Table 1 Inhibitory effects of C-terminally extended Leu -enkephalins on the electrically induced contractions of the guinea pig myenteru plexus-longitudinal muscle and the vasa deferentia of the mouse, rat and rabbitIC50 (nM) C-terminal extensions Guinea pig Mouse vas Rat vas Rabbit vasof Leu5-enkephalin myenteric plexus deferens deferens deferens _ 28.65.4 1.70.3 55062>10,000 Lys6 10729.1 25.64.9 72045>10,000 Arg6 10217.2 10.91.1 790 46 3,000 930Arg6-Arg7 17.32.52 25.54.2 7,5002,000 43.011.5 Arg6-Arg7-Ile8 4.920.53 9.21.30>10,000 11.71.27 Arg6-Arg7-Ile8-Arg9 2.360.67 6.50.34>10,000 6.10.45 Dynorphini_13 0.310.16 0.33 0.08>10,000 2.43 0.49 Dynorphini_i7 0.29 0.09 0.910.13>10,000 3.011.6 a-Neo-endorphin 2.98 0.44 7.7 0.98>10,000 21.44.5 The values shown are the means.e.m. of three to four observations. The activity of peptidases has been inhibited by bestatin (10 p,M, or 30 |xM in the rat and rabbit), L-leucyl-L-leucine (2mM), thiorphan (0.3 |jtM) and captopril (10 M-M); in the rabbit vas deferens, this treatment increased the potency of the enzyme-sensitive peptides, but not of dynorphini_13 or dynorphini_i7, 60-270-fold. The sequence of dynorphin is Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gln and of a-neo-endorphin is Tyr-Gly-Gly-Phe-Leu-Arg-Lys-Tyr-Pro-Lys. The peptides were obtained from Peninsula, Bachem and Cambridge Research Biochemicals or were gifts from Drs A. Goldstein, J. Morley and S. Udenfriend.Our results were obtained from four in vitro pharmacological assays in which the preparations were made to contract by electrical stimulation. The preparations respond differently to activation of the /u,-, 6- and K-receptors. The inhibitory potencies of the peptides are given as their 50% inhibitory concentration (IC50; nM) values. In the myenteric plexus-longitudinal muscle of the guinea pig ileum, an inhibitory effect is readily observed with ligands that activate fjL- and /c-receptors, whereas the mouse vas deferens responds preferentially to ligands acting on 8-receptors4; the vas deferens of the rat has a low sensitivity to K-ligands5 in contrast to the vas deferens of the rabbit in which K -ligands, but not \L- or -ligands, are inhibitory6'7. In these assays, the activity of peptidases was inhibited by a mixture of bestatin, L-leucyl-L-leucine, thiorphan and captopril (Table 1 and A.T.McK., A.D.C. and H.W.K., unpublished observations). The peptides used in this investigation had agonist, but no antagonist, action. The binding of the peptides was measured by determining the inhibition constants (Ki, nM) in three assays on homogen-ates of guinea pig brain. For this purpose, 3H-labelled D-Ala2, MePhe4, Gly-ol5-enkephalin was used as a highly selective /Lt-ligand and 3H-D-Ala2, D-Leu5-enkephalin as a 5-ligand which, although selective, has a cross-reactivity of -10% at the /it-binding site8. The most suitable ligand for the assay of the /c-binding site was found to be 3H-(-)bremazocine9 after suppression of the binding at the /LC- and 5-sites by addition of unlabelled /- and 5-ligands at a concentration of 100 nM each.To reduce degradation by peptidases, the binding assays were carried out at 0 C for 150 min (Table 2). Two groups of C-terminal extensions of Leu5-enkephalin have been shown to occur in the central nervous system (CNS). They are dynorphini_8 (Leu5-enkephalyl-Arg-Arg-Ile; refs 10, 11) and dynorphhii_i7 (refs 12, 13), which have two arginine residues in positions 6 and 7, and a- and #-neoendorphin14'15, which are nona- and decapeptides respectively, with arginine and lysine residues in positions 6 and 7.In the pharmacological assays (Table 1), the introduction of Arg6 or Lys6 at the C-terminal end of Leu5-enkephalin reduced its potency without altering the relative activity pattern. However, lengthening the chain from residue 7 onwards increased the ratio of IC50 for the mouse vas deferens to IC50 for the myenteric plexus. The most important effects of the extensions by Arg6-Arg7 or Arg6-Lys7 and beyond are the complete loss of potency in the rat vas deferens, which probably has no K-binding sites5, and the progressively increasing activity in the rabbit vas deferens which responds to ketazocine- or K-like compounds but not to pt,- or 6-like ligands6'7. The most potent C-extended forms of Leu5-enkephalin are dynorphin^is, dynorphin!_17 and dynorphin^, Leu5-enkaphalyl-Arg-Arg-Ile-Arg (Table 1). Table 2 The inhibitory effects of C-terminally extended Leu5-enkephalins on the binding in homogenates of guinea pig brain of 3H-D-Ala2, MePhe4, Gly-ol5-enkephalin (1.0 nM), 3H-D-Ala2, D-Leu5-enkephalin (1.0 nM) and 3H-bremazocine (0.30 nM)ATi (nM) 3H-(-)bremazocine3H-D-Ala2, MePhe2, 3H-D-Ala2, after suppression of C-terminal extensions Gly-ol5-enkephalin D-Leu5-enkephalin H- and 5-bindingof Leu5-enkephalin (/Li-site) (6-site) (K-site) _ 18.81.77 1.180.20 8,210 1,090LVf dynorphhii_7, dynorphini_8 and dynorphin^ were, respectively, 367, 30.8 and 21.5 nM before treatment with the peptidase inhibitors and 17.3, 4.9 and 2.4 nM after treatment, an average potentiation of about 12, while there was no potenti-ation of either dynorphhii_i3 or dynorphini_i7. Furthermore, in four experiments in the presence of the peptidase inhibitors, the recovery of the effect of dynorphini_9 on the contraction of the myenteric plexus-longitudinal muscle was rapid (fi/2 = 48 6 s) and the recovery after dynorphhii_17 was slow (t\/2 - 178 8s). Finally, in the rabbit vas deferens pretreated with peptidase inhibitors, the IC50 value of dynorphini_8 is 11.7 nM and that of dynorphini_i3 2.43 nM, while the corresponding KI values at the K-binding site are 1.34 and 0.045, respectively. Thus, the 'small' dynorphins are liable to be degraded by peptidases, are easily washed out and have moderate potency at the /c-site while the 'large' dynorphins are peptidase resistant, do not wash out readily and are "extraordinarily potent"17. The small dynorphins seem to have the qualities of a neurotrans-mitter or neuromodulator of short and rapid action, whereas the large dynorphins have the characteristics of a highly potent hormonal action of long duration. Moreover, it is of interest that the small dynorphin found in neuronal tissue is dynor-phini_8, which is a less selective /c-ligand than dynorphhii_9. Note that the K-binding site is very demanding in its choice of ligands as Met5-enkephalin, Leu5-enkephalin and jS-endorphin have only low affinities18; thus, dynorphin^, which has affinities to the /u,- and 5-binding sites, is sufficiently selective as far as the K-binding site is concerned. An urgent requirement is confirmation or otherwise that dynorphini-8 and possibly dynorphin i_9 are strong candidates for the role of an endogenous ligand at the K-binding site mediating neurotransmission or neuromodulation. Lastly, it will be important to correlate the putative K-like actions of the small dynorphins with the pharmacological effects of the ketazocine-like compounds or K-agonists19'20 which are antinociceptive agents and yet do not substitute for morphine in the morphine-dependent rhesus monkey21"23. It may now become possible to exploit the therapeutic potentials of this group of compounds8. This work was supported by grants from the MRC and the US National Institute on Drug Abuse (DA 00662).