At the cutting edge

Melatonin binding sites: Are they receptors?

Prevention of dementia is important, because it is a leading cause of disability in elderly people. We previously reported that acute intraperitoneal treatment with N-acetyl-5-methoxy kynuramine (AMK), a melatonin (MEL) metabolite, enhanced long-term object recognition memory in ICR mice, a MEL deficient strain. Despite the presumable availability of AMK for dementia, its effects on cognitive performance have not been elucidated. It is unclear whether endogenous AMK is responsible for modulating long-term memory performance. To address this question, we assessed the effects of endogenous AMK on learning and memory using an object recognition test. C3H mice, a MEL-proficient strain, showed peak MEL levels at zeitgeber times (ZT) 19 and 22. Object recognition memory at ZT20 was superior to that at ZT8. Norharmane (NHM, 100 mg/kg), an indoleamine-2,3-dioxygenase (IDO) inhibitor, prevented the transformation of MEL to AMK, thereby suppressing AMK synthesis at ZT20. NHM (100 mg/kg) and another IDO inhibitor, 1-methyl-L-tryptophan (1-MT, 100 mg/kg), disrupted elevated cognitive performance at ZT20. These data imply that endogenous AMK may play a physiological role in the modulation of cognitive function. We also investigated the effects of pharmacological doses of MEL and AMK on object recognition memory in young C3H mice. MEL administration of 0.1 mg/kg, but not 0.01 mg/kg, enhanced object recognition memory, whereas 0.01 and 1 mg/kg AMK enhanced object recognition memory. Administration of 0.1 and 1 mg/kg AMK also enhanced object recognition memory in old C3H mice. These findings in MEL-proficient mice should be confirmed in other learning and memory tests before encouraging the clinical use of AMK.

Melatonin is a skin lightening agonist of the toadBufo ictericus.The hormone also exhibits an autodesensitizing action as well as an inhibitory activity on the darkening response to alpha-MSH. In an attempt to pharmacologically characterize the melatonin receptor of the toad pigment cells, serotonin and N-acetylserotonin (intermediate products of melatonin biosynthesis) biological activities were compared to melatonin effects. Serotonin (6.4 × 10⁻⁵to 10⁻⁹M) exhibited no lightening or inhibitory activity on MSH-elicited darkening responses or melatonin-elicited lightening responses.N-acetylserotonin, although less potent than melatonin, elicited a significant lightening response on previously MSH-darkened skins. Previous exposure of the skins withN-acetylserotonin reduced both MSH-induced darkening and melatonin-induced lightening in MSH-darkened skins. These results suggest a competitive inhibition of melatonin byN-acetylserotonin and indicate that melatonin receptors inB. ictericuspigment cells may be similar to ML-1 type found in chicken retina.

We now describe the preparation and characterization of a novel radioligand, 2-[¹²⁵I]]iodo-5-methoxy-carbonylamino-N-acetyltryptamine (2-[¹²⁵I]MCA-NAT), with high affinity and pharmacological selectivity for melatonin ML₂ receptor sites. 2-[¹²⁵I]MCA-NAT was prepared by introducing an [¹²⁵I]iodine molecule on carbon 2 of 5-MCA-NAT (5-methoxycarbonylamino N-acetyltryptamine), a selective melatonin ML₂ receptor ligand. The specific binding (88%) of 2-[¹²⁵I]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-[¹²⁵I]MCA-NAT showed no affinity for melatonin ML₁ receptors of chicken retina. Competition curves of various melatonin analogues for 2-[¹²⁵I]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 ML₂ sites [2-iodomelatonin (0.77) > 6-chloro-2-methyl-melatonin (2.56) > 6-chloromelatonin (6.8) > prazosin (21.7) ≥ N-acetylserotonin (23.3 nM) ≥ 5-MCA-NAT (29.5) ≥ melatonin (83.9) > luzindole (1687) > serotonin (2120)]. Affinity constants for competition of melatonin analogues on [¹²⁵I]MCA-NAT binding to hamster brain, testes, and kidney correlated significantly [r = 0.962, P < 0.001, n = 9; r = 0.892, P < 0.0001, n = 13; r = 0.975, P < 0.0001, n = 9, respectively) with the affinities determined on 2-[¹²⁵I]iodomelatonin binding to ML₂ sites (hamster brain) but not to ML₁ sites (chicken retina, r = 0.33, P > 0.05, n = 16). In conclusion, 2-[¹²⁵I]MCA-NAT is a specific radioligand for the identification and characterization of ML₂ binding sites in brain and peripheral tissues.

There is some indirect evidence that the pineal hormone melatonin can suppress plasma levels of cholesterol in hypercholesterolemic rats. We have examined the effects of the hormone on cellular cholesterol metabolism in freshly isolated human mononuclear leukocytes. Incubation of cells for up to 20 h in a lipid-free medium resulted in an increase in the rate of cholesterol synthesis from [¹⁴C]acetate and the high affinity accumulation and degradation of [¹²⁵I]labeled low density lipoprotein (LDL). Addition of melatonin in increasing concentrations to the incubation medium at zero time inhibited cholesterol synthesis and the specific accumulation and degradation of [¹²⁵I]labeled LDL; at a concentration of 100 μM, the inhibitions were 38 %, 42 %, and 48 %, respectively. Similar results were obtained using [¹⁴C]mevalonate as precursor. Fatty acid synthesis was not altered under these conditions. In contrast to cholesterol the synthesis of the first cyclic compound lanosterol was not affected by the pineal hormone. These results implicate that melatonin inhibits this pathway between lanosterol and cholesterol. The action of melatonin on LDL receptor activity appeared to be mediated by a decrease in the number of LDL receptors and not by a change in binding affinity. Pharmacological characterization of the potential melatonin receptor site using several analogs hire tryptamine, 5-hydroxytryptamine, N-acetyl-5-hydroxtryptamine, 5-methoxytryptamine, and 6-chloromelatonin indicated that the 5-methoxy group is indispensible for the hormone action on cholesterol synthesis. The data provide evidence that melatonin can modulate cholesterol metabolism in human cells.

Literature is reviewed indicating that aging is characterized by changes in circadian rhythms and sleep quality. The most marked change is an attenuation of amplitude. An advance of phase, a shortening of period, and a desynchronization of rhythms are also evident. The mechanisms underlying these changes are unknown. However, age-related changes in the retina, suprachiasmatic nucleus, and pineal gland seem relevant along with behavioral changes such as a reduction in physical activity and exposure to photic stimulation. Changes in circadian rhythms are frequently associated with a reduction in nighttime sleep quality, a decrease in daytime alertness, and an attenuation in cognitive performance; reversing such changes could enhance the quality of life for a large and rapidly increasing percentage of the population. Reversal appears possible by increasing melatonin levels with either appropriately timed exposure to photic stimulation and/or appropriately timed administration of exogenous melatonin. These interventions may increase aspects of genetic expression that have changed with aging. A hypothesis concerning the potential benefits of enhanced circadian amplitude is also offered.