S-NITROSOTHIOLS INHIBIT NEURONAL NOREPINEPHRINE TRANSPORT

Although it has been recently shown that nitric oxide (NO) and its con geners (NOx), including nitrosothiols, may modify catecholamine turnov er in the brain, it is not known whether NOx affect norepinephrine (NE ) uptake by sympathetic neurons. The nitrosothiol NO donor S-nitroso-a cetylpenicillamine (SNAP, 100 mu M for 1 h) elicited a concentration-d ependent reduction in desipramine-sensitive [H-3]NE uptake into PC-12 cells (66 +/- 3%; P < 0.01) or cultured rat superior cervical ganglia (74 +/- 5%; P < 0.001), whereas desipramine-insensitive [H-3]NE uptake was unaffected, indicating a selective effect on uptake-1-mediated tr ansport. Short-term coculture of PC-12 cells with microvascular endoth elial cells expressing the cytokine-inducible NO synthase (NOS2) also exhibited a reduction in [H-3]NE uptake (33 +/- 3%, P < 0.001) that co uld be prevented by the addition of the NOS inhibitor N-monomethyl-L-a rginine (L-NMMA, 1 mM). Endogenous production of NOx by nerve growth f actor-pretreated PC-12 cells also exhibited an L-NMMA-inhibitable redu ction in [H-3]NE uptake. Whereas SNAP resulted in a 10-fold elevation of PC-12 guanosine 3',5'-cyclic monophosphate (cGMP) content (P < 0.01 ), its effect on [H-3]NE uptake was not mimicked by exposure to 8-brom o-cGMP However, the inhibitory effect of SNAP on uptake-1-mediated [H- 3]NE transport could be attenuated by 1 mM cysteine, a sulfhydryl comp ound that could act as a sink for NOx-mediated nitrosation reactions, although cysteine did not affect the increase in intracellular cGMP wi th SNAP. These data suggest that an endogenous NOx source(s) modifies the activity of the uptake-1 catecholamine transporter in postganglion ic sympathetic neurons, which, as we demonstrate, express both NOS1 an d NOS3 isoforms, possibly by S-nitrosothiol-mediated nitrosation of re gulatory sites on the transporter.