Ncuropeptides 5: 81-84, 1984

OPIATE BINDING IN THE RAT SPINAL CORD: EVIDENCE FOR MU AND DELTA SITES

J.R. Traynor and M.J. Rance. Department of Chemistry, University of

Technology, Loughborough, Leics. LEll 3TU, U.K. and ICI Pharmaceuticals
Division, Alderley Park, Macclesfield, Cheshire SK10 4TG, U.K. (Reprint
requests to JRT)

ABSTRACT

The presence of binding sites for ligands of mu and delta, in

addition to kappa selectivity, have been demonstrated in the
lymbo-sacral spinal cord of the rat by direct binding of
[ Hlligands. These findings have been confirmed by competition
studies. This information helps explain the observed pharmacology
of opioids after intrathecal administration.

INTRODUCTION

There is much evidence to suggest the dorsal horn of the spinal cord as
an important site for the modulation of nociceptive information. In
particular, attention has been focussed on the importance of this area for
the action of kappa agonists (1,2). However, intrathecal (i.t.) and
epidural morphine and other mu agonists are used to achieve analgesia in
humans (3) and in animal models i.t. mu and delta agonists are effective in
heat nociception tests (4) and are able to block the actions of i.t.
Substance P in mice (5). These findings are difficult to reconcile with
reports (6,7) that lumbo-sacral spinal tissue from several species lacks mu
and delta receptors.

To explain this apparent discrepancy we have sought to further

characterise spinal binding sites for opioids. In particular we have
investigate the ability of the mu selective [3H]GLYOL and the delta-
1HI-DADLE to specifically bind to the lumbo-sacral spinal cord of
selective I:
t e rat, and compared this with the binding of the kappa agent
[9HlBremazocine.

MATERIALS AND METHODS

The following labelled_\igangs were used. [3H]Tsr-D-Ala-Gly-MePhe-NH

(CH )pH(GLYOL)(60Ci rmnol ), C H]Tyr-D-Al$2,D-Leu -enkephalin (DADLE) (32Ci
f- ) (Amersham International plc) and [ H]Brematocine (3OCi mrol'l) (New
am10
England Nuclear). Unlabelled ligands used were: DADLE (Sigma, U.K.),
GLYOL,N,N-diallyl-Tyr-Aib-Aib-Phe-LeuOH(ICI 174864) and
ethylketocyclazocine (EKC) (Sterling Winthrop).

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 Lumbo-sacral cords were obtained from male Alderley Park strain rats.
Homogenates were prepared as in earlier studies (8) but included a
pre-incubation period (37 for 45 min) to destroy endogenous enkephalins.
Binding assays were performed as previously described (8) for 60 min at 37O.
Specific binding defined by parallel incubations in the presence of
diprenorphine (1pM) or naloxone (10uM) was >60%.

RESULTS

C3H]Opioid ligands with mu and delta pharmacological profiles exhibited

saturable binding to lumbo-sacral spinal tissue of the rat . The data (Fig
1) show the highly selective mu ligand C3H]GLYOb bound to approximately
twice the number of sites as the delta ligand [:HIDADLE which was assayed in
the presence of cold GLYOL WnW$ [3~]~~~~~) to suppres its mu component.
Both [3H]GLYOL and C3H1DADLE bound with high affinity. [3H]Brematocine
bound to many more sites than the mu and delta ligands and afforded
curvilinear Scatchard plots suggesting recognition of more than one receptor
site.

I a

Fi . 1: Scatchard plots of the binding of a) C3H]GLYOL and b) c3H]DADLE,

9t;-
in t e presence of 1OnM GLYOL/K DADLE, to rat lumbo-sacral cord. Points
represent means obtained from tR ree separate experiments.

GLYOL and the selective delta antagonist ICI 174864 displaced bound C3H]
Bremazocine (2nM) in a bi-phasic manner. The first part of the displacement
curves represented 34% and 18% respectively of specifically bound material.
Approximate 1~50 values for this first phase for GLYOL of 6nM and for ICI
174864 of 200nM confirmed these compounds were displacing from mu and delta
sites. Indeed in the presence of suitable concentrations of these ligands
to suppress binding to mu and delta sites (15nM GLYOL and l$l ICI 174864/KD

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C3H]Bremazocine) thf binding of bremazocine was reduced to give a B-max of
45fmols.mg protein' .

Competition binding experiments (Table 1) further confirm the identity of

the mu and delta sites.

Table 1 I hibitory effects of opioids on the binding of C3H]GLYOL and

+H]DADLE*

Ki (nM)

Competing Ligand C3H]GLYOL C3H]~A~~E

GLYOL 2.5 f 0.1 438 f 86

DADLE 11.1 * 1.5 4.4 f 0.9
ICI 174864 6192 f 834 223 2 43
EKC 2.5 f 0.1 5.8 -+2.2

Values are means f s.e.m. of 3 observations

*After suppression of mu component with 1OnM GLYOL/KD [3H]DADLE

DISCUSSION

The results amply demonstrate the presence of mu and delta binding sites
in rat lumbo-sacral spinal cord, thereby supporting our previous findings
(8). The affinities of GLYOL and DADLE for their respective sites more
closely reflect those observed in brain (9) than our previous work. This
discrepancy is probably due to the improved specific binding, and therefore
ease of interpretation, obtained in this present study. The density of mu
and delta receptors, 30% and 14% respectively of total opioid sites, is much
lower than that reported in rat brain (46% and 42%) (9).

The presence of each sub-type in the cord supports pharmacological

evidence for a spinal action of opioids of varying specificity. Furthermore,
the relative proportions of subtypes may help to explain some of the
armacological observations. Thus the high percentage of sites labelled by
E3HlBremazocine supports the role of the cord as an important target for the
action of kappa agents (1,2). On the other hand the paucity of delta sites
could be the reason why DADLE is less effective after i.t. administration
then expected if delta receptors mediate spinal nociception (4).

REFERENCES

1. Martin, W.R., Eades, C.G., Thompson, R.E., Huppler, R.E. and Gilbert, J.
(1976). The effects of morphine- and nalorphine-like drugs in the
non-dependent and morphine-dependent chronic spinal dog. J. Phannac. exp.
Ther. 197: 517-532.

2. Wood, P.L., Rackham, A. and Richard, J. (1981). Spinal analgesia:

comparison of the mu agonist morphine and the kappa agonist
ethylketocyclazocine. Life Sci. 28: 2119-2125.

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NELJR --- E
3. Yaksh, T.L. (1981). Spinal opiate analgesia: characteristics and
principles of action. Pain 11: 293-346.

4. Tung, A.S. and Yaksh, T.L. (1982) In vivo evidence for multiple opiate
receptors mediating analgesia in the rat spinal cord. Brain Res. 247:
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5. Hylden, J.L.K. and Wilcox, G.L. (1983) Intrathecal opioids block a

spinal action of Substance P in mice: functional importance of both mu and
delta receptors. Eur. J. Pharmacol. 86: 95-98.

6. Gouarderes, C., Attali, B., Audiger, Y. and Cros, J. (1981) Opiate

binding sites in the lumbo-sacral spinal cord from various species. In:
Takagi, H. (ed.) Advances in endogenous and exogenous opioids, Kodansha
Ltd., Tokyo ~18-20.

7. Gouarderes, C., Attall, B., Audiger, Y. and Cros, J. (1983) Interaction

of selective mu and delta ligands with the kappa2 subtype of opiate binding
sites. Life Sci. 33, Sup. 1: 175-178.

8. Traynor, J.R., Kelly, P.D. and Rance, M.J. (1982) Multiple opiate
binding sites in rat spinal cord. Life Sci. 31: 1377-1380.

9. Gillan, M.G.C. and Kosterlitz, H.W. (1982). Spectrum of the mu, delta
and kappa binding sites in homogenates of rat brain. Brit. J. Pharmac. 77:
461-469.

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