We now describe the preparation and characterization of a novel radioligand, 2-[I-125]iodo-5-methoxy-carbonylamino-N-acetyltryptamine (2-[I-125]MCA-NAT), with high affinity and pharmacological selectivity for melatonin ML(2) receptor sites. 2-[I-125]MCA-NAT was prepared by introducing an [I-125]iodine molecule on carbon 2 of 5-MCA-NAT (5-methoxycarbonylamino N-acetyltryptamine), a selective melatonin ML(2) receptor ligand. The specific binding (88%) of 2-[I-125]MCA-NAT (50 pM) to whole washed hamster brain membranes showed rapid kinetics of association/dissociation, and was of high affinity and saturable (K-d value = 116 +/- 14 pM; B-max value = 15.5 +/- 1.8 fmol/mg protein, n = 3). 2-[S-125]]MCA-NAT showed no affinity for melatonin ML, receptors of chicken retina. Competition curves of various melatonin analogues for 2-[I-125]MCA-NAT binding to hamster brain, testes and kidney were monophasic and showed a pharmacological order of affinities (K-i values for brain, nM) identical to that of the ML(2) sites [2-iodomelatonin (0.77) > 6-chloro-2-methyl-melatonin (2.56) > 6-chloromelatonin (6.8) > prazosin (21.7) greater than or equal to N-acetylserotonin (23.3 nM) greater than or equal to 5-MCA-NAT (29.5) greater than or equal to melatonin (83.9) > luzindole (1687) > serotonin (2120)]. Affinity constants for competition of melatonin analogues on [I-125]MCA-NAT binding to hamster brain, testes, and kidney correlated significantly [r = 0.962, P < 0.001, n = 9; r = 0.982, P < 0.0001, n = 13; r = 0.975, P < 0.0001, n = 9, respectively) with the affinities determined on 2-[I-125]iodomelatonin binding to ML(2) sites (hamster brain) but not to ML(1) sites (chicken retina, r = 0.33, P > 0.05, n = 16). In conclusion, 2-[I-125]MCA-NAT is a specific radioligand for the identification and characterization of ML(2) binding sites in brain and peripheral tissues.
