The observation that SDT fatty rats showed blunted circadian rhythms of plasma melatonin led us to investigate whether the expression of Aanat and Asmt mRNA, enzymes important for melatonin synthesis, in the pineal gland of SDT fatty rats was impaired compared with that of control SD rats at night, when melatonin expression was highest.
Unexpectedly, there was no significant difference in the expression of Aanat and Asmt between the SDT fatty and control SD rats, while there was a tendency for higher expression in the SDT fatty rats.
TNF[alpha] inhibits AANAT mRNA expression as well as the synthesis of the melatonin precursor N-acetylserotonin .
Our results also indicate that inflammation may suppress the expression of AANAT in the ovine pineal gland both at the transcriptional and posttranscriptional levels.
Therefore, while AANAT is the key enzyme for determining the circadian rhythm of melatonin, ASMT is the photoperiodic enzyme; that is, it determines the higher melatonin amplitude in the winter when compared to summer nights.
The NF-[kappa]B dimer translocated to the nuclei (p50/p50) in pinealocytes has no transactivating domain (TAD) and impairs Aanat transcription .
Oligonucleotide primers for amplification of Aanat and b-actin were designed using Primer 5.0 software based on the published nucleotide sequence information of Aanat gene (GenBank accession number NM_001077437) and b-actin gene of Canis lupus familiaris (GenBank accession number AF484115).
Western blot analysis of MT1 receptor and AANAT protein expression levels.
AANAT activity was measured by a radiometric assay (Parfitt et al., 1975).
TPH and AANAT activity results are expressed in pmol of radioactive product/200 mg of protein/h.
The enzyme AANAT
should readily take up this molecule, they reasoned, because it looks so much like the serotonin-acetyl-CoA combination.
However, ASMT and AANAT
were detected in the retina of Macaca mulatta, but the transcript concentration of the former is very low and detected only at night .