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

Abstract

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±0.34M). This effect was inhibited by non-selective antagonists of receptors and melatonin binding sites N-acetyl-2-benzyltryptamine (luzindole, 5μM), but it was not inhibited by the Mel1b melatonin receptor antagonist 4-phenyl-2-propionamidotetralin (4-P-PDOT, 10nM). The QR2 cosubstrate, N-methyl-dihydronicotinamide (NMH) (−logEC50=6.74±0.26M), increased endogenous dopamine levels in controls and in retinas stimulated with 5-MCA-NAT (3nM). The QR2 inhibitor benzo[e]pyrene inhibited endogenous dopamine levels in both control (−logIC50=7.4±0.28M) and NMH-stimulated (at 100nM and 1μM 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.