Characteristics of [3H]diprenorphine binding to kappa opioid receptors in human cortex: Kappa opioid receptor heterogeneity

149 CHARACTERISTICS OF [3H]DIPRENORPHINE BINDING TO KAPPA OPIOID RECEPTORS IN HUMAN CORTEX: KAPPAOPIOID RECEPTOR HETEROGENEITY K.W Kim 1, S.M. Soh 1, H.I. Kim2, H.W. Rho 3 and K.P. Cho I Dept. of Pharmacol. 1, Neurosurg. 2 and Biochem. 3, Chonbuk Natl. Univ. Medical Sch., Chonju 560-182, Chonbuk, Republic of Korea Experimental data have shown that there are considerable species differences in the d i s t r i b u t i o n of opioid receptor types in m a m m a l i a n brain. We have evaluated the properties of non-ix, non-8 binding of [3H]-diprenorphine([3H]DIP), a nonselective opioid antagonist, in h u m a n cortex membranes. Bindings to Ix and ~ sites were inhibited by the use of an excess of competing selective agonists, DAMGO(1 IxM) and DPDPE ( 1 IxM), for these sites. Dynorphin A(1-13)(Dyn-A), U69,593(U69) and U50,488H(U50), selective ~l-agonists, (-)ethylketocyclazocine(EKC), bremazocine(BREM) and norbinaltorphimine(nor-BNI), ~1 and K2 opioid ligands, inhibited the [3H]DIP binding to h u m a n cortex membranes with high affinities. In the presence of blocking ligands for Ix, 8 and ~1 sites, 1 IXMeach of DAMGO, DPDPE and U69,593, [3H]DIP labeled another single p o p u l a t i o n of b i n d i n g sites. Specific b i n d i n g of [3H]DIP to these sites were not i n f l u e n c e d by U-50,488H or opioid peptides even in micromolar dose range, but inhibited by DIP, bremazocine, (-)ethylketocyclazocine a n d nor-binaltorphimine. The concentration-inhibition curves of these ligands in the presence of DAMGO and DPDPE were displaced further to right by the addition of U69,593. These data suggest that there are at least 2 kinds of ~:-opioid binding sites(~l and ~2) in h u m a n cortex. Heterogeneity of opioid receptors in mammalian brain are well established. Significant interspecies differences are reported in the distribution of 3 major types of opioid receptor in b r a i n ( l ) . There are, also, species differencies in subtypes of ~: opioid receptors(2). Discrepancies are exist in subtypes of ~ opioid receptor in h u m a n brain(3,4). We are attempted to clarify the subtypes of ~ opioid receptor existed in h u m a n cerebral cortex by employing [3H]DIP, a non-selective opioid antagonist that possess appreciable affinities for ~1 and ~2 sites. Preparation of cortical membranes from drug-resistant epilepsy patients who underwent to surgery (Both sexes, age 20-30) and binding assays were performed as described previously(5). Binding assays were conducted at 37 oC in modified Krebs-HEPES containing 125 mM Na+ in the presence of blocking ligands, DAMGO(1 IXM) and DPDPE(1 ~tM), for IXand ~ opioid receptors. In some experiments, DAMGO, DPDPE and U69593(1 IXM) were included in incubation media to block Ix, 8 and ~1 opioid receptors. Inhibitors of peptidase, 10 IXM bacitracin and 1 IXM leupeptin, were included for binding assay with opioid peptides. Triplicate samples of m e m b r a n e suspension were p r e i n c u b a t e d for 5 min with or without nonladioactive competing drug. [3H]DIP(0.5 nM, specific activity; 44 Ci/mmol) was then added and the incubation continued for 20 min. The incubation was terminated by the addition of icecold buffer a n d rapid filtration through W h a t m a n GF/B filter membrane. Specific binding was defined as the difference between the binding in the presence and absence of 10 IXM naloxone. Displacement curves for cold ligand were analyzed using LIGAND program(6). In the presence of blocking ligands for ~t and 8 opioid receptors, [3H]DIP labeled binding sites w h i c h showed h i g h e r a p p a r e n t a f f i n i t i e s for b e n z o m o r p h a n s , n o r b i n a l t o r p h i m i n e (nor-BNI), a r y l a c e t a m i d e s a n d d y n o r p h i n - A ( D y n - A ) , m o d e r a t e affinity for DADLE, lower affinity for morphine, and little affinity for B-endorphin(131). Analysis of competition curves indicated a single population of non-ix/8 [3H]DIP binding sites, with a KD of about 3.5 nM. Meanwhile, [3H]DIP labeled a n o t h e r single population of binding sites which showed KD of 9.3 nM in the presence of 1 ~tM each of DAMGO, DPDPE and U69. USO, a selective agonist for K1 opioid receptor, could not inhibit [3H]DIP binding to these sites even in micromolar dose range. However, BREM, (-)EKC and nor-BNI effectively inhibited [3H]DIP b i n d i n g with varying a p p a r e n t affinities. These sites showed little affinities for opioid peptides, Dyn-A, DADLE and ~-endorphin.

150 The Ki value of m o r p h i n e was decreased by the addition of U69. Apparent affinities of tested opioid ligands from competition assay are shown in Table 1. Table 1. Affinity constant (Ki, nM) of various opioid ligands for specific binding site of [3H]diprenorphine in the presence of blocking ligands for I~ and 8, or for ~t, 8 and r l sites in h u m a n cortex membranes Blocking Ligands

1 ixlvlDAMGO 1 ~M DPDPE

1 ~¢1DAMGO 1 la/vlDPDPE 1 IJ-MU69593

.fald_iae.an~ Diprenorphine

3.5

9.3

Bremazocine

5.0

22.5

(-)Ethylketocyclazocine nor-Binaltorphimine

25.2

147.5

3.2

428.8

U69593

63.7

> 10,000

US0488H

32.3

> 10,000

Morphine Dynorphin-A DADLE ~-Endorphin (1-31 )

1415.7

786.2

57.6

2,286

612.2

2,693

> 10,000

3690

Each value represents the mean of two or more independent experiments with varying SEM less than + 11 9/o. These results suggest that there are heterogeneity of • opioid receptors in h u m an cerebral cortex. Its physiological roles are remained to be elucidated. REFERENCES 1. L.E. Robson, M.G.C. Gillzm mad H.W. Kosterlitz(1985) Eur. J. Pharmacol. 112, 65-71. 2. R. S. Zukin, M. Eghbali, D. Olive, E. M. Unterwald and A. Tempel (1988) Proc. Natl. Acad. Sci. USA 85, 4061-4065 3. B.A. Vogt, M.D. Plager, P.B. Crino and E.D. Bird(1990) Neurosci. 36, 165-174 4. K.C. Rice, J.E. Kleinman and LS. Brady(1992) Peptides 13, 977-987 5. L L Werling, P. S. Puttfarcken and B. M. Cox(1988) Mol. Pharmacol. 33, 423-431 6. P. J. Munson and D. Rodbard (1980) Puaal. Biochem. 107, 220-226 Supported by a KOSEF Grant 941-0700-008-1