Neurochemical Research, Vol. 15, No. 9, 1990, pp. 899-904 Characterization of Kappa1 and Kappa20pioid Sites in Frog (Rana eseulenta) Brain Membrane Preparation Binding Sandor Benyhe 1, Eva Varga 1, Jozsef Hepp 2, Anna Magyar 2, Anna Borsodi 1, and Maria Wollemann 1 (Accepted June 7, 1990) The distribution and properties of frog brain kappa-opioid receptor subtypes differ not only from those of the guinea pig brain, but also from that of the rat brain. In guinea pig cerebellum the kappas is the dominant receptor subtype, frog brain contains mainly the kappa2 subtype, and the distribution of the rat brain subtypes is intermediate between the two others. In competition ex- periments it has been established that ethylketocyclazocine and N-cyclopropylmethyl-norazido- morphine, which are nonselective kappa-ligands, have relatively high affinities to frog brain membranes. The kappa2 ligands (MetS)enkephalin-Arg6-Phe v and etorphine also show high affinities to the frog brain. Kappa 1 binding sites measured in the presence of 5 txM/D-Ala2-LeuS/enkephalin represent 25-30% of [3H]ethylketocyclazocine binding in frog brain membranes. The kappa 2 sub- type in frog brain resembles more to the mu subtype than the delta subtype of opioid receptors, but it differs from the mu subtype in displaying low affinity toward beta-endorphin and/D-AIa 2- (Me)Phe4-GlyS-ol/enkephalin (DAGO). From our data it is evident that the opioid receptor subtypes are already present in the amphibian brain but the differences among them are less pronounced than in mammalian brain. KEY WORDS: Opioid receptor subtypes; frog brain; kappa receptors; equilibrium binding experiments; [3H]ethylketocyclazocine. INTRODUCTION Opioid receptors of amphibian brain are character- ized mainly by their relatively high-affinity binding to i Institute of Biochemistry, Biological Research Center of Hungarian Academy of Sciences, P.O. Box 521. 6701 Szeged, Hungary. 2CentraI Research Institute for Chemistry of Hungarian Academy of Sciences, P.O. Box 17. 1025 Budapest, Hungary. Abbreviations used: DAGO = /D-Ala2-(Me)Phe4-GlyS-ol/enke- phalin, DALE = /D-Ala2-L-LeuS/-enkephalin, EKC = ethylketocy- clazocine, DHM = dihydromorphine, CAM = N-cyclopropylmethyl- norazidomorphine, nor-BNI = nor-binaltorphimine, MR2034 = ( - ) - (1R,5R,9R) - 5, 9 - dimethy] - 2 ( L - tetrahydrofuryI - 2' - hydroxy- 6,7benzomorphan), MR2035 = ( + ) - (1R,5R,gR) - 5,9 - dimethyl - 2 ( L - tetrahydrofuryl - 2' - hydroxy- 6,7- benzomorphan), U50488H = 3,4- dichloro-N-/2- (1 - pyrrolidinyl) - cyclohexo/- benzene- acetamide, PD 117302 = trans - N - methyl - N - / 2 - (1 - pyrmlidinyl) - cyclohexyl/- benzo (b) thiophene - 4- acetamide 899 benzomorphan drugs such as [3H]bremazocine and [3H]ethylketocyclazocine (EKC) and by their low affin- ity to mu and delta ligands when/D-Ala2-(Me)Phe4-Gly 5- ol/enkephalin (DAGO) and /D-Ala2-LeuS/enkephalin (DALE) are used (1-7). Morphinan derivatives (e.g., [3H]naloxone, [3H]oxymorphazone), and oripavine com- pounds ([3H]etorphine, [3H]diprenorphine) also bind with high affinity to membranes prepared from frog (Rana sp.) brain (4,8-10). Earlier it was proposed that kappa opioid receptors can be divided in two subgroups, namely kappa I and kappa2 (11-14). Therefore we attempted to characterize the frog brain opioid receptor with different radio-la- beled and non-labeled subtype- specific opioid ligands. 0364-3190/90/0900-0899506.00/0 9 1.990 Plenum Publishlng Corporatilm