European Journal of Pharmacology, 98 (1984) 149-150 149 Elsevier Rapid communication BINDING OF [3H]DYNORPHIN A TO APPARENT K OPIOID RECEPTORS IN DEEP LAYERS OF GUINEA PIG CEREBRAL CORTEX MICHAEL E. LEWIS *, ELIZABETH A. YOUNG, RICHARD A. HOUGHTEN **, HUDA AKIL and STANLEY J. WATSON Mental Health Research Institute, University of Michigan, Ann Arbor, Michigan 48109, and ** Scripps Clinic and Research Institute, Department of Immunopathology, La Jolla, California 92037, U.S.A. Received 21 December 1983, accepted 27 December 1983 Dynorphin A and its fragments (e.g. dynorphin A-(1-13)) potently displace prototypical alkaloid ligands for the ~ subtype of opioid receptor in rat (Quirion and Pert, 1981) and guinea pig (Chavkin et al., 1982; Corbett et al., 1982) brain membranes. However, we and others (Quirion and Pert, 1981; Corbett et al., 1982) have found that dynorphin A-related peptides also exhibit substantial potency in displacing prototypical/~ and 6 receptor ligands from brain membranes. Since these ligands are not completely selective, it is difficult to distinguish whether dynorphin acts at the/~ and 8 sites or if the radioligands bind to the K receptor. This issue has been clarified for guinea pig brain: [3H]dy- norphin A binds specifically to guinea pig brain membranes and appears to selectively label x sites (Young et al., 1983). These sites, when labelled by [3H]ethylketocyclazocine or [3H]bremazocine, are concentrated in the deep layers of guinea pig cerebral cortex (Goodman and Snyder, 1982). Since these tritiated alkaloids exhibit little receptor selec- tivity, excess concentrations of ~ and ~ ligands are included in the incubation medium to ensure that only x sites are available for labelling (Corbett et al., 1982; Goodman and Snyder, 1982). However, the concentration of morphine (30 nM) used by Goodman and Snyder (1982) may have been insuf- ficient to saturate the /~ sites (cf. Corbett et al., 1982). Since [3H]dynorphin A exhibits high x selectivity in guinea pig brain (Young et al., 1983), we carried out the present study to determine whether specific binding sites for this ligand are distributed in the deep cortical layers. * To whom all correspondence should be addressed. Adult male Hartley guinea pigs were deeply anesthetized with pentobarbital and their brains were rapidly removed and frozen. Twenty-/~m coronal sections of the brains were cut in a Bright cryostat at -16°C and thaw-mounted onto gela- tin-coated microscope slides. The sections were dried at 0°C under vacuum and stored at -70°C before being warmed to 4°C for assay. The sec- tions were covered with 200/~1 of 0.05 M Tris. HC1 (pH 7.55) containing 2.5 nM [3H]dynorphin A for 90 min at 4°C (Young et al., 1983). Nonspecific binding was evaluated by incubating adjacent sec- tions in the same medium also containing 10/~M UM-1071 (the active stereoisomer of MR 2034, a potent K agonist; see Young et al., 1983). The slides were then washed by continuous agitation in 4 changes (2 rain each) of 200 ml of 0.05 M Tris HC1 (pH 7.55) at 4°C, rapidly dried under a stream of cool air, and exposed in an X-ray cas- sette to tritium-sensitive LKB Ultrofilm for 10 weeks. The film was then developed for autoradi- ography according to the manufacturer's direc- tions, and photographed using an enlarger. [3H]Dynorphin A binding to guinea pig brain sections, as determined by liquid scintillation counting of the sections after agitation for 1 h in scintillant, was displaced approximately 45 percent by 10 FM UM-1071, a x ligand previously shown to specifically (and selectively) displace [3H]dy- norphin A binding in guinea pig brain membranes (Young et al., 1983). Autoradiographs of the sec- tions revealed a concentration of [3H]dynorphin A binding sites in the deeper layers (predominantly V and VI) of cerebral cortex (fig. 1A), although there were some regional variations in binding site 0014-2999/84/$03.00 © 1984 Elsevier Science Publishers B.V.