The synthesis of the pineal hormone, melatonin, is finely regulated by the pineal enzyme serotonin N-acetyltransferase (NAT). In the absence of light, the activity of NAT is markedly enhanced by the release of nor-adrenaline from sympathetic nerve endings in the pineal. Exposure of animals to light during darkness causes a sudden and dramatic reduction in the activity of NAT. The present study investigated a possible mechanism for this sudden decline in NAT activity. These investigations included the determination of the effects of S-adenosylmethionine (SAM), adenosine nucleotides and calcium on NAT activity. In vitro experiments using SAM showed that pineals pre-incubated with SAM prior to adrenergic stimulation did not significantly alter NAT activity or pineal indoleamine metabolism. However, measurement of pineal cyclic AMP showed that SAM exposure reduced the adrenergic-induced rise in pineal cyclic AMP. Experiments using adenosine 5'-monophosphate (5'-AMP) showed that this nucleotide enhanced both dark- and isoproterenol-induced NAT activity. Adenosine 5'-triphosphate (A TP), on the other hand, reduced NAT activity with a concomitant reduction in pineal indoleamine metabolism. Exposure of isoproterenol-stimulated pineals in organ culture to propranolol resulted in a marked rise in ATP and adenosine 5'-diphosphate (ADP) synthesis accompanied by a decline in 5'-AMP levels as compared with pineals treated with isoproterenol alone. This then implies that exposure of animals to light could cause a change in pineal nucleotide levels. Since nucleotide levels are also controlled by calcium, experiments were carried out to determine the effect of calcium on pineal NAT activity. These experiments showed that ethyleneglycol-bis-N,N,N,N,-tetraacetic acid (EGTA) enhanced NAT activity whilst calcium reduced the activity in pineal homogenates, implying that calcium may act directly on NAT to regulate its activity. Exposure of pineal glands in organ culture to the calmodulin antagonist R24571 caused a rise in pineal cyclic AMP levels with a concomitant decrease in cAMP-phosphodiesterase activity. This was, however, accompanied by a decline in Nacetyl serotonin and melatonin synthesis. These findings implicate a number of factors in the regulation of pineal NAT activity. A mechanism for the regulation of pineal NAT is proposed.

Indiana University-Purdue University Indianapolis (IUPUI) / Serotonin (5-hydroxytryptamine, 5-HT) is a monoamine neurotransmitter that is synthesized from the amino acid L-tryptophan and is reported to localize in mitochondria of embryonic stem cells. Even before its role as a neurotransmitter in mature brain was discovered, 5-HT has been shown to play an important role in regulating brain development. However, there is a lack of knowledge about the downstream target genes regulated by serotonin in embryonic stem (ES) cells. Towards this end, our study helps in understanding transcriptional regulatory mechanisms of 5-HT responsive genes in ES cells. By combining the gene expression data with motif prediction algorithms, literature validation and comparison with public domain data, gene targets specific to endogenous or exogenous 5-HT in ES cells were identified. By performing one-way ANOVA, and volcano plots using GeneSpring software, we identified 44 5-HT induced and 29 5-HT suppressed genes, likely to be transcriptionally regulated by 4 & 2 TFs respectively. Motif enrichment analysis on these target genes using MotifScanner revealed that the transcription factor TFAP2A plays a key role in regulating the expression of 5-HT responsive genes. Furthermore, by comparing our dataset with published expression profiles of ES cells, we observed a number of 5-HT responsive target genes showing enrichment in ES cells. Genes such as Nanog, Slc38a5, Hoxb1 and Eif2s1 from this analysis have been observed to be components of ‘stemness’ phenotypes reported in literature. Functional annotation of the 5-HT responsive genes identified gene ontologies such as regulation of translation in response to stress and energy derivation by oxidation, suggesting a regulatory role for 5-HT in mitochondrial functions of ES cells. Additionally, enrichment of other biological process terms such as development of various parts of nervous system, cell adhesion, and apoptosis suggests that 5-HT target genes may play an important role in ES cell differentiation. Our study implemented a new combinatorial approach for identifying gene regulatory mechanisms involved in 5-HT responsive genes and proposed potential mediatory role for serotonin in ES cell differentiation and growth. Thus, this study provides potential 5-HT target genes in ES cells for biological validation.