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We showed previously, using in vitro microdialysis, that activation of the nitric oxide (NO)/cyclic GMP pathway was the underlying mechanism of exogenous NO-induced dopamine (DA) secretion from PC12 cells. In this study, infusion of the potential peroxynitrite generator 3-morpholinosydnonimine (SIN-1, 1.0 mM for 60 min) induced a long-lasting decrease in dialysate DA+3-methoxytyramine (3-MT) in dialysates from PC12 cell suspensions. Ascorbic acid (0.2 mM) co-infusion allowed SIN-1 to increase dialysate DA+3-MT. SIN-1+ascorbic acid effects were abolished by Ca 2+ omission. Infusion of high K + (75 mM) induced a 2.5-fold increase in dialysate DA+3-MT. The increase was inhibited by SIN-1 co-infusion. Conversely, co-infusion of ascorbic acid (0.2 mM) with SIN-1+high K + resulted in a 3.5 fold increase in dialysate DA+3-MT. The L-type Ca 2+ channel inhibitor nifedipine selectively inhibited the DA+3-MT increase pertaining to high K + , while the soluble guanylate cyclase (sGC) inhibitor 1 H -[1,2,4]-oxadiazolo[4,3]quinoxalin-1-one selectively inhibited the increase pertaining to SIN-1 effects. These results suggest that activation of the NO/sGC/cyclic GMP pathway is the underlying mechanism of extracellular Ca 2+ -dependent effects of SIN-1 on DA secretion from PC12 cells. Extracellular Ca 2+ entry occurs through nifedipine-insensitive channels. Ascorbic acid is a key determinant in modulating the distinct profiles of SIN-1 effects.

In vitro microdialysis was used to investigate the mechanism of nitric oxide (NO) donor-induced changes in dopamine (DA) secretion from PC12 cells. Infusion of the NO-donor S-nitrosoN-acetylpenicillamine (SNAP, 1.0 mM) induced a long-lasting increase in DA and 3-methoxytyramine (3-MT) dialysate concentrations. SNAP-induced increases were inhibited either by pre-infusion of the soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4] oxadiazolo[4,3]quinoxalin-1-one (ODQ, 0.1 mM) or by Ca 2+ omission. Ca 2+ re-introduction restored SNAP effects. SNAP-induced increases in DA + 3-MT were unaffected by co-infusion of the L-type Ca 2+ channel inhibitor nifedipine. The NO-donor (+ / –)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro3-hexenamide (NOR-3, 1.0 mM) induced a short-lasting decrease in dialysate DA + 3-MT. Ascorbic acid (0.2 mM) co-infusion allowed NOR-3 to increase dialysate DA + 3-MT. ODQ pre-infusion inhibited NOR-3 + ascorbic acid-induced DA + 3-MT increases. Infusion of high K + (75 mM) induced a 2.5-fold increase in dialysate DA + 3-MT. The increase was abolished by NOR-3 co-infusion. Conversely, co-infusion of ascorbic acid (0.2 mM) with NOR-3 + high K + restored high K + effects. Co-infusion of nifedipine inhibited high K + -induced DA + 3-MT increases. These results suggest that activation of the NO / sGC / cyclic GMP pathway may be the underlying mechanism of extracellular Ca 2+ -dependent effects of exogenous NO on DA secretion from PC12 cells. Extracellular Ca 2+ entry may occur through nifedipine-insensitive channels.

A capillary apparatus for in vitro microdialysis was used to investigate melatonin and ascorbic acid effects on dopamine (DA) autoxidation or nitric oxide (NO)-mediated oxidation in suspended PC12 cells. Following high K+ (KCl 75 mm) infusion, secreted DA underwent a partial autoxidation or peroxynitrite-mediated oxidation when the potential peroxynitrite generator 3-morpholinosydnonimine (SIN-1, 1.0 mm) was co-infused with KCl. Ascorbic acid was supplied to the medium by means of intracellular reduction of infused dehydroascorbic acid (DHAA) (5.0 mm). Melatonin (50 microm) and DHAA showed a synergistic effect in inhibiting DA autoxidation and peroxynitrite-mediated DA oxidation. Moreover, melatonin increased dialysate recovery of ascorbic acid released from PC12 cells. Endogenous melatonin was depleted in rats maintained on a 24-hr light cycle for 1 wk. In melatonin-depleted rats, baseline levels of dialysate ascorbic acid were lower than controls, while those of DA were unaffected. In these rats, intrastriatal infusion of 5.0 mm SIN-1 induced DA increases significantly lower than in controls; in addition, dialysate ascorbic acid concentrations exhibited significant decreases. Melatonin co-infusion restored SIN-1 effects on dialysate DA and antagonized SIN-1-induced ascorbic acid decreases. Melatonin-depleted rats were allowed to recover. In these rats, striatal baseline ascorbic acid, as well as SIN-1-induced increases in dialysate DA did not differ from controls. Taken together, these findings suggest that endogenous melatonin is an active component of the striatal extracellular antioxidant pool, as it maintains endogenous ascorbic acid in its reduced status and co-operates with ascorbic acid in protecting extracellular DA from exogenous NO-mediated oxidation.

We showed previously, using in vitro microdialysis, that the activation of the soluble guanylate cyclase (sGC)/cyclic GMP pathway was the underlying mechanism of the extracellular Ca(2+)-dependent effects of exogenous NO on dopamine (DA) secretion from PC12 cells. In this study, the co-infusion of the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3] quinoxalin-1-one (ODQ) failed to affect the NO donor 3-morpholinosydnonimine (SIN-1, 5.0 mM)-induced DA increase (sevenfold baseline) in dialysates from the striatum of freely moving rats. Ca(2+) omission from the perfusion fluid abolished baseline DA release but did not affect SIN-1-induced DA increases. The reintroduction of Ca(2+) in the perfusion fluid restored the baseline dialysate DA; however, when Ca(2+) reintroduction was associated with the infusion of either SIN-1 or the NO-donor S-nitrosoglutathione (SNOG), a sustained DA overflow was observed. DA overflow was selectively inhibited by the co-infusion of the store-operated channel blocker 2-aminoethoxydiphenyl borate. The chelation of intracellular Ca(2+) by co-infusing 1,2-bis (o-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM, 0.2 mM) greatly potentiated both SIN-1- and SNOG-induced increases in dialysate DA. BAPTA-AM-induced potentiation was inhibited by Ca(2+) omission. We conclude that the sGC/cyclic GMP pathway is not involved in the extracellular Ca(2+)-independent exogenous NO-induced striatal DA release; however, when intracellular Ca(2+) is either depleted (by Ca(2+) omission) or chelated (by BAPTA-AM co-infusion), exogenous NO does promote Ca(2+) entry, most likely through store-operated channels, with a consequent further increase in DA release.