| Dopamine has a wide range of important physiological roles in humans, but elevated levels due to disruption in its metabolic pathway has been implicated in the etiology of several neurodegenerative disorders. Excessive production of nitric oxide (NO) resulting in "nitrative stress" may lead to the generation of the highly reactive free radical, peroxynitrite, which is also known to be a risk factor for certain inflammatory conditions and neurodegenerative abnormalities. Sulfation, as mediated by the sulfotransferase enzymes, has been reported to play a key role in the regulation of dopamine and its metabolite, 3-methyldopamine, which are predominantly excreted in the sulfated form. Thus, the present study was aimed at finding out whether nitrative stress, as simulated by two NO donors, may affect the homeostasis of dopamine and its metabolite, 3-methyldopamine; and to elucidate possible mechanisms underlying this phenomenon. The sulfotransferase enzyme, SULT1A3, was identified as the main cytosolic sulfotransferase responsible for catalyzing the sulfation of dopamine and 3-methyldopamine. Kinetic studies using [35S]sulfate-labeled SK-N-MC cells revealed that the inhibitory effect of two NO donors, 3-morpholinosydnonimine (SIN-1) and diethylenetriamine NONOate (DETA NONOate), on the generation and release of dopamine 3-O-[35S]sulfate and 3-methyldopamine 4- O-[35S]sulfate occurred in a time-dependent manner. Metabolic labeling experiments using increasing concentrations of SIN-1 and DETA NONOate showed a dramatic decrease in the generation and release of dopamine 3-O-[35S]sulfate and 3-methyldopamine 4-O-[ 35S]sulfate by [35S]sulfate-labeled SK-N-MC cells. Furthermore, cell lysates prepared from SK-N-MC cells treated with increasing concentrations of DETA NONOate showed significant decrease in dopamine- and 3-methyldopamine-sulfating activities, compared to cell lysates prepared from untreated SK-N-MC cells. Cell viability assay using SK-N-MC cells treated with increasing concentrations of SIN-1 or DETA NONOate revealed no significant cytotoxic effect of the NO donors compared to untreated cells. |
