Melatonin receptor antagonists that differentiate hetween the human Mel(1a) and Mel(1b) recombinant subtypes are used to assess the pharmacological profile of the rabbit retina ML(1) presynaptic heteroreceptor

We have identified subtype selective agonists, partial agonists and antagonists, which distinguish the human recombinant Me(1a), and Mel(1b) melatonin receptors expressed in COS-7 cells. Melatonin receptor agonists showed higher affinity for competition of 2-[¹²⁵I]-iodomelatonin binding for the Mel(1b) than the Mel(1a) melatonin receptor. The dissociation constants (Ki) of 16 agonists determined on the recombinant human Mel(1a) and Mel(1b) melatonin receptor subtypes showed a significant correlation (r² = 0.851 slope 0.97, P < 0.0001, n = 16). However, six agonists showed 10 to 60 fold higher affinity for the Mel(1b) melatonin receptor as indicated by the affinity selectivity ratios (Mel(1a)/Mel(1b)) [8-methoxy-2-acetamidotetraline (11); S20098 (14); 8-methoxy-2- propionamidotetraline (20); 6, 7 di-chloro-2-methyl-melatonin (21); 6-chloromelatonin (57); 6-methoxymelatonin (59)]. Dissociation constants for competition of 11 partial agonists and antagonist for 2-[¹²⁵I]-iodomelatonin binding were between 15.5 (luzindole, pKi: 7.7) to 362 (2-phenyl-2-chloroacetamidotetraline, pKi: 9.1) fold higher for the Mel(1b) than for the Mel(1a) melatonin receptor. The lack of correlation between the pKi values (r² = 0.23, P > 0.1, n = 11) strongly suggest that the two human melatonin receptor subtypes can be distinguished pharmacologically. The partial agonist: 5-methoxyluzindole (pKi: 9.6) and the competitive melatonin receptor antagonists: GR128107 (pKi: 9.6), 4-phenyl-2-chloroacetamidotetraline (pKi: 9.1), 4-phenyl-2-acetamidotetraline (pKi: 8.9) and 4-phenyl-2-propionamidotetraline (pKi: 8.8) are selective Mel(1b) melatonin receptor analoguesas their affinity selectivity ratios (Mel(1a)/Mel(1b)) are bigger than 100. We conclude that the 40% overall amino acid difference in the sequence of the human recombinant Mel(1a), and Mel(1b) melatonin receptors is reflected in distinct pharmacological profiles for the subtypes. We compared the pharmacological profile of the presynaptic ML₁ melatonin heteroreceptor of rabbit retina mediating inhibition of the calcium-dependent release of dopamine to that of the recombinant Mel(1a) and Mel(1b) melatonin receptors. Melatonin inhibited [³H]dopamine release by 50% (IC₅₀) at 20 pM with a maximal inhibitory effect (80%) at 1 nM. The partial agonists, i.e., N-acetyltryptamine (IC₅₀: 5.6, maximal inhibition 55%) and 5-methoxyluzindole (IC₅₀: 1.3, maximal inhibition 40%) showed various degrees of efficacy while none of the competitive melatonin receptor antagonists did inhibit [³H]dopamine release on their own. The potency (IC₅₀) of full melatonin receptor agonists significantly correlated with their affinity to compete for 2-[¹²⁵I]-iodomelatonin binding to either the Mel(1a) (r² = 0.76, slope = 0.77, P < 0.0001, n = 17) or Mel(1b) (r² = 0.63, slope = 0.75, P < 0.001, n = 17) human melatonin receptors. BY contrast, the apparent dissociation constants (K(B)) for partial agonists and antagonists to antagonize the inhibition of [³H]dopamine release mediated by activation of the ML₁ heteroreceptor by melatonin, significantly correlated with the affinity constants (Ki) for 2-[¹²⁵I]-iodomelatonin binding determined on the Mel(1b) (r² = 0.77, slope = 0.55, P < 0.001; n = 11) but not the Mel(1a), (r² = 0.27, P < 0.1, n = 11) subtype. Together these results demonstrate that the pharmacological profile of the human recombinant Mel(1b) melatonin receptor is similar to that of the functional presynaptic melatonin heteroreceptor of rabbit retina, which we referred as an ML(1B) subtype. We conclude that the selective Mel(1b) melatonin partial agonists and antagonists described here can be used to identify melatonin receptor subtypes in native tissues and to search for subtype selective analogues with therapeutic potential.