British Journal of Pharmacology (2001) 132, 941 ± 949 ã 2001 Nature Publishing Group All rights reserved 0007 ± 1188/01 $15.00 www.nature.com/bjp Analysis of S-nitroso-N-acetylpenicillamine e€ects on dopamine release in the striatum of freely moving rats: role of endogenous ascorbic acid and oxidative stress Pier Andrea Serra, 1Giovanni Esposito, 1M. Rosaria Delogu, 1Rossana Migheli, 1Gaia Rocchitta, Egidio Miele, 1Maria S. Desole & *,1,2Maddalena Miele 1 1 1 Department of Pharmacology, University of Sassari, viale S.Pietro 43B, 07100 Sassari, Italy and 2The Bethlem and Maudsley NHS Trust, Bethlem Royal Hospital, Monks Orchard Road, Beckenham, Kent BH3 3BX 1 We showed previously that interaction between NO and iron(II), both released following decomposition of sodium nitroprusside (SNP), accounted for the late SNP-induced dopamine (DA) increase in dialysates from the striatum of freely moving rats. 2 In this study, intrastriatal infusion of the NO-donor S-nitroso-N-acetylpenicillamine (SNAP) (0.2 mM for 180 min) induced a moderate increase in dialysate DA and decreases in ascorbic acid dialysate concentrations; in contrast, SNAP 1 mM infusion induced a long-lasting decrease in both DA and ascorbic acid dialysate concentrations. 3-Methoxy-tyramine (3-MT), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and uric acid levels were una€ected. 3 Co-infusion of ferrous sulphate [iron(II), 1 mM for 40 min] with SNAP either 1 or 0.2 mM (for 180 min), produced a signi®cant increase in both DA and 3-MT dialysate concentrations, but it did not a€ect decreases in dialysate ascorbic acid levels. All other dialysate neurochemicals were una€ected. 4 Co-infusion of ascorbic acid (0.1 mM) with SNAP (1 mM) for 180 min did not modify SNAPinduced decreases in dialysate DA levels. In contrast, co-infusion of uric acid (1 mM) reversed SNAP-induced decreases in dialysate DA; co-infusion of a superoxide dismutase mimetic delayed SNAP-induced DA decreases for a short period, while co-infusion of the antioxidant Nacetylcysteine (NAC, 0.1 mM) signi®cantly increased dialysate DA. 5 The results of this study show that SNAP induces concentration-related changes in DA dialysate levels. At higher concentrations, SNAP induces non-enzymatic DA oxidation, which is inhibited by uric acid and NAC; ascorbic acid failed to protect dialysate DA from oxidation, probably owing to its promoting e€ect on SNAP decomposition; exogenous iron(II) may react with NO generated from SNAP decomposition, with a consequent increase in dialysate DA and 3-MT, therefore mimicking SNP e€ects on striatal DA release. British Journal of Pharmacology (2001) 132, 941 ± 949 Keywords: NO-donor SNAP; dopamine; in vivo release; ascorbic acid; iron; microdialysis; rat striatum Abbreviations: ANOVA, analysis of variance; DA, dopamine; DOPAC, dihydroxyphenylacetic acid; HVA, homovanillic acid; MnTBP, manganese(III) tetrakis (4-benzoic acid) porphyrin; 3-MT, 3-methoxytyramine; NAC, n-acetylcysteine; NO, nitric oxide; SIN-1, 3-morpholinosydnonimine; SNAP, S-nitroso-N-acetylpenicillamine; SNP, sodium nitroprusside Introduction Nitric oxide (NO) is a widespread and versatile biological messenger molecule. Roles proposed for NO in CNS pathophysiology are increasingly multiform and range from intracellular signaling (Garthwaite & Boulton, 1995; Park et al., 1998), through neurotransmitter release, neural development, synapse plasticity, killing of cells and invading pathogens, apoptosis, to tissue damage or protection (Yun et al., 1996; Murphy, 2000). It is evident that either damaging, protective, or signalling e€ects of NO depend on both the source of NO (neuronal, glial, extracellular, exogenous) and the timing of NO production (Murphy, *Author for correspondence at: Department of Pharmacology, Gynaecology and Obstetrics, Faculty of Medicine, University of Sassari, Viale S.Pietro 43B, 07100 Sassari, Italy E-mail: Pharmaco@ssmain.uniss.it 2000). This assumption appears to be also true when dealing with the biological e€ects of NO-generating drugs. These e€ects vary according to the chemical structure of NOgenerating drugs (Bates et al., 1991; Holm et al., 1998; Menconi et al., 1998) and the composition of the endogenous environment in which NO is generated (Millar, 1995; Reiser et al., 1999; Serra et al., 2000a). Ascorbic acid is a very important component of the endogenous environment, since NO readily reacts with ascorbic acid. Neuronal ascorbic acid concentrations (10 mM) are about 10 times higher than glial and, respectively, 20 ± 25 times higher than extracellular concentrations (Miele & Fillenz, 1996; Rice, 2000). Extracellular ascorbic acid levels are also dynamically modulated by glutamate-mediated activity (Rice, 2000), via glutamateascorbate heteroexchange (Miele et al., 1994). The close relationship between NO and ascorbic acid has been outlined 942 P.A. Serra et al in several biological systems (Millar, 1995; Lilley & Gibson, 1997). Ascorbic acid may either generate NO from nitrite ions (NO27) in the extracellular space (Millar, 1995), protect NO from destruction by superoxide anions (Dudgeon et al., 1998; Jackson et al., 1998), or scavenge it (Whiteman & Halliwell, 1996). In this regard, Karanth et al. (2000) claimed that ascorbic acid may even act as an inhibitory transmitter in the hypothalamus by scavenging NO. Neal et al. (1999) showed that dopamine (DA) released from the retina is oxidized by NO and that endogenous ascorbic acid protects DA from oxidation by scavenging NO. Ascorbic acid may trigger decomposition of the NO-donor sodium nitroprusside (SNP) both in biological tissue in vitro (Bates et al., 1991; Reiser et al., 1999) and in the striatal extracellular space in vivo (Serra et al., 2000a). In addition, ascorbic acid potentiates decomposition of S-nitroso-N-acetylpenicillamine (SNAP) in striatal slices in vitro, but not that of 3morpholinosydnonimine (SIN-1) (Reiser et al., 1999). In a previous study (Serra et al., 2000a), we showed that intrastriatal infusion of either SNI-1 or SNP produced a NOmediated increase in DA concentration in dialysates from the striatum of freely moving rats. In addition, we showed that only SNP decreased dialysate ascorbic acid levels. This decrease was related to the ascorbic acid triggering of SNP decomposition (Bates et al., 1991; Reiser et al., 1999) in the striatal extracellular space. The e€ects of the NO-donor SNAP on DA release in terminal ®elds of the dopaminergic system in vivo have been often con¯icting. West & Galloway (1996; 1997) showed that SNAP increased DA e‚ux from the striatum of chloral hydrate-anaesthetized rats. In contrast, Guevara-Guzman et al. (1994) showed that SNAP decreased extracellular DA concentration in urethane-anaesthetized rat, as did NO gas, given directly by dissolution in degassed perfusion ¯uid. Segieth et al. (2000) showed that SNAP promoted DA release from the rat hippocampus in vivo at low concentrations, whilst high concentrations induced longlasting DA decreases. More recently, Trabace & Kendrick (2000) have shown that short-lasting intrastriatal infusion of SNAP induced increases in dialysate DA at low concentration, and decreases in dialysate DA at high concentrations; these DA changes were attributed to SNAP-induced peroxynitrite formation; at high levels, peroxynitrite would reduce extracellular DA concentration through oxidation, while at low levels it increases DA levels in a cyclic GMP-dependent manner. The study of the role of endogenous ascorbic acid and superoxide anion in SNAP-induced changes in DA release and metabolism in the striatum of freely moving rats was therefore deemed of interest. Methods Animals Male Wistar rats (Morini, R. Emilia, Italy), weighing between 280 ± 330 g were used in all experiments. The rats were maintained under standard animal care conditions (12 : 12 h light/dark cycle, lights coming on at 0700 h; room temperature 218C), with food and water ad libitum. Prior to the start of any experiment, the health of the rat was assessed according to published guidelines (Morton & Griths, 1985). All procedures were speci®cally licensed under the British Journal of Pharmacology vol 132 (4) Exogenous NO and striatal oxidative stress European Community directive 86/609 included in Decreto No. 116/1992 of the Italian Ministry of Public Health. Drugs SNAP, SIN-1, N-acetylcysteine (NAC), ascorbic acid, uric acid, and ferrous sulphate [FeSO4, iron(II)] were purchased from Sigma-Aldrich (Milan, Italy); manganese(III) tetrakis (4-benzoic acid) porphyrin (MnTBP) from Calbiochem (Darmstadt, Germany). Drug administration Iron(II), ascorbic acid and SIN-1 concentrations were chosen according to Serra et al. (2000a); SNAP according to Guevara-Guzman et al. (1994) and Trabace & Kendrick (2000); NAC according to Serra et al. (2000b); MnTBP according to Petersen et al. (2000) Microdialysis probe construction The striatal probe combined two independent microdialysis probes of concentric design with two separate inlets and a shared outlet, as previously described (Miele et al., 2000; Serra et al., 2000b). The probes were constructed using two section of plastic-coated silica tubing (diameter 0.15 mm; Scienti®c Glass Engineering, Milton Keynes, U.K.) each placed in the centre of a semi-permeable polyacrylonitrile dialysis ®bres (molecular cut-o€ weight of 12 KD, Filtral 16 Hospal Industrie, France). Each probe had a ®nal diameter of 0.22 mm. The tips of the dialysis ®bres were sealed and joined using quick-drying epoxy glue. The two sections of silica tubing served as inlets; the outlet was made also with a section of plastic-coated silica tubing, positioned in the centre of polythene tubing. The semipermeable membrane was coated with epoxy leaving an active length of 4 mm. The diameter of the ®nal probe was approximately 0.50 mm. The striatal probe combining two microdialysis probes of concentric design with two separate inlets and a shared outlet, allowed separate co-infusion of drugs (Serra et al., 2000b). Surgery Stereotaxic surgery was performed under chloral hydrate (400 mg kg71 i.p.) anaesthesia. The microdialysis probes were implanted in the right striatum using the following coordinates from the atlas of Paxinos & Watson (1986): A/P +0.5 mm from bregma, +2.5 mm M/L, and 76.0 mm D/V from dura. Body temperature during anaesthesia was maintained at 378C by means of an isothermal-heating pad (Harvard Apparatus, Kent, U.K.). Following surgery the animals were placed in large plastic bowls (50655 cm), and maintained in a temperature- and light-controlled environment, with free access to food and water. Experiments were carried out 24 h after probe implantation with the animal in its home bowl. This arrangement allowed the rats free movement. Microdialysis procedure The composition of the Ringer solution used was as follows (in mM): NaCl 147, KCl 4, CaCl2 1.2, MgCl2 1 (pH 6.0). A P.A. Serra et al microinfusion pump (CMA/100, Microdialysis, Sweden) pumped Ringer solution at a ¯ow rate of 1.0 ml min71 using two separate syringes connected to the inlets by a length of polythene tubing; every 20 min, 40 ml dialysate samples were collected manually in 250 ml micro-centrifuge tubes (Alpha Laboratories, U.K.) attached to the outlet. Subsequently, a 20 ml aliquot of collected dialysate was injected into the analytical system. Drugs were added to the Ringer solution and infused via the striatal probe implanted in the striatum. Chromatographic analysis Exogenous NO and striatal oxidative stress 943 induced by the higher SNAP concentration (Figure 1C). 3MT levels were una€ected (Figure 1B), as were other neurochemicals (data not shown). Effect of intrastriatal co-infusion of ascorbic acid on SNAP-induced changes in dialysate neurochemical concentrations We showed previously (Serra et al., 2000a) that the peroxynitrite generator SIN-1 increased dialysate DA without changes in dialysate ascorbic acid concentration; in DA, 3-methoxytyramine (3-MT), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), ascorbic acid and uric acid were quanti®ed by high performance liquid chromatography with electrochemical detection (HPLC-EC) as previously described (Serra et al., 2000a), using an Alltech 426 HPLC pump equipped with a Rheodyne injector, column 15 cm64.6 mm i.d. Alltech Adsorbsphere C18 5U, electrochemical detector Antec CU-04-AZ and Varian Star Chromatographic Workstation. The mobile phase was citric acid 0.5 M, Na acetate 1 M, EDTA 12.5 mM, MeOH 10% and sodium octylsulphate 650 mg l71 (pH=3.0); the ¯ow rate was 1.3 ml min71. The ®rst sample was collected after 60 min of stabilization (time 0), then dialysates were collected, at 20 min intervals, for 40 min prior to the start of experiments. Histology Following the experiments, rats were killed with an overdose of chloral hydrate (800 mg kg71 i.p.). The location of each microdialysis probe was con®rmed by post-mortem histology. Brains were ®xed in formal saline and 50 mm coronal sections were made with a cryostat. The slices were stained with cresyl violet and examined under a microscope. Statistical analysis The concentrations in the dialysate were expressed in nM (DA, 3-MT) or mM (DOPAC, HVA, ascorbic acid, uric acid) and given as mean+s.e.mean. Drug e€ects on neurochemicals were statistically evaluated in terms of changes in absolute dialysate concentrations. Statistical signi®cance was assessed using analysis of variance (ANOVA) for di€erence between groups and over time. Di€erence within or between groups were determined by paired or unpaired t-tests with Bonferroni multiple comparison adjustment. Results Effect of intrastriatal infusion of t SNAP on DA, 3-MT, DOPAC, HVA, ascorbic acid and uric acid dialysate levels Intrastriatal infusion of SNAP (1 mM for 180 min, n=4) induced a long-lasting decrease in DA and ascorbic acid dialysate concentrations (Figure 1A,C), whilst 3-MT (Figure 1B), DOPAC, HVA and uric acid (Figure 2) were una€ected. Intrastriatal infusion of SNAP (0.2 mM for 180 min, n=3) induced increases in dialysate DA (Figure 1A) and a decrease in dialysate ascorbic acid (ANOVA P50.02) lower than that Figure 1 E€ect of intrastriatal infusion of SNAP 1 mM (n=4) or 0.2 mM (n=3) on DA (A), 3-MT (B), and ascorbic acid (C) dialysate concentrations. Dialysates were collected, at 20 min intervals, for 180 min during drug infusion (horizontal black bar) and for 80 min after discontinuation of drug infusion. Values are given as mean+s.e. mean. *P50.05 compared with baseline values. British Journal of Pharmacology vol 132 (4) P.A. Serra et al 944 addition, we showed that intrastriatal ascorbic acid coinfusion inhibited SIN-1-induced increases in dialysate DA. The inhibition was related to NO scavenging by ascorbic Figure 2 E€ect of intrastriatal infusion of SNAP 1 mM (n=4) on DOPAC, HVA and uric acid dialysate concentrations. Dialysates were collected, at 20 min intervals, for 180 min during drug infusion (horizontal black bar) and for 80 min after discontinuation of drug infusion. Values are given as mean+s.e.mean. Exogenous NO and striatal oxidative stress acid. In the present study, SNAP induced a concentrationrelated decrease in dialysate ascorbic acid. The study of ascorbic acid co-infusion on SNAP-induced decreases in DA dialysate concentrations was therefore deemed of interest. Co-infusion of ascorbic acid (0.1 mM for 180 min, n=3) did not a€ect SNAP (1 mM)-induced decreases in dialysate DA (Figure 3A). In addition, ascorbic acid co-infusion a€ected neither 3-MT (Figure 3B), uric acid (Figure 3D), nor DOPAC and HVA dialysate levels (data not shown). Co-infusion of ascorbic acid (0.1 mM for 180 min, n=3) inhibited SNAP (0.2 mM)-induced increases in dialysate DA; during co-infusion, dialysate levels of DA were always in the range of baseline values. All other neurochemicals were una€ected (data not shown). Dialysate ascorbic acid concentrations attained during ascorbic acid/SNAP (1 mM) co-infusion were compared with those attained with ascorbic acid/SIN-1 (1 mM) co-infusion (n=3). As shown in Figure 4, dialysate ascorbic acid concentrations attained during 180 min co-infusion with SNAP (1 mM) were signi®cantly lower (by about 28 ± 30%) than those attained during 180 min co-infusion with SIN-1. Dialysate ascorbic acid concentrations attained during coinfusion with SNAP 0.2 mM did not statistically di€er from those attained during 180 min co-infusion with SIN-1 (data not shown). Figure 3 E€ect of intrastriatal co-infusion of ascorbic acid 0.1 mM (n=3), uric acid 1 mM (n=3), or MnTBP 0.1 mM (n=4) on SNAP-induced changes in DA (A), 3-MT (B), ascorbic acid (C), and uric acid (D) dialysate concentrations. Dialysates were collected, at 20 min intervals, for 180 min during drug infusion (horizontal black bar) and for 80 min after discontinuation of drug infusion. Values are given as mean+s.e.mean. *P50.05 compared with baseline values. Thin horizontal black bar in (A) indicates signi®cant decreases for both SNAP/ascorbic acid SNAP/MnTBP groups; in (C), it indicates signi®cant decreases for both SNAP/ uric acid and SNAP/MnTBP groups. +P50.05 compared with SNAP/MnTBP group. British Journal of Pharmacology vol 132 (4) P.A. Serra et al Exogenous NO and striatal oxidative stress 945 (Patel & Day, 1999), in order to assess the role of peroxynitre in SNAP-induced oxidation of dialysate DA. Co-infusion of MnTBP (0.1 mM for 180 min, n=3) shortly delayed SNAP (1 mM)-induced decreases in dialysate DA (Figure 3A) and attenuated ascorbic acid decreases (Figure 3C). MnTBP co-infusion a€ected neither 3-MT (Figure 3B), nor DOPAC and HVA dialysate levels (data not shown); in contrast, MnTBP signi®cantly increased uric acid levels (up to 60% of baseline at end of co-infusion) (Figure 3D). Effect of NAc-cysteine co-infusion on SNAP-induced changes in DA and ascorbic acid dialysate concentrations Figure 4 Dialysate ascorbic acid concentrations following intrastriatal co-infusion of ascorbic acid 0.1 mM with SIN-1 1 mM (n=3) or SNAP 1 mM (n=3). Dialysates were collected, at 20 min intervals, for 180 min during drug infusion (horizontal black bar) and for 80 min after discontinuation of drug infusion. Values are given as mean+s.e.mean. +P50.05 compared with ascorbic acid/SIN-1 group. Effect of uric acid co-infusion on SNAP-induced changes in DA and ascorbic acid dialysate concentrations In their very recent paper, Trabace & Kendrick (2000) suggest that SNAP induces peroxynitrite formation which results, according to the peroxynitrite level attained, either in extracellular DA oxidation, or in increases in DA release from the striatum of freely moving rats. Uric acid is a natural strong scavenger of peroxynitrite (Hooper et al., 1998). In addition, one of the scavenging activity of uric acid is to maintain ascorbic acid in its reduced form in biological ¯uids (Sevanian et al., 1991). Although in the present study SNAP infusion failed to modify dialysate uric acid concentrations (Figure 2), the study of uric acid co-infusion on SNAP-induced changes in DA and ascorbic acid dialysate concentrations was deemed of interest. In preliminary experiments, it was found that uric acid dialysate concentrations ranging from 80 to 100 mM could be attained with the infusion of uric acid 1 mM. Co-infusion of uric acid 1 mM with SNAP 1 mM for 180 min (n=3) reverted SNAP-induced decreases in dialysate DA levels (Figure 3A), whilst 3-MT levels were una€ected (Figure 3B); in addition, uric acid co-infusion attenuated SNAP-induced decreases in dialysate ascorbic acid (Figure 3C); DOPAC and HVA levels were una€ected (data not shown). Effect of superoxide dismutase (SOD) mimetic MnTBP co-infusion on SNAP-induced changes in DA and ascorbic acid dialysate concentrations Peroxynitrite is formed by reaction of NO with superoxide anions. Ascorbic acid, at high physiological concentrations, is a natural inhibitor of this reaction (Jackson et al., 1998); however, in this study, co-infusion of ascorbic acid failed to protect dialysate DA from SNAP-induced oxidation. SOD is too large a molecule to cross the dialysis membrane used; therefore, we used the cell-permeant SOD mimetic MnTBP We showed previously (Serra et al., 2000b) that intrastriatal infusion of NAC protected DA and L-DOPA, which are both catechol-containing compounds, from autoxidation. It is well known that O-methylation of either DA or L-DOPA protects these compounds from non-enzymatic oxidation (Miller et al., 1996). The fact that SNAP 1 mM induced decreases in dialysate DA concentration leaving una€ected that of 3-MT, the DA O-methylated derivative, prompted us to evaluate the e€ect of NAC co-infusion on the SNAP-induced dialysate DA decrease. Co-infusion of NAC 0.1 mM with SNAP 1 mM for 180 min (n=4) resulted in a signi®cant increase in dialysate DA levels (Figure 5A), whilst 3-MT levels were una€ected (Figure 5B); in addition, NAC co-infusion attenuated the decrease in dialysate ascorbic acid (Figure 5C), whilst DOPAC, HVA and uric acid levels were una€ected (data not shown). Effect of iron(II) intrastriatal co-infusion on SNAP-induced changes in dialysate neurochemicals concentrations We showed previously (Serra et al., 2000a) that interaction between NO and iron(II), both released following decomposition of SNP, accounted for the late but quite substantial SNP-induced dopamine (DA) increase in dialysates from the striatum of freely moving rats. In addition, we showed (unpublished observation) that the late SNP-induced increase in dialysate DA was mimicked by co-infusing the NO-donor SIN-1 with either iron(II), or potassium ferrocyanide, the iron-carrier of SNP, therefore con®rming that an exogenous NO/exogenous iron interaction was responsible for the late great SNP-induced increase in dialysate DA. The evaluation of iron(II) co-infusion on SNAP-induced changes in DA dialysate concentrations was therefore deemed of interest. Intrastriatal infusion of iron(II) (1 mM for 40 min, n=3) induced a late moderate increase in dialysate DA concentration, whilst all other neurochemicals (3-MT, DOPAC, HVA, ascorbic acid, and uric acid) dialysate concentrations were una€ected (data not shown). When SNAP (1 mM for 180 min) was co-infused with iron(II) (1 mM for 40 min, n=4), dialysate DA greatly increased progressively to a peak (316% of baseline) at the end of 180 min SNAP infusion (Figure 5A). Similarly, iron(II) co-infusion signi®cantly increased 3-MT dialysate levels, but in this case the peak (407% of baseline) occurred early (Figure 5B). Iron(II) co-infusion did not a€ect SNAPinduced dialysate ascorbic acid decreases (Figure 5C). All other neurochemicals (DOPAC, HVA and uric acid) were una€ected (data not shown). British Journal of Pharmacology vol 132 (4) 946 P.A. Serra et al Exogenous NO and striatal oxidative stress Figure 5 E€ect of intrastriatal co-infusion of NAC (0.1 mM for 180 min, n=4) or iron(II) (1 mM for 40 min, n=4), on SNAP 1 mMinduced changes in DA (A), 3-MT (B), and ascorbic acid (C) dialysate concentrations Dialysates were collected, at 20 min intervals, for 180 min during drug infusion (horizontal black bar) and for 80 min after discontinuation of drug infusion. Values are given as mean+s.e.mean (n=3). *P50.05 compared with baseline values. Figure 6 E€ect of intrastriatal co-infusion of iron(II) (1 mM for 40 min, n=3) on SNAP 0.2 mM-induced changes in DA (A), 3-MT (B), and ascorbic acid (C) dialysate concentrations. SNAP 0.2 mM, same group as in Figure 1. Dialysates were collected, at 20 min intervals, for 180 min during drug infusion (horizontal black bar) and for 80 min after discontinuation of drug infusion. Values are given as mean+s.e.mean. *P50.05 compared with baseline values; +P50.05 compared with SNAP group. When SNAP (0.2 mM for 180 min) was co-infused with iron(II) (1 mM for 40 min, n=3), the early SNAP-induced increase in dialysate DA concentration was inhibited during 40 min iron(II) co-infusion. Following discontinuation of iron(II) co-infusion, dialysate DA signi®cantly increased to a peak (340% of baseline) at the end of 180 min SNAP infusion (Figure 6A). Similarly, iron(II) co-infusion signi®cantly increased 3-MT dialysate levels, but in this case the peak (265% of baseline) occurred early (Figure 6B). Dialysate ascorbic acid concentration signi®cantly decreased during 40 min of iron(II) co-infusion. Following discontinuation of iron(II) co-infusion, dialysate ascorbic acid showed a trend to recovery (Figure 6C). All other neurochemicals (DOPAC, HVA, uric acid) were una€ected (data not shown). British Journal of Pharmacology vol 132 (4) Discussion The results of the present study con®rm the results of a previous study (Serra et al., 2000a) that both ascorbic acid P.A. Serra et al and iron(II) play a key role in DA changes induced by NOdonors in dialysates from the striatum of freely moving rats. Ascorbic acid promotes degradation of both SNAP (Reiser et al., 1999) and SNP (Bates et al., 1991; Reiser et al., 1999), but not that of SIN-1 (Reiser et al., 1999). In this study, SNAP induced a concentration-dependent decrease in dialysate ascorbic acid levels; we showed previously (Serra et al., 2000a) that SNP infusion induced time-dependent decreases in dialysate ascorbic acid levels, whilst SIN-1 did not a€ect dialysate ascorbic acid. These ®nding are consistent with the role of endogenous ascorbic acid as an active promoter of both SNAP and SNP degradation in vivo. Following SNAP degradation, and the ensuing NO release, DA dialysate levels showed concentration-dependent changes: long-lasting decreases at higher SNAP concentration (1 mM), and short lasting increases at lower concentration (0.2 m). These ®ndings are in agreement with those of Trabace & Kendrick (2000). Decrease in DA dialysate induced by the higher concentration of SNAP is undoubtedly due to DA non-enzymatic oxidation (Trabace & Kendrick, 2000), since dialysate levels of 3-MT, the extracellular O-methylated DA metabolite which is resistant to oxidation (Miller et al., 1996), were una€ected by SNAP. The question arises as to whether SNAP-induced oxidation of DA might be mediated by excess of NO generated from SNAP degradation, by peroxynitrite formed following endogenous superoxide anion reaction with NO generated from SNAP degradation, or both. Intrastrial infusion of either SIN-1, a well-known peroxynitrite generator (Menconi et al., 1998), or SNP, which also induces peroxynitrite formation in vitro (Keller et al., 1998), resulted in a long-lasting increase in dialysate DA (Serra et al., 2000a). These ®ndings seem to exclude a peroxynitrite role in DA oxidation. However, both the chemical structure of NOgenerating drugs and the composition of the endogenous environment in which NO is generated must be taken in consideration. The NO generated in vitro by SNAP is far greater than that by SIN-1 (Holm et al., 1998). In addition, NO generation from SNAP degradation, in the present study in vivo, is likely to be increased by the potentiating e€ect of endogenous ascorbic acid on SNAP degradation (Reiser et al., 1999). NO released in excess by the higher SNAP concentration might act both as a free radical to promote extracellular DA oxidation, and as a promoter of peroxynitrite formation by reacting with endogenous superoxide anion. The SOD mimetic MnTBP delayed SNAP-induced decreases in dialysate DA for a short period, attenuated decreases in dialysate ascorbic acid, and increased dialysate uric acid levels. These convergent ®ndings suggest an initial formation of peroxynitrite, which might initially participate to extracellular DA oxidation. Thereafter, NO continuously generated from SNAP degradation would take the place of peroxynitrite in the extracellular DA oxidation. This hypothesis is supported by the fact that: (1) Co-infusion with uric acid protected DA from SNAP-induced oxidation. Uric acid is a natural strong scavenger of peroxynitrite, but not of NO (Hooper et al., 1998). Uric acid is an active component of the neuronal antioxidant pool (Becker, 1993). It is capable of inhibiting SNP-and free radical-initiated lipid peroxidation and DNA damage (Cohen et al., 1984; Keller et al., 1998); in addition, it forms strong complexes with iron ions, particularly Fe3+ (Cohen et al., 1984), and inhibits DA autoxidation (Church & Ward, 1994); (2) Co-infusion of the Exogenous NO and striatal oxidative stress 947 antioxidant NAC with SNAP completely protected DA from oxidation with a consequent signi®cant increase in dialysate DA levels. Wang et al. (1998) showed that NAC is an active antioxidant also in presence of NO. Co-infusion of SNAP with iron(II), which is known to react readily with NO (Stamler et al., 1992; Le Brun et al., 1997), signi®cantly increased dialysate DA and 3-MT levels. The latter ®nding further highlights the role of iron in the NO-donor drug-induced increase in dialysate DA. In a previous study (Serra et al., 2000a), we showed that NO released following SIN-1 decomposition increased dialysate DA. In contrast to SNAP (Holm et al., 1998), SIN-1 also generates the superoxide anion; thus, as a potential peroxynitrite generating drug (Menconi et al., 1998), SIN-1 should promote DA oxidation (to 6-hydroxyindole-5-one, according to Kerry & Rice-Evans, 1999), as SNAP does. Evidently, this peroxynitrite-induced DA oxidation does not occur in striatal dialysates following SIN-1 infusion. In a submitted study, in order to explain the fact that the iron chelator deferoxamine inhibited the SIN-1-induced increase in dialysate DA (unpublished observations), we suggested that the superoxide anion generated by SIN-1 decomposition would preferentially release iron from storage proteins and enzymic [4Fe-4S] clusters, rather than react with NO, with a consequent elevation in free iron levels (Keyer & Imlay, 1996) The endogenous iron would then react with NO released by SIN-1 decomposition, to form NO-iron complexes (Stamler et al., 1992; Le Brun et al., 1997), therefore mimicking the e€ect of SNP (which notoriously releases both NO and iron) on striatal DA release (Serra et al., 2000a). Endogenous ascorbic, acting as a NO scavenger (Whiteman & Halliwell, 1996), may either protect DA from oxidation (Neal et al., 1999) or even act as an inhibitory transmitter in the hypothalamus by scavenging NO (Karanth et al., 2000). In a previous study (Serra et al., 2000a) we hypothesized that exogenous ascorbic acid inhibited SIN-1induced increases in dialysate DA by scavenging NO. The results of this study con®rm this hypothesis, since the DA releasing e€ect of the lower SNAP concentration (0.2 mM) was completely blocked by ascorbic acid co-infusion. It is likely that exogenous ascorbic acid, besides promoting further the degradation of SNAP (Reiser et al., 1999), had scavenged the ensuing NO. Exogenous ascorbic acid exerts antidopaminergic action on striatal function in vivo (Desole et al., 1987; Rebec & Pierce, 1994; Gulley & Rebec, 1999). The NO scavenging activity of ascorbic acid might be one of the mechanisms of this antidopaminergic action. The results of this study raise the question as to whether NO-generating drugs might be useful tools for the in vivo study of the role of endogenous NO in striatal dopaminergic transmission. The composition of the endogenous environment in which NO is generated may play a key role. When NO is generated intraneuronally, it has to face an ascorbic acid concentration of about 10 mM (Rice, 2000), which is 20 ± 25 times higher than that found in vivo in the striatal extracellular space (Miele & Fillenz, 1996). Therefore, the neuronal ascorbic acid concentration far exceeds the extracellular one at which exogenous ascorbic acid acts as a scavenger of exogenous NO. In conclusion, NO generated from low SNAP concentrations increases DA dialysate levels; endogenous extracellular ascorbic acid may promote SNAP decomposition, with a British Journal of Pharmacology vol 132 (4) 948 P.A. 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