little regional selectivity, displacing morphine with similar IC~,)'s across the various structures. In order to more clearly demonstrate the characteristics of the endogenous opioids in binding to the ~ and 6 sites, we compared the ratio of potencies of dynorphin(l-13), fl-endorphin, morphine, and D A D L E in displacing the two labeled ligands in brain regions (Fig. 1). This comparison is particularly meaningful since breakdown should affect the potency of the endogenous opioids equally against the two labeled ligands. One can see that both morphine and dynorphin(1-13) are more potent against 3H-morphine than against 3H-DADLE while D A D L E and to a lesser extent /3-endorphin are more potent against the tritiated enkephalin analogue. These differences in potency are most pronounced in striatum and frontal cortex where both # and 6 sites are abundant and where the tritiated ligands can preferentially label their respective binding sites. In these regions where there are high and roughly equivalent amounts of 3Hmorphine and ~H-DADLE binding sites, we can assess crossreactivity of the '~H-ligands at the inappropriate sites. In striatum morphine is fifty times and dynorphin(l-13) is eight times more potent vs. 3H-morphine than against 3HDADLE. Thus morphine has a very low tendency to bind to 3H-DADLE high affinity sites. D A D L E on the other hand is less discriminating and shows a preference for ~H-DADLE labeled sites by only a factor of about six, indicating that 3H-DADLE will partially label the # binding site in rat brain.

DISCUSSION Our approach in these studies has been to obtain full competitive curves of morphine, DADLE,/3-endorphin and dynorphin(1-13) against 3H-morphine and '~H-DADLE in freshly prepared tissue of different brain regions. Statements of differential potencies are thus based on relative potencies against both ligands in any area. This approach has advantages in that it partially controls for differential regional occupany of receptors by endogenous ligands and for differential regional breakdown since the selectivity of an opioid towards a given receptor is based on competition within the s a m e brain region against the two labeled ligands, on comparisons with other competitors in the same area, as well as on differences across regions. F o r example, if in a given area, dynorphin has a lower IC.~0 against 3H-morphine than against 3H-DADLE, this cannot be due to the breakdown of dynorphin. If this difference is not apparent in another region, it is difficult to invoke breakdown as the only explanation. We demonstrate that the potency of an unlabeled ligand against a given labeled ligand can vary remarkably from brain region to brain region, giving five separate receptor preparations with which to characterize opiate ligands. Both 3H-morphine and 3H-DADLE binding sites are in greatest concentrations in striatum and frontal cortex, but the 3Hmorphine (/x) binding site is preponderate in midbrain and hypothalamus. While morphine has a high selectivity for its own site, 3H-DADLE is less selective and can interact at/x sites in brain (Fig. 1). Thus in regions like hypothalamus and midbrain 3H-DADLE may largely be cross-labeling/x binding sites which predominate in these regions while in striatum 3H-DADLE will preferentially label its own 6 sites.

I

I

[

I

I

i

MORE POTENT vs 3H MORPHINE 50

20

:~ 10 " 5,0 c~ O i

~

\

~ 2.0 -

EOUIPOTENT

1.0

%

///.o

\,1,/

0 I--

,~

0.5

n,-

0.2 0.1 0

I WB

I ST

I FC

MORE POTENT vs 3H D A D L E I I I HC HT MB

BRAIN REGIONS

FIG. 1. Ratio of potencies for different unlabeled ligands against '~H-morphine and 3H-DADLE computed as a ratio of (IC~, against :~H-DADLE)/(IC:,) against '~H-morphine). Morphine and dynorphin(1-13) are more potent in displacing :'H-morphine binding while DADLE, and to a lesser extent/3-endorphin, are more potent in displacing :~H-DADLE binding. WB (whole brain), ST (slriatum/, FC (frontal cortex) HC (hippocampus), HT (hypothalamus), MB (midbrain). I--m--unlabeled morphine, D--~--unlabeled DADLE, O----O--dynorphin(l-13), 5) --,~--/3-endorphin.

This would explain why morphine is significantly more potent in displacing 3H-DADLE binding in hypothalamus than in striatum. The present studies with unlabeled fl-endorphin suggest that /3-endorphin may interact potently with both ~ and g~ receptors, although it shows a slight constant preference for 3H-DADLE labeled sites. Dynorphin(1-13) is also potent in displacing both 3H-DADLE and 3H-morphine binding despite its potential breakdown in these preparations. In general, the pattern of displacement for dynorphin(1-13) against both 3H-DADLE and 3H-morphine (Fig. 1) is most similar to that of unlabeled morphine. In fact, in rat brain the pattern of displacement of dynorphin(1-13) against :~H-morphine, 3HD A D L E , and even the benzomorphan 3H-EKC in these regions is more similar to the pattern of unlabeled morphine than to that of unlabeled EKC [16], a ligand with k activity [24], and dynorphin(1-13) is more potent against '~Hmorphine than against 3H-EKC in whole rat brain and across rat brain regions [16]. While it is clear from the work of several groups [2, 7, 9, 29, 36] that dynorphin has highest affinity and specificity for the k opiate receptor, the number of specific '~H-benzomorphan binding sites is low relative to the numbers ofp~ and 6 sites in this tissue [6,16] and our data suggests that along the remaining p~-6 continuum, dynorphin is more potent at ~t sites than 8 sites. This finding is consistent with the work of others who have examined the/~ and 6 properties of dynorphin in guinea pig ilium [7] and brain [91. The ability of dynorphin to act at ~ opiate receptors is sup-

/z A N D 6 B I N D I N G O F O P I O I D P E P T I D E S

857

ported also by in v i v o w o r k showing that dynorphin(1-13) will substitute for morphine in the m o r p h i n e - d e p e n d e n t rhesus m o n k e y [1] although, paradoxically, dynorphin does not produce any o t h e r morphine-like effects in these animals [1]. Thus the question as to w h e t h e r dynorphin can act endogenously or e x o g e n o u s l y at /z receptors needs to be further explored. It is not known how many different populations of opiate receptors exist in the rat brain. N u m e r o u s studies [4, 10, 14, 15, 20, 21,27, 31, 34] indicate that at l e a s t two populations of opiate receptors exist in the rat brain. We interpret our binding data here in terms of a m i n i m u m , two receptor, p,-6 model and c o n s i d e r interactions only on t h e / z - & c o n t i n u u m while recognizing that other opiate receptors may exist, in particular a b e n z o m o r p h a n binding site, with which our labeled ligands do not appreciably interact. There is a possibility that # and 6 sites are not distinct physical entities but are different conformational states of the same macromolecule ]30]. While the data obtained in these studies do not exclude this possibility, the findings suggest that certain e n d o g e n o u s peptides do have a preference for one conformation or site o v e r the other and that certain regions are s e l e c t i v e l y e n r i c h e d in one site or c o n f o r m a t i o n with similar treatment of all the areas in v i t r o . It is also possible that dynorphin(1-13) has been degraded in our prepartion to a fragment with different opiate properties. H o w e v e r , Leslie

et al. [2] have shown that at 4°C d y n o r p h i n ( l - 1 3 ) is largely

deactivated in m e m b r a n e prepartions by an aminopeptidase to a non-opiate des-tyr metabolite. In addition, shorter dynorphin fragments have relatively i n c r e a s e d 8 activity relative to ~z activity [9] indicating a greater/x-like character of the original dynorphin(1-13). There are also numerous factors which can alter the opiate binding characteristics of these peptides including the presence of ions I28,32] and nucleotides [3,8]. While dynorphin(1-13) has been shown to mimic the pharmacologic b e h a v i o r of the native d y n o r p h i n ( l - 1 7 ) [12,13], W e b e r et al. 135] have now shown that the fragment, dynorphin(1-8), is present in approximately ten-fold higher concentrations than dynorphin(l-17) in brain, indicating that this smaller peptide may play an important physiological role. Thus, while the data presented here clearly indicate a preference of d y n o r p h i n ( l - 1 3 ) for all-morphine labeled sites o v e r a H - D A D L E labeled sites in these five receptor preparations under the conditions of these experiments, the effects of small modulating molecules on this b e h a v i o r and the pharmacology of dynorphin(1-8) in this system remain to be examined. ACKNOWLEDGEMENTS This work was supported in part by NIDA grant DA 01207. We thank Dr. Huda Akil for helpful discussions and Sue Poage for her secretarial assistance in the preparation of this manuscript.

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