The melatonin analog 5-MCA-NAT increases endogenous dopamine levels by binding NRH:quinone reductase enzyme in the developing chick retina.

NRH:quinone reductase (QR2) is present in the retinas of embryonic and post-hatched (PH) chicks. 5-Methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT) is a QR2 ligand that increases cAMP levels in developing retinas, but it does not affect cAMP levels in CHO-QR2 cells. The dopamine quinone reductase activity of QR2 retrieves dopamine, which increases cAMP levels in developing retinas. The objective of the present study was to investigate whether 5-MCA-NAT increases endogenous dopamine levels in retinas from chick embryos and post-hatched chicks. Endogenous dopamine was measured by enzyme-linked immunosorbent assay (ELISA). 5-MCA-NAT increased retinal endogenous dopamine levels at all developmental stages studied and in PH chicks (-logEC50=11.62±0.34 M). This effect was inhibited by non-selective antagonists of receptors and melatonin binding sites N-acetyl-2-benzyltryptamine (luzindole, 5 µM), but it was not inhibited by the Mel1b melatonin receptor antagonist 4-phenyl-2-propionamidotetralin (4-P-PDOT, 10 nM). The QR2 cosubstrate, N-methyl-dihydronicotinamide (NMH) (-logEC50=6.74±0.26 M), increased endogenous dopamine levels in controls and in retinas stimulated with 5-MCA-NAT (3 nM). The QR2 inhibitor benzo[e]pyrene inhibited endogenous dopamine levels in both control (-logIC50=7.4±0.28 M) and NMH-stimulated (at 100 nM and 1 µM benzo[e]pyrene concentrations) retinas. Theoretical studies using Molegro Virtual Docking software corroborated these experimental results. We conclude that 5-MCA-NAT increases the level of endogenous dopamine via QR2. We suggest that this enzyme triggers double reduction of the dopamine quinone, recovering dopamine in retinal development.

Melatonin and the time window for the expression of the alpha8 nicotinic acetylcholine receptor in the membrane of chick retinal cells in culture.

We have previously shown that melatonin influences the development of alpha8 nicotinic acetylcholine receptor (nAChR) by measurement of the acetylcholine-induced increase in the extracellular acidification rate (ECAR) in chick retinal cell cultures. Cellular differentiation that takes place between DIV (days in vitro) 4 and DIV 5 yields cells expressing alpha8 nAChR and results in a significant increase in the ECAR acetylcholine-induced. Blocking melatonin receptors with luzindole for 48h suppresses the development of functional alpha8 nAChR. Here we investigated the time window for the effect of melatonin on retinal cell development in culture, and whether this effect was dependent on an increase in the expression of alpha8 nAChR. First, we confirmed that luzindole was inhibiting the effects of endogenous melatonin, since it increases 2-[(125)I] iodomelatonin (23pM) binding sites density in a time-dependent manner. Then we observed that acute (15, 60min, or 12h) luzindole treatment did not impair acetylcholine-induced increase in the ECAR mediated by activation of alpha8 nAChR at DIV 5, while chronic treatment (from DIV 3 or DIV 4 till DIV 5, or DIV 3.5 till DIV 4.5) led to a time-dependent reduction of the increase in the acetylcholine-induced ECAR. The binding parameters for [(125)I]-alpha-bungarotoxin (10nM) sites in membrane were unaffected by melatonin suppression that started at DIV 3. Thus, melatonin surges in the time window that occurs at the final stages of chick retinal cell differentiation in culture is essential for development of the cells expressing alpha8 nAChR subtype in full functional form.

An unexpected effect of 5-MCA-NAT in chick retinal development.

Luzindole is an unselective antagonist of the melatonin receptors and melatonin's other binding sites, although some exceptions have been observed in chick retinal neurodevelopment, where this unselective antagonist does not block melatonin's inhibitory effect on the adenylate cyclase enzyme, probably due to the presence of some other melatonin receptor(s) or binding site(s). The present study investigated the modulation of cyclic adenosine 3'-5'-monophosphate (cAMP) levels via MT3 melatonin-binding sites, located within the QR2 (dihydronicotinamide riboside: quinone oxidoreductase 2) enzyme, by observing the response to luzindole. Embryonic and post-hatch retinas, incubated with a selective agonist for the MT3 melatonin-binding site 5-methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT, 10 or 100 nM), had an increase in cAMP accumulation relative to control retinas. Luzindole (5microM) inhibited the 5-MCA-NAT stimulatory effect at all ages tested. The agonist 5-MCA-NAT enhanced the melatonin inhibitory effect on cAMP levels stimulated by forskolin (5microM), but not the stimulatory forskolin effect. The results suggest that MT3 melatonin-binding sites are present in embryonic and post-hatch chick retinas and that luzindole more selectively blocks the 5-MCA-NAT effect on cAMP accumulation than it blocks the melatonin inhibitory effect via G protein-coupled receptors in chick retinal neurodevelopment.

Melatonin inhibitory effect on cAMP accumulation in the chick retina development.

During vertebrate neurodevelopment, neuritogenesis and synaptogenesis are modulated by intracellular cAMP rises. Melatonin, which is implicated in neuronal differentiation, mainly inhibits this pathway. Here, an investigation about the profile of this effect during the vertebrate neurodevelopment is reported. In the embryonic chick retinas at days 8, 12, 14, 16 and at 2 days post-hatched (E8, E12, E14, E16 and PH), those control embryonic retinas incubated only with the phosphodiesterase inhibitor at days corresponding to commencement of neuronal differentiation (E8, E12) and PH, presented cAMP levels inhibited by melatonin. While the cAMP accumulation stimulated by forskolin was inhibited in the embryonic retinas at all testing days. Neither the unselective antagonist N-acetyl-2-benziltryptamine (luzindole) nor the selective Mel(1b) antagonist 4-phenyl-2-propionamidotetralin (4-P-PDOT) blocked the melatonin concentration-dependent inhibitory effect on cAMP accumulation in the retinas initiating differentiation (E7-E9), suggesting a tight binding between melatonin and their receptors. However, 4-P-PDOT competitively reverted the melatonin effect on cAMP stimulated by forskolin during synaptogenesis stages. Together, the melatonin effect on cAMP levels in chick retina, which is mainly through melatonin receptors, is depending on the developmental period observed, probably taking part in the mechanisms surrounding the melatonin action on neuronal differentiation.