The circadian rhythm of 5-HT biosynthetic and degradative enzymes in immortalized mouse neuroendocrine pineal cell line - A model for studying circadian rhythm

Serotonin (5-HT) plays an important role in circadian rhythm and its level decreases at night and increases during day time in pineal gland. This study investigates the role of 5-HT biosynthetic and metabolic genes in the circadian rhythm using the mouse neuroendocrine pineal cell line (PT811). We first determined which type of monoamine oxdiase (MAO) was present in PT811 cells. Our results showed that only MAO A, a key which enzyme degrades 5-HT, was present in these cells because both 5-HT (the MAO A preferred substrate) and PEA (the MAO B preferred substrate) oxidation were sensitive to clorgyline but insensitive to deprenyl inhibition. This finding was supported by Northern blot analysis in which MAO A, but not MAO B, transcript was detectable. More sensitive quantitative RT-PCR showed the presence of both MAO A and MAO B, however there was no difference between day and night time. In contrast, the expression of tryptophan hydroxylase (TPH, the 5-HT synthetic enzyme), arylalklamine N-acetyltransferase (AANAT) and Arc (activity regulated, cytoskeleton associated protein) genes showed diurnal cycle. AANAT converts 5-HT to N-acetylserotonin, leading to the synthesis of melatonin. The Arc gene is an effector early immediate gene, regulated by 5-HT. This study suggested that the 5-HT circadian rhythm is regulated by TPH and AANAT but not the MAO A gene in this cell line. This cell line will be a valuable model for studying the molecular mechanisms of circadian rhythm.