La arilaquilamina n-acetiltransferasa ha sido considerada la enzima limitante en la ruta de síntesis de melatonina en los vertebrados. El objetivo de la tesis doctoral es el conocimiento del papel que juega esta enzima en la síntesis de melatonina en la retina de los anfibios anuros, su regulación por factores ambientales correlacionándose con los mecanismos endógenos del animal. La nat muestra un ritmo diario en su actividad mostrando apareciendo los valores máximos durante la noche, la exposición a la luz reduce los niveles de la nat hasta valores equivalentes a los basales diurnos. La aparición del ritmo ocular de la actividad nat durante la ontogenia de los anuros coincide con el comienzo de la función visual. Las variaciones estacionales del fotoperiodo y la temperatura determinan el perfil diario de la melatonina y nat ocular. La temperatura regula la amplitud de la oscilación diaria de melatonina, y el fotoperiodo la duración del máximo nocturno. La cinética de la nat muestra una modulación térmica positiva, y una inhibición por exceso de serotonina dependiente de la temperatura, la regulación endogena del ritmo de melatonina y nat se lleva a cabo por medio de la dopamina a través de receptores d2 los mensajeros intracelulares responsables del ritmo de melatonina son el ampc y el calcio
Velarde, Elena, Cerdá-Reverter, Jose Miguel, Alonso-Gómez, Angel Luis, Sánchez, Elisa, Isorna, Esther, and Delgado, María Jesús
Chronobiology International: The Journal of Biological & Medical Rhythm Research; Jul2010, Vol. 27 Issue 6, p1178-1201, 24p, 2 Diagrams, 3 Charts, 6 Graphs
MELATONIN, ENZYMES, TRYPTAMINE, MESSENGER RNA, and ENZYMOLOGY
It has been suggested that melatonin is synthesized in nonphotosensitive organs of vertebrates in addition to the well-known sites of the pineal gland and retina. However, very few studies have demonstrated the gene expression of melatonin-synthesizing enzymes in extrapineal and extraretinal locations. This study focuses on the circadian expression of the two key enzymes of the melatoninergic pathway, arylalkylamine N-acetyltransferase (AANAT) and hydroxyindole- O-methyltransferase (HIOMT), in central and peripheral locations of a teleost fish, the goldfish ( Carassius auratus). First, the full-length cDNA sequences corresponding to the goldfish AANAT-2 (g Aanat-2) and HIOMT-2 (g Hiomt-2) were cloned, showing high similarity with other teleost sequences. Two forms of AANAT exist in teleosts. Here, for the first time, two isoforms of HIOMT are deduced from phylogenetic analysis. Moreover, both HIOMT and AANAT were detected in several peripheral locations, including liver and gut, the present results being the first to find HIOMT in nonphotosensitive structures of a fish species. Second, quantitative real-time polymerase chain reaction (PCR) studies were performed to investigate regulation of g Aanat-2 in pineal and peripheral locations of goldfish maintained under different lighting conditions. The current results show circadian rhythms in Aanat-2 and Hiomt-2 transcripts in liver and hindgut, suggesting a local melatonin synthesis in goldfish. Moreover, the analysis of daily expression of g Aanat-2 under different lighting conditions, including continuous light (24L) and darkness (24D) revealed light-dependent rhythms in the pineal and retina, as expected, but also in liver and hindgut. The persistence in hindgut of these g Aanat-2 rhythms under both constant conditions, 24L and 24D, suggests expression of this transcript is governed by a circadian clock and entrained by nonphotic cues. Finally, the current results support the existence of melatonin synthesis in gut and liver of the goldfish. (Author correspondence: ) [ABSTRACT FROM AUTHOR]
Oliveira, Catarina, López‐Olmeda, José Fernando, Delgado, María Jesus, Alonso‐Gómez, Angel Luis, and Sánchez‐Vázquez, Francisco Javier
Chronobiology International: The Journal of Biological & Medical Rhythm Research; 2008, Vol. 25 Issue 4, p645-652, 8p, 1 Graph
MELATONIN, BRAIN, HYPOTHALAMUS, CEREBELLUM, TELENCEPHALON, and AQUACULTURE
We localized melatonin binding sites in different brain regions (optic tectum, telencephalon, cerebellum, hypothalamus, olfactory bulbs, and medulla oblongata) of Senegal sole, a species of aquaculture interest, and checked day/night changes in density (Bmax) at mid-light (ZT06) and mid-dark (ZT18). Plasma melatonin was measured using a radioimmunoassay, while binding assays were performed using 2-[125I]iodomelatonin as a radioligand. Plasma melatonin concentrations were significantly lower at mid-light (189.5±46 pg/ml) than mid-dark (455.5±163 pg/ml). Values of Bmax were statistically significantly higher in the optic tectum (5.6±0.6 and 12.3±1 fmol/mg prot, at mid-light and mid-dark, respectively) and in the cerebellum (7.7±1.1 and 10.6±1.3 fmol/mg prot, at mid-light and mid-dark, respectively). Significant day/night differences were only observed in these two tissues. These results show for the first time the distribution of melatonin binding sites within the brain of a flatfish species and their lack of down-regulation. [ABSTRACT FROM AUTHOR]
Melatonin is a key neuroendocrine transducer in the circadian organization of vertebrates. However, its role in gastrointestinal physiology has not been explored in depth. In goldfish, a role for melatonin as a modulator of intestinal motility has been reported, whereby it attenuates the cholinergic contraction. The aim of the present work was to investigate this relaxation induced by melatonin in the gut smooth muscle of the goldfish, studying the possible involvement of nitric oxide. An in vitro model of isolated goldfish intestine was used to test the effects on intestinal motility. The addition of melatonin (10pM–100μM) to the organ bath relaxed acetylcholine- and serotonin-stimulated gut strips, but no effect was observed on KCl-contracted preparations. The addition of L-NAME (nitric oxide synthase inhibitor) increased the amplitude of the spontaneous slow waves, while sodium nitroprusside (SNP, nitric oxide donor) abolished them. All these results support a role for the nitrergic system in goldfish gut motility. However, neither L-NAME, nor SNP nor the nitric oxide precursor, l-arginine, modified the melatonin relaxing effect. These results highlight the existence of a basal nitrergic tone in the gut of goldfish, where melatonin would exert a calcium-dependent, nitric oxide-independent relaxing effect on serotonergic and cholinergic contraction.
Ghrelin is the only known hormone posttranslationally modified with an acylation. This modification is crucial for most of ghrelin's physiological effects and is catalyzed by the polytopic enzyme ghrelin O-acyltransferase (GOAT). The aim of this study was to characterize GOAT in a teleost model, goldfish (Carassius auratus). First, the full-length cDNA sequence was obtained by RT-PCR and rapid amplification of cDNA ends methods. Two highly homologous cDNAs of 1491 and 1413 bp, respectively, named goat-V1 and goat-V2 were identified. Deduced protein sequences (393 and 367 amino acids, respectively) are predicted to present 11 and 9 transmembrane regions, respectively, and both contain two conserved key residues proposed to be involved in catalysis: asparagine 273 and histidine 304. RT-qPCR revealed that both forms of goat mRNAs show a similar widespread tissue distribution, with the highest expression in the gastrointestinal tract and gonads and less but considerable expression in brain, pituitary, liver and adipose tissue. Immunostaining of intestinal sections showed the presence of GOAT immunoreactive cells in the intestinal mucosa, some of which colocalize with ghrelin. Using an in vitro approach, we observed that acylated ghrelin downregulates GOAT gene and protein levels in cultured intestine in a time-dependent manner. Finally, we found a rhythmic oscillation of goat mRNA expression in the hypothalamus, pituitary and intestinal bulb of goldfish fed at midday, but not at midnight. Together, these findings report novel data characterizing GOAT, and offer new information about the ghrelinergic system in fish.