Pharmacology of a new tritiated endomorphin-2 analog containing the proline mimetic cis-2-aminocyclohexanecarboxylic acid.
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 Pro(2) is replaced by 2-aminocyclohexanecarboxylic acid, ACHC. [(3)H][(1S,2R)ACHC](2)EM-2 (specific activity 63.49Ci × mmol(-1)) bound specifically to its binding sites with high affinity (K(D) = 0.55 ± 0.06 nM) and saturably, yielding a receptor density, B(max) 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 [(3)H][(1S,2R)ACHC](2)EM-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.
Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary.
Attila
A
Keresztes
Erika
E
Birkás
Annamária
A
Páhi
Géza
G
Tóth
Lidia
L
Bakota
Károly
K
Gulya
Mária
M
Szücs
D000818:Animals; D001345:Autoradiography; D002264:Carboxylic Acids / Q000737:chemistry*; D003503:Cyclobutanes / Q000737:chemistry*; D007700:Kinetics; D018716:Molecular Mimicry; D009842:Oligopeptides / Q000737:chemistry / Q000494:pharmacology*; D011392:Proline / Q000737:chemistry*; D011869:Radioligand Assay; D051381:Rats
C542744:2-aminocyclobutanecarboxylic acid; D002264:Carboxylic Acids; D003503:Cyclobutanes; D009842:Oligopeptides; C105294:endomorphin 2; D011392:Proline