S-nitrosothiols inhibit neuronal norepinephrine transport

Although it has been recently shown that nitric oxide (NO) and its congeners (NOx), including nitrosothiols, may modify catecholamine turnover in the brain, it is not known whether NOx affect norepinephrine (NE) uptake by sympathetic neurons. The nitrosothiol NO donor S-nitroso-acetylpenicillamine (SNAP, 100 mu M for 1 h) elicited a concentration-dependent 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 transport. Short-term coculture of PC-12 cells with microvascular endothelial cells expressing the cytokine-inducible NO synthase (NOS2) also exhibited a reduction in [H-3]NE uptake (33 +/- 3%, P < 0.001) that could be prevented by the addition of the NOS inhibitor N-monomethyl-L-arginine (L-NMMA, 1 mM). Endogenous production of NOx by nerve growth factor-pretreated PC-12 cells also exhibited an L-NMMA-inhibitable reduction 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-bromo-cGMP However, the inhibitory effect of SNAP on uptake-1-mediated [H-3]NE transport could be attenuated by 1 mM cysteine, a sulfhydryl compound that could act as a sink for NOx-mediated nitrosation reactions, although cysteine did not affect the increase in intracellular cGMP with SNAP. These data suggest that an endogenous NOx source(s) modifies the activity of the uptake-1 catecholamine transporter in postganglionic sympathetic neurons, which, as we demonstrate, express both NOS1 and NOS3 isoforms, possibly by S-nitrosothiol-mediated nitrosation of regulatory sites on the transporter.