Endomorphin-1 (Tyr-Pro-Trp-Phe-NH 2 ) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH 2 ) are two recently isolated m-opioid selective peptides with a potent antinociceptive activity, involved in a number of physiological processes, including food intake, vasomotricity, sexual behavior, as well as neuroendocrine and cardiorespiratory functions. The neuroanatomical distribution of endomorphins prompted us to study their antidepressant activity in two animal behavioral models of depression: forced-swimming and tail-suspension tests. In both tests, the intracerebroventricular (i.c.v.) injection of either endomorphin-1 or endomorphin-2 significantly decreased the duration of immobility, interpreted as an expression of 'behavioral despair', which could be related to the depression syndrome. These effects of endomorphins did not result from the stimulation of the animal motor activity. We have also demonstrated that the antidepressant-like effect of endomorphins was antagonized by the universal opioid antagonist, naloxone and the m-opioid receptor selective antagonist, b-funaltrexamine. In contrast, this effect was not antagonized by d-and k-opioid receptor selective antagonists, naltrindole and nor-binaltorphimine, respectively. The results of the present study demonstrate that endomorphin-1 and endomorphin-2 produce potent antidepressant-like effects after i.c.v. injection in mice. We may suggest that endomorphins and the m-opioid receptors might be involved in the physiopathology of depressive disorders, and that the endomorphinergic system could serve as a novel target for the development of antidepressant drugs. Neuropsychopharmacology (2007) 32, 813-821.

A functional assay, based on aequorin-derived luminescence triggered by receptor-mediated changes in intracellular calcium levels, was used to examine relative potency and efficacy of the -opioid agonists endomorphin-1, endomorphin-2, morphiceptin, and their position 3-substituted analogs, as well as the ␦-agonist deltorphin-II. The results of the aequorin assay, performed on recombinant cell lines, were compared with those obtained in the functional assay on isolated tissue preparations (guinea pig ileum and mouse vas deferens). A range of nine opioid peptide ligands produced a similar rank order of potency for the -and ␦-opioid receptor agonists in both functional assays. The highest potency at the -receptor was observed for endomorphin-1, endomorphin-2, and [D-1-Nal 3 ]morphiceptin, whereas deltorphin-II was the most potent ␦-receptor agonist. In the aequorin assay, the -and ␦-agonist-triggered luminescence was inhibited by the opioid antagonists naloxone and naltrindole, respectively. We can conclude that the use of the aequorin assay for new -and ␦-receptor-selective opioid analogs gives pharmacologically relevant data and allows high-throughput compound screening, which does not involve radioactivity or animal tissues. This is the first study that validates the application of this assay in the screening of opioid analogs.Current methods for the identification and characterization of new ligands at the three main opioid receptor types (, ␦, ) typically use binding assays, where a radioligand with high affinity to one receptor type is displaced by a tested analog. Apart from binding studies, different functional assays can also be used to determine relative potency and efficacy of new analogs at the opioid receptors. The most popular functional assay used for opioid activity determination in vitro is performed on isolated tissue preparations. This assay is based on inhibition of electrically evoked contractions of guinea pig ileum (GPI) and mouse vas deferens (MVD). The opioid effect in the GPI preparations is mainly mediated by the -receptors, whereas the predominant receptors of the MVD are of the ␦-type. The GPI/MVD assay makes it possible to determine the -and ␦-receptor interactions, which is the main focus in standard tests for opioid activity.In the present study, we used an alternative functional assay, which does not involve radioactivity or animal tissues and allows high-throughput screening of opioid peptide analogs. This assay is based on the recombinant mammalian cell line expressing an opioid receptor together with a luminescent reporter protein.Opioid receptors belong to a large family of the G proteincoupled receptors (GPCR), which on stimulation by a ligand induce the activation of the phospholipase C, with the subsequent generation of the primary second messenger metabolites: diacylglycerol and inositol 1,4,5-trisphosphate, followed by a release of calcium from intracellular stores. These agonist-induced receptor-mediated changes in calcium levels can be quantitatively assessed and ...

Urokinase plasminogen activator plays a key role in tumor-associated processes, increasing cancer cell invasion and metastasis, and is therefore used as a marker in cancer prognosis. In this study, we have determined the effect of mu-opioid receptor agonists and antagonists on the urokinase plasminogen activator secretion in MCF-7 cell line. It was shown that mu-opioid receptor agonists, such as morphine and endomorphins, greatly stimulate urokinase plasminogen activator secretion, while naloxone and MOR-selective antagonists elicit the opposite effect. The same tendency was observed also on the urokinase plasminogen activator mRNA level. However, neither agonists nor antagonists had any effect on proliferation of MCF-7 cells. The findings reported in this study may be useful in designing further experiments aimed at elucidating the role of the opioid system in cancer cells.

The aim of the study was to investigate the presence of opioid receptor types in human breast adenocarcinoma MCF-7 cells and to characterize the changes in MOR expression induced by opioid agonist and antagonist treatment. We have shown that all three types of opioid receptors, but predominantly MOR, are expressed in MCF-7 cells. Selective MOR agonists, morphine, endomorphin-1, and endomorphin-2 downregulated MOR mRNA levels in a concentration- and time-dependent manner, but the effect produced by endomorphins was much stronger. Downregulation was blocked by the opioid antagonist naloxone. Naloxone alone produced a slight increase in MOR gene expression. Immunoblotting with antiserum against MOR-1 confirmed these results at the protein level. The results of our study indicate that, in MCF-7 cells, MOR gene expression is downregulated by opioid agonists and upregulated by opioid antagonists. We propose that the opioid-induced regulation of MOR mRNA expression is mediated by reduced binding of the transcription factors NFkappaB and AP-1 to the promoter region on the MOR gene.

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