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Serotonin-N-acetyltransferase (arylalkylamine-N-acetyltransferase, AANAT) is the key enzyme in the biosynthesis of melatonin in the pineal gland and retinal photoreceptors. Rhythmic AANAT activity drives rhythmic melatonin production in these tissues. The presence of two AANATs, AANAT1 and AANAT2, has been previously demonstrated in three fresh water teleosts. This duality, the result of early gene duplication, is unique to teleost species. In this study, the cDNAs encoding for AANAT1 and AANAT2 were cloned from a marine fish, the gilthead seabream (sb, Sparus aurata). Northern blot hybridization analysis indicates that sbAANAT1 and sbAANAT2 are exclusively expressed in the retina and pineal gland, respectively. Bacterially expressed recombinant sbAANATs exhibit differential enzyme kinetics. Recombinant retinal sbAANAT1 has relatively high substrate affinity and low activity rate; it is inhibited by high substrate and product concentrations. In contrast, recombinant pineal sbAANAT2 exhibits low substrate affinity and high activity rate and is not inhibited by substrates or products. The two recombinant enzymes also exhibit differential substrate preference. Retinal sbAANAT1 acetylates a range of arylalkylamines while pineal sbAANAT2 preferentially acetylates indoleethylamines, especially serotonin. The different spatial expression patterns, enzyme kinetics, and substrate preferences of the two sbAANATs support the hypothesis that, as a consequence of gene duplication, teleosts have acquired two AANATs with different functions. Pineal AANAT2 specializes in the production of large amounts of melatonin that is released into the circulation and exerts an endocrine role. Retinal AANAT1, on the other hand, is involved in producing low levels of melatonin that execute a paracrine function. In addition, retinal AANAT1 may carry out an as yet unknown function that involves acetylation of arylalkylamines other than serotonin.

Distribution and metabolism of the thyroid hormone 3,5, 3'-l-triiodothyronine (T3) were studied in several ways to gain insights into these processes in the warm water fish tilapia Oreochromis mossambicus. Trace doses of 125I-labeled T3 (T*3)1 were injected intraarterially, extraarterially, or intraperitoneally in freshwater-reared male tilapia to explore plasma clearance kinetic responses to these different input modalities. Multicompartmental analysis of the plasma clearance data indicated a kinetic distribution of T*3 much like that reported for the rat and human, with about 2% of total body T*3 in plasma, 5% in rapidly exchanging tissues such as kidney and liver, and 93% in slowly exchanging tissues such as muscle. However, plasma clearance rates (PCR, 5.37 mL/h . 100 g body wt) and plasma appearance rates (PAR3 = PCR x [T3] plasma = 36.3 ng/h . 100 g body wt) were quite different than these indices in rat and human and 5 to 50 times larger than values reported for rainbow trout. On a whole-body basis, normalized for body weight, the tilapia we studied produced and accumulated much more T3 than rat, human, or rainbow trout. Enzymatic and chromatographic analyses of the plasma clearance data samples indicated substantial production of labeled glucuronide, but not sulfate, conjugates of iodothyronines (TiG) of unknown origin appearing in plasma. The TiG appeared beginning a few hours postinjection, peaked at 6 hours, and yielded a predicted steady-state TiG level of 8.3% of the T3 level in plasma. In contrast, in published studies, no conjugates were detected in rainbow trout plasma from 2 to 24 h after iv injection of T*3, T*4, or reverse-T*3, although conjugates of all were present in bile. To our knowledge, although T3 and T4 sulfate conjugates are present in the sera of several mammals, this is the first quantification of iodothyronine glucuronides reported in blood of any species under normal conditions. This might have physiological significance for the tilapia, with T3G providing a reversible storage form of T3 in blood, as has been suggested for sulfate conjugates of T3 and T4 in blood of several mammals.