Departamento de Fisiología (Fisiología Animal II), Facultad de Biología, Universidad Complutense de Madrid, Spain.
Chronobiol Int. 2010 Jul;27(6):1178-201. doi: 10.3109/07420528.2010.496911.
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 (gAanat-2) and HIOMT-2 (gHiomt-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 gAanat-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 gAanat-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 gAanat-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.
有人认为，褪黑素除了在众所周知的松果体和视网膜部位外，还在脊椎动物的非感光器官中合成。然而，很少有研究证明褪黑素合成酶在松果体和视网膜外的位置的基因表达。本研究集中于生物钟在鱼类金鱼（Carassius auratus）的中枢和外周位置的两种关键褪黑素能途径的酶，芳香族烷基胺 N-乙酰转移酶（AANAT）和羟基吲哚-O-甲基转移酶（HIOMT）的表达。首先，克隆了金鱼 AANAT-2（gAanat-2）和 HIOMT-2（gHiomt-2）的全长 cDNA 序列，与其他鱼类序列具有高度相似性。鱼类中有两种 AANAT 存在。在这里，首次从系统发生分析中推断出两种 HIOMT 同工型。此外，HIOMT 和 AANAT 都在几个外周位置被检测到，包括肝脏和肠道，这是首次在鱼类的非感光结构中发现 HIOMT。其次，进行了定量实时聚合酶链反应（PCR）研究，以研究在不同光照条件下维持的金鱼松果体和外周位置的 gAanat-2 调节。目前的结果表明，肝脏和后肠中的 Aanat-2 和 Hiomt-2 转录物具有节律性，表明金鱼中存在局部褪黑素合成。此外，分析不同光照条件下 gAanat-2 的日常表达，包括连续光照（24L）和黑暗（24D），除了预期的在松果体和视网膜中发现光依赖性节律外，还在肝脏和后肠中发现了光依赖性节律。在 24L 和 24D 两种恒定条件下，后肠中这些 gAanat-2 节律的持续存在表明该转录物的表达受生物钟控制并受非光线索的影响。最后，目前的结果支持在金鱼的肠道和肝脏中存在褪黑素合成。
