Biochemical characterization and regional quantification of μ, δ and κ opioid binding sites in rat spinal cord

Brain Research, 551) ( 1991 ) 77-85 © 1991 Elsevier Science Publishers B.V. (~X)6-8993/91/$03.50 A DONIS 0(106899391167176

77

BRES 16717

Biochemical characterization and regional quantification of/ , 6 and 7c opioid binding sites in rat spinal cord Craig W. Stevens*, Carolyn B. Lacey, Kenneth E. Miller**, Robert P. Elde and

Virginia S. Seybold D~Tmrtment of('ell Biology and Neuroanatomy, University of Minnesota, Minneapolis, MN 55455 (U. S. A. )

(Acccpted 22 January 1991) Key words: ()pioid receptor" Autoradiography: Rat spinal cord: Antinociception; Dorsal horn; Pain pathway

The spinal cord contains lt. b and K opioid receptors which mediate the antinociccptive effects of opioid agonists administered onto the spinal cord. In this study, wc characterized the binding sites for highly-selective/~, 6 and ~:opioid radioligands and quantified the distribution of opioid binding sites in rat lumbosacral spinal cord using autoradiography. In sections of rat brain mounted on glass slides, the l~ ligand, [3H]sufentanil, bound with high affinity with an apparent Kj of (I.46 nM. The b ligand, [3H]DPDPE ([o-Pen25]-enkephalin), bound with a Kd of 4.31 nM, and the ~:-ligand, [3H]U69593, bound with a K,j of 2.27 nM. Three regions of the spinal gray were targeted for quantification of binding sites by autoradiography. The data indicate that when considered as a percentage of the total opioid binding capacity within a region, the contribution ofg sites in laminae I-II was about 90c~, with ,4 and ~: sites 7% and 3%, respectively. In lamina V, the/~ sites comprised about 70% of the total opioid sites, with c~and h- sites comprising 28~ and 2%, respectively. In the area adjacent to the central canal, u sites contributed about 65% of the total opioid sites followed by ~ sites at 33% and ~c sites at 2% of total opioid sites. These results demonstrate a differential distribution of u, b and ~¢binding sites with respect to the organization of the spinal gray matter. The preferential occurrence of all 3 opioid binding sites in the superficial dorsal horn is notcworthy since many fine caliber primary afferent fibers mediating nociccption establish synaptic contact in this region. INTRODUCTION Numerous pharmacological studies utilizing the spinal administration of selective opioid agonists have shown that/~, 6, and ~¢opioid receptors mediate the suppression of nociccptive transmission in a variety of vertebrates. including humans 124"52"~. A m o n g the 3 types of opioid receptors, spinal intrathecal administration of ~ agonists produce the most potent antinociceptive effects on cutaneous thermal and visceral chemical algesiometric tests, whereas 6 agonists arc more potent than h: agonists on cutaneous thermal tests, and h" agonists more potent than 6 agonists on visceral chemical tests 47"49'53'~. Radioligand binding experiments have confirmed the existence o f ~ , 6, and h" opioid receptors in homogenates of rat spinal cord tissue ~''t~'13.lg"2n'29"39.S6.57. Consistent with the potent antinociceptive action of spinally administered ,tt agonists noted above, these binding studies have shown that the spinal tissue contains g opioid binding sites in great excess c o m p a r e d to 6 and ~c sites. Although

binding studies in tissue h o m o g e n a t e s have characterized the spinal ,u, b, and 1¢ opioid sites with regard to ligand specificity, binding affinity, and r e c e p t o r density, binding studies lack the anatomical resolution to determine where opioid receptors are distributed in the spinal cord and the density of opioid receptors in specific regions of the spinal cord. R e c e p t o r a u t o r a d i o g r a p h y with highly-selective opioid radioligands, however, can provide both increased spatial resolution and quantitative methods to provide detailed information of opioid binding sites with respect to regions of primary afferent terminals and neuronal organization of the spinal cord. Previous autoradiographic studies of rat spinal cord d e m o n s t r a t e d high concentrations of putative/~, 6, and ~: binding sites within the superficial laminae (I-11) of the dorsal horn, with lesser amounts of opioid binding observed throughout the r e m a i n d e r of the spinal cord 3' ii1.1~.21.22.42.44.4~.51.59 Although these studies have provided consistent qualitative data on the overall distribution of opioid sites, quantitative a u t o r a d i o g r a p h i c stud-

" Present address: Department of Physiology and Pharmacology, College of Osteopathic Medicine, Oklahoma State University, Tulsa, OK 741(17-1898, U.S.A ** Present address: Department of Anatomical Sciences, Medical School, University of Oklahoma, Oklahoma City, OK, U.S.A. Correwondence: V.S. Scybold, Department of Cell Biology and Ncuroanatomy, 4-'135 Jackson Hall, 321 Church St., S.E., University of Minnesota. Minneapolis. MN 55455. U.S.A.

78 ies 3a'3b''t2 d i s a g r e e as to the relative a m o u n t s of ~, O, and o p i o i d sites within specific regions of the spinal cord. W i t h r e s p e c t to l a m i n a e I and 1I, r e p o r t s oft~ sites range f r o m a p p r o x i m a t e l y 7 0 - 9 0 % , b sites f r o m 3 - 2 5 % , and r: sites are c o n s i s t e n t at 5 - 7 % o f the total o p i o i d binding. O n e r e a s o n for the d i s c r e p a n c i e s in t h e s e data m a y be that t h e studies e m p l o y e d m o d e r a t e l y selective radiolig a n d s in c o n j u n c t i o n with selective u n l a b e l e d ligands to label 6 and K o p i o i d sites in s o m e cases. W e sought to r e s o l v e t h e s e d i s c r e p a n c i e s by e m p l o y i n g c u r r e n t l y available, highly selective, radioligand, a , b, and r o p i o i d r a d i o l i g a n d s in a u t o r a d i o g r a p h i c studies. In the p r e s e n t p a p e r , we e m p l o y e d the highly-selective ligands, [3H]sufentanil3~, [ 3 H } D P D P E ([D-Pen2'S]-en kephalin43), and [3H]U69593 ( U p j o h n compound31), to

below. The brain was minced in an embedding mold before freezing to optimize homogeneous tissue sections. Brain tissue was cut on a cryostat at a thickness of 10 ~um. Slides with sections of rat brain were incubated in triplicate for determination of total and nonspecific binding for each radioligand. At the end of the last cold wash, tissues were wiped from the slides with a glass Cdter disc (GF/A. Whatman) and placed into scintillation vials with 0.5 ml absolute ethanol and 10 ml Ecoscint A (National Diagnostics) for liquid scintillation spectroscopy. Equilibrium dissociation constants (Kj) and receptor capacity (Bm~) from multiple experiments were determined from Scatchard plots of data for 7 concentrations of radioligands incubated in triplicate. Determination of inhibitory constants (K,) and concentrations producing 50% blockade of binding (IC~,) for unlabelled opioid ligands (see Table II) were performed in a similar manner. Each parameter was determined with a minimum of 3 rat brains in a minimum of 3 experiments for estimation of Kj, or 2 experiments for determination of inhibitory constants. Data were analyzed by the LIGAND program adapted for an IBM P C 37 and are presented as the mean + S.E.M. of values determined in separate experiments

d e t e r m i n e o p t i m a l assay c o n d i t i o n s and to c h a r a c t e r i z e # , 6, and r o p i o i d binding sites in tissue sections of rat brain. S u b s e q u e n t l y , we q u a n t i f i e d the r e l a t i v e a m o u n t of o p i o i d b i n d i n g a m o n g s e l e c t e d regions of rat l u m b a r spinal c o r d f r o m a u t o r a d i o g r a m s of tissue sections using i m a g e analysis and c o m p u t e r i z e d grain c o u n t i n g . T h e p r e s e n t results c o n f i r m the selectivity of the radioligands on

slide-mounted

tissue

sections

and

demonstrate

a

differential distribution of # , 6, and ~c binding sites with respect to the r e g i o n s of the spinal gray k n o w n to possess b o t h o p i o i d n e u r o n s and t e r m i n a l s of n o c i c e p t i v e p r i m a r y afferents. T h e s e results f u r t h e r e l u c i d a t e the n e u r o c h c mical o r g a n i z a t i o n o f e n d o g e n o u s o p i o i d systems in the rat spinal c o r d with respect to n o c i c e p t i v e processing. MATERIALS AND METHODS Characterization o f ligand binding The following radiolabelled opioid ligands were used to identify #, 6, or r opioid binding sites: [3H]sufentanil (15 Ci/mmol, Janssen Pharmaceutica, Belgium), [3H]DPDPE (31 Ci/mmol [D-Pen2'S]enkephalin, Amersham), and [3H]U69593 (68 Ci/mmol, Amersham). The protocol for the binding of [3H]dihydromorphine, a non-selective opioid agonist, on slide-mounted tissue sections in vitro has been previously described m , and this protocol was adapted for use in the present experiments as outlined below. The binding protocols for all 3 ligands differed only in the time of radioligand incubation. Each protocol included an initial 15-rain pre-incubation in 50 mM Trizma, pH 7.6 (assay buffer) at 25 °C with the addition of 100 mM NaCI and 50 /tM NaGTP to enhance dissociation of endogenous ligands. Following this pro-incubation. two 5-min washes in the assay buffer were performed. The tissue sections were incubated with the radioligand in the assay buffer with the addition of 0.2% BSA and peptidase inhibitors (0.002% bacitracin and 1 /.tM CPAB [N-(l-carboxy-2-phenylethyl)-PhepAB]; see ref. 2). The following incubation times were determined empirically as the minimum time required to yield equilibrium binding conditions: 60, 90, and 30 min for the u, b, and radioligands, respectively. Following incubation with radioligand. sections were rinsed 3 × 10 min in 50 mM Trizma, pH 7.7 at 4 *C. Studies to determine optimal binding conditions, equilibrium dissociation constants, and inhibitory constants of opioid ligands were carried out on 10-~m sections of minced rat brain. Data were collected on individual brains. Sections of rat brain were obtained of the whole brain minus cerebellum from animals as described

Preparation of autoradiograms Spinal cord samples were taken from adult, male rats (SpragueDawley, 200-220 g). To minimize the usage of animals, these animals served as the untreated control group for other experiments. Tissues were obtained after anesthetizing the animals (12.6 mg chloral hydrate and 2.9 mg pentobarbital/100 g body weight) and transcardially perfusing each animal with 180 ml of 0.16 M sucrose in 0.1 M phosphate-buffered saline solution. Rat brains were removed for ligand characterization studies as described above. Following a rapid laminectomy, spinal segment L4 was identified, excised, blocked and frozen for cryostat sectioning. Serial cryostat sections (10 #m) of transverse orientation were thaw-mounted onto gelatin-coated slides and stored for l-4 weeks at -17 °C until use in binding studies. Autoradiographic data were collected from a total of 3 animals Near adjacent spinal cord sections through segment L4 were incubated in [3H]sufentanil, [3H]DPDPE, and [3H]U69593 to label 1~. b, and K binding sites, respectively. [~H]Sufentanil was used at a concentration of 0.3 nM; [3H]DPDPE at 4.7 nM, and [aH]U69593 at 3.0 nM. Thus, each radioligand was used near its calculated equilibrium dissociation constant for its respective receptor (see Table I). Non-specific binding of ,u. ~, and r radioligands was defined by the addition of 1 #M of unlabeled DAMGO [t,Ala2-Met-Phe4-Gly-olS]-enkephalin), DADLE ([D-Ala2-o-LeuS} enkephalin), or U50488H, respectively, to the solution containing the radiolabeled ligand. Spinal cord sections were organized such that samples for total and non-specific binding for each opioid ligand were adjacent tissue sections, and each pair of .sections followed sequentially for the 3 ligands. Autoradiograms were prepared as described by Young and Kuhar 6~ using NTB-3 nuclear emulsion (Kodak) diluted I:1 with distilled H20. The permanent apposition between the coverslip and slide on one end of the slide and therefore

TABLE 1 Binding characterLstics of I~, t~, and ~c opioid radioligand3 on tissue sections of rat brain

Data are expressed as mean + S.E.M. B m,,~ (fmol/mg prot.)

Hill coefficient

Radioligand

n"

Ka (nM)

[3H]Sufentanil [3H]DPDPE [3H]U69593

9 9 5

11.46 ___11.(19 97.4_+ 10.2 0.93 + 0.07 1.03+0.03 4.31 +_11.57 34.5+4.1 5.3 + 0.6 0.94 + 0.07 2.27 +__I).38

" Number of experiments performed.

79 precise registration of the emulsion-coated coverslip to the tissue allowed analysis of specific regions in adjacent tissue scctions incubated under conditions of total binding or non-specific binding. Autoradiograms for quantification were developed after an exposure time of 13 weeks at 4 °C for each ligand. For illustrative purposes (scc Fig. 3) some autoradiograms were exposed for 2(1 weeks. Autoradiograms of Tritium microscalc standards (Amersham) wcrc exposed with each group of tissue to ensure that the autoradiographic data fell within the linear response range of the emulsion and to allow conversion of the densities of autoradiographic grains to fmol of ligand bound/rag tissue equivalent (t.e.) for each opioid ligand

Quantification of autoradiograms For each ligand, an area of 27,5(~) um 2 was analyzed within the substantia gelatinosa, the lateral reticulated area of the deep dorsal horn, and dorsolatcral to the central canal. The 3 regions analyzed for each side of each spinal cord section arc shown in a schematic diagram (Fig. 1). For convenicnce, these regions of analysis were noted as laminae I-II. lamina V, and lamina X, respectively, as determined by reference to the cytoarchitcctural studies of rat spinal cord a~. Due to the variation in the preservation of spinal cord shape in fresh-frozen spinal segments, internal landmarks were used to define consistent areas within different cord samples (see legend of Fig. 1). Four pairs of adjacent sections (one section of each pair incubated for total and one section for non-specific binding) wcrc analyzed for each ligand from each spinal segment obtained. "l'hcsc 3 regions were sclcctcd fi)r analysis as these areas of the spinal cord contain the highest densities of opioid peptide immunoreactive terminals and neurons that express opioid peptides 4<4s as well as opioid receptors 5". These same regions also receive a major projection of nociccptivc primary afferent fibers and have been shown to be areas of processing of nociceptive information TM. A video image of autoradiographic grains in the emulsion above each region was obtained under brightficld microscopy at a magnification of x 125. The video image was digitized on a Macll computer with

an image proccssing program (Image v. 1.13b, kindly supplied by Dr. Rasband at NIH). The contrast of the images of silver grains was enhanced to facilitate selective quantification of the autoradiographic grains. A threshold function was used to sum the pixels representing silver grains. This procedure produced a linear relationship between pixcls and amount of radioactivity when applied to the autoradiograms of tritium microscale standards. As the nonspecific binding was greater than 5% in many cases, the specific binding of each region of each slide was determined by the subtraction of the non-specific binding of the corresponding region on the adjacent section. For each region examined in the autoradiograms, the specific binding, expressed in density of autoradiographic grains, was converted to nCi/mg tissue equivalents (t.e.) using the equation obtained from the linear regression of data for the microscale standards. The amount of bound ligand in fmol/mg t.e. was calculated from the specific activity of the radioligand. Estimations of the total binding capacity for each ligand was made by consideration of the percent of receptor occupancy occurring at the concentration of the radioligand used to incubate the tissues and the equilibrium dissociation constant according to the law of massaction.

Statistical analyses Differences in the density of binding sitcs among the regions wcrc assessed by one-way ANOVA followed by a post-hoe NeumanKculs multiple comparisons test. Comparisons were made at the P < 0.05 level of significance. RESULTS

Characterization of opioid ligand binding T h e binding characteristics of e a c h r a d i o l i g a n d are s u m m a r i z e d in Table I. In s l i d e - m o u n t e d sections of rat brain,

the .u-specific ligand,

[3H]sufentanil,

exhibited

high-affinity binding with an a p p a r e n t e q u i l i b r i u m dissociation c o n s t a n t

(Kd) o f 0.46

+

0.09

nM

(mean

_+

S . E . M . ) . T h e binding c a p a c i t y (B . . . . ) of [SH]sufentanil was 97.4 _+ 10.2 f m o l / m g p r o t e i n . T h e ¢~-specific ligand, [ ~ H ] D P D P E , b o u n d with an a p p a r e n t K d of 4.31 _+ 0.57 nM

and

B ..... of 34.5

_+ 4.1

fmol/mg protein.

The

~c-spccific ligand, [3H]U69593, b o u n d with an a p p a r e n t K a of 2.27 + 0.38 nM and B . . . . of 5.3 + 0.6 f m o l / m g protein. C o m p e t i t i o n curves for each specific o p i o i d radioligand arc shown in Fig. 2. I n h i b i t o r y c o n s t a n t s (Ki) and concentrations

producing

50%

inhibition

of

binding

(ICso) d e r i v e d f r o m c o m p e t i t i o n studies are given in Table I1. In the c o m p e t i t i o n e x p e r i m e n t s with [3H]sufcntanil, D A M G O "'l

region of analysis (27.500 sq ~.m)

e x h i b i t e d an a p p a r e n t

K i o f 0.77

nM. T h e r e m a i n i n g u n l a b e l e d o p i o i d ligands e x h i b i t e d 1 mm

Fig. I. Regions of autoradiograms analyzed with respect to laminar organization of the gray matter of the rat lumbar spinal cord. Laminae I-!I: at V2 the length of a line from the medial curvature of the superficial dorsal horn to the dorsal root entry zone (*). Lamina V: at .% the length of a line from the ventral aspect of the dorsal columns to the lateral curvature of the superficial dorsal horn. Lamina X: at the intersection of 2 lines; the first line extending ventrally from the medial curvature of the dorsal horn, the second line extending laterally from the midpoint between the central canal and the ventral aspect of the dorsal columns.

the following rank o r d e r of p o t e n c y , f r o m g r e a t e s t to least, in c o m p e t i t i o n with I3H]sufentanil: DSLET > DPDPE

DADLE

>

> U 5 0 4 8 8 H (sec Fig. 2 A and Table

11 for identification of ligands). T h e s e d a t a are consistent with the high selectivity of [3H]sufentanii for the ~ o p i o i d site. In c o m p e t i t i o n with the delta ligand, [ 3 H I D P D P E , selective o p i o i d ligands e x h i b i t e d a rank o r d e r of D S L E T > ENK-NH 2 > DADI,E

> sufentanii > D A M G O

>

U69593, consistent with the selectivity of [ ~ H ] D P D P E for

80 the 6 opioid site (Fig. 2B, Table II). In order of potency of opioid agonists in competition with the r-selective ligand, [3H]U69593, was U50488H > sufentanil > D A M G O > D A D L E > DPDPE, as expected for the binding of [3H]U69593 at the r opioid site (Fig. 2C, Table If). Distribution o f opioid binding sites in rat spinal cord Mu binding sites. The binding of [3H]sufentanil to transverse sections of rat spinal cord revealed a high density of autoradiographic grains over the superficial dorsal horn, with much fewer grains observed throughout the remainder of the spinal cord (Fig, 3A). Incubation under conditions for non-specific binding decreased the autoradiographic signal (Fig. 3B). Specific binding for [3H]sufentanil was 89.7 + 0.9% for laminae I - I I , 63.1 _+ [3 H ] - S U F E N T A N I L

80

\

"%\

"%"i

"

60

•

(~ Z

.~0

-9

-8

-7

-6

-5

U$0488H

.4

[3H].DPDPE

3.7% for lamina V, and 66.6 _+ 4.8% for lamina X (n = 10 paired sections). Analysis of the density of autoradiographic grains representing [3H]sufentanil binding in the 3 regions examined showed that laminae I - l I had a binding capacity of 579.8 _+ 21.6 fmol/mg i.e., lamina V had 74.0 _+ 4.8 fmol/mg t.e., and lamina X had 76.4 + 2.5 fmol/mg t.e. (Fig. 4). The density of [3H]sufentanil binding sites in laminae l - l l of the spinal cord was approximately 8 times that of laminae V and X. These comparisons, however, are somewhat biased by the differential quenching of the autoradiographic signal among the regions. Delta binding sites. Autoradiography of the binding of [-~H]DPDPE demonstrated a slightly higher density of grains over the superficial aspect of the dorsal horn of the rat spinal cord with fewer autoradiographic grains dispersed throughout the remainder of the spinal cord (Fig. 3C). However the distribution of 5 opioid sites was more homogenous throughout the spinal gray compared to that of either the /, or ~¢ opioid sites. Incubation of [3H]DPDPE under conditions for non-specific binding resulted in a low autoradiographic signal (Fig. 3D). The specific binding of [3H]DPDPE was 60.4 + 3.5% in laminae 1/I1, 50.4 _+ 0.7% in lamina V, and 58.3 + 6.5% in lamina X (n = 12 paired sections). Quantification of grains in selected regions of the spinal gray matter showed that the binding capacity of [3H]DPDPE was 44.9 + 2.0 fmol/mg t.e. in laminae VII, 29.9 + 2.0 fmol/mg I.e. in lamina V, and 37.2 + 2.1 fmol/mg t.e. in for lamina

TABLE I1 Competition of opioid ligands for [¢H]sufentanil, ff H]DPDPE, and [~H]U69593 binding sites o --,-% . . . . . . . . . . . . . . . Z UJ

0

O n-

-10

.g

.8

7.. .7

-6

.5

, 4

[3H]-U69593

W a.

{.0

Radioligand

Opioid ligand

K, (nM)

IC w (nM)

[3H]Sufentanil

DAMGO DADLE DSLET DPDPE U50488H

0.77 51 21.2 506 549

1.61 10.5 44.11 1110 121X)

[3H]DPDPE

DSLET ENK-NH2 DADLE sufentanit DAMGO U69593

0.55 0.68 3.7 21.0 111.9 " 10,000

1.11 1.41 7.7 41/.9 212.9 ">10.000

[3H]U69593

U50488H sufentanil DAMGO DADLE DPDPE

3.31 49.3 1470 28111 > 10,000

5.95 86.0 2690 4940 > 10,000

80

40 20 o

C

l0

.9

.8

.7

.6

5

4

LOG CONCENTRATION DISPLACER

Fig. 2. Competition curves of opioid ligands against p, b, and K-selective opioid radioligands on sections of rat brain, Slidemounted tissue sections were incubated as described in the Materials and Methods section. Radioligand concentrations used were 0.3 nM [or [3H]sufcntanil, 4.7 nM for [3H]DPDPE, or 3.0 nM for [3H]U69593. Symbols for unlabelied opioid compounds arc defined in the key in the top panel, and definitions of acronyms arc given in Table If. Competition curves of ligands with Ki's greater than 10 p M arc not drawn.

Abbreviations: DAMGO, [D-Alag-MetPhe4-Gly-olS]-enkephalin; DADLE, [o-Ala2-D-Leuq-e~halin; DSLET, [D-Sera-Leu~-ThP]enkephalin; DPDPE, [D-Pen • ]-enkephalin; ENK-NH> leucineenkephalinamide.

81

13

,a

C

.

.

.

...

.

.

lt'/

F

Fig. 3. Autoradiograms of u, ,6. and x opioid binding in adjacent tissue sections from rat spinal cord segment L4. A: tissue section was incubated with [~H]sufentanil (0.3 nM). B: tissue section was incubatcd in [~H]sufentanil + 1 I~M DAMGO. C: tissue section was incubated with I~II]DPDPE (4.7 nM). D: tissue section was incubated in I3H]DPDPE + 1 I*M DADLE. E: tissue section was incubated with [~H]U69593 (3.0 nM). F: tissuc section was incubated in [3HIU69593 + 1 I~M U504881t. Scale bar = 4(~),um.

X (Fig. 4). Thus, of the 3 opioid radioligands, the density of [~H]DPDPE binding sites were the most uniformly distributed among the 3 regions analyzed.

Kappa binding sites. A u t o r a d i o g r a m s of K binding from spinal cord sections arc shown in Fig. 3E. The binding of [3H]U69593 gave a relatively m o d e r a t c signal of a u t o r a d i o g r a p h i c grains along the superficial aspect of the dorsal horn and a lesser amount of grains throughout

the remainder of the spinal cord (Fig. 3E). Incubation of sections under conditions for non-specific binding substantially decreased this signal (Fig. 3F). The specific binding of [3H]U69593 was 79.4 + 1.7% in laminae 1-II, 52.9 _+ 1.4% in lamina V, and 55.6 + 4.2%. in lamina X (n = 12 paired sections). Analysis of the 3 regions showed that thc binding capacity of [3H]U69593 was 11.1 + 0.9 fmol/mg t.e. in lamine I - I I , 3.5 _+ 0.6 fmol/mg t.e.

82

E I

• [] []

150

MU DELTA KAPPA

o

E 100 co w I-

50 r.,

5 i

El. O

LAM

I-II

LAM V

LAM X

REGION OF ANALYSIS

Fig. 4. Binding capacity of ~, 6, and K opioid sites within selected regions of rat spinal cord. Histograms reresent values for mean _+ S.E.M. of tissue sections from 2-4 replicate sections each from 3 animals. Mu sites (solid bars) were significantly greater than 6 (striped bars) or K sites (stippled bars) in each region examined (one-way A N O V A followed by Neuman-Keuls, P < 0.05).

in lamina V, and 2.9 _+ 0.3 fmol/mg t.e. in lamina X (Fig. 4). Thus, the density of [3H]U69593 binding sites in laminae I-II was approximately 3 times that of laminae V and X.

Comparison of I~ t), and K binding sites in spinal cord As illustrated in Fig. 4, the density of/~ sites was significantly greater than both b and K sites within all 3 regions examined (ANOVA with Neuman-Keuls posthoc test, P < 0.05). Additionally, for all 3 ligands, the binding capacity observed in laminae 1-II was significantly greater than the binding capacity found in laminae V and X for each iigand (Fig. 4, ANOVA followed by Neuman-Keuis, P < 0.05). When considered as a percentage of the total opioid binding capacity (g + ~ + K) observed within each region analyzed and assuming a Z o

0U.I er

100

-

• []

91

[]

er

m O.

MU DELTA KAPPA

80 69

66

fJ)

__. (/}

60

.J

~O

32

40-

&

Iii

O

lz 14,1

o n,, u,l

200 I.AM I-II

iL

LAM V

I.AM X

n

REGION OF ANALYSIS

Fig. 5. Percentage of/~, b, or K binding capacity in rat spinal cord sections for each region examined. Average values for each opioid ligand are plotted as percentage of total opioid binding capacity (u + 6 + K) of each region for u (solid), b (striped), or K (stippled) sites.

uniform density throughout each region, the binding capacity of g sites contributed to more than 90% of the total opioid binding capacity of laminae I-II. However, the/~ binding capacity decreased to 69% in lamina V and to 66% of total opioid binding capacity of lamina X (Fig. 5). In contrast, the percentage of 6 binding capacity was less than 10% of total opioid binding capacity of laminae I-II, but increased to 28% in lamina V and to 32% of total opioid binding capacity of lamina X. The K binding capacity maintained an overall low 2-3% of total opioid binding capacity observed in all 3 regions (Fig. 5). DISCUSSION

Using highly selective, radiolabeled ligands for/~, 6 and K opioid receptor, we have found a differential distribution of these receptors with respect to the laminar organization of the gray matter of the rat spinal cord. Of the previous quantitative autoradiographic studies of the spinal cord 3a'3b'42, our results are most consistent with those of Morris and Herz 42. Together, these studies underscore the sizeable mismatch between the high density of ~ opioid binding sites in laminae I and II and the absence of an endogenous ligand with high affinity for /~ receptors. Furthermore, the consistencies between our data (obtained with highly selective, radiolabeled opioid ligands) and those of Morris and Herz (obtained by blocking nonselective radiolabeiod opioid ligands with selective, unlabeled ligands) demonstrate that both direct and indirect approaches to the localization of opioid receptors are valid.

Characterization of opioid binding sites The characteristics and selectivity of [3H]sufentanil; [3H]DPDPE, and [3H]U69593 binding in rat brain homogenates has been previously described a. To ensure that these radioligands maintained their selectivity under the experimental conditions needed for receptor autoradiography, binding characterization was performed on slide-mounted tissue sections. Due to the relatively low capacity of opioid binding in spinal cord sections, experiments on ,sections of rat brain were used to optimize the incubation conditions and characterize the binding selectivity. The results of the binding experiments in the present study are in general agreement with previous studies using these same radioligands on rat brain homogenates. The Kj values of the present study are within the range of those previously reported for [3H]sufentaniiS.33, [3H]DPDPES"22"36 and [3H]U6959331 45. The results of the competition experiments with a variety of selective opioid ligands (Table I1) confirmed the selectivity of [3H]sufentanil, ['~H]DPDPE, and [3HIU69593 for t~, 6, and K opioid sites, respectively.

83 Therefore, the results of the characterization experiments validated the high selectivity of these opioid radioligands under conditions used in the present study for receptor autoradiography.

Distribution of opioid sites in spinal gray matter The overall autoradiographic signals obtained from the incubation of rat spinal cord sections with [3H]sufentanil, [3H]DPDPE and [3H]U69593 were consistent with the qualitative results of similar studies previously reported for rat spinal cord using generally less selective ligands 3" 1o.18,21.22.44.46.51.5o. The present results are also in good agreement with a previous quantitative study examining the distribution of opioid binding sites in rat spinal cord utilizing [3HIDAMGO, [3H]DADLE and [3H]bremazocine under conditions selective for visualization of/~, d, and ~¢ sites a2. The results of our quantitative analysis demonstrate a preponderance of /~ opioid sites within all regions analyzed. From estimates of total binding capacity, the density of/~ sites was greater than 500 fmol/mg t.e. within the region of laminae I-II examined. Although it is not possible to convert these values to fmol/mg protein for comparison to homogenate studies, an area of the same size was examined in regions of laminae 1-II, V and X. Thus, it is possible to make comparisons across the rcgions analyzed and among the different types of opioid binding sites. Our results show that the binding capacity of all 3 opioids among the 3 regions analyzed was greatest in laminae I-II (Fig. 4). Consideration of the percentage of opioid binding capacity for each ligand with respect to total opioid binding capacity observed within each region rcveals that although/~ binding comprised over 90% of total opioid binding in laminae 1-11, the relative amount ofl~ binding capacity decreased to about 60% in laminae V and X (Fig. 5). In contrast, 6 binding sites comprised less than 1(1% of total opioid binding capacity in laminae I-II, but d binding capacity increased to about 30% in laminae V and X. Kappa binding sites maintained an ovcrall low percentage of total opioid binding capacity (2-3~,) in all 3 regions examined. Givcn the differential distribution of/~, 6, and ~copioid binding sites within the gray matter of the dorsal horn of the rat spinal cord, how does this distribution relate to the localization of spinal endogenous opioid peptides? The 3 regions of spinal gray matter analyzed were selected on the basis of the high density of terminals and perikarya immunoreactive for opioid peptides. With regard to the distribution of Mct-enkephalin- and dynorphin-likc immunorcactivity, both opioid peptides are found in ncuronal cell bodies and fibers throughout thc dorsal half of the spinal cord, with the greatest densities in lamina 1/II and lamina V 4'12'14"38"40'48. There are also

moderate amounts of Met-enkephalin- and dynorphinimmunoreactive perikarya and fibers in lamina X 17'48. The comparatively high density of opioid immunoreactive varicosities and perikarya in laminae I/I1 is consistent with the high density of opioid binding sites seen in this region for all 3 opioid ligands in the present study. As Met-enkephalin shows the greatest potency among the naturally-occurring endogenous opioid peptides at d receptors and dynorphin at ~c receptors 25"2s, the most parsimonious explanation for the preponderance of 6 and ~copioid binding sites in laminae 1-1I is that these binding sites mediate the action of the opioid peptides also found in this region. However, a number of caveats limit this broad interpretation. First, while d and ~¢sites may serve to mediate the effects of Met-enkephalin and dynorphin, respectively, our results show that binding capacity of/~ sites in laminae I-I1 greatly exceeds that for the 6 and ~c sites. Thus, there is the enigma of an overwhelming density of It opioid sites in the superficial dorsal horn without an identified highly-selective endogenous/~ opioid ligand for the/~ opioid binding site within the spinal cord. The opioid peptide, fl-endorphin, is more selective for/~ opioid sites than 6 or ~ sites 25"2x, however, the localization of fl-endorphin within the adult rat spinal cord is limited to a sparse distribution of immunoreactive fibers of supraspinal origin terminating around the central canal 16'5s. Dermorphin, an endogenous opioid iigand first isolated from frog skin, binds with high affinity and selectivity to /~ receptors in homogenate preparations 3° and exhibits a pharmacological profile of a potent .u-selective agonist after intrathecal administration in rats 54. Dermorphin immunoreactive elements have been visualized immunohistochemically in mammalian CNS, but have not yet been detected in spinal cord tissue 5. Finally, morphine itself has been isolated from rat and human CNS and is present in high concentrations in lumbar rat spinal cord where it is increased in arthritic rats ~1. Whether any of these substances or as yet undiscovered factors is the primary endogenous ~ opioid ligand in the adult vertebrate spinal cord remains to be proven. Another caveat limiting the hypothesis of a one-to-one correlation of opioid ligand and receptor type is that the actual opioid peptide sequence released and active at opioid synapses in the spinal cord is not known. The length of an opioid peptide sequence dramatically alters the selectivity and potency of the peptide at the different opioid receptors 2~'2s. For example, the spinal cord contains dynorphin-converting enzyme which degrades dynorphin A to leu-enkephalin-Arg ~, a sequence which is more potent at d receptors than ~c receptors 35. Furthermore, the availability of opioid receptor type(s) at the synapse may prevent a one-to-one correlation of opioid

84 receptor type to e n d o g e n o u s ligand. While the endogenous opioid peptides show a relatively high degree of selectivity in well-defined isolated tissues preparations in vitro, the concentration of any e n d o g e n o u s opioid ligand

basis for the pharmacological effects of intraspinally administered p, b, and K selective opioids 47'49'5~, the most

in the synapse may be high enough to allow interaction at any of the opioid receptor types. Thus, the above

will come from functional studies of identified spinal

considerations preclude a one-to-one correlation of any spinal opioid peptide with a given type of opioid

well-defined electrophysiological studies has suggested that only the nociceptive n e u r o n s in the marginal zone of the rat spinal cord are inhibited by b-selective opioids 15.

receptor. Finally, the more general consideration of receptor mismatch has gained much support from numerous anatomical studies of the rat central nervous system 23. For example, although opioids inhibit the release of putative transmitters from the central terminations of primary afferents 27 and opioid receptors are present on primary afferent fibers t3'32, direct enkephalinergic axo-axonic synapses have not been observed at the ultrastructural level 26's5. This suggests that, in general, the location of opioid binding sites and the endogenous ligands for those sites may not be precisely correlated. Although the presence of opioid sites demonstrated in this and previous studies provides a neuroanatomical REFERENCES 1 Akil, H., Watson, S.J., Young, E., Lewis, M.E. and Khatchaturian, H., Endogenous opioids: biology and function, Annu. Rev. Neurosci., 7 (1984) 223-255. 2 Almenoff. J. and Orlowski, M., Membrane-bound kidney neutral metalloendopeptidase: interaction with synthetic sub° strates, natural peptides, and inhibitors, Biochemistry, 22 (1983) 590-599. 3 Atweh, S.E and Kuhar, M.J., Autoradiographic localization of opiate receptors in rat brain, I. Spinal cord and lower medulla. Brain Research, 124 (1977) 53-67. 3a. Besse, D., Lombard, M.C., Zajac, J.M., Roques, B.P. and Besson, J.M., Pre- and postsynaptic location of mu, delta and kappa opioid receptors in the superficial layers of the dorsal horn of the rat spinal cord. In: Proceedings of the International Narcotics Research Conference (INRC) '89, Alan R. Liss, New York, 1990, pp. 183-186. 3b. Besson, J.M., Lombard, M.C., Zajac, J.M., Besse, D.. Peschanski, M., Roques, B.P., Opioid receptors in the dorsal horn of intact and deafferented rats: autoradiographic and electrophysiologicai studies. In: E Cervero G.J. Bennett, P.M. Headley (Eds.), Processing of Sensory Information in the Superficial Dorsal Horn of the Spinal Cord, Plenum Press, New York, 1989, pp. 415-428. 4 Botticelli, L.J., Cox, B.M. and Goldstein, A., lmmunoreactive dynorphin in mammalian spinal cord and dorsal root ganglia. Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 7783-7786. 5 Buffa, R., Solcia, E., Magnoni, E., Rindi, G., Negri, L. and Melchiorri, P., Immunohistochemical demonstration of a dermorphin-like peptide in the rat brain, Histochemistry, 76 (1982) 273-276. 6 Castanas, E., Blanc, D., Bourhim, N., Cupo, A., Cantau, P. and Giraud, P., Reassessment of opioid binding sites in the rat brain, Neuropeptides, 7 (1986) 369-380. 7 Cervero, F., Dorsal horn neurons and their sensory inputs. In: T.L. Yaksh (Ed.), Spinal Afferent Processing, Plenum Press, New York, 1986, pp. 197-213. 8 Clark, M.J., Carter, B.D. and Medzihradsky, F., Selectivity of ligand binding to opioid receptors in brain membranes from the rat, monkey, and guinea pig, Eur. J. Pharrnacol.. 148 (1988)

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Much work along these avenues of investigation will be needed to further our nascent understanding of the detailed neurochemical organization of spinal endogenous opioid systems with respect to the nociceptive processing.

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