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Signaling pathways in the nitric oxide and iron-induced dopamine release in the striatum of freely moving rats: Role of extracellular Ca2+ and L-type Ca2+ channels
Brain Research 1047 (2005) 18 – 29 www.elsevier.com/locate/brainres
Research report
Signaling pathways in the nitric oxide and iron-induced dopamine release in the striatum of freely moving rats: Role of extracellular Ca2+ and L-type Ca2+ channels Gaia Rocchittaa, Rossana Mighelia, Maria P. Muraa, Giuseppe Grellab, Giovanni Espositoa, Bianca Marchettia,c, Egidio Mielea, Maria S. Desolea, Maddalena Mieled, Pier Andrea Serraa,* a Department of Pharmacology, University of Sassari, viale S.Pietro 43B, 07100 Sassari, Italy Department of Pharmaco-chemical Toxicology, University of Sassari, via Muroni 29, 07100 Sassari, Italy c OASI Institute for Research and Care on Mental Retardation and Brain Aging (IRCCS), Neuropharmacology Section, 94018 Troina, Italy d Department of Psychiatry, St. Mary’s Hospital, London, UK b
Accepted 1 April 2005 Available online 10 May 2005
Abstract We showed previously that exogenous iron potentiated nitric oxide (NO) donor-induced release of striatal dopamine (DA) in freely moving rats, using microdialysis. In this study, the increase in dialysate DA induced by intrastriatal infusion of the NO-donor 3morpholinosydnonimine (SIN-1, 1.0 mM for 180 min) was scarcely affected by Ca2+ omission. N-methyl-d-glucamine dithiocarbamate (MGD) is a thiol compound whose NO trapping activity is potentiated by iron(II). Intrastriatal co-infusion of MGD either alone or associated with iron(II), however, potentiated SIN-1-induced increases in dialysate DA. In contrast, co-infusion of the NO trapper 4-(carboxyphenyl)4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide (carboxy-PTIO) significantly attenuated the increase in dialysate DA induced by SIN-1 (5.0 mM for 180 min). SIN-1+MGD+iron(II)-induced increases in dialysate DA were inhibited by Ca2+ omission or co-infusion of either deferoxamine or the L-type (Cav 1.1 – 1.3) Ca2+ channel inhibitor nifedipine; in contrast, the increase was scarcely affected by co-infusion of the N-type (Cav 2.2) Ca2+ channel inhibitor N-conotoxin GVIA. These results demonstrate that exogenous NO-induced release of striatal DA is independent on extracellular Ca2+; however, in presence of the NO trapper MGD, NO may preferentially react with either endogenous or exogenous iron to form a complex which releases striatal DA with an extracellular Ca2+-dependent and nifedipine-sensitive mechanism. D 2005 Elsevier B.V. All rights reserved. Theme: Calcium channel physiology, pharmacology and modulation Topic: Mechanism of neurotransmission release Keywords: Exogenous nitric oxide; Iron; Complex; Calcium channels; Striatal dopamine release; Parkinson’s disease
1. Introduction Nitric oxide (NO) is a versatile and widespread biological messenger molecule. NO signaling plays an important role in the functioning of the central nervous system (CNS) [11]. Activation of soluble guanylate cyclase (sGC) is one of the main intracellular effects of NO. The resulting increase in cyclic GMP modulates, among other activities, * Corresponding author. Fax: +39 79 228525. E-mail address: [email protected] (P.A. Serra). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.04.008
that of ion channels [20]. The modulation of ion channels, including Ca2+ channels and pores, is emerging as a general mechanism by which NO exerts biological signaling [18]. Several types of Ca2+ channels coexist to regulate neurotransmitter release in the CNS [54]. Physiological dopamine (DA) release from dopaminergic terminals has been shown to be largely dependent on N-type (Cav 2.2) and P/Q-type (Cav 2.1) voltage-dependent Ca2+ channels [5,35]. A variety of in vivo studies, in which NO-generating drugs were preferentially employed, have shown that NO modulates extracellular levels of DA in the rat striatum [12,44,46–
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48,58]. In a previous study, we demonstrated that exogenous NO-induced striatal DA release from the rat striatum in vivo was independent of both sGC/cyclic GMP pathway activation and extracellular Ca2+ [44]. Understanding the chemistry of NO is important in order to clarify the activity of NO in the striatal DA release in vivo. NO is a simple hydrophobic gaseous molecule that is highly diffusible and reactive. The following forms are important for its biological action: NOI radical, which can be oxidized to nitrosonium cation (NO+), or reduced to nitronyl anion (NO ) [53]. NO readily reacts with either iron(II) to form Fe(III)– NO complexes, or with iron(III) to form Fe(II) – NO+ complexes [23,53]. Iron is a transitional metal involved in many catalytic and regulatory neuronal processes [60], but unless appropriately shielded, it can promote oxidative stress through reactive oxygen species formation [16]. NO may behave either as pro-oxidant [2] or antioxidant [17] in the iron-mediated oxidative stress. In turn, iron may protect tumor cell from pro-apoptotic effects of NO [9]. Interestingly, either NO-donors, with the exception of sodium nitroprusside (SNP), or NO gas in Ringer’s solution, protected nigral neurones from iron(II)-induced oxidative stress [25,29,37]. The composition of the endogenous environment in which NO is generated [28,39,50,51], as well as the timing of NO generation [59] may also regulate its biological actions. In this regard, ascorbic acid is a very important component of the endogenous environment. Neuronal ascorbic acid concentrations (10 mM) are about 10 times higher than glial and, respectively, 20– 25 times higher than extracellular concentrations [27,40]. The close relationship between NO and ascorbic acid has been outlined in several biological systems [24,28]. NO is readily oxidized to nitrite (NO2). Ascorbic acid may reduce nitrite ions (NO2 ) back to NO in the extracellular space [28]. In addition, ascorbic acid protects NO from destruction by superoxide anion (O2 ) [8,15]. We showed previously that interaction between NO and iron(II), both released following the decomposition of SNP, accounted for the late SNP-induced DA increase in dialysates from the striatum of freely moving rats [46]; in addition, we showed that co-infusion of iron(II) with either the NO-donor S-nitroso-N-acetylpenicillamine [47] or the NO-donor and potential peroxynitrite generator 3-morpholinosydnonimine (SIN-1) [48] mimicked SNP effects on striatal DA release. Adding information on the mechanism of the iron/NO complex-induced release of striatal DA has been the aim of the present research.
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nonimine (SIN-1), S-nitroso-N-acetylpenicillamine (SNAP), deferoxamine, and ferrous sulphate [FeSO4, iron(II)] were purchased from Sigma-Aldrich (Milano, Italy); N-methyld-glucamine dithiocarbamate (MGD) was synthesized by one of us (G. G.); N-conotoxin GVIA (N-C-GVIA) from was purchased from Tocris Cookson (Bristol Avon, UK). 2.2. Animals Male Wistar rats (Morini, R. Emilia, Italy), weighing between 280 and 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 7 a.m.; room temperature 21 -C), with food and water ad libitum. Prior to the start of any experiment, the health of the rat was assessed according to published guidelines [31]. All procedures were specifically licensed under the European Community directive 86/609 included in Decreto No. 116/ 1992 of the Italian Ministry of Public Health. 2.3. Microdialysis probe construction The striatal probe (Fig. 1), which combines two independent microdialysis probes of concentric design with two separate inlets and two separate outlets, has been previously described in detail [43,44]. The two inlets with two corresponding separate outlets permit separate coinfusion of drugs and separate dialysate sample collection from the same intrastriatal site. Separate sample collection is useful when one or more drugs which may have either pro-
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2. Materials and methods 2.1. Sources of compounds Nifedipine (NIF), 4-(Carboxyphenyl)-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide (carboxy-PTIO), 3-morpholinosyd-
Fig. 1. Drawing of a striatal probe combining two independent microdialysis probes of concentric design with two separate inlets and two corresponding outlets. Each probe has a final diameter of 0.22 mm. The semipermeable membrane has an active length of 4.0 mm. The diameter of the final probe is approximately 0.45 mm. The main drug is infused through the inlet 1A, conventionally indicated as ipsilateral inlet, while another drug may be co-infused through the inlet 1B, conventionally indicated as contralateral inlet. Both drugs diffuse through the 4 mm of the pertinent dialytic membrane and reach the extracellular compartment. Thereafter, the concentration of neurochemicals is determined separately in dialysates collected from both outlets (2A, conventionally indicated as ipsilateral outlet, and 2B, conventionally indicated as contralateral outlet).
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oxidant or antioxidant properties are infused. In fact, separate sample collection may permit the distinction of neurochemical changes which occur in the extracellular compartment (‘‘intra-striatum’’) from those which may occur in the collected sample (‘‘post-striatum’’, i.e., in vitro), as a result of a chemical reaction. The probe was constructed using two sections of plastic-coated silica tubing (diameter 0.15 mm; Scientific Glass Engineering, Milton Keynes, UK) each placed in the centre of semi-permeable polyacrylonitrile dialysis fibres (molecular cut-off weight of 12 KD, Filtral 16 Hospal Industrie (Meyzieu Cedex, France). Each probe had a final diameter of 0.22 mm. The tips of the dialysis fibbers were sealed and joined using quick-drying epoxy glue. The two sections of silica tubing served as inlets; the outlets were also made with a section of plastic-coated silica tubing, positioned in the centre of the polythene tubing. The semi-permeable membrane was coated with epoxy leaving an active length of 4 mm. The diameter of the final probe was approximately 0.50 mm. 2.4. Stereotaxic surgery Stereotaxic surgery was performed under chloral hydrate (400 mg kg/i.p.) anesthesia. The microdialysis probes were implanted in the right striatum using the following coordinates from the atlas of Paxinos and Watson [34]: A/P +0.5 mm from bregma, 2.5 mm M/L, and 6.0 mm D/V from dura. Body temperature during anesthesia was maintained at 37 -C by means of an isothermal-heating pad (Harvard Apparatus, Kent, UK). Following surgery, the animals were placed in large plastic bowls (50 55 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. 2.5. Microdialysis procedure The composition of the Ringer solution used was as follows, in mM: NaCl 147.0, KCl 4.0, CaCl2 1.2, MgCl2 1.0 (pH 6.0). A microinfusion pump (CMA/100, Microdialysis, Sweden) pumped Ringer solution at a flow rate of 1.5 Al/min using two separate syringes connected to the inlets by a length of polythene tubing; every 20 min, two 30-Al dialysate samples were collected manually in 250 Al micro-centrifuge tubes (Alpha Laboratories, UK) attached to the outlets. Subsequently, a 20-Al aliquot of each collected dialysate was injected into each of two parallel analytical systems. Drugs were added to the Ringer solution and infused via the striatal probe implanted in the striatum. 2.6. Chromatographic analysis DA (detection limit 0.1 nM) and ascorbic acid (detection limit 0.05 AM) were quantified by high performance liquid
chromatography with electrochemical detection (HPLC-EC) as previously described [43,44], using an Alltech 426 HPLC pump equipped with a Rheodyne injector (mod. 7725), column 15 cm4.6 mm i.d. (Toso Haas ODS80TM C18), electrochemical detector BAS mod. LC4B and a PC-based analog-to-digital converter system (Varian Star Chromatographic Workstation). The mobile phase was citric acid 0.1 M, Na acetate 0.1 M, EDTA 1.0 mM, MeOH 9% and sodium octylsulphate 50 mg/l (pH = 2.9); the flow rate was 1.3 ml/ min. The first sample was collected after 60 min of stabilization (time 0), then dialysates were collected at 20min intervals for 40 min prior to the start of experiments. 2.7. Histology Following the experiments, rats were killed with an overdose of chloral hydrate (800 mg/kg i.p.). The location of each microdialysis probe was confirmed by post-mortem histology. Brains were fixed in formal saline and 50 Am coronal sections were made with a cryostat. The slices were stained with cresyl violet and examined under a microscope. 2.8. NO detection system in phosphate buffered saline (PBS) in vitro NO generated from decomposition of NO-donors in phosphate-buffered saline (PBS) at 37 -C was measured according to the procedure previously described [44,51]. The same procedure was used in order to assess the NOtrapping activity of MGD. Briefly, a 7-Am carbon fibre electrode was modified with O-phenylenediamine (OPD) and Nafion by combining three previously described protocols [3,10,61]. After OPD and Nafion coating, only electrodes with NO detection limits >50 nM and selectivity against ascorbate (>500:1), dopamine (>250:1) and nitrite (>800:1) were used. The calibration of selective NO microelectrodes was performed by adding known volumes of a standard SNAP solution in a saturated CuCl solution [61]. NO electrodes displayed excellent linearity in the range of 0 –100 AM (r > +0.969). A three-electrode potentiostat (BAS CV-37 voltammograph) was used for constant potential amperometric oxidation of NO at +865 mV vs. Ag/AgCl reference electrode [61]. 2.9. Statistical analysis The concentrations in the dialysate were expressed in nM (DA) or AM (ascorbic acid) and given as mean T SEM Drug effects on neurochemicals were statistically evaluated in terms of changes in absolute dialysate concentrations. Statistical significance was assessed using analysis of variance (ANOVA) for difference between groups and over time. Difference within or between groups were determined by paired or unpaired t tests with Bonferroni multiple comparison adjustment.
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3. Results 3.1. NO-trapping activity of MGD in vitro MGD is a thiol compound with NO trapping activity [21,22]; the latter is potentiated by iron, as a consequence of a MGD/iron(II) adduct formation [21,22,56]. Thus, MGD appeared to be most suitable compound for purposes of this study. We deemed necessary the assessment of the NO trapping activity of MGD, since the compound was synthesized by one of us. As shown in Fig. 2, the electrochemical detection of NO generated from the decomposition of the NO-donor SNAP 1.0 mM [51] was completely abolished by MGD 5.0 mM. 3.2. Effects of intrastriatal co-infusion of MGD on SIN-1-induced increases in dialysate DA Intrastriatal infusion of SIN-1 (5.0 mM for 180 min, n = 4) induced a long-lasting increase in DA concentrations in dialysates collected from the ipsilateral outlet, with a peak (897% of baseline) after 60 min (Fig. 3, panel A); in dialysates from the contralateral outlets, the increase reached a lower peak (395% of baseline) after 80 min (Fig. 3, panel B). SIN-1 co-infusion did not significantly affect ascorbic acid concentrations in dialysates from both ipsilateral (baseline value 9.28 T 1.23 AM) (Fig. 3, panel B) and contralateral (baseline value 8.84 T 1.36 AM, data not shown) outlets. SIN-1 infusion did not induce behavioral changes, such as increases in motor activity or stereotypy. Preliminary experiments showed that infusion of MGD alone (up to concentrations of 5.0 mM) for 200 min did not modify all striatal neurochemical parameters (data not shown). MGD 5.0 mM co-infusion was initiated through the contralateral inlet 20 min before SNI-1 infusion (5.0 mM for 180 min through the ipsilateral inlet, n = 4), to allow MGD diffusion in the extracellular compartment.
Fig. 3. Effect of intrastriatal infusion of SIN-1 on DA concentrations in dialysates from the striatum of freely moving rats and effects of MGD coinfusion on SIN-1-induced changes. SIN-1 was infused for 180 min (solid horizontal bar a) through the ipsilateral inlet (n = 4); MGD (n = 4) was infused for 200 min (solid horizontal bar b) through the contralateral inlet. MGD co-infusion was initiated 20 min before SIN-1 infusion. Dialysates were collected, at 20 min intervals, during drug infusion and for 80 min after discontinuation of drug infusion. Values are given as mean T SEM and refer to concentrations in dialysates from the ipsilateral outlet (panel A) or the contralateral outlet (panel B). *P < 0.05 compared with pertinent baseline values of all groups (thin horizontal bars); +P < 0.05 compared with SIN-1 group.
MGD co-infusion caused a significant potentiation of SINinduced increases in DA concentrations in dialysates from both ipsilateral (with a peak of 1.100% of baseline after 60 min) (Fig. 3, panel A) and contralateral (with a peak of 742% of baseline after 20 min) outlets (Fig. 3, panel B). MGD co-infusion did not significantly affect ascorbic acid concentrations in dialysates from both outlets (data not shown). 3.3. Effects of intrastriatal co-infusion of the NO trapper carboxy-PTIO on SIN-1-induced changes in dialysate DA
Fig. 2. Electrochemical detection of NO following the decomposition of SNAP 1.0 mM in PBS at 37 -C (a) and effects of MGD 5.0 mM on NO electrochemical detection (b). The data are representative of three independent experiment.
The unexpected finding of a further increase in dialysate DA following MGD co-infusion with SIN-1 prompted us to evaluate the effects of carboxy-PTO, a well known [1] NO trapper, on SIN-1 induced increases in dialysate DA.
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Intrastriatal infusion of carboxy-PTIO (0.5 mM for 200 min, n = 3) induced the following: a) in dialysates collected from the opposite outlet (currently indicated as ipsilateral outlet): lack of changes in dialysate DA (baseline levels 4.04 T 1.03 nM) (Fig. 4, panel A) and significant decreases (by about 37%) in ascorbic acid concentrations (Fig. 4, panel B); b) in dialysates collected from the pertinent outlet (currently indicated as contralateral outlet): lack of changes in baseline DA concentrations (baseline values 3.88 T 0.96 nM) and a greater decrease (by about 85%) in ascorbic acid concentrations (data not shown). The decrease in dialysate ascorbic acid concentrations reveals that carboxy-PTIO has
a pro-oxidant activity which may occur both in the extracellular space (intra-striatum), as revealed by the decrease in dialysates from the opposite outlet, and during dialysate collection from the pertinent outlet (post-striatum). When carboxy-PTIO was infused at concentrations 1.0 mM, baseline DA concentrations were greatly decreased, while ascorbic acid was not detectable, in dialysates from the pertinent outlet; in dialysates from the opposite outlet, DA baseline concentrations were scarcely affected while ascorbic acid was still not detectable (data not shown). Carboxy-PTIO (0.5 mM, n = 4) co-infusion was initiated through the contralateral inlet 20 min before SIN-1 5.0 mM 180 min infusion (ipsilateral inlet), to allow carboxy-PTIO diffusion in the extracellular compartment. Carboxy-PTIO co-infusion significantly attenuated SIN-1induced increases in DA concentrations in dialysates from both the ipsilateral (Fig. 4, panel A) and contralateral outlet (data not shown). Carboxy-PTIO co-infusion with SIN-1 resulted in a greater decrease in ascorbic acid concentrations (by about 89%) in dialysates from the ipsilateral outlet (Fig. 4, panel B), while ascorbic acid was not detectable in dialysates from the contralateral outlet (data not shown). 3.4. Effects of Ca2+ omission on MGD+SIN-1-induced increase in dialysate DA
Fig. 4. Effect of intrastriatal infusion of SIN-1 or carboxy-PTIO on DA (panel A) and ascorbic acid (panel B) concentrations in dialysates from the striatum of freely moving rats, and effects of carboxy-PTIO co-infusion on SIN-1-induced changes. SIN-1 (n = 4, same group as in Fig. 2) was infused for 180 min (solid horizontal bar a) through the ipsilateral inlet for 180 min, while carboxy-PTIO 0.5 mM was infused (solid horizontal bar b) through the contralateral inlet for 200 min either alone (n = 3) or with SIN-1 (ipsilateral inlet, n = 4). Carboxy-PTIO co-infusion was initiated 20 min before SIN-1 infusion. Dialysates were collected, at 20 min intervals, during drug infusion and for 80 min after discontinuation of drug infusion. Values are given as mean T SEM and refer to concentrations in dialysates from the ipsilateral outlet. *P < 0.05 compared with pertinent baseline values in all groups (thin horizontal bars); +P < 0.05 compared with SIN-1 group (thin horizontal bar).
In a previous study [44], we demonstrated that SIN-1induced DA increases in dialysates from the striatum of freely moving rats was independent of extracellular Ca2+. Therefore, we deemed of interest the evaluation of the role of extracellular Ca2+ in the further increase in dialysate DA following MGD co-infusion with SIN-1, Ca2+-free Ringer solution was infused through both inlets from the beginning of the experiments. Omission of Ca2+ resulted in a decrease by about 95 –100% of baseline DA concentrations. SIN-1 (5.0 mM for 180 min, n = 4), was thereafter infused through the ipsilateral inlet. Ca2+ omission scarcely affected SIN-1-induced dialysate DA increases (Fig. 5). MGD 5.0 mM co-infusion was initiated through the contralateral inlet 20 min before SNI-1 1.0 mM (n = 4) infusion for 180 min through the ipsilateral inlet. Omission of Ca2+ resulted in a fully inhibition of the MGD effects on SIN-1-induced DA increases; in addition, dialysate DA return toward baseline occurred significantly earlier than in SIN-1 group (Fig. 5). 3.5. Effects of intrastriatal co-infusion of the N-type (Cav 2.2) voltage-sensitive Ca2+ channel inhibitor x-C-GVIA or the L-type (Cav 1.1– 1.3) voltage-sensitive Ca2+ channel inhibitor nifedipine on MGD+SIN-1-induced DA release The finding that MGD effects are dependent on extracellular Ca2+ prompted us to assess the type of channel
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Fig. 5. Effects of Ca2+ omission on MGD+SIN-1-induced increases in DA concentrations in dialysates from the striatum of freely moving rats. SIN-1 was infused for 180 min (solid horizontal bar a) through the ipsilateral inlet either in Ringer (same group as in Fig. 3) or in Ca2+-free Ringer (open horizontal bar c) (n = 4); MGD was co-infused (solid horizontal bar b) for 200 min through the contralateral inlet. MGD infusion was initiated 20 in before SIN-1 infusion either in Ringer (same group as in Fig. 3) or in Ca2+free Ringer (n = 4). Dialysates were collected, at 20 min intervals, during drug infusion and for 80 min after discontinuation of drug infusion. Values are given as mean T SEM and refer to concentrations in dialysates from the ipsilateral outlet. *P < 0.05 compared with pertinent baseline values in all groups (thin horizontal bar); +P < 0.05 compared with SIN-1+MGD group (thin horizontal bar); #P < 0.05 compared with SIN-1 in Ca2+-free Ringer group.
involved in the MGD+SIN-1-induced Ca2+ entry in dopaminergic terminals. For this purpose, either the N-type Ca2+ channel inhibitor N-C-GVIA or the L-type Ca2+ channel inhibitor nifedipine was co-infused with SIN-1 1.0 mM+MGD 5.0 mM. The concentrations of N-C-GVIA and nifedipine and were chosen according to Rocchitta et al. [43]. N-C-GVIA 10 AM 200 min infusion (n = 3) significantly decreased (up to 80 –90%) baseline levels of dialysate DA (Fig. 6). Co-infusion of N-C-GVIA (10 AM, n = 4) with MGD 5.0 mM was initiated through the contralateral inlet 20 min before SIN-1 5.0 mM 180 min infusion through the ipsilateral inlet. N-C-GVIA scarcely affected MGD+SIN-1induced increases in dialysate DA (Fig. 6). In a previous study [43], we showed that infusion of nifedipine (10 AM) did not affects baseline levels of DA. Nifedipine (10 AM, n = 4) co-infusion with MGD 5.0 mM was initiated through the contralateral inlet 20 min before SIN-1 5.0 mM 180 min infusion through the ipsilateral inlet. Nifedipine co-infusion significantly inhibited MGD+SIN-1induced increases in dialysate DA (Fig. 6). 3.6. Effects of intrastriatal co-infusion of iron(II) on MGD+SIN-1-induced changes in dialysate DA and effects Ca2+ omission The effects of exogenous iron(II) on SIN-1-induced DA increases in dialysates from the striatum of freely moving rats have been thoroughly addressed in a previous study [48]. We demonstrated that a short-lasting co-infusion of iron(II) (1.0 mM) potentiated the DA dialysate increase
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given by SIN-1 in a range of concentrations from 0.2 to 5.0 mM. Therefore, we deemed of interest the evaluation of exogenous iron(II) effects on SIN-1+MGD-induced DA increases in dialysate DA. The iron(II) concentration of 1.0 mM proved to be not suitable for the purposes of this study. Solutions of either MGD 5 mM or iron(II) 0.1 mM in Ringer are colorless. However, a solution of MGD 5.0 mM with iron(II) 0.1 mM in Ringer yields an intense brown color, probably as a consequence of a MGD/iron(II) complex in vitro formation [56]. For this reason, we had to infuse MGD and iron(II) through separate inlets. MGD 5.0 mM was co-infused for 200 min through the contralateral inlet, while iron(II) 0.1 mM and SIN-1 5.0 mM were co-infused for 180 min through the ipsilateral inlet. The infusion of MGD started 20 min before SIN-1+iron(II) co-infusion, to allow MGD diffusion in the extracellular compartment. Co-infusion of iron(II) 0.1 mM with SIN-1 5.0 mM for 180 min (n = 3) induced increases in DA concentrations in dialysates from the ipsilateral outlet, with a peak (895% of baseline) after 140 min (Fig. 7, panel A); these increases did not statistically differ from those induced by SIN-1 5.0 mM alone; in contrast, the increase was greater than that induced by SIN-1 alone in dialysates from the contralateral outlet, with a peak of 491% of baseline after 120 min (data not shown). Ascorbic acid concentrations were unaffected in dialysates from both outlets (Fig. 8, panels A, B).
Fig. 6. Effects of intrastriatal infusion of x-C-GVIA or nifedipine on MGD+SIN-1-induced increases in DA concentrations in dialysates from the striatum of freely moving rats. x-C-GVIA (n = 3) alone was infused through the contralateral inlet for 200 min. Either x-C-GVIA (n = 4) or nifedipine (n = 4) was co-infused with MGD for 200 min through the contralateral inlet for 200 min, while SIN-1 was infused for 180 min through the ipsilateral inlet. Either x-C-GVIA+MGD or nifedipine+MGD co-infusion was initiated 20 in before SIN-1 infusion. Dialysates were collected, at 20-min intervals, during drug infusion and for 80 min after discontinuation of drug infusion. Values are given as mean T SEM and refer to concentrations in dialysates from the ipsilateral outlet. *P < 0.05 compared with pertinent baseline values in all groups (thin horizontal bars); + P < 0.05 compared with SIN-1+MGD group (thin oblique bar).
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ipsilateral outlet had a light brown color, while those from the contralateral outlet were colorless. Ascorbic acid concentrations fell below the detection limit in dialysates from the ipsilateral outlet (Fig. 8, panel A); in contrast, ascorbic acid concentrations were unaffected in dialysates from the contralateral outlet (Fig. 8, panel B). Ca2+-free Ringer solution was infused through both inlets from the beginning of the experiments. Thereafter, MGD 5.0 mM 200 min was infused for 200 min trough the contralateral inlet while SIN-1 5.0 mM+iron(II) 0.1 mM were infused for 180 min trough the ipsilateral inlet (n = 4). Ca2+-omission fully inhibited iron(II)+MGD-induced potentiation of SIN-1-induced DA release (Fig. 7, panel A). Ca2+omission did not affect iron(II)+MGD+SIN-1-induced changes in dialysate ascorbic acid concentrations (data not shown). 3.7. Effects of the iron chelator deferoxamine or nifedipine co-infusion on iron(II)+MGD+SIN-1-induced changes in dialysate DA Preliminary experiments showed that deferoxamine co-infusion for 180 up to 5.0 mM affected neither DA
Fig. 7. Effects of intrastriatal infusion of MGD on iron(II)+SIN-1-induced increases in DA concentrations in dialysates from the striatum of freely moving rats and effects of Ca2+ omission, deferoxamine or nifedipine coinfusion on iron(II)+MGD+SIN-1-induced changes. Panel A: SIN-1 mM) iron(II) (n = 4) were co-infused for 180 min (solid horizontal bar a) through the ipsilateral inlet for 180 min. MGD 5.0 mM was infused for 200 min through the contralateral inlet (solid horizontal bar b), while SIN-1 5.0 mM+iron(II) 0.1 mM were co-infused for 180 min through the ipsilateral inlet for 180 min (solid horizontal bar a), either in Ca2+containing Ringer (n = 4) or in Ca2+-free Ringer (open horizontal bar c, n = 4). Panel B: MGD 5.0 mM was co-infused with either deferoxamine (n = 4) or nifedipine (n = 4) (solid horizontal bar b) through the contralateral inlet for 200 min, while SIN-1 5.0 mM+iron(II) 0.1 mM were co-infused for 180 min through the ipsilateral inlet for 180 min (solid horizontal bar a). Dialysates were collected, at 20 min intervals, during drug infusion and for 80 min after discontinuation of drug infusion. Values are given as mean T SEM and to refer concentrations in dialysates from the ipsilateral outlet. *P < 0.05 compared with pertinent baseline values for all groups (thin horizontal bar, panels A, B). Panel A: +P < 0.05 compared with MGD+SIN-1+iron(II) group (thin horizontal bars). Panel B: deferoxamine and nifedipine groups: +P < 0.05 compared with iron(II)+SIN1+MGD group (panel A) (thin horizontal bar).
Co-infusion of MGD 5.0 mM 200 min with SIN-1 5.0 mM+iron(II) 0.1 mM for 180 min (n = 4) potentiated SIN1+iron(II)-induced increases in DA concentration in dialysates from both ipsilateral (peak 1623% of baseline after 60 min (Fig. 7, panel A) and contralateral (peak 985% of baseline, data not shown) outlets. Dialysates from the
Fig. 8. Effects of intrastriatal infusion of deferoxamine on MGD+iron(II)+ SIN-1-induced changes in ascorbic acid concentrations in dialysates from the striatum of freely moving rats. Same groups as in Fig. 7 (panel A). Values are given as mean T SEM and refer to concentrations in dialysates from the ipsilateral outlet (panel A) or the contralateral outlet (panel B). *P < 0.05 compared baseline values; +P < 0.05 compared with MGD+SIN-1+iron(II) group (thin horizontal bar).
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nor ascorbic acid dialysate concentrations (data not shown). Deferoxamine 2.0 mM attenuated at the borderline of statistical significance the iron(II)+MGD+SIN-1-induced increase in dialysate DA (data not shown). Deferoxamine 5.0 mM co-infusion with MGD 5.0 mM (n = 4) was initiated through the contralateral inlet 20 min before SIN-1 5.0 mM+ iron(II) 0.1 mM 180 min coinfusion (ipsilateral inlet), to allow deferoxamine and MGD diffusion in the extracellular compartment. Deferoxamine co-infusion fully inhibited the iron(II)+MGDinduced potentiation of SIN-1-induced increases in DA concentrations in dialysates from both ipsilateral (Fig. 7, panel B) and contralateral (data not shown) outlets. Dialysates from the ipsilateral outlet were colorless. In addition, deferoxamine co-infusion greatly inhibited (Fig. 8, panel A) the MGD+SIN-1+iron(II)-induced fall of ascorbic acid concentrations below the detection limit in dialysates from the ipsilateral outlet, which were colorless. Nifedipine 10 AM co-infusion with MGD 5.0 mM (n = 4) was initiated through the contralateral inlet 20 min before SIN-1 5.0 mM + iron(II) 0.1 mM 180 min coinfusion (ipsilateral inlet). Nifedipine co-infusion fully inhibited the iron(II)+MGD-induced potentiation of SIN1-induced increases in DA concentrations in dialysates from both ipsilateral (Fig. 7, panel B) and contralateral (data not shown) outlets. Dialysates from the ipsilateral outlet had a light brown color. Nifedipine co-infusion, however, failed to inhibit the MG+SIN-1+iron(II)-induced fall of ascorbic acid concentrations below the detection limit in dialysates from the ipsilateral outlet (data not shown). 3.8. Effects of deferoxamine co-infusion on MGD+SIN-1-induced increases in dialysate DA The finding that deferoxamine fully inhibited iron(II)+ MGD effects on SIN-1-induced increases in dialysate DA prompted us to investigate the effects of deferoxamine coinfusion on MGD+SIN-1-induced increases in dialysate DA. The working hypothesis was that exogenous MGD might form a complex with endogenous iron; such a complex per se is unable to trigger striatal DA release, since infusion of MGD alone did not induce changes in dialysate DA. However, when NO is generated by co-infusion of the NO-donor, exogenous NO might react preferentially with the exogenous MGD/endogenous iron complex, to form the well-known MGD/iron/ NO adduct. Exogenous NO itself might render available endogenous iron by releasing it from storage proteins [38,45]. Deferoxamine 2.0 mM was co-infused with MGD 5.0 mM through the contralateral inlet 20 min before SIN-1 5.0 mM (n = 4) infusion for 180 min through the ipsilateral inlet. Co-infusion of deferoxamine significantly
25
Fig. 9. Effects of intrastriatal infusion of deferoxamine on MGD+SIN-1induced increases in DA concentrations in dialysates from the striatum of freely moving rats. MGD+SIN-1 group (n = 4), same group as in Fig. 2. Deferoxamine was co-infused with MGD (horizontal bar b) for 200 min through the contralateral inlet 20 min before SIN-1 (n = 4) infusion for 180 min through the ipsilateral inlet (horizontal bar a). Values are given as mean T SEM and refer to concentrations in dialysates from the ipsilateral outlet. *P < 0.05 compared with pertinent baseline values for both groups (thin horizontal bar). +P < 0.05 compared with MGD+SIN-1 group (thin horizontal bar).
inhibited MGD effects on SIN-1-induced increases in dialysate DA (Fig. 9).
4. Discussion The key findings of this study are the following: (i) spontaneous release of striatal DA is dependent on extracellular Ca2+ and it is inhibited by the N-type (Cav 2.2) voltage sensitive Ca2+ channel inhibitor N-C-GVIA; in contrast, SIN-1-induced striatal DA release appears to be independent of external Ca2+; (ii) SIN-1-induced striatal DA release is inhibited by the NO trapper carboxy-PTIO; in contrast, the NO trapper MGD, whose trapping activity is increased by iron, further increases SIN-1-induced striatal DA release, probably as a consequence of an iron(II)/MGD/ NO adduct formation; (iii) striatal DA release induced by the iron(II)/MGD/NO adduct is dependent on extracellular Ca2+ and it is inhibited by the L-type (Cav 1.1– 1.3) voltage sensitive Ca2+ channel inhibitor nifedipine. The NO/sGC/cyclic GMP pathway plays an important role in Ca2+ cycling [18]. In a previous study [44], we demonstrated that exogenous NO-induced striatal DA release was independent of both the activation of the sGC/ cyclic GMP pathway and extracellular Ca2+; however, when intracellular Ca2+ stores were depleted, exogenous NO promoted Ca2+ entry through store-operated channels with a consequent great increase in DA release. The current view on the release of neurotransmitters by Ca2+-triggered synaptic vesicle exocytosis is that the process involves several steps and is controlled by a protein machinery (SNARE proteins) [42]. On the basis of the inhibition of exogenous NO-induced release of striatal DA by means of
26
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either the alkylating agent N-ethylmaleimide or clostridial neurotoxins [44], we suggested that exogenous NO might target directly SNARE proteins and promote DA release with a Ca2+-independent and N-ethylmaleimide-sensitive mechanism (Fig. 10). Decomposition of SIN-1 gives rise to NO and O2 ; NO reacts readily with O2I to form peroxynitrite [14], which is a powerful DA oxidizing agent [7]. Peroxynitrite downstream formation following SIN-1 decomposition does occur in vitro [50]; most likely, however, it does not occur in the striatal extracellular compartment [44,48], owing to the presence of high concentration of ascorbic acid (0.2 – 0.4 mM), which are kept constant by an efficient homeostatic mechanism [27]. NO is a simple hydrophobic gaseous molecule that is highly diffusible and reactive [53]; therefore, when NO is released in the extracellular compartment, it is not surprising if NO can in part diffuse through the neuronal membrane and in part react with NO-trapper molecules (Fig. 9). However, while the trapper carboxy-PTIO decreased SIN-1-induced release of DA, MGD actually further increased it. Such discrepancy may be related to the ability of MGD to react also with iron(II), with a consequent increase in its NO-trapping activity. When exogenous iron(II) was supplied, most likely it generated a NO/MGD/
I
iron(II) adduct, which proved to be a powerful striatal DA releaser. The adduct-induced increase in DA release was dependent on extracellular Ca2+ and was inhibited by either nifedipine or deferoxamine co-infusion. The finding that deferoxamine co-infusion inhibited also the SIN-1+MGDinduced increase in DA release, strongly support the hypothesis that, in absence of exogenous iron(II), endogenous iron might also generate the NO/MGD/iron(II) adduct formation. Exogenous NO itself might render available endogenous iron by releasing it from storage proteins [38,45]. Taken together, these results allow us to suggest that the adduct-induced DA release is Ca2+-dependent and Ca2+ entry occurs through L-type (Cav 1.1 – 1.3) channels; it is relevant, to this regard, that microinjections of L-type Ca2+ channel activators in the caudate putamen of freely moving rats are known to elicit release of large amount of DA [32]. Ca2+ cycling occurs through a variety of definite channels [26]. In striatal dopaminergic terminals, the Ca2+ entry which ensures the physiological release of DA [5] occurs through N-type (Cav 2.2) channels [35]. Therefore, the striatal L-type Ca2+ channels activated by our pharmacological manipulation should be regarded as silent channels. The MGD/iron(II) adduct formation occurs both in vitro [56] and in vivo [21,22]. The adduct can undergo complex
Fig. 10. Dynamics of pathways involved in the SIN-1 and iron(II)-induced striatal dopamine release in the striatum of freely moving rats. Spontaneous firing of nigral neurons (1) induces membrane depolarization of terminals and activates Ca2+ entry through N-type (Cav 2.2) channels, with a consequent SNAREmediated dopamine (DA) release. Decomposition of SIN-1 in the extracellular compartment gives rise (2) to NO and O2 . Endogenous ascorbic acid inhibits downstream peroxynitrite formation [48,50], thus allowing NO to enter the membrane ending and promote (3) Ca2+-independent SNARE-mediated [44] DA release. NO-induced DA release is inhibited by the NO-trapper carboxy-PTIO. In presence of the NO-trapper MGD, NO released from SIN-1 decomposition preferentially reacts with MGD and iron(II), either endogenous or exogenous, to form a NO/MGD/iron(II) adduct (4), which activates a Ca2+-dependent DA release. The iron chelator deferoxamine, by inhibiting the adduct formation (5), selectively blocks the pertinent quota of DA release. The inhibitory effect of the L-type (Cav 1.1 – 1.3) Ca2+ channel blocker nifedipine (6) allows us to hypothesize that the adduct might promote Ca2+ entry through an activation of silent L-type Ca2+ channels.
I
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redox chemistry [57]; in fact, it is rapidly oxidized under aerobic conditions to form MGD/iron(III) complex [56,57]. Reducing agents such as ascorbic acid, hydroquinone or cysteine, reduce MGD/iron(III) back to MGD/iron(II) complex [57]. The redox chemistry of the MGD/iron(II) complex may account for the disappearance of ascorbic acid in dialysates collected from the ipsilateral outlet during SIN1+iron(II) and MGD co-infusion. An oxidation of the MGD/ iron(II)/NO adduct to MGD/iron(III)/NO most likely occurred ‘‘post-striatum’’ (i.e., in vitro, during dialysate collection), and dialysate ascorbic acid most likely reduced the adduct back to MGD/iron(II)/NO. In fact, the iron chelator deferoxamine, which binds both iron(II) and iron(III) ions [13], by inhibiting the adduct formation and the consequent oxidation, restored dialysate ascorbic acid concentrations. Ascorbic acid concentrations were unaffected in colorless dialysates from the contralateral outlet, most likely since either they were restored by the efficient homeostatic mechanism quoted above, or owing to the lack of adduct oxidation in the extracellular compartment. The question arises as to whether the coupling of NO and iron with silent striatal L-type Ca2+ channels might have relevance to the physiopathology of dopaminergic terminals. Parkinson’s disease (PD) is characterized by an important loss of nigral dopaminergic neurons. The concept of a threshold of DA depletion for onset of parkinsonian symptoms is widely accepted. Parkinsonian symptoms appear when dopaminergic neuronal death exceeds a critical threshold, 70 –80% of striatal nerve terminals and 50 – 60% of nigral perikarya [41]. These findings clearly support the activation of nigro-striatal endogenous compensatory mechanisms. Neuritic sprouting [52], activation of either tyrosine hydroxylase immunoreactive striatal intrinsic neurons [6] or silent nigro-striatal dopaminergic neurons [55], and appearance of new dopaminergic neurons within the striatum itself [36] are indicated as putative endogenous compensatory mechanisms. Nigro-striatal microglia activation, with a consequent up-regulation of inducible NO synthase, is believed to be the main culprit for dopaminergic neuron demise [19,30,49]. In addition, misregulation of iron metabolism, iron-induced oxidative stress, and iron accumulation in the nigro-striatal structures are also widely believed to be important pathogenetic mechanisms of neuronal death in PD [16,60]; moreover, iron is known to increase NO production from activated microglia [22]. Intraneuronal NO is released in short controlled burst, probably in the range of picomolar concentrations, and most likely, it does not reach the extracellular compartment [20]. In the striatum, however, it has been described a distinct type of interneurons which produces and releases NO in the extracellular compartment [4]. Moreover, large amounts of NO, in the range of micromolar concentrations, are generated for long period and released in the extracellular striatal compartment by striatal microglia/macrophages and astrocytes, as demonstrated in vivo by Oyoshi et al. [33], using microdialysis in freely moving rats; in addition,
27
microglial/macrophages and astrocyte activation may further increase (by more that 100%) NO production and release [33]. Taken together, the above findings allow us to hypothesize that the coupling of NO and iron with silent striatal L-type Ca2+ channels, as well as the coupling of NO with striatal store-operated Ca2+ channels [44], and the consequent release of striatal DA from surviving dopaminergic terminals and striatal dopaminergic neurons either intrinsic, silent or newly appeared, could play a role in maintaining an adequate post-synaptic dopaminergic input in the pre-symptomatic phase of PD.
Acknowledgment The research was supported in part by the University of Sassari (ex 60% fund).
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Research report
Signaling pathways in the nitric oxide and iron-induced dopamine release in the striatum of freely moving rats: Role of extracellular Ca2+ and L-type Ca2+ channels Gaia Rocchittaa, Rossana Mighelia, Maria P. Muraa, Giuseppe Grellab, Giovanni Espositoa, Bianca Marchettia,c, Egidio Mielea, Maria S. Desolea, Maddalena Mieled, Pier Andrea Serraa,* a Department of Pharmacology, University of Sassari, viale S.Pietro 43B, 07100 Sassari, Italy Department of Pharmaco-chemical Toxicology, University of Sassari, via Muroni 29, 07100 Sassari, Italy c OASI Institute for Research and Care on Mental Retardation and Brain Aging (IRCCS), Neuropharmacology Section, 94018 Troina, Italy d Department of Psychiatry, St. Mary’s Hospital, London, UK b
Accepted 1 April 2005 Available online 10 May 2005
Abstract We showed previously that exogenous iron potentiated nitric oxide (NO) donor-induced release of striatal dopamine (DA) in freely moving rats, using microdialysis. In this study, the increase in dialysate DA induced by intrastriatal infusion of the NO-donor 3morpholinosydnonimine (SIN-1, 1.0 mM for 180 min) was scarcely affected by Ca2+ omission. N-methyl-d-glucamine dithiocarbamate (MGD) is a thiol compound whose NO trapping activity is potentiated by iron(II). Intrastriatal co-infusion of MGD either alone or associated with iron(II), however, potentiated SIN-1-induced increases in dialysate DA. In contrast, co-infusion of the NO trapper 4-(carboxyphenyl)4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide (carboxy-PTIO) significantly attenuated the increase in dialysate DA induced by SIN-1 (5.0 mM for 180 min). SIN-1+MGD+iron(II)-induced increases in dialysate DA were inhibited by Ca2+ omission or co-infusion of either deferoxamine or the L-type (Cav 1.1 – 1.3) Ca2+ channel inhibitor nifedipine; in contrast, the increase was scarcely affected by co-infusion of the N-type (Cav 2.2) Ca2+ channel inhibitor N-conotoxin GVIA. These results demonstrate that exogenous NO-induced release of striatal DA is independent on extracellular Ca2+; however, in presence of the NO trapper MGD, NO may preferentially react with either endogenous or exogenous iron to form a complex which releases striatal DA with an extracellular Ca2+-dependent and nifedipine-sensitive mechanism. D 2005 Elsevier B.V. All rights reserved. Theme: Calcium channel physiology, pharmacology and modulation Topic: Mechanism of neurotransmission release Keywords: Exogenous nitric oxide; Iron; Complex; Calcium channels; Striatal dopamine release; Parkinson’s disease
1. Introduction Nitric oxide (NO) is a versatile and widespread biological messenger molecule. NO signaling plays an important role in the functioning of the central nervous system (CNS) [11]. Activation of soluble guanylate cyclase (sGC) is one of the main intracellular effects of NO. The resulting increase in cyclic GMP modulates, among other activities, * Corresponding author. Fax: +39 79 228525. E-mail address: [email protected] (P.A. Serra). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.04.008
that of ion channels [20]. The modulation of ion channels, including Ca2+ channels and pores, is emerging as a general mechanism by which NO exerts biological signaling [18]. Several types of Ca2+ channels coexist to regulate neurotransmitter release in the CNS [54]. Physiological dopamine (DA) release from dopaminergic terminals has been shown to be largely dependent on N-type (Cav 2.2) and P/Q-type (Cav 2.1) voltage-dependent Ca2+ channels [5,35]. A variety of in vivo studies, in which NO-generating drugs were preferentially employed, have shown that NO modulates extracellular levels of DA in the rat striatum [12,44,46–
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48,58]. In a previous study, we demonstrated that exogenous NO-induced striatal DA release from the rat striatum in vivo was independent of both sGC/cyclic GMP pathway activation and extracellular Ca2+ [44]. Understanding the chemistry of NO is important in order to clarify the activity of NO in the striatal DA release in vivo. NO is a simple hydrophobic gaseous molecule that is highly diffusible and reactive. The following forms are important for its biological action: NOI radical, which can be oxidized to nitrosonium cation (NO+), or reduced to nitronyl anion (NO ) [53]. NO readily reacts with either iron(II) to form Fe(III)– NO complexes, or with iron(III) to form Fe(II) – NO+ complexes [23,53]. Iron is a transitional metal involved in many catalytic and regulatory neuronal processes [60], but unless appropriately shielded, it can promote oxidative stress through reactive oxygen species formation [16]. NO may behave either as pro-oxidant [2] or antioxidant [17] in the iron-mediated oxidative stress. In turn, iron may protect tumor cell from pro-apoptotic effects of NO [9]. Interestingly, either NO-donors, with the exception of sodium nitroprusside (SNP), or NO gas in Ringer’s solution, protected nigral neurones from iron(II)-induced oxidative stress [25,29,37]. The composition of the endogenous environment in which NO is generated [28,39,50,51], as well as the timing of NO generation [59] may also regulate its biological actions. In this regard, ascorbic acid is a very important component of the endogenous environment. Neuronal ascorbic acid concentrations (10 mM) are about 10 times higher than glial and, respectively, 20– 25 times higher than extracellular concentrations [27,40]. The close relationship between NO and ascorbic acid has been outlined in several biological systems [24,28]. NO is readily oxidized to nitrite (NO2). Ascorbic acid may reduce nitrite ions (NO2 ) back to NO in the extracellular space [28]. In addition, ascorbic acid protects NO from destruction by superoxide anion (O2 ) [8,15]. We showed previously that interaction between NO and iron(II), both released following the decomposition of SNP, accounted for the late SNP-induced DA increase in dialysates from the striatum of freely moving rats [46]; in addition, we showed that co-infusion of iron(II) with either the NO-donor S-nitroso-N-acetylpenicillamine [47] or the NO-donor and potential peroxynitrite generator 3-morpholinosydnonimine (SIN-1) [48] mimicked SNP effects on striatal DA release. Adding information on the mechanism of the iron/NO complex-induced release of striatal DA has been the aim of the present research.
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nonimine (SIN-1), S-nitroso-N-acetylpenicillamine (SNAP), deferoxamine, and ferrous sulphate [FeSO4, iron(II)] were purchased from Sigma-Aldrich (Milano, Italy); N-methyld-glucamine dithiocarbamate (MGD) was synthesized by one of us (G. G.); N-conotoxin GVIA (N-C-GVIA) from was purchased from Tocris Cookson (Bristol Avon, UK). 2.2. Animals Male Wistar rats (Morini, R. Emilia, Italy), weighing between 280 and 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 7 a.m.; room temperature 21 -C), with food and water ad libitum. Prior to the start of any experiment, the health of the rat was assessed according to published guidelines [31]. All procedures were specifically licensed under the European Community directive 86/609 included in Decreto No. 116/ 1992 of the Italian Ministry of Public Health. 2.3. Microdialysis probe construction The striatal probe (Fig. 1), which combines two independent microdialysis probes of concentric design with two separate inlets and two separate outlets, has been previously described in detail [43,44]. The two inlets with two corresponding separate outlets permit separate coinfusion of drugs and separate dialysate sample collection from the same intrastriatal site. Separate sample collection is useful when one or more drugs which may have either pro-
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2. Materials and methods 2.1. Sources of compounds Nifedipine (NIF), 4-(Carboxyphenyl)-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide (carboxy-PTIO), 3-morpholinosyd-
Fig. 1. Drawing of a striatal probe combining two independent microdialysis probes of concentric design with two separate inlets and two corresponding outlets. Each probe has a final diameter of 0.22 mm. The semipermeable membrane has an active length of 4.0 mm. The diameter of the final probe is approximately 0.45 mm. The main drug is infused through the inlet 1A, conventionally indicated as ipsilateral inlet, while another drug may be co-infused through the inlet 1B, conventionally indicated as contralateral inlet. Both drugs diffuse through the 4 mm of the pertinent dialytic membrane and reach the extracellular compartment. Thereafter, the concentration of neurochemicals is determined separately in dialysates collected from both outlets (2A, conventionally indicated as ipsilateral outlet, and 2B, conventionally indicated as contralateral outlet).
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oxidant or antioxidant properties are infused. In fact, separate sample collection may permit the distinction of neurochemical changes which occur in the extracellular compartment (‘‘intra-striatum’’) from those which may occur in the collected sample (‘‘post-striatum’’, i.e., in vitro), as a result of a chemical reaction. The probe was constructed using two sections of plastic-coated silica tubing (diameter 0.15 mm; Scientific Glass Engineering, Milton Keynes, UK) each placed in the centre of semi-permeable polyacrylonitrile dialysis fibres (molecular cut-off weight of 12 KD, Filtral 16 Hospal Industrie (Meyzieu Cedex, France). Each probe had a final diameter of 0.22 mm. The tips of the dialysis fibbers were sealed and joined using quick-drying epoxy glue. The two sections of silica tubing served as inlets; the outlets were also made with a section of plastic-coated silica tubing, positioned in the centre of the polythene tubing. The semi-permeable membrane was coated with epoxy leaving an active length of 4 mm. The diameter of the final probe was approximately 0.50 mm. 2.4. Stereotaxic surgery Stereotaxic surgery was performed under chloral hydrate (400 mg kg/i.p.) anesthesia. The microdialysis probes were implanted in the right striatum using the following coordinates from the atlas of Paxinos and Watson [34]: A/P +0.5 mm from bregma, 2.5 mm M/L, and 6.0 mm D/V from dura. Body temperature during anesthesia was maintained at 37 -C by means of an isothermal-heating pad (Harvard Apparatus, Kent, UK). Following surgery, the animals were placed in large plastic bowls (50 55 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. 2.5. Microdialysis procedure The composition of the Ringer solution used was as follows, in mM: NaCl 147.0, KCl 4.0, CaCl2 1.2, MgCl2 1.0 (pH 6.0). A microinfusion pump (CMA/100, Microdialysis, Sweden) pumped Ringer solution at a flow rate of 1.5 Al/min using two separate syringes connected to the inlets by a length of polythene tubing; every 20 min, two 30-Al dialysate samples were collected manually in 250 Al micro-centrifuge tubes (Alpha Laboratories, UK) attached to the outlets. Subsequently, a 20-Al aliquot of each collected dialysate was injected into each of two parallel analytical systems. Drugs were added to the Ringer solution and infused via the striatal probe implanted in the striatum. 2.6. Chromatographic analysis DA (detection limit 0.1 nM) and ascorbic acid (detection limit 0.05 AM) were quantified by high performance liquid
chromatography with electrochemical detection (HPLC-EC) as previously described [43,44], using an Alltech 426 HPLC pump equipped with a Rheodyne injector (mod. 7725), column 15 cm4.6 mm i.d. (Toso Haas ODS80TM C18), electrochemical detector BAS mod. LC4B and a PC-based analog-to-digital converter system (Varian Star Chromatographic Workstation). The mobile phase was citric acid 0.1 M, Na acetate 0.1 M, EDTA 1.0 mM, MeOH 9% and sodium octylsulphate 50 mg/l (pH = 2.9); the flow rate was 1.3 ml/ min. The first sample was collected after 60 min of stabilization (time 0), then dialysates were collected at 20min intervals for 40 min prior to the start of experiments. 2.7. Histology Following the experiments, rats were killed with an overdose of chloral hydrate (800 mg/kg i.p.). The location of each microdialysis probe was confirmed by post-mortem histology. Brains were fixed in formal saline and 50 Am coronal sections were made with a cryostat. The slices were stained with cresyl violet and examined under a microscope. 2.8. NO detection system in phosphate buffered saline (PBS) in vitro NO generated from decomposition of NO-donors in phosphate-buffered saline (PBS) at 37 -C was measured according to the procedure previously described [44,51]. The same procedure was used in order to assess the NOtrapping activity of MGD. Briefly, a 7-Am carbon fibre electrode was modified with O-phenylenediamine (OPD) and Nafion by combining three previously described protocols [3,10,61]. After OPD and Nafion coating, only electrodes with NO detection limits >50 nM and selectivity against ascorbate (>500:1), dopamine (>250:1) and nitrite (>800:1) were used. The calibration of selective NO microelectrodes was performed by adding known volumes of a standard SNAP solution in a saturated CuCl solution [61]. NO electrodes displayed excellent linearity in the range of 0 –100 AM (r > +0.969). A three-electrode potentiostat (BAS CV-37 voltammograph) was used for constant potential amperometric oxidation of NO at +865 mV vs. Ag/AgCl reference electrode [61]. 2.9. Statistical analysis The concentrations in the dialysate were expressed in nM (DA) or AM (ascorbic acid) and given as mean T SEM Drug effects on neurochemicals were statistically evaluated in terms of changes in absolute dialysate concentrations. Statistical significance was assessed using analysis of variance (ANOVA) for difference between groups and over time. Difference within or between groups were determined by paired or unpaired t tests with Bonferroni multiple comparison adjustment.
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3. Results 3.1. NO-trapping activity of MGD in vitro MGD is a thiol compound with NO trapping activity [21,22]; the latter is potentiated by iron, as a consequence of a MGD/iron(II) adduct formation [21,22,56]. Thus, MGD appeared to be most suitable compound for purposes of this study. We deemed necessary the assessment of the NO trapping activity of MGD, since the compound was synthesized by one of us. As shown in Fig. 2, the electrochemical detection of NO generated from the decomposition of the NO-donor SNAP 1.0 mM [51] was completely abolished by MGD 5.0 mM. 3.2. Effects of intrastriatal co-infusion of MGD on SIN-1-induced increases in dialysate DA Intrastriatal infusion of SIN-1 (5.0 mM for 180 min, n = 4) induced a long-lasting increase in DA concentrations in dialysates collected from the ipsilateral outlet, with a peak (897% of baseline) after 60 min (Fig. 3, panel A); in dialysates from the contralateral outlets, the increase reached a lower peak (395% of baseline) after 80 min (Fig. 3, panel B). SIN-1 co-infusion did not significantly affect ascorbic acid concentrations in dialysates from both ipsilateral (baseline value 9.28 T 1.23 AM) (Fig. 3, panel B) and contralateral (baseline value 8.84 T 1.36 AM, data not shown) outlets. SIN-1 infusion did not induce behavioral changes, such as increases in motor activity or stereotypy. Preliminary experiments showed that infusion of MGD alone (up to concentrations of 5.0 mM) for 200 min did not modify all striatal neurochemical parameters (data not shown). MGD 5.0 mM co-infusion was initiated through the contralateral inlet 20 min before SNI-1 infusion (5.0 mM for 180 min through the ipsilateral inlet, n = 4), to allow MGD diffusion in the extracellular compartment.
Fig. 3. Effect of intrastriatal infusion of SIN-1 on DA concentrations in dialysates from the striatum of freely moving rats and effects of MGD coinfusion on SIN-1-induced changes. SIN-1 was infused for 180 min (solid horizontal bar a) through the ipsilateral inlet (n = 4); MGD (n = 4) was infused for 200 min (solid horizontal bar b) through the contralateral inlet. MGD co-infusion was initiated 20 min before SIN-1 infusion. Dialysates were collected, at 20 min intervals, during drug infusion and for 80 min after discontinuation of drug infusion. Values are given as mean T SEM and refer to concentrations in dialysates from the ipsilateral outlet (panel A) or the contralateral outlet (panel B). *P < 0.05 compared with pertinent baseline values of all groups (thin horizontal bars); +P < 0.05 compared with SIN-1 group.
MGD co-infusion caused a significant potentiation of SINinduced increases in DA concentrations in dialysates from both ipsilateral (with a peak of 1.100% of baseline after 60 min) (Fig. 3, panel A) and contralateral (with a peak of 742% of baseline after 20 min) outlets (Fig. 3, panel B). MGD co-infusion did not significantly affect ascorbic acid concentrations in dialysates from both outlets (data not shown). 3.3. Effects of intrastriatal co-infusion of the NO trapper carboxy-PTIO on SIN-1-induced changes in dialysate DA
Fig. 2. Electrochemical detection of NO following the decomposition of SNAP 1.0 mM in PBS at 37 -C (a) and effects of MGD 5.0 mM on NO electrochemical detection (b). The data are representative of three independent experiment.
The unexpected finding of a further increase in dialysate DA following MGD co-infusion with SIN-1 prompted us to evaluate the effects of carboxy-PTO, a well known [1] NO trapper, on SIN-1 induced increases in dialysate DA.
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Intrastriatal infusion of carboxy-PTIO (0.5 mM for 200 min, n = 3) induced the following: a) in dialysates collected from the opposite outlet (currently indicated as ipsilateral outlet): lack of changes in dialysate DA (baseline levels 4.04 T 1.03 nM) (Fig. 4, panel A) and significant decreases (by about 37%) in ascorbic acid concentrations (Fig. 4, panel B); b) in dialysates collected from the pertinent outlet (currently indicated as contralateral outlet): lack of changes in baseline DA concentrations (baseline values 3.88 T 0.96 nM) and a greater decrease (by about 85%) in ascorbic acid concentrations (data not shown). The decrease in dialysate ascorbic acid concentrations reveals that carboxy-PTIO has
a pro-oxidant activity which may occur both in the extracellular space (intra-striatum), as revealed by the decrease in dialysates from the opposite outlet, and during dialysate collection from the pertinent outlet (post-striatum). When carboxy-PTIO was infused at concentrations 1.0 mM, baseline DA concentrations were greatly decreased, while ascorbic acid was not detectable, in dialysates from the pertinent outlet; in dialysates from the opposite outlet, DA baseline concentrations were scarcely affected while ascorbic acid was still not detectable (data not shown). Carboxy-PTIO (0.5 mM, n = 4) co-infusion was initiated through the contralateral inlet 20 min before SIN-1 5.0 mM 180 min infusion (ipsilateral inlet), to allow carboxy-PTIO diffusion in the extracellular compartment. Carboxy-PTIO co-infusion significantly attenuated SIN-1induced increases in DA concentrations in dialysates from both the ipsilateral (Fig. 4, panel A) and contralateral outlet (data not shown). Carboxy-PTIO co-infusion with SIN-1 resulted in a greater decrease in ascorbic acid concentrations (by about 89%) in dialysates from the ipsilateral outlet (Fig. 4, panel B), while ascorbic acid was not detectable in dialysates from the contralateral outlet (data not shown). 3.4. Effects of Ca2+ omission on MGD+SIN-1-induced increase in dialysate DA
Fig. 4. Effect of intrastriatal infusion of SIN-1 or carboxy-PTIO on DA (panel A) and ascorbic acid (panel B) concentrations in dialysates from the striatum of freely moving rats, and effects of carboxy-PTIO co-infusion on SIN-1-induced changes. SIN-1 (n = 4, same group as in Fig. 2) was infused for 180 min (solid horizontal bar a) through the ipsilateral inlet for 180 min, while carboxy-PTIO 0.5 mM was infused (solid horizontal bar b) through the contralateral inlet for 200 min either alone (n = 3) or with SIN-1 (ipsilateral inlet, n = 4). Carboxy-PTIO co-infusion was initiated 20 min before SIN-1 infusion. Dialysates were collected, at 20 min intervals, during drug infusion and for 80 min after discontinuation of drug infusion. Values are given as mean T SEM and refer to concentrations in dialysates from the ipsilateral outlet. *P < 0.05 compared with pertinent baseline values in all groups (thin horizontal bars); +P < 0.05 compared with SIN-1 group (thin horizontal bar).
In a previous study [44], we demonstrated that SIN-1induced DA increases in dialysates from the striatum of freely moving rats was independent of extracellular Ca2+. Therefore, we deemed of interest the evaluation of the role of extracellular Ca2+ in the further increase in dialysate DA following MGD co-infusion with SIN-1, Ca2+-free Ringer solution was infused through both inlets from the beginning of the experiments. Omission of Ca2+ resulted in a decrease by about 95 –100% of baseline DA concentrations. SIN-1 (5.0 mM for 180 min, n = 4), was thereafter infused through the ipsilateral inlet. Ca2+ omission scarcely affected SIN-1-induced dialysate DA increases (Fig. 5). MGD 5.0 mM co-infusion was initiated through the contralateral inlet 20 min before SNI-1 1.0 mM (n = 4) infusion for 180 min through the ipsilateral inlet. Omission of Ca2+ resulted in a fully inhibition of the MGD effects on SIN-1-induced DA increases; in addition, dialysate DA return toward baseline occurred significantly earlier than in SIN-1 group (Fig. 5). 3.5. Effects of intrastriatal co-infusion of the N-type (Cav 2.2) voltage-sensitive Ca2+ channel inhibitor x-C-GVIA or the L-type (Cav 1.1– 1.3) voltage-sensitive Ca2+ channel inhibitor nifedipine on MGD+SIN-1-induced DA release The finding that MGD effects are dependent on extracellular Ca2+ prompted us to assess the type of channel
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Fig. 5. Effects of Ca2+ omission on MGD+SIN-1-induced increases in DA concentrations in dialysates from the striatum of freely moving rats. SIN-1 was infused for 180 min (solid horizontal bar a) through the ipsilateral inlet either in Ringer (same group as in Fig. 3) or in Ca2+-free Ringer (open horizontal bar c) (n = 4); MGD was co-infused (solid horizontal bar b) for 200 min through the contralateral inlet. MGD infusion was initiated 20 in before SIN-1 infusion either in Ringer (same group as in Fig. 3) or in Ca2+free Ringer (n = 4). Dialysates were collected, at 20 min intervals, during drug infusion and for 80 min after discontinuation of drug infusion. Values are given as mean T SEM and refer to concentrations in dialysates from the ipsilateral outlet. *P < 0.05 compared with pertinent baseline values in all groups (thin horizontal bar); +P < 0.05 compared with SIN-1+MGD group (thin horizontal bar); #P < 0.05 compared with SIN-1 in Ca2+-free Ringer group.
involved in the MGD+SIN-1-induced Ca2+ entry in dopaminergic terminals. For this purpose, either the N-type Ca2+ channel inhibitor N-C-GVIA or the L-type Ca2+ channel inhibitor nifedipine was co-infused with SIN-1 1.0 mM+MGD 5.0 mM. The concentrations of N-C-GVIA and nifedipine and were chosen according to Rocchitta et al. [43]. N-C-GVIA 10 AM 200 min infusion (n = 3) significantly decreased (up to 80 –90%) baseline levels of dialysate DA (Fig. 6). Co-infusion of N-C-GVIA (10 AM, n = 4) with MGD 5.0 mM was initiated through the contralateral inlet 20 min before SIN-1 5.0 mM 180 min infusion through the ipsilateral inlet. N-C-GVIA scarcely affected MGD+SIN-1induced increases in dialysate DA (Fig. 6). In a previous study [43], we showed that infusion of nifedipine (10 AM) did not affects baseline levels of DA. Nifedipine (10 AM, n = 4) co-infusion with MGD 5.0 mM was initiated through the contralateral inlet 20 min before SIN-1 5.0 mM 180 min infusion through the ipsilateral inlet. Nifedipine co-infusion significantly inhibited MGD+SIN-1induced increases in dialysate DA (Fig. 6). 3.6. Effects of intrastriatal co-infusion of iron(II) on MGD+SIN-1-induced changes in dialysate DA and effects Ca2+ omission The effects of exogenous iron(II) on SIN-1-induced DA increases in dialysates from the striatum of freely moving rats have been thoroughly addressed in a previous study [48]. We demonstrated that a short-lasting co-infusion of iron(II) (1.0 mM) potentiated the DA dialysate increase
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given by SIN-1 in a range of concentrations from 0.2 to 5.0 mM. Therefore, we deemed of interest the evaluation of exogenous iron(II) effects on SIN-1+MGD-induced DA increases in dialysate DA. The iron(II) concentration of 1.0 mM proved to be not suitable for the purposes of this study. Solutions of either MGD 5 mM or iron(II) 0.1 mM in Ringer are colorless. However, a solution of MGD 5.0 mM with iron(II) 0.1 mM in Ringer yields an intense brown color, probably as a consequence of a MGD/iron(II) complex in vitro formation [56]. For this reason, we had to infuse MGD and iron(II) through separate inlets. MGD 5.0 mM was co-infused for 200 min through the contralateral inlet, while iron(II) 0.1 mM and SIN-1 5.0 mM were co-infused for 180 min through the ipsilateral inlet. The infusion of MGD started 20 min before SIN-1+iron(II) co-infusion, to allow MGD diffusion in the extracellular compartment. Co-infusion of iron(II) 0.1 mM with SIN-1 5.0 mM for 180 min (n = 3) induced increases in DA concentrations in dialysates from the ipsilateral outlet, with a peak (895% of baseline) after 140 min (Fig. 7, panel A); these increases did not statistically differ from those induced by SIN-1 5.0 mM alone; in contrast, the increase was greater than that induced by SIN-1 alone in dialysates from the contralateral outlet, with a peak of 491% of baseline after 120 min (data not shown). Ascorbic acid concentrations were unaffected in dialysates from both outlets (Fig. 8, panels A, B).
Fig. 6. Effects of intrastriatal infusion of x-C-GVIA or nifedipine on MGD+SIN-1-induced increases in DA concentrations in dialysates from the striatum of freely moving rats. x-C-GVIA (n = 3) alone was infused through the contralateral inlet for 200 min. Either x-C-GVIA (n = 4) or nifedipine (n = 4) was co-infused with MGD for 200 min through the contralateral inlet for 200 min, while SIN-1 was infused for 180 min through the ipsilateral inlet. Either x-C-GVIA+MGD or nifedipine+MGD co-infusion was initiated 20 in before SIN-1 infusion. Dialysates were collected, at 20-min intervals, during drug infusion and for 80 min after discontinuation of drug infusion. Values are given as mean T SEM and refer to concentrations in dialysates from the ipsilateral outlet. *P < 0.05 compared with pertinent baseline values in all groups (thin horizontal bars); + P < 0.05 compared with SIN-1+MGD group (thin oblique bar).
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ipsilateral outlet had a light brown color, while those from the contralateral outlet were colorless. Ascorbic acid concentrations fell below the detection limit in dialysates from the ipsilateral outlet (Fig. 8, panel A); in contrast, ascorbic acid concentrations were unaffected in dialysates from the contralateral outlet (Fig. 8, panel B). Ca2+-free Ringer solution was infused through both inlets from the beginning of the experiments. Thereafter, MGD 5.0 mM 200 min was infused for 200 min trough the contralateral inlet while SIN-1 5.0 mM+iron(II) 0.1 mM were infused for 180 min trough the ipsilateral inlet (n = 4). Ca2+-omission fully inhibited iron(II)+MGD-induced potentiation of SIN-1-induced DA release (Fig. 7, panel A). Ca2+omission did not affect iron(II)+MGD+SIN-1-induced changes in dialysate ascorbic acid concentrations (data not shown). 3.7. Effects of the iron chelator deferoxamine or nifedipine co-infusion on iron(II)+MGD+SIN-1-induced changes in dialysate DA Preliminary experiments showed that deferoxamine co-infusion for 180 up to 5.0 mM affected neither DA
Fig. 7. Effects of intrastriatal infusion of MGD on iron(II)+SIN-1-induced increases in DA concentrations in dialysates from the striatum of freely moving rats and effects of Ca2+ omission, deferoxamine or nifedipine coinfusion on iron(II)+MGD+SIN-1-induced changes. Panel A: SIN-1 mM) iron(II) (n = 4) were co-infused for 180 min (solid horizontal bar a) through the ipsilateral inlet for 180 min. MGD 5.0 mM was infused for 200 min through the contralateral inlet (solid horizontal bar b), while SIN-1 5.0 mM+iron(II) 0.1 mM were co-infused for 180 min through the ipsilateral inlet for 180 min (solid horizontal bar a), either in Ca2+containing Ringer (n = 4) or in Ca2+-free Ringer (open horizontal bar c, n = 4). Panel B: MGD 5.0 mM was co-infused with either deferoxamine (n = 4) or nifedipine (n = 4) (solid horizontal bar b) through the contralateral inlet for 200 min, while SIN-1 5.0 mM+iron(II) 0.1 mM were co-infused for 180 min through the ipsilateral inlet for 180 min (solid horizontal bar a). Dialysates were collected, at 20 min intervals, during drug infusion and for 80 min after discontinuation of drug infusion. Values are given as mean T SEM and to refer concentrations in dialysates from the ipsilateral outlet. *P < 0.05 compared with pertinent baseline values for all groups (thin horizontal bar, panels A, B). Panel A: +P < 0.05 compared with MGD+SIN-1+iron(II) group (thin horizontal bars). Panel B: deferoxamine and nifedipine groups: +P < 0.05 compared with iron(II)+SIN1+MGD group (panel A) (thin horizontal bar).
Co-infusion of MGD 5.0 mM 200 min with SIN-1 5.0 mM+iron(II) 0.1 mM for 180 min (n = 4) potentiated SIN1+iron(II)-induced increases in DA concentration in dialysates from both ipsilateral (peak 1623% of baseline after 60 min (Fig. 7, panel A) and contralateral (peak 985% of baseline, data not shown) outlets. Dialysates from the
Fig. 8. Effects of intrastriatal infusion of deferoxamine on MGD+iron(II)+ SIN-1-induced changes in ascorbic acid concentrations in dialysates from the striatum of freely moving rats. Same groups as in Fig. 7 (panel A). Values are given as mean T SEM and refer to concentrations in dialysates from the ipsilateral outlet (panel A) or the contralateral outlet (panel B). *P < 0.05 compared baseline values; +P < 0.05 compared with MGD+SIN-1+iron(II) group (thin horizontal bar).
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nor ascorbic acid dialysate concentrations (data not shown). Deferoxamine 2.0 mM attenuated at the borderline of statistical significance the iron(II)+MGD+SIN-1-induced increase in dialysate DA (data not shown). Deferoxamine 5.0 mM co-infusion with MGD 5.0 mM (n = 4) was initiated through the contralateral inlet 20 min before SIN-1 5.0 mM+ iron(II) 0.1 mM 180 min coinfusion (ipsilateral inlet), to allow deferoxamine and MGD diffusion in the extracellular compartment. Deferoxamine co-infusion fully inhibited the iron(II)+MGDinduced potentiation of SIN-1-induced increases in DA concentrations in dialysates from both ipsilateral (Fig. 7, panel B) and contralateral (data not shown) outlets. Dialysates from the ipsilateral outlet were colorless. In addition, deferoxamine co-infusion greatly inhibited (Fig. 8, panel A) the MGD+SIN-1+iron(II)-induced fall of ascorbic acid concentrations below the detection limit in dialysates from the ipsilateral outlet, which were colorless. Nifedipine 10 AM co-infusion with MGD 5.0 mM (n = 4) was initiated through the contralateral inlet 20 min before SIN-1 5.0 mM + iron(II) 0.1 mM 180 min coinfusion (ipsilateral inlet). Nifedipine co-infusion fully inhibited the iron(II)+MGD-induced potentiation of SIN1-induced increases in DA concentrations in dialysates from both ipsilateral (Fig. 7, panel B) and contralateral (data not shown) outlets. Dialysates from the ipsilateral outlet had a light brown color. Nifedipine co-infusion, however, failed to inhibit the MG+SIN-1+iron(II)-induced fall of ascorbic acid concentrations below the detection limit in dialysates from the ipsilateral outlet (data not shown). 3.8. Effects of deferoxamine co-infusion on MGD+SIN-1-induced increases in dialysate DA The finding that deferoxamine fully inhibited iron(II)+ MGD effects on SIN-1-induced increases in dialysate DA prompted us to investigate the effects of deferoxamine coinfusion on MGD+SIN-1-induced increases in dialysate DA. The working hypothesis was that exogenous MGD might form a complex with endogenous iron; such a complex per se is unable to trigger striatal DA release, since infusion of MGD alone did not induce changes in dialysate DA. However, when NO is generated by co-infusion of the NO-donor, exogenous NO might react preferentially with the exogenous MGD/endogenous iron complex, to form the well-known MGD/iron/ NO adduct. Exogenous NO itself might render available endogenous iron by releasing it from storage proteins [38,45]. Deferoxamine 2.0 mM was co-infused with MGD 5.0 mM through the contralateral inlet 20 min before SIN-1 5.0 mM (n = 4) infusion for 180 min through the ipsilateral inlet. Co-infusion of deferoxamine significantly
25
Fig. 9. Effects of intrastriatal infusion of deferoxamine on MGD+SIN-1induced increases in DA concentrations in dialysates from the striatum of freely moving rats. MGD+SIN-1 group (n = 4), same group as in Fig. 2. Deferoxamine was co-infused with MGD (horizontal bar b) for 200 min through the contralateral inlet 20 min before SIN-1 (n = 4) infusion for 180 min through the ipsilateral inlet (horizontal bar a). Values are given as mean T SEM and refer to concentrations in dialysates from the ipsilateral outlet. *P < 0.05 compared with pertinent baseline values for both groups (thin horizontal bar). +P < 0.05 compared with MGD+SIN-1 group (thin horizontal bar).
inhibited MGD effects on SIN-1-induced increases in dialysate DA (Fig. 9).
4. Discussion The key findings of this study are the following: (i) spontaneous release of striatal DA is dependent on extracellular Ca2+ and it is inhibited by the N-type (Cav 2.2) voltage sensitive Ca2+ channel inhibitor N-C-GVIA; in contrast, SIN-1-induced striatal DA release appears to be independent of external Ca2+; (ii) SIN-1-induced striatal DA release is inhibited by the NO trapper carboxy-PTIO; in contrast, the NO trapper MGD, whose trapping activity is increased by iron, further increases SIN-1-induced striatal DA release, probably as a consequence of an iron(II)/MGD/ NO adduct formation; (iii) striatal DA release induced by the iron(II)/MGD/NO adduct is dependent on extracellular Ca2+ and it is inhibited by the L-type (Cav 1.1– 1.3) voltage sensitive Ca2+ channel inhibitor nifedipine. The NO/sGC/cyclic GMP pathway plays an important role in Ca2+ cycling [18]. In a previous study [44], we demonstrated that exogenous NO-induced striatal DA release was independent of both the activation of the sGC/ cyclic GMP pathway and extracellular Ca2+; however, when intracellular Ca2+ stores were depleted, exogenous NO promoted Ca2+ entry through store-operated channels with a consequent great increase in DA release. The current view on the release of neurotransmitters by Ca2+-triggered synaptic vesicle exocytosis is that the process involves several steps and is controlled by a protein machinery (SNARE proteins) [42]. On the basis of the inhibition of exogenous NO-induced release of striatal DA by means of
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either the alkylating agent N-ethylmaleimide or clostridial neurotoxins [44], we suggested that exogenous NO might target directly SNARE proteins and promote DA release with a Ca2+-independent and N-ethylmaleimide-sensitive mechanism (Fig. 10). Decomposition of SIN-1 gives rise to NO and O2 ; NO reacts readily with O2I to form peroxynitrite [14], which is a powerful DA oxidizing agent [7]. Peroxynitrite downstream formation following SIN-1 decomposition does occur in vitro [50]; most likely, however, it does not occur in the striatal extracellular compartment [44,48], owing to the presence of high concentration of ascorbic acid (0.2 – 0.4 mM), which are kept constant by an efficient homeostatic mechanism [27]. NO is a simple hydrophobic gaseous molecule that is highly diffusible and reactive [53]; therefore, when NO is released in the extracellular compartment, it is not surprising if NO can in part diffuse through the neuronal membrane and in part react with NO-trapper molecules (Fig. 9). However, while the trapper carboxy-PTIO decreased SIN-1-induced release of DA, MGD actually further increased it. Such discrepancy may be related to the ability of MGD to react also with iron(II), with a consequent increase in its NO-trapping activity. When exogenous iron(II) was supplied, most likely it generated a NO/MGD/
I
iron(II) adduct, which proved to be a powerful striatal DA releaser. The adduct-induced increase in DA release was dependent on extracellular Ca2+ and was inhibited by either nifedipine or deferoxamine co-infusion. The finding that deferoxamine co-infusion inhibited also the SIN-1+MGDinduced increase in DA release, strongly support the hypothesis that, in absence of exogenous iron(II), endogenous iron might also generate the NO/MGD/iron(II) adduct formation. Exogenous NO itself might render available endogenous iron by releasing it from storage proteins [38,45]. Taken together, these results allow us to suggest that the adduct-induced DA release is Ca2+-dependent and Ca2+ entry occurs through L-type (Cav 1.1 – 1.3) channels; it is relevant, to this regard, that microinjections of L-type Ca2+ channel activators in the caudate putamen of freely moving rats are known to elicit release of large amount of DA [32]. Ca2+ cycling occurs through a variety of definite channels [26]. In striatal dopaminergic terminals, the Ca2+ entry which ensures the physiological release of DA [5] occurs through N-type (Cav 2.2) channels [35]. Therefore, the striatal L-type Ca2+ channels activated by our pharmacological manipulation should be regarded as silent channels. The MGD/iron(II) adduct formation occurs both in vitro [56] and in vivo [21,22]. The adduct can undergo complex
Fig. 10. Dynamics of pathways involved in the SIN-1 and iron(II)-induced striatal dopamine release in the striatum of freely moving rats. Spontaneous firing of nigral neurons (1) induces membrane depolarization of terminals and activates Ca2+ entry through N-type (Cav 2.2) channels, with a consequent SNAREmediated dopamine (DA) release. Decomposition of SIN-1 in the extracellular compartment gives rise (2) to NO and O2 . Endogenous ascorbic acid inhibits downstream peroxynitrite formation [48,50], thus allowing NO to enter the membrane ending and promote (3) Ca2+-independent SNARE-mediated [44] DA release. NO-induced DA release is inhibited by the NO-trapper carboxy-PTIO. In presence of the NO-trapper MGD, NO released from SIN-1 decomposition preferentially reacts with MGD and iron(II), either endogenous or exogenous, to form a NO/MGD/iron(II) adduct (4), which activates a Ca2+-dependent DA release. The iron chelator deferoxamine, by inhibiting the adduct formation (5), selectively blocks the pertinent quota of DA release. The inhibitory effect of the L-type (Cav 1.1 – 1.3) Ca2+ channel blocker nifedipine (6) allows us to hypothesize that the adduct might promote Ca2+ entry through an activation of silent L-type Ca2+ channels.
I
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redox chemistry [57]; in fact, it is rapidly oxidized under aerobic conditions to form MGD/iron(III) complex [56,57]. Reducing agents such as ascorbic acid, hydroquinone or cysteine, reduce MGD/iron(III) back to MGD/iron(II) complex [57]. The redox chemistry of the MGD/iron(II) complex may account for the disappearance of ascorbic acid in dialysates collected from the ipsilateral outlet during SIN1+iron(II) and MGD co-infusion. An oxidation of the MGD/ iron(II)/NO adduct to MGD/iron(III)/NO most likely occurred ‘‘post-striatum’’ (i.e., in vitro, during dialysate collection), and dialysate ascorbic acid most likely reduced the adduct back to MGD/iron(II)/NO. In fact, the iron chelator deferoxamine, which binds both iron(II) and iron(III) ions [13], by inhibiting the adduct formation and the consequent oxidation, restored dialysate ascorbic acid concentrations. Ascorbic acid concentrations were unaffected in colorless dialysates from the contralateral outlet, most likely since either they were restored by the efficient homeostatic mechanism quoted above, or owing to the lack of adduct oxidation in the extracellular compartment. The question arises as to whether the coupling of NO and iron with silent striatal L-type Ca2+ channels might have relevance to the physiopathology of dopaminergic terminals. Parkinson’s disease (PD) is characterized by an important loss of nigral dopaminergic neurons. The concept of a threshold of DA depletion for onset of parkinsonian symptoms is widely accepted. Parkinsonian symptoms appear when dopaminergic neuronal death exceeds a critical threshold, 70 –80% of striatal nerve terminals and 50 – 60% of nigral perikarya [41]. These findings clearly support the activation of nigro-striatal endogenous compensatory mechanisms. Neuritic sprouting [52], activation of either tyrosine hydroxylase immunoreactive striatal intrinsic neurons [6] or silent nigro-striatal dopaminergic neurons [55], and appearance of new dopaminergic neurons within the striatum itself [36] are indicated as putative endogenous compensatory mechanisms. Nigro-striatal microglia activation, with a consequent up-regulation of inducible NO synthase, is believed to be the main culprit for dopaminergic neuron demise [19,30,49]. In addition, misregulation of iron metabolism, iron-induced oxidative stress, and iron accumulation in the nigro-striatal structures are also widely believed to be important pathogenetic mechanisms of neuronal death in PD [16,60]; moreover, iron is known to increase NO production from activated microglia [22]. Intraneuronal NO is released in short controlled burst, probably in the range of picomolar concentrations, and most likely, it does not reach the extracellular compartment [20]. In the striatum, however, it has been described a distinct type of interneurons which produces and releases NO in the extracellular compartment [4]. Moreover, large amounts of NO, in the range of micromolar concentrations, are generated for long period and released in the extracellular striatal compartment by striatal microglia/macrophages and astrocytes, as demonstrated in vivo by Oyoshi et al. [33], using microdialysis in freely moving rats; in addition,
27
microglial/macrophages and astrocyte activation may further increase (by more that 100%) NO production and release [33]. Taken together, the above findings allow us to hypothesize that the coupling of NO and iron with silent striatal L-type Ca2+ channels, as well as the coupling of NO with striatal store-operated Ca2+ channels [44], and the consequent release of striatal DA from surviving dopaminergic terminals and striatal dopaminergic neurons either intrinsic, silent or newly appeared, could play a role in maintaining an adequate post-synaptic dopaminergic input in the pre-symptomatic phase of PD.
Acknowledgment The research was supported in part by the University of Sassari (ex 60% fund).
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