European Journal of Pharmacology, 192 (1991) 371-375 © 1991 Elsevier Science Publishers B.V. (Biomedical Division) 0014-2999/91/$03.50 ADONIS 0014299991001221 371 EJP 51671 Pharmacological characterization of [D-Ala2,LeuS,Ser 6]enkephalin (DALES): antinociceptive actions at the 6non.complexed-Opioid receptor Antonia Mattia, Todd Vanderah, Henry I. Mosbcrg 1, John R. Omnaas 1, Wayne D. Bowen 2 and Frank Porrcca Department of Pharmacology, University of Arizona Health Sciences Center, Tucson, A Z 85724, U.S.A., I College of Pharmacy, University of Michigan, Ann Arbor, M1 48109, U.S.A. and e Section of Biochemistry, Division of Biology and Medicine, Brown University, Providence, RI 02912, U.S.A. Received 30 July 1990, revised MS received 12 October 1990, accepted 23 October 1990 Substantial evidence has been accumulated which suggests that opioid 8 receptors may be distinguished on the basis of their involvement in the modulation (i.e., increase or decrease in potency) of/x-mediated antinociception. On this basis, it has been hypothesized that some opioid 8 receptors exist within a functional complex with /L receptors (8~omplexe d (8c~) receptors) while other 8 sites do not (8....... plex,d (Sncx) receptors). Recent work with [D-Ala2,LeuS,Cys6]enkephalin (DALCE) has demonstrated that this compound produces initial antinociceptive actions, does not modulate morphine antinociception and appears to bind irreversibly to the 8no x site, presumably by means of thiol-disulfide exchange between the receptor and the cysteine sulfhydryl group. To determine if a structural basis exists for actions at the hypothesized 8n~ x receptor, in the present study we report the synthesis and pharmacological characterization of [D-Ala2,LeuS,Ser6]enkephalin (DALES), a close structural analogue of DALCE. If a structural basis for action at the 8no x site exists, then DALES would be predicted to produce antinociception, fail to modulate morphine antinociception and, since it lacks the free sulfhydryl group present in DALCE, fail to exhibit irreversible antagonistic actions; these predictions were supported. Additionally, pretreatment with DALCE at -24 h, but not with DALES, blocked DALES-induced antinociception. These observations in vivo support the concept of a structural basis for activity at the hypothesized 8nCx site and suggest that DALES, like DALCE, may be a useful probe for pharmacological characterization of putative 8 receptor subtypes. Opioid antinociception; 8-Opioid receptors; /~-Opioid receptors; Enkephalins; DALES ([D-Ala2,LeuS,Ser6]enkephalin); (Intracerebroventricular) 1. Introduction The multiplicity of opioid receptors has been well accepted since the initial suggestion of multiple opioid receptors by Martin et al. (1976). While uncertainty still exists as to the specific physiological functions of opioid receptors, pharmacological evidence has accumulated which strongly implicate 3, as well as /~, receptors in supraspinal antinociception in the mouse (Heyman et al., 1987; Heyman et al., 1988). Additionally, data have also been accumulated which indicate that in some cases, opioid bt and ~ receptors may be involved in functional interactions in vitro (e.g., Rothman and Westfall, 1982) and in vivo (Heyman et al., 1989a,b; Jiang et al., 1990a,b; and see Holaday et al., 1985 for review). Early observations demonstrated that the proposed endogenous ligands for the opioid 8 receptor, [LeuS]en- Correspondence to: F. Porreca, Department of Pharmacology, College of Medicine, University of Arizona, Tucson, AZ 85724, U.S.A. kephalin and [MetS]enkephalin, respectively produced an increase and decrease in morphine antinociceptive potency (Vaught and Takemori, 1979; Lee et al., 1980). These observations have been confirmed with other synthetic ~ ligands in vivo (Heyman et al., 1989a,b) as well as with radioligand binding techniques in vitro (Rothman et al., 1988) and have supported the sugges- tion that endogenous opioids may function in a modula- tory capacity for ~t-mediated analgesia (Barrett and Vaught, 1982). The hypothesis of functional or physical interactions between/~ and 8 opioid receptors has been extended to suggest that some opioid 8 receptors may exist within a /~-8 complex (i.e., ~complexed (~cx) recep- tors) while other 8 receptors do not (i.e., 8....... plexea (Sncx) receptors) (Rothman et al., 1988). Substantial data in vivo are consistent with such a hypothesis. In this regard, doses of 8 agonists such as [D-Pen2,D-PenS]enkephalin (DPDPE) or [D-Ala 2, MetS]enkephalinamide (DAMA) which do not produce antinociceptive actions alone, respectively increase and decrease the antinociceptive potency of intracerebro- ventricular (i.c.v.) morphine in mice (Heyman et al.,