ABSTRACT3,-Endorphin-(1-27), a naturally occurring fragment of human f-endorphin (ph-endorphin), diminishes the analgesic effect of ph-endorphin when coinjected intracerebroventricularly into mice. A parallel shift in the doseresponse curve of 3h-endorphin in the presence of (3h-endorphin-(1-27) suggests competition at the same site. The potency of ,,-endorphin-(1-27) in antagonizing analgesia is >4 times greater than that of the opiate antagonist naloxone.

Six naturally occurring homologs of (3-endorphin were tested for their potency in inhibiting the binding of documented in a number ofprevious studies (1-3). The affinity determined in vitro may not be simply related to that in vivo due to differences in assay conditions or the status of the tissue preparation. Alternatively, there may be another step in the receptor-endorphin interaction that is not detected by binding measurements but that is more closely related to the biological response and sensitive to structural differences in ,B-EPs.The existence ofpotent alkaloid antagonists for opiate receptors demonstrates that binding to the receptor is not sufficient to elicit analgesic activity, and it seems likely that structural differences among 3-EP analogs and homologs could differentially affect not only binding potency but also efficacy (4) in producing analgesia once bound to the receptor. In addition, it is well established that Na+ favors binding of opiate alkaloid antagonists, while decreasing binding ofagonists (5, 6). In order to gain more information on the biological behavior of 3EP analogs and homologs, the potency of six naturally occurring ,& EP homologs [human (h), camel (c), equine (e), turkey (0), ostrich (j,) and des-acetyl salmon (,J) ,B-EPs; for amino acid sequences of these homologs, see Fig. 1] in inhibiting the binding of 3H-labeled naloxone (Nx) and [3H]-Ph-EP was determined in the presence or absence of Na+.MATERIALS AND METHODS Human (7), camel (8), equine (9), turkey (10), ostrich (11), and des-acetyl salmon (10) f3-EPs were synthesized as described. Nx was a gift from Endo Laboratories (Garden City, NY). [Tyr27-3H2]-ph-EP (50 Ci/mmol; 1 Ci = 3.7 x 1010 becquerels) was prepared as described (12).[3H]Nx (50 Ci/mmol) was purchased from New England Nuclear. Rat brain membranes were prepared and stored as described (3, 13). Membranes obtained by Polytron (Brinkmann) homogenization of decerebellate rat brains (180-g Sprague-Dawley rats) were suspended in 50 mM Tris HCl, pH 7.5, and washed by three cycles of centrifugation at 27,000 X g for 20 min and resuspension in the same buffer. Washed membranes were stored at -800C in 50 mM Tris HCl, pH 7.5, with 20% (vol/vol) glycerol as cryoprotective agent (14).Preliminary experiments confirmed that the binding of Nx is enhanced by incubation at 40C in the presence of Na', asdescribed (5, mM NaCl was performed at 24°C for 30 min because very little binding occurred at 40C in the presence of Na+. Control experiments were also done at 4°C in the absence of Na' in order to check the possible effects of the temperature on the binding ofB EPs. All assays were terminated by filtration at 40C through glass fiber filters (Whatman GF/B) coated with myelin basic protein and washing with 10 ml ofice-cold 50 mM Tris HCl, pH 7.5/0.1% bovine serum albumin buffer and measuring radioactivity as described (3,13). IC50 values were obtained from linear regression of that portion of the log(dose) vs. logit plot corresponding to 10-80% total specific binding. A...