cAMP regulation of arylalkylamine N-acetyltransferase (AANAT, EC 2.3.1.87)- A new cell line (1E7) provides evidence of intracellular AANAT activation

Arylalkylamine N-acetyltransferase (serotonin N-acetyltransferase, AANAT, E C 2.3.1.87) is the penultimate enzyme in melatonin synthesis. As described here, a cell line (1E7) expressing human AANAT (hAANAT) has been developed to study the human enzyme. 1E7 hAANAT is detectable in immunoblots as a 23- kDa band and is immunocytochemically visualized in the cytoplasm. The speci fic concentration of hAANAT in homogenates is comparable to that of the nig ht rat pineal gland. Kinetics of AANAT extracted from 1E7 cells are the sam e as those of bacterially expressed hAANAT; both preparations of hAANAT are equally sensitive to the inhibitor CoA-S-N-acetyltryptamine. Studies of cA MP regulation indicate that treatment with forskolin, dibutyryl cAMP, isobu tylmethylxanthine, or isoproterenol activate cellular hAANAT within intact 1E7 cells similar to8-fold without markedly increasing the abundance of AAN AT protein or the activity of AANAT in broken cell preparations; and, that forskolin, isobutylmethylxanthine and isoproterenol elevate cyclic AMP prod uction. These observations extend our understanding of cAMP regulation of A ANAT activity, because it is currently thought that this only involves chan ges in the steady-state levels of AANAT protein. This previously unrecogniz ed switching mechanism could function physiologically to control melatonin production without changing AANAT protein levels.