Binding of buprenorphine to opiate receptors

Neuropharmacology Vol. 23, No. 3, pp. 373-375, Printed in Great Britain. All rights reserved

BINDING

1984

0028-3908/84$3.00+ 0.00 Copyright 0 1984Pergamon Press Ltd

OF BUPRENORPHINE RECEPTORS

REGULATION

TO OPIATE

BY GUANYL NUCLEOTIDES AND METAL IONS

J. W. VILLIGER Department of Pharmacology and Clinical Pharmacology, School of Medicine, University of Auckland, Private Bag, Auckland, New Zealand (Accepted 26 July 1983) Summary-The effects of guanosine-5’-triphosphate (GTP), sodium chloride and manganese chloride on the binding of buprenorphine to opiate receptors present in rat brain has been studied. Manganese chloride significantly decreased the affinity of binding of both [3H]buprenorphine and unlabelled buprenorphine to morphine and enkephalin receptors. Guanosine-5’-triphosphate increased the affinity of buprenorphine for morphine sites, but had no effect on binding of buprenorphine to enkephalin or benzomorphan sites, or binding of [3H]buprenorphine. Sodium chloride had no effect on binding of buprenorphine. Control studies indicated similar apparent affinities of buprenorphine for morphine (K, = 0.30 nM) and enkephalin (Ki = 0.31 nM) sites, and lower affinity for benzomorphan sites (Ki = 4.16 nM). No evidence could be obtained for a differential effect of ions or guanosine-5’-

triphosphate on binding of buprenorphine to opiate receptor subtypes. Key words: buprenorphine, opiate receptors, GTP, metal ions.

In vivo, partial opiate agonists may display agonistic pharmacological characteristics in small doses and these either plateau or become antagonistic at larger doses. The oripavine derivative, buprenorphine, clearly produces such bell-shaped doseresponse curves in animal tests of analgesia (e.g. Cowan, Lewis and McFarlane, 1977; Dum and Herz, 1981). Recent evidence indicates the presence of three opiate receptor subtypes; the morphine (p), enkephalin (S) and benzomorphan (K) sites, in brain (Chang, Hazum and Cuatrecasas, 1981; Kosterlitz, Paterson and Robson, 198 1). Furthermore, Sadee, Rosenbaum and Herz (1982) have suggested that buprenorphine produces its characteristic bellshaped, dose-response curve by a process of noncompetitive autoinhibition, i.e. activation of one receptor subtype at small doses produces an agonist response which is then antagonized by activation of a different receptor subtype at larger doses. Since the affinity of buprenorphine for p, 6 and K receptors has not been accurately determined in vitro, and since the agonist and antagonist properties of opiates may be revealed by the effect of guanyl nucleotides and metal cations on the affinity of the drug (Childers and Snyder, 1980; Pasternak, Snowman and Snyder, 1975), this study was designed to examine the effect of guanosine-5’-triphosphate (GTP), sodium (Na+) and manganese (Mn’+) ions on the binding of buprenorphine to opiate receptors.

METHODS

[3H]opiate binding assays were performed using homogenates (10 mg tissue/ml unless otherwise stated) of whole rat brain (minus cerebellum), prepared and assayed as described previously (Villiger and Taylor, 1982). Morphine @) and enkephalin (6) sites were labelled by incubating a 10 mg/ml homogenate with 0.4 nM [3H]naloxone (II sites) or 0.5 nM [ ‘HI-D-Ala2, D-Leu’-enkephalin (DADLE; 6 sites). Non-specific binding was defined as that occurring in the presence of 1 PM levorphanol. Benzomorphan sites were identified according to the method of Chang et al. (1981) using 0.5 nM [‘Hldiprenorphine, 10 p M morphiceptin and 0.1 PM DADLE incubated with a 5 mg/ml homogenate. Diprenorphine (1 PM) was used to assess non-specific binding. All assays were incubated at 23°C for 60min. It should be noted that these assay conditions are not completely selective for the respective opiate receptor subtypes. Preliminary studies with the p-selective ligand morphiceptin (up to lo-’ M) indicated that approx. 35% of the binding of [3H]DADLE and 95% of [‘Hlnaloxone was to p receptors. The residual 65% of [3H]DADLE binding was almost certainly to 6 receptors since this peptide has an extremely low affinity for benzomorphan sites (Chang et al., 1981). The residual 5% of binding of [3H]naloxone may have been to either enkephalin or benzomorphan sites. There is also a slight cross373

374

J. w.

tical significance test (two-tailed).

0-9 Control

26 a ;

VILLIGER

0-0 ImM

MnCI,

was determined

using Student’s

t-

24

0 1 0 E

20

,a

16

RESULTS

Scatchard

analysis of saturation isotherms for revealed no significant effect of either 100 nM NaCl of 0.1 mM guanosine-5’triphosphate on binding of [ ‘Hlbuprenorphine (Fig. 1). In contrast, the addition of 1 mM MnCl, resulted in an almost 3-fold (P < 0.01) decrease in the affinity of [3H]buprenorphine for its binding site. These results are consistent with those obtained by Sadee et al. (1982) who found that 1 mM MnCl, resulted in a 2-fold decrease in the affinity of buprenorphine for [3H]diprenorphine binding sites, but found the addition of 100mM NaCl to have no effect. However, since [3H]buprenorphine is a nonselective opiate, subtle effects of guanosine-5’triphosphate and ions on the binding of buprenorphine to p, 6 and K receptors might have been masked in the above experiment. These effects were therefore examined using assay conditions designed to label the opiate receptor subtypes selectively. The binding of buprenorphine to p receptors was clearly modulated by guanosine-S-triphosphate and ions. Guanosine5’-triphosphate resulted in a 3-fold increase (P < 0.025) in the affinity of buprenorphine while MnCli induced a 4-fold decrease (P < 0.01) in affinity (Table 1). Sodium ions resulted in a 1.8-fold increase in affinity which was not statistically significant. These changes in affinity resulted from parallel shifts in the displacement curves as indicated by nH values v 1 under all experimental conditions (data not shown). Sodium ions and guanosine-5’triphosphate had no significant effect on binding of buprenorphine to enkephalin and benzomorphan receptors, while MnCl, produced a 2-fold decrease (P < 0.01) in the affinity of buprenorphine for enkephalin sites (Table 1).

[ ‘Hlbuprenorphine

L 0

I

I

J

I

I

I

I

4

6

12

16

20

24

26

C3Hl buprenorphlne

bound

lb 32

34

(pmol/g)

Fig. 1. Scatchard plots of the binding of [ ‘Hlbuprenorphine in the absence and presence of 1 mM MnCl,. Plots of the binding of [ 3H]buprenorphine in the presence of 0.1 mM guanosine-5’-triphosphate (G’TP) or 100 mM NaCl were not significantly different from control. Linear regression of Scatchard plots yielded the following binding parameters (mean _+SD for 3 experiments) in the absence and presence of guanosine-5’-triphosphate and ions; control: B,,, = 33.9 * 4.1 pmol/g; 1OOmM KD=l.2k0.1nM, NaCI: K, = 1.3 k 0.2 nM, B,,, = 36.8 + 3.7 pmol/g; 1 mM MnCl,: K, = 3.5 f 2 nM, B,,, = 36.3 + 4.0 pmol/g; 0.1 mM GTP: K, = 1.5 k 0.1 nM, B,,, = 37.3 + 3.4 pmol/g.

reactivity between enkephalin and benzomorphan receptors (Chang et al., 1981). Drugs and their sources were as follows: [3H]naloxone (38.6 Ci/mmol, New England Nu[3H]DADLE (39.5 Ci/mmol, clear), NEN), [3H]diprenorphine (7.5 Ci/mmol, Radiochemical Center), [3H]buprenorphine (30 Ci/mmol, Reckitt & Colman) buprenorphine and diprenorphine (Reckitt & Colman), guanosine-5’-triphosphate, DADLE and morphiceptin (Sigma), ethylketocyclazocine (Sterling-Winthrop) and levorphanol (HoffmanLaRoche). Scatchard and Hill plots were analysed using a computerized linear regression programme (Standard Pat, Hewlett-Packard 85 Desktop computer). StatisTable 1.

The effects of N&l, MnCl, and guanosine-5’striphosphate (G’Tp) on the affinity of buprenorphine for morphine, enkephalin and benzomorphan binding sites Affinity

Condition

Morphine

of buprenorphine

for binding

Enkephalin

sites (nM) Benzomorphan

Control

0.43 f 0.25 (1 I) K, = 0.31

0.38 f 0.05 (6) K, = 0.3

12.48 +_4.5 (4) K, = 4.16

IOOmM NaCl

0.24 k 0.25 (I 1) K, = 0.17

0.3OiO.l2(4) K, = 0.24

12.15 f 4.2 (3) K, = 4.05

0.1 nM GTP

0.14f0.10(4) K,=O.lO

0.29f0.12(5) K, = 0.23

12.33 +_8.55 (3) K,=4.11

I mM MnCI,

1.76 f 0.34(4)** K, = 1.26

0.75 * 0.25 (3)** K, = 0.75

Not determined

Mean f standard deviation (n) IC,, values are given. Mean K, values are given beneath each IC,,. The K,‘s were determined according to the equation: K, = IC,,/[l + L/K,] where L = [‘Hlligand concentration and K, is the apparent equilibrium dissociation constant previously determined from saturation experiments (Villiger and Taylor, 1982 and unpublished data). Effects of MnCl, on binding of buprenorphine to benzomorphan sites could not be accurately determined because M&I, significantly suppressed the binding of [‘Hldiprenorphine to benzomorphan sites. *P < 0.025: **P < 0.01.

Regulation of buprenorphine

binding of buprenorphine to morphine, or buprenorphine receptors.

DISCUSSION

These results indicate that under conditions equilibrium, in vitro, buprenorphine behaves as antagonist at p, 6 and K opiate receptors, i.e. binding affinity is either increased or not affected

375

binding

of an

the by

guanosine-S-triphosphate and NaCl, and MnCl, reduces rather than increases affinity. This differentiates buprenorphine from other partial agonists which show moderate decreases in their affinity for p and 6 receptors in the presence of NaCl and guanosine-5’triphosphate (Chang, Hazum and Cuatrecasas, 1980). The reason for this difference is not clear. The most consistent finding in the present study was the marked reduction of the affinity of buprenorphine induced by MnCl,. Such an effect on opiate binding has not previously been reported (Chapman and Way, 1980), and it suggests that the binding of buprenorphine to the opiate receptor differs in some fundamental way from that of other opiate drugs. The present study has also delineated the relative affinity of buprenorphine for ,u, 6 and K receptors. Buprenorphine possessed similar affinities for p and 6 sites (K,‘s = 0.31 and 0.30 nM respectively) with a receptors somewhat lower affinity for K (K, = 4.16 nM). Buprenorphine is therefore relatively non-selective for the opiate receptor subtypes and is in this respect similar to other partial agonists (Chang et al., 1981). In conclusion, a consistent effect of MnCl, on the binding of buprenorphine to opiate receptors was obtained. No evidence was obtained for a differential effect of ions of guanosine-5’-triphosphate on the

enkephalin

thank Reckitt 8~ Colman, Hull, England for the gift of [‘Hlbuprenorphine and Carolyn

Acknowledgements-1

Bunkall for excellent technical assistance. This research was supported by a grant from the Medical Research Council of

New Zealand. REFERENCES K-J., Hazum E. and Cuatrecasas P. (1980) Possible role of distinct morphine and enkephalin receptors in

Chang

mediating actions of benzomorphan drugs (putative K and D agonists). Proc. natn. Acad. Sci. U.S.A. 77: 4469-4473. Chang K-J., Hazum E. and Cautrecasas P. (1981) Novel opiate binding sites selective for benzomorphan drugs. Proc. natn. Acad. Sci. U.S.A. 78: 41414145. Chapman D. B. and Way E. L. (1980) Metal ion interactions with opiates. A. Rev. Pharmac. Toxic. 20: 553-557. Childers S. R. and Snyder S. H. (1980) Differential regulation by guanine nucleotides of opiate agonist and antagonist interactions. J. Neurochem. 34: 584-594. Cowan A., Lewis J. W. and MacFarlane I. R. (1977) Agonist and antagonist properties of buprenorphine, a new antinociceptive agent. Br. J. Pharmac. 60: 537-545. Dum J. E. and Herz A. (1981) In vivo receptor binding of the opiate partial agonists, buprenorphine, correlated with its agonistic and antagonistic actions. Br. J. Pharmat. 14: 621-633. Kosterlitz H. W., Paterson S. J. and Robson L. E. (1981) Characterization of the K-subtype of the opiate receptor in the guinea-pig brain. Br. J. Pharmac. 73: 939-949. Pasternak G. W., Snowman A. S. and Snyder S. H. (1975) Selective enhancement of opiate agonists by divalent cations Molec. Pharmac. 11: 735-744. Sad&e W., Rosenbaum J. S. and Herz A. (1982) Buprenorphine: Differential interaction with opiate receptor subtypes in vivo. J. Pharmac. exp. Ther. 223: 157-162. Villiger J. W. and Taylor K. M. (1982) Buprenorphine: High-affinity binding to dorsal spinal cord. J. Neurochem. 38: 1771-1773.