Release of substance P in the periaqueductal grey in anaesthetized and awake rats

58 Novel, Delta Opioid Receptor-Selective Peptide Antagonists: The Demonstration of a Possible Opioid Interaction in the Absence of a Protonated Nitrogen A. Z. R6nai*, J. Botyhnszki*, J. Hepp*, A. Magyar*, A. Borsodit, M. Holl6si* and K. Medzihradszky* *Department of Organic Chemistry, L. EiitvGs University, Budapest, tBiologica1 Research Center, Szeged, Hungary The design, synthesis and biological data are presented for novel opioid peptides, which act as pure, delta receptor antagonists and contain an ‘opioid’ nitrogen which cannot exist in protonated form while interacting with the receptor. The K, values of Boc-Tyr-Pro-Gly-Phe-Leu, Boc-Tyr-Pro-Gly-Phe-Leu-Thr, Boc-Tyr-Pro-Gly-PheLeu-Thr (tBu) and phenylacetyl- or diphenylacetyl-TyrPro-Gly-Phe-Lou-Thr determined against [Mets]enkephalin or DADLE in the mouse vas deferens bioassay fall into the l&‘-4 x 10” M range, with a 50-350 fold delta over mu receptor selectivity. Displacement data in the receptor binding assay in rat brain membranes against ‘H-DALE, ‘H-DSLET, 3H-DAG0 and 3H-DHM matched the results of bioassay. Since not only the Nblocked derivatives but even the ones with a free N terminus were devoid of significant opioid agonist activity, it is suggested that these novel derivatives do not interact with the conventional ‘opioid’ nitrogen binding site although the hydrophobic binding site involved must be located in a reasonable vicinity. Conformational analyses of peptides appear to support this assumption.

Release of Substance P in the Periaqueductal Grey in Anaesthetized and Awake Rats A. RosCn and E. Brodin Dept. of Pharmacology, Karolinska Institute, Stockholm, Sweden The extracellular level of substance P (SP) during basal and stimulated conditions was monitored in the periaqueductal grey (PAG) in anaesthetized and awake male Sprague-Dawiey rats (300-350 g body weight). In the first group of animals, a microdialysis probe (membrane length 2 mm) was stereotaxically implanted in the PAG under halothanelnitrous oxide anaesthesia, which was then continued throughout the experiment. The probe was perfused with Krebs-Ringer solution (7 pl/min) and collection of 15 min samples started 1 h after implantation. In the other group, the same surgical technique was used to implant a guide. Two days later a microdialysis probe was inserted in the guide and the animal placed in a freely moving animal system and dialysate samples collected. SP-like immunoreactivity was measured by RIA. In all 7 experiments on awake animals, a basal level of SP in the dialysates could be detected (mean value 1.65 f 0.45

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fmol/ml, S.E.M.), while in the anaesthetized animals (n = 14), SP could only occasionally be detected under basal conditions. Stimulation with potassium (100 p_Min the perfusion fluid during 15 min) increased the SP concentration to 23.86 + 5.48 and 21.53 + 3.38 fmoliml in anaesthetized and awake animals, respectively. A second stimulation, which was initiated 90 min later. induced a similar release in the anaesthetized rats and a somewhat less pronounced release in the awake animals. Morphine (1 mg/kg, s.c.) given 60 min after the first stimulation in anaesthetized animals tended to reduce the release in response to the second stimulation (S2/Sl ratio reduced from 0.96 to 0.52), suggesting an inhibition of the SP release in the PAG which may be in accordance with the previously demonstrated increase in tissue level of SP in the PAG following acute administration of morphine.

Structure-Activity Study of R 396, an NK-2 Receptor Antagonist Selective for the NK-2s Receptor Subtype P. Rovero*, M. Astolfit, D. Jukicl, N. Rouissif, D. Regoliz and C. A. Maggig *Ist. di Mutagenesi e Differenziamento, CNR, Pisa, Italy; tMenarini Sud, Pomezia, Italy; $Univ. of Sherbrooke, Canada; §A. Menarini Pharmaceuticals, Firenze, Italy R 396 is a linear hexapeptide (Ac-Leu-Asp-Gln-Trp-PheGly-NH,) derived from the tachykinin antagonist L 659,874 (Ac-Leu-Met-Gin-Trp-Phe-Gly-NHz) by replacing Asp for Met in position 2. R 396, as compared to L 659,874, shows a tenfold greater antagonist affinity in the hamster trachea (HT), while maintaining the same affinity in the rabbit pulmonary artery (RPA). This marked difference of affinity between two NK-2 preparations was instrumental for the discovery of the NK-2 receptor subtypes. We report here on a structure-activity study of R 396 : Asp2, Trp4 and the C-terminal glycinamide have been challenged by classical amino acid substitutions with the aim of elucidating the structural requirements responsible for NK-2 subtype selectivity. The biological activities of R 396 analogues indicate that the Asp residue in position 2 has a crucial role for the high affinity of this peptide at the NK-2, subtype (HT): none of the analogues substituted in position 2 display higher affinity in the HT, as compared to R 396, regardless of the nature of the residue introduced. Trp residue in position 4 has been replaced by other aromatic residues: while Phe and His yielded completely inactive analogues, introduction of Tyr in position 4 gave rise to a quite interesting analogue which maintains antagonistic activity at the NK-2B receptor subtype (pA, = 6.9 in the HT) while being a fnll agonist in the RPA (NK-2,). Finally, the C-terminal amide appears to be crucial for affinity, the free acid analogue being devoid of biological activity on both HT and RPA. On the contrary, the Gly residue do not have per se a key role: antagonistic activ-