Morphinomimetic activity of synthetic fragments of f 3 - lipotropin and analogs ( peptides / endorphins / enkephalins / myenteric plexus / hypothalamus )

In the myenteric plexus-longitudinal muscle bioassay, ,'-endorphin, i.e., fl-ipotropin (-LPH){81-91J, has a potency of 450 with confidence limits of 281when Met5enkephalin is used as a reference standard with a potency of 100. The primary amide and the ethylamide of Met5-enkephalin have potencies statistically overlapping with that of f.endorphin. The primary amide of a-endorphiu has twice the potency of the free acid form of a-endorphin. An intact NH2-terminal tyrosine is not necessary for full intrinsic activity. The shortest fragment of g$-LPH with morphinomimetic activity is P-LPH{61-64J. Two laboratories have recently reported the isolation and primary structure of novel peptides with morphine-like activity isolated from whole brain or hypothalamus-neurohypophysis extracts (1, 2). Hughes et al. (1) recognized that Met5-enkephalin isolated from whole pig brain has a primary structure identical to that of the fragment Tyr61-Met65 of the fl-lipotropins (f3-LPH) (3), while Leu5-enkephalin would share at least the sequence Tyr6l-Phe64 of the known fl-lipotropins. Guillemin et al. (2) similarly called attention to the fact that the a-endorphin isolated from extracts of porcine hypothalamus-neurohypophysis was identical to the sequence Tyr61-Thr76 of the various fl-lipotropins, Met5-enkephalin thus being the NH2terminal pentapeptide of a-endorphin. These results and early evidence (2, 4) that synthetic #l-LPH-[61-69] and f,-LPH[61-91] showed opiate-like activity led to the proposal (1, 2, 4) that ,B-LPH could be a prohormone for the various endorphins and enkephalins. Indeed, Lazarus et al. recently reported (5) that while ,B-LPH-[1-91] has no morphinomimetic activity in the bioassay or opiate-receptor binding assays classically used in these studies, its incubation with the high-speed supernatant of an aqueous extract of rat brain rapidly generates (peptide fragments with) morphinomimetic activity. Moreover, we now have characterized y-endorphin as being identical to ,BLPH-[61-77]. The purpose of this short note is to describe the primary structures of a series of fragments of fl-LPH and several analogs, all prepared by synthesis, and to report their morphinomimetic activity in the myenteric plexus bioassay. MATERIALS AND METHODS Bioassay. The assay for opiates is the myenteric plexuslongitudinal muscle of the guinea pig's ileum as described by Paton and Zar (6); a response reversed or prevented by the opiate-antagonist naloxone is classically recognized to indicate specific involvement of "opiate receptors" (6, 7). Synthetic Peptides. All peptides were synthesized as described previously (8, 9) by the solid phase technique; (-endorphin, i.e., f3-LPH-[61-91], was prepared with a Beckman synthesizer model no. 990 (with the collaboration of Dr. jean Rivier). Abbreviations: ,B-LPH, f3-lipotropin; f3-LPH-[61-911, etc., the peptide consisting of residues 61-91 of fl-LPH. Statistical Analysis of Bioassays. Potencies shown in Table 1 were calculated, in multiple four-point assays, by factorial analysis, following an analysis of variance of all the responses obtained on several tissue strips. RESULTS AND DISCUSSION Results of the various bioassays are presented in Table 1. The smallest peptide sequence related to ,B-LPH, with morphinelike activity in the assay described here, is the tetrapeptide III, H-Tyr-Gly-Gly-Phe-OH, in agreement with the data of Bradbury et al. (10). While its specific activity (potency) is ca. 1-10-3 that of the next longest peptide, V, i.e., Met5-enkephalin, the tetrapeptide III has full intrinsic activity. Three peptides, VI, VII, and XVII, have strikingly higher potency than any other of the series reported here; the most potent substance in this series is ,B-endorphin, XVII, i.e., ,BLPH-[61-91], a substance isolated earlier by several groups from extracts of the pituitary gland (3, 13) on the basis of its chemical behavior. Our finding of a statistically greater potency for fl-endorphin (fl-LPH-[61-91]) than for Met5-enkephalin is at variance with the results recently reported by Cox et al. (4) using the same bioassay as used here. Greater potency of f3-endorphin than that of aand y-endorphins as seen by us here is in keeping with earlier results (in Fig. 2 of ref. 5) using displacement of [3H]etorphine from a synaptosomal preparation of rat brain and is also in agreement with the recent results and conclusions of Bradbury et al. (10) using a similar opiate-receptor binding assay. The other two most potent substances in the series reported here are VI and VII, the two synthetic analogs of Met5-enkephalin amidated at the COOH-terminal. Considering the overlapping of fiducial limits of the bioassays, the two amidated pentapeptides are statistically of similar potency as ,B-LPH[61-91]. Similarly, the COOH-terminal amide of a-endorphin (XV) has increased potency when compared to that of the free acid form (XIV). It is somewhat puzzling to observe (Table 1) that extending the peptide chain at the COOH-terminal end of the tetrapeptide III would produce a considerable (100OX) increase in potency as in V (Met5-enkephalin), followed by a lowering of potency (M2, O) upon further extension (XIII, + 4 residues; XIV, + 11 residues; XVI, + 12 residues), but producing a new increase in potency (5X) upon more extension (XVII, + 26 residues). Before attempting to explain these variations of potency, it may be wise to ascertain their existence on a statistical basis in a large number of quantitative assays, in several laboratories. Discussions have recently appeared in the literature (4, 11, 12, 17) emphasizing the requirement of a tyrosine as the NH2-terminal for any peptide with opiate-like activity, in view of its similarity to the A-ring of morphine. The lower specific