Maria Rosaria Delogu

Enhancing effect of manganese on L-DOPA-induced apoptosis in PC12 cells: role of oxidative stress.

Abstract : L‐DOPA and manganese both induce oxidative stress‐mediated apoptosis in catecholaminergic PC12 cells. In this study, exposure of PC12 cells to 0.2 mM MnCl2 or 10‐20 μM L‐DOPA neither affected cell viability, determined by the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay, nor induced apoptosis, tested by flow cytometry, fluorescence microscopy, and the TUNEL technique. L‐DOPA (50 μM) induced decreases in both cell viability and apoptosis. When 0.2 mM MnCl2 was associated with 10, 20, or 50 μM L‐DOPA, a concentration‐dependent decrease in cell viability was observed. Apoptotic cell death also occurred. In addition, manganese inhibited L‐DOPA effects on dopamine (DA) metabolism (i.e., increases in DA and its acidic metabolite levels in both cell lysate and incubation medium). The antioxidant N‐acetyl‐L‐cysteine significantly inhibited decreases in cell viability, apoptosis, and changes in DA metabolism induced by the manganese association with L‐DOPA. An increase in autoxidation of L‐DOPA and of newly formed DA is suggested as a mechanism of manganese action. These data show that agents that induce oxidative stress‐mediated apoptosis in catecholaminergic cells may act synergistically.

Glucocorticoid receptor deficiency increases vulnerability of the nigrostriatal dopaminergic system: critical role of glial nitric oxide.

Glucocorticoids (GCs) exert via glucocorticoid receptors (GRs) potent anti‐inflammatory and immunosuppressive effects. Emerging evidence indicates that an inflammatory process is involved in dopaminergic nigro‐striatal neuronal loss in Parkinsons disease. We here report that the GR deficiency of transgenic (Tg) mice expressing GR antisense RNA from early embryonic life has a dramatic impact in “programming” the vulnerability of dopaminergic neurons to 1‐ methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP). The GR deficiency of Tg mice exacerbates MPTP‐induced toxicity to dopaminergic neurons, as revealed by both severe loss of tyrosine hydroxylase positive nigral neurons and sharp decreases in striatal levels of dopamine and its metabolites. In addition, the late increase in dopamine oxidative metabolism and ascorbic acid oxidative status in GR‐deficient mice was far greater than in wild‐type (Wt) mice. Inducible nitric oxide synthase (iNOS) was sharply increased in activated astrocytes, macrophages/microglia of GR‐deficient as compared with Wt mice. Moreover, GR‐deficient microglia produced three‐ to fourfold higher nitrite levels than Wt mice; these increases preceded the loss of dopaminergic function and were resistant to GR the inhibitory effect of GC, pointing to peroxynitrites as candidate neurotoxic effectors. The iNOS inhibitor N6‐(1‐ iminoethyl)‐L‐lysine normalized vulnerability of Tg mice, thus establishing a novel link between genetic impairment of GR function and vulnerability to MPTP.

Role of the nitric oxide/cyclic GMP pathway and extracellular environment in the nitric oxide donor-induced increase in dopamine secretion from PC12 cells: a microdialysis in vitro study

In vitro microdialysis was used to investigate the mechanism of nitric oxide (NO) donor‐induced changes in dopamine (DA) secretion from PC12 cells. Infusion of the NO‐donor S‐nitroso‐N‐acetylpenicillamine (SNAP, 1.0 mm) induced a long‐lasting increase in DA and 3‐methoxytyramine (3‐MT) dialysate concentrations. SNAP‐induced increases were inhibited either by pre‐infusion of the soluble guanylate cyclase (sGC) inhibitor 1H‐[1,2,4] oxadiazolo[4,3]quinoxalin‐1‐one (ODQ, 0.1 mm) or by Ca2+ omission. Ca2+ re‐introduction restored SNAP effects. SNAP‐induced increases in DA + 3‐MT were unaffected by co‐infusion of the l‐type Ca2+ channel inhibitor nifedipine. The NO‐donor (+/–)‐(E)‐4‐ethyl‐2‐[(E)‐hydroxyimino]‐5‐nitro‐3‐hexenamide (NOR‐3, 1.0 mm) induced a short‐lasting decrease in dialysate DA + 3‐MT. Ascorbic acid (0.2 mm) co‐infusion allowed NOR‐3 to increase dialysate DA + 3‐MT. ODQ pre‐infusion inhibited NOR‐3 + ascorbic acid‐induced DA + 3‐MT increases. Infusion of high K+ (75 mm) induced a 2.5‐fold increase in dialysate DA + 3‐MT. The increase was abolished by NOR‐3 co‐infusion. Conversely, co‐infusion of ascorbic acid (0.2 mm) with NOR‐3 + high K+ restored high K+ effects. Co‐infusion of nifedipine inhibited high K+‐induced DA + 3‐MT increases. These results suggest that activation of the NO/sGC/cyclic GMP pathway may be the underlying mechanism of extracellular Ca2+‐dependent effects of exogenous NO on DA secretion from PC12 cells. Extracellular Ca2+ entry may occur through nifedipine‐insensitive channels. NO effects and DA concentrations in dialysates largely depend on both the timing of NO generation and the extracellular environment in which NO is generated.

Role of the nitric oxide/cyclic GMP pathway and ascorbic acid in 3-morpholinosydnonimine (SIN-1)-induced increases in dopamine secretion from PC12 cells. A microdialysis in vitro study

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

Glutamate and catabolites of high-energy phosphates in the striatum and brainstem of young and aged rats subchronically exposed to manganese.

The degradation of high-energy phosphates was recently shown to precede manganese-induced cellular death. We evaluated hypoxanthine, xanthine, uric acid and glutamate levels in the striatum and brainstem of 3- and 20-month-old rats after subchronic oral exposure to manganese (MnCl2, 200 mg/kg/day in young rats, and 50–100 or 200 mg/kg/day in aged rats). Aged rats had higher basal levels of hypoxanthine, xanthine, and glutamate both in the striatum and brainstem than young rats; conversely, basal uric acid levels were lower in the striatum, but higher in the brainstem. Manganese induced a significantly greater increase in hypoxanthine, xanthine, uric acid and gluta-mate levels in aged rats than in young rats in both brain regions. These findings depict a greater manganese-induced energetic impairment (increases in hypoxanthine and xanthine levels), xanthine oxidase-induced free radical generation (increases in xanthine and uric acid levels), and excitotoxic status (increases in glutamate levels) in aged rats than in young rats. In addition, these findings may also account for a greater manganese toxicity to the nigro-striatal dopaminergic system in aged than in young rats, as shown in a previous work.

Glutathione deficiency potentiates manganese-induced increases in compounds associated with high-energy phosphate degradation in discrete brain areas of young and aged rats

Aging is a factor known to increase neuronal vulnerability to oxidative stress, which is widely accepted as a mechanism of manganese-induced neuronal damage. We previously showed that subchronic exposure to manganese induced greater energy impairment (as revealed by increases in hypoxanthine, xanthine and uric acid levels) in the striatum and brainstem of aged rats vs young rats. This study shows that inhibition of glutathione (GSH) synthesis, by means of buthionine (SR) sulfoximine, decreased GSH levels and increased the ascorbic acid oxidation status in the striatum and limbic forebrain of both young and aged rats. In addition, inhibition of GSH synthesis greatly potentiated the manganese-induced increase in inosine, hypoxanthine, xanthine and uric acid levels in both regions of aged rats; moreover, inhibition of GSH synthesis significantly increased inosine, hypoxanthine, xanthine and uric acid levels in both regions of young rats, compared with the manganese-treated group. These results suggest that an impairment in the neuronal antioxidant system renders young rats susceptible to manganese-induced energetic impairment, and further support the hypothesis that an impairment in this system plays a permissive role in the increase of neuronal vulnerability that occurs with aging.