OBJECTIVE: Melatonin not only plays an important role in regulating circadian rhythms, but is also involved in antioxidative defense and immunomodulation. Circulating melatonin levels are derived primarily from the pineal gland while other sources of melatonin have also been reported. Recently, we reported that cultured rat cortical astrocytes and glioma C6 cells synthesize melatonin. In addition, apolipoprotein E genotype influences melatonin biosynthesis by regulating NAT and MAOA expression in C6 cells.
METHODS: Here, we investigated the expression of genes and enzymes that is responsible for the multistep conversion of tryptophan to serotonin and further to melatonin in mouse embryonic fibroblasts NIH3T3 cells by radioimmunoassay, Immunofluorescence staining, real-time PCR and Western blotting techniques.
RESULTS: Our results showed that cultured NIH3T3 cells could synthesize melatonin and serotonin. Serotonin N-acetyltransferase (NAT), the key enzyme in the pathway of melatonin synthesis, was also detectable using both by western blot and PCR methods. In addition, two other key enzymes, tryptophan hydroxylase (TPH1 and TPH2) for serotonin synthesis and the metabolic enzyme monoamine oxidase A (MAOA) for 5-HT, were present in NIH3T3 cell line.
CONCLUSIONS: In conclusion, we provided evidence that the NIH3T3 cells can synthesize intrinsic serotonin and melatonin and express key enzymes related biosynthetic pathways.