European Journal of Pharmacology, 64 (1980) 1--8 1 © Elsevier/North-Holland Biomedical Press BINDING OF 3H-~-ENDORPHIN TO RAT BRAIN MEMBRANES: CHARACTERIZATION OF OPIATE PROPERTIES AND INTERACTION WITH ACTH HUDA AKIL #, WILLIAM A. HEWLETT *, JACK D. BARCHAS * and CHOH HAO LI Mental Health Research Institute, University of Michigan, Ann Arbor, Michigan 48109, *Nancy Pritzker Laboratory of Behavioral Neurochemistry, Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, California 94305 and Hormone Research Laboratory, University of California, San Francisco, California 94148, U.S.A. Received 20 February 1980, accepted 25 February 1980 H. AKIL, W.A. HEWLETT, J.D. BARCHAS and C.H. LI, Binding of 3H-~-endorphin to rat brain membranes: characterization of opiate properties and interaction with A CTH, European J. Pharmacol. 64 (1980) 1--8. The binding of tritiated ~-endorphin (3H-~-EP) to brain homogenates is described. This had been difficult to achieve due to the lack of availability of 3H-~-EP and to technical difficulties associated with high non-specific binding of ~-EP. We now report that 3H-~-EP binding is saturable, stereospecific, has high affinity and is inhibited by sodium. Its dissociation rate is ten-fold longer than that of naloxone. Its regional distribution exhibits interest- ing differences from naloxone and enkephalin binding. ACTHt.24 appears to displace it more effectively than it displaces 3H-naloxone. The results are discussed in terms of multiple transmitter systems and the multiple opiate receptor hypothesis. 3H-~-Endorphin binding Opiate receptors Peptide binding 1. Introduction While the endogenous opioid, ~-endorphin (~-EP) was first discovered in the pituitary (Li and Chung, 1976; Bradley et al., 1976), it is also present within an anatomically distinct neuronal system in mammalian brain. We and others (Watson et al., 1978; Bloom et al., 1978) have shown to existence of ~-EP and its precursors in a well-defined hypothalamic cell group which projects to a number of limbic and midbrain structures. This system is inde- pendent of the pituitary ~-EP system, and is readily distinguished from enkephalin path- ways (Watson et al., 1978). Interestingly, the synthetic machinery characteristic of pitui- tary ~-EP -- including the 31K prescursor #Address all correspondence to H. Akil, Mental Health Research Institute, University of Michigan, Ann Arbor, Michigan 48109, U.S.A. described by Mains et al. (1977) -- appears to exist within the brain. This conclusion is based primarily on immunohistochemical evi- dence demonstrating ~-LPH, ACTH and a number ofothex 3iK fragments (Watson et al., 1978) within these ~-EP-containing neurons. ~-EP is known to exhibit opiate-like proper- ties both in vitro (Cox et al., 1976) and in vivo (Loh et al., 1976). However, in pharma- cological doses, it also produces effects which differ, in part, from those of morphine. These include the induction of limbic seizures and rigidity at subanalgesic doses (Bloom et al., 1976). While 3H-enkephalin binding had been studied and characterized, we know little about the specific properties of ~-EP binding. 3H-~:EP has not been commercially available to date, preventing direct studies of its recep- tor interactions. Yet, the unique anatomical and biosynthetic properties of the brain ~-EP system, and the distinct pharmacological pro-