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Pharmacology of a new tritiated endomorphin-2 analog containing the proline mimetic cis-2-aminocyclohexanecarboxylic acid

2011; Elsevier BV; Volume: 32; Issue: 4 Linguagem: Inglês

10.1016/j.peptides.2011.01.017

1873-5169

Attila Keresztes, Erika Birkás, Annamária Páhi, Géza Tóth, Lidia Bakota, Károly Gulya, Mária Szűcs,

Receptor Mechanisms and Signaling

As part of ongoing work aimed at generating proteolytically stable, readily applicable, radiolabeled endomorphin-2 (EM-2) analogs for elucidation of the topological requirements of peptide binding to μ-opioid receptors, we report here on the synthesis, radiolabeling, binding kinetics and binding site distribution of an EM-2 analog in which Pro2 is replaced by 2-aminocyclohexanecarboxylic acid, ACHC. [3H][(1S,2R)ACHC]2EM-2 (specific activity 63.49 Ci × mmol−1) bound specifically to its binding sites with high affinity (KD = 0.55 ± 0.06 nM) and saturably, yielding a receptor density, Bmax of 151 ± 4 fmol × mg protein−1 in rat brain membranes. A similar affinity value was obtained in kinetic assays. Both Na+ and Gpp(NH)p decreased the affinity, proving the agonist character of the radioligand. Specific μ-opioid ligands displaced the radioligand with much higher affinities than did δ- and κ-ligands. The autoradiographic distribution of the binding sites of [3H][(1S,2R)ACHC]2EM-2 agreed well with the known locations of the μ-opioid receptors in the rat brain. In consequence of its high affinity, selectivity and enzymatic resistance [19], the new radioligand will be a good tool in studies of the topographical requirements of μ-opioid-specific peptide binding.