RESUMEN
Highly sensitive detection of ethanol concentrations in discrete brain regions of rats voluntarily accessing ethanol, with high temporal resolution, would represent a source of greatly desirable data in studies devoted to understanding the kinetics of the neurobiological basis of ethanol's ability to impact behavior. In the present study, we present a series of experiments aiming to validate and apply an original high-tech implantable device, consisting of the coupling, for the first time, of an amperometric biosensor for brain ethanol detection, with a sensor for detecting the microvibrations of the animal. This device allows the real-time comparison between the ethanol intake, its cerebral concentrations, and their effect on the motion when the animal is in the condition of voluntary drinking. To this end, we assessed in vitro the efficiency of three different biosensor designs loading diverse alcohol oxidase enzymes (AOx) obtained from three different AOx-donor strains: Hansenula polymorpha, Candida boidinii, and Pichia pastoris. In vitro data disclosed that the devices loading H. polymorpha and C. boidinii were similarly efficient (respectively, linear region slope [LRS]: 1.98 ± 0.07 and 1.38 ± 0.04 nA/mM) but significantly less than the P. pastoris-loaded one (LRS: 7.57 ± 0.12 nA/mM). The in vivo results indicate that this last biosensor design detected the rise of ethanol in the nucleus accumbens shell (AcbSh) after 15 minutes of voluntary 10% ethanol solution intake. At the same time, the microvibration sensor detected a significant increase in the rat's motion signal. Notably, both the biosensor and microvibration sensor described similar and parallel time-dependent U-shaped curves, thus providing a highly sensitive and time-locked high-resolution detection of the neurochemical and behavioral kinetics upon voluntary ethanol intake. The results overall indicate that such a dual telemetry unit represents a powerful device which, implanted in different brain areas, may boost further investigations on the neurobiological mechanisms that underlie ethanol-induced motor activity and reward.
Asunto(s)
Técnicas Biosensibles/instrumentación , Etanol/farmacología , Actividad Motora/efectos de los fármacos , Núcleo Accumbens/efectos de los fármacos , Administración Oral , Consumo de Bebidas Alcohólicas/metabolismo , Oxidorreductasas de Alcohol/química , Animales , Espacio Extracelular , Técnicas In Vitro , Masculino , Núcleo Accumbens/ultraestructura , Ratas , Ratas Sprague-Dawley , TelemetríaRESUMEN
A deficiency of striatal dopamine (DA) is generally accepted as an expression of manganese (Mn) toxicity in experimental animals. Since compromised cellular defence mechanisms may be involved in Mn neurotoxicity, we investigated the response of the neuronal antioxidant system [ascorbic acid (AA) oxidation, glutathione (GSH) and uric acid levels] and neurochemical changes in the striatum in aged rats exposed to Mn. Levels of dopamine (DA), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), AA, dehydroascorbic acid (DHAA), GSH and uric acid were determined after subchronic oral exposure to MnCl2 200 mg/kg (3-month-old rats) and 30-100-200 mg/kg (20-month-old-rats). Aged rats had basal levels of striatal DA, DOPAC, HVA, 5-HT, 5-HIAA, GSH and AA lower than those of young rats. In the striatum of aged rats, Mn induced biphasic changes in the levels of DA, DOPAC, HVA (an increase at the lower dose and a decrease at the higher dose) and DHAA (opposite changes). Mn decreased GSH levels and increased uric acid levels both in the striatum and in synaptosomes in all groups of aged rats. All of these parameters were affected to a lesser extent in young rats. In conclusion, the response of cellular defence mechanisms in aged rats is consistent with a Mn-induced increase in the formation of reactive oxygen species. An age-related impairment of the neuronal antioxidant system may play an enabling role in Mn neurotoxicity.
Asunto(s)
Envejecimiento/fisiología , Intoxicación por Manganeso , Neostriado/metabolismo , Estrés Oxidativo/efectos de los fármacos , Animales , Ácido Ascórbico/metabolismo , Dopamina/metabolismo , Glutatión/metabolismo , Masculino , Neostriado/citología , Neostriado/efectos de los fármacos , Estrés Oxidativo/fisiología , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Serotonina/metabolismo , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismo , Ácido Úrico/metabolismoRESUMEN
1. We showed previously that interaction between NO and iron(II), both released following decomposition of sodium nitroprusside (SNP), accounted for the late SNP-induced dopamine (DA) increase in dialysates from the striatum of freely moving rats. 2. In this study, intrastriatal infusion of the NO-donor S-nitroso-N-acetylpenicillamine (SNAP) (0.2 mM for 180 min) induced a moderate increase in dialysate DA and decreases in ascorbic acid dialysate concentrations; in contrast, SNAP 1 mM infusion induced a long-lasting decrease in both DA and ascorbic acid dialysate concentrations. 3-Methoxy-tyramine (3-MT), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and uric acid levels were unaffected. 3. Co-infusion of ferrous sulphate [iron(II), 1 mM for 40 min] with SNAP either 1 or 0.2 mM (for 180 min), produced a significant increase in both DA and 3-MT dialysate concentrations, but it did not affect decreases in dialysate ascorbic acid levels. All other dialysate neurochemicals were unaffected. 4. Co-infusion of ascorbic acid (0.1 mM) with SNAP (1 mM) for 180 min did not modify SNAP-induced decreases in dialysate DA levels. In contrast, co-infusion of uric acid (1 mM) reversed SNAP-induced decreases in dialysate DA; co-infusion of a superoxide dismutase mimetic delayed SNAP-induced DA decreases for a short period, while co-infusion of the antioxidant N-acetylcysteine (NAC, 0.1 mM) significantly increased dialysate DA. 5. The results of this study show that SNAP induces concentration-related changes in DA dialysate levels. At higher concentrations, SNAP induces non-enzymatic DA oxidation, which is inhibited by uric acid and NAC; ascorbic acid failed to protect dialysate DA from oxidation, probably owing to its promoting effect on SNAP decomposition; exogenous iron(II) may react with NO generated from SNAP decomposition, with a consequent increase in dialysate DA and 3-MT, therefore mimicking SNP effects on striatal DA release.
Asunto(s)
Ácido Ascórbico/fisiología , Cuerpo Estriado/efectos de los fármacos , Dopamina/metabolismo , Donantes de Óxido Nítrico/farmacología , Estrés Oxidativo , Penicilamina/farmacología , Acetilcisteína/farmacología , Animales , Cuerpo Estriado/metabolismo , Hierro/metabolismo , Masculino , Microdiálisis , Penicilamina/análogos & derivados , Ratas , Ratas Wistar , S-Nitroso-N-AcetilpenicilaminaRESUMEN
1. We showed previously that interaction between NO and iron (II), both released following the decomposition of sodium nitroprusside (SNP), accounted for the late SNP-induced dopamine (DA) increase in dialysates from the striatum of freely moving rats; in addition, we showed that co-infusion of iron (II) with the NO-donor S-nitroso-N-acetylpenicillamine mimicked SNP effects on striatal DA release. 2. In the present study, intrastriatal co-infusion of iron (II) (given as FeSO(4), 1 mM for 40 min) with the NO-donor and potential peroxynitrite generator 3-morpholinosydnonimine (SIN-1) (0.2, 0.5, 1.0 or 5.0 mM for 180 min), potentiated the SIN-1-induced increase in DA concentration in dialysates from the striatum of freely moving rats. Neither alone nor associated with iron (II) did SIN-1 induce changes in dialysate ascorbic acid or uric acid concentrations. 3. Neither co-infusion of a superoxide dismutase mimetic nor uric acid affected SIN-1-induced increases in dialysate DA concentration. 4. Infusion of the iron chelator deferoxamine (0.2 mM for 180 min) decreased dialysate DA and attenuated SIN-1-induced increases in dialysate DA concentrations. 5. These results suggest that iron plays a key role in SIN-1-induced release of striatal DA and do not support any role for either peroxynitrite or superoxide anion in SIN-1-induced release of striatal DA.
Asunto(s)
Cuerpo Estriado/efectos de los fármacos , Dopamina/metabolismo , Hierro/farmacología , Molsidomina/análogos & derivados , Molsidomina/farmacología , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico/fisiología , Ácido 3,4-Dihidroxifenilacético/metabolismo , Acetilcisteína/farmacología , Animales , Ácido Ascórbico/metabolismo , Cuerpo Estriado/metabolismo , Deferoxamina/farmacología , Soluciones para Diálisis/química , Relación Dosis-Respuesta a Droga , Depuradores de Radicales Libres/farmacología , Ácido Homovanílico/metabolismo , Masculino , Metaloporfirinas/farmacología , Movimiento , Ratas , Ratas Wistar , Ácido Úrico/metabolismo , Ácido Úrico/farmacologíaRESUMEN
The effects of intrastriatal infusion of 3-morpholinosydnonimine (SIN-1) or sodium nitroprusside (SNP) on dopamine (DA), 3-methoxytyramine (3-MT), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), L-dihydroxyphenylalanine (L-DOPA), ascorbic acid and uric acid concentrations in dialysates from the striatum of freely moving rats were evaluated using microdialysis. SIN-1 (1 mM) infusion for 180 min increased microdialysate DA and 3-MT concentrations, while L-DOPA, DOPCA+HVA, ascorbic acid and uric acid levels were unaffected. Co-infusion with ascorbic acid (0.1 mM) inhibited SIN-1-induced increases in DA and 3-MT dialysate concentration. SNP (1 mM) infusion for 180 min increased greatly the dialysate DA concentration to a peak (2950% of baseline) at the end of the infusion, while increases in 3-MT were negligible. In addition, SNP decreased ascorbic acid and L-DOPA but increased uric acid concentration in the dialysate. Co-infusion with deferoxamine (0.2 mM) inhibited the late SNP-induced increase in DA dialysate concentration, but did not affect the decrease in ascorbic acid and increase uric acid dialysate concentrations. SNP (1 mM) infusion for 20 min moderately increased uric acid, DA and 3-MT, but decreased L-DOPA levels in the dialysate. Ascorbic acid concentration increased at the end of SNP infusion. Co-infusion with ascorbic acid (0.1 mM) inhibited the SNP-induced increase in DA and 3-MT, but did not affect the decrease in L-DOPA and increase in uric acid dialysate concentrations. These results suggest that NO released from SIN-1 may account for the increase in the dialysate DA concentration. NO released following decomposition of SNP may account for the early increase in dialysate DA, while late changes in microdialysate composition following SNP may result from an interaction between NO and the ferrocyanide moiety of SNP. Exogenous ascorbic acid inhibits the effect of exogenous NO on DA release probably by scavenging NO, suggesting that endogenous ascorbic acid may modulate the NO control of DA release from 300 striatal dopaminergic terminals.
Asunto(s)
Ácido Ascórbico/farmacología , Cuerpo Estriado/metabolismo , Dopamina/metabolismo , Hierro/fisiología , Molsidomina/análogos & derivados , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico/fisiología , Nitroprusiato/farmacología , Animales , Deferoxamina/farmacología , Masculino , Microdiálisis , Molsidomina/farmacología , Ratas , Ratas WistarRESUMEN
1. The effects of systemic, intrastriatal or intranigral administration of d-amphetamine on glutamate, aspartate, ascorbic acid (AA), uric acid, dopamine (DA), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) concentrations in dialysates from the striatum of freely-moving rats were evaluated using microdialysis. 2. d-Amphetamine (2 mg kg-1) given subcutaneously (s.c.) increased DA, AA and uric acid and decreased DOPAC + HVA, glutamate and aspartate dialysate concentrations over a 3 h period after d-amphetamine. 5-HIAA concentrations were unaffected. Individual changes in glutamate and AA dialysate concentrations were negatively correlated. 3. d-Amphetamine (0.2 mM), given intrastriatally, increased DA and decreased DOPAC + HVA and aspartate dialysate concentrations, but failed to change those of glutamate, AA uric acid or 5-HIAA, over a 2 h period after d-amphetamine. Haloperidol (0.1 mM), given intrastriatally, increased aspartate concentrations without affecting those of glutamate or AA. 4. d-Amphetamine (0.2 mM), given intranigrally, increased AA and uric acid dialysate concentrations and decreased those of glutamate, aspartate and DA; DOPAC + HVA and 5-HIAA concentrations were unaffected. 5. These results suggest that d-amphetamine-induced increases in AA and uric acid and decreases in glutamate concentrations are triggered at nigral sites. The changes in aspartate levels may be evoked by at least two mechanisms: striatal (mediated by inhibitory dopaminergic receptors) and nigral (activation of amino acid carrier-mediated uptake).
Asunto(s)
Ácido Ascórbico/metabolismo , Dextroanfetamina/farmacología , Inhibidores de Captación de Dopamina/farmacología , Ácido Glutámico/metabolismo , Neostriado/metabolismo , Ácido Úrico/metabolismo , Ácido 3,4-Dihidroxifenilacético/metabolismo , Animales , Dextroanfetamina/administración & dosificación , Dopamina/metabolismo , Antagonistas de Dopamina/farmacología , Inhibidores de Captación de Dopamina/administración & dosificación , Haloperidol/farmacología , Técnicas In Vitro , Inyecciones , Masculino , Microdiálisis , Actividad Motora/efectos de los fármacos , Neostriado/efectos de los fármacos , Oxidación-Reducción , Ratas , Ratas Wistar , Técnicas Estereotáxicas , Conducta Estereotipada/efectos de los fármacosRESUMEN
We have previously shown that manganese enhances L-dihydroxyphenylanine (L-DOPA) toxicity to PC12 cells in vitro. The supposed mechanism of manganese enhancing effect [an increase in L-DOPA and dopamine (DA) auto-oxidation] was studied using microdialysis in the striatum of freely moving rats. Systemic L-DOPA [25 mg kg(-1) intraperitoneally (i.p.) twice in a 12 h interval] significantly increased baseline dialysate concentrations of L-DOPA, dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and uric acid, compared to controls. Conversely, DA and ascorbic acid concentrations were significantly decreased. A L-DOPA oxidation product, presumptively identified as L-DOPA semiquinone, was detected in the dialysate. The L-DOPA semiquinone was detected also following intrastriatal infusion of L-DOPA. In rats given L-DOPA i.p. , intrastriatal infusion of N-acetylcysteine (NAC) significantly increased DA and L-DOPA dialysate concentrations and lowered those of L-DOPA semiquinone; in addition, NAC decreased DOPAC+HVA and uric acid dialysate concentrations. In rats given L-DOPA either systemically or intrastriatally, intrastriatal infusion of manganese decreased L-DOPA dialysate concentrations and greatly increased those of L-DOPA semiquinone. These changes were inhibited by NAC infusion. These findings demonstrate that auto-oxidation of exogenous L-DOPA occurs in vivo in the rat striatum. The consequent reactive oxygen species generation may account for the decrease in dialysate DA and ascorbic acid concentrations and increase in enzymatic oxidation of xanthine and DA. L-DOPA auto-oxidation is inhibited by NAC and enhanced by manganese. These results may be of relevance to the L-DOPA long-term therapy of Parkinson's disease.
Asunto(s)
Cuerpo Estriado/efectos de los fármacos , Levodopa/metabolismo , Manganeso/farmacología , Ácido 3,4-Dihidroxifenilacético/metabolismo , Acetilcisteína/farmacología , Animales , Ácido Ascórbico/metabolismo , Cloruros/farmacología , Cromatografía Líquida de Alta Presión , Cuerpo Estriado/metabolismo , Soluciones para Diálisis/química , Dopamina/metabolismo , Ácido Homovanílico/metabolismo , Bombas de Infusión , Levodopa/farmacología , Levodopa/uso terapéutico , Masculino , Compuestos de Manganeso/farmacología , Microdiálisis , Movimiento , Oxidación-Reducción/efectos de los fármacos , Enfermedad de Parkinson/tratamiento farmacológico , Ratas , Ratas Wistar , Factores de Tiempo , Ácido Úrico/metabolismoRESUMEN
Oxidative stress is thought to play a key role in the apoptotic death of several cellular systems, including neurons. Oxidative stress is proposed also as a mechanism of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)- and manganese (Mn)-induced neuronal death. We have recently shown that Mn and the MPTP analogue 1-methyl-4-(2'-ethylphenyl)-1,2,3,6-tetrahydropyridine (2'Et-MPTP), which is metabolized by MAO-A to 1-methyl-4-(2'-ethylphenyl)-pyridinium ion, induce apoptosis in PC12 cells. In the present study, we evaluated the effects of deprenyl and the antioxidant drugs N-acetylcysteine (NAC) and ascorbic acid (AA) on Mn- and 2'Et-MPTP-induced apoptosis in PC12 cells. Apoptosis was tested by terminal deoxynucleotidyl transferase-mediated 2'-deoxy-uridine-5'-triphosphate nick end labelling (TUNEL) technique, flow cytometry and fluorescence microscopy. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Mn-induced apoptosis and decrease in cell viability was inhibited by the antioxidants NAC and AA. Deprenyl failed to inhibit the above Mn effects. Neither NAC, AA nor deprenyl were able to inhibit both 2'Et-MPTP-induced apoptosis and decrease in cell viability. These results confirm that apoptosis may be an important mechanism of cell death in MPTP- and Mn-induced parkinsonism. However, an oxidative stress mechanism may be recognized, at least in vitro, only in the Mn-induced apoptosis.
Asunto(s)
1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/análogos & derivados , Apoptosis/fisiología , Manganeso/farmacología , Estrés Oxidativo , Células PC12/efectos de los fármacos , Células PC12/fisiología , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , Acetilcisteína/farmacología , Animales , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Ácido Ascórbico/farmacología , Supervivencia Celular/efectos de los fármacos , RatasRESUMEN
Recent findings have shown that systemic morphine increases extracellular dopamine (DA), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), ascorbic acid (AA) and uric acid concentrations in the striatum of freely moving rats. The morphine-induced increase in DA oxidative metabolism is highly correlated with that of xanthine. In the present study, we evaluated the effects of subcutaneous (s.c.) naloxone (1 mg/kg) on morphine-induced changes in DA, DOPAC, HVA, 5-hydroxyindoleacetic acid (5-HIAA), AA, uric acid and glutamate in the striatum of freely moving rats using microdialysis. Dialysates were assayed by high performance liquid chromatography with electrochemical detection or (glutamate) ultraviolet detection. Morphine (5-20 mg/kg) given s.c. increased DA, DOPAC+HVA, 5-HIAA, AA and uric acid and decreased glutamate dialysate concentrations over a 3 h period after morphine. Morphine (1 mM), given intrastriatally, did not affect all the above parameters, with the exception of an early short-lasting decrease in AA concentration. Naloxone antagonised all morphine-induced changes with the exception of AA increase and glutamate decrease in dialysate concentrations. Systemic or intrastrial (0.2-2 mM) naloxone increased AA and decreased glutamate dialysate concentrations. When given intranigrally, morphine (1 mM) increased DOPAC+HVA, AA and uric acid and decreased glutamate dialysate concentrations over a 2 h period after morphine; DA and 5-HIAA concentrations were unaffected. These results suggest that: (i) morphine increases striatal DA release and 5-hydroxytryptamine oxidative metabolism by a micro-opioid receptor-mediated mechanism mainly at extranigrostriatal sites; (ii) morphine increases DA and xanthine oxidative metabolism and affects glutamate and AA release by a micro-opioid receptor mediated mechanism acting also at nigral sites; and (iii) a micro-opioid receptor-mediated mechanism tonically controls at striatal sites extracellular AA and glutamate concentrations.
Asunto(s)
Analgésicos Opioides/farmacología , Ácido Ascórbico/metabolismo , Dopamina/metabolismo , Ácido Glutámico/metabolismo , Morfina/farmacología , Ácido Úrico/metabolismo , Ácido 3,4-Dihidroxifenilacético/metabolismo , Animales , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Ácido Homovanílico/metabolismo , Ácido Hidroxiindolacético/metabolismo , Locomoción/fisiología , Masculino , Microdiálisis , Naloxona/farmacología , Antagonistas de Narcóticos/farmacología , Ratas , Ratas Wistar , Sustancia Negra/efectos de los fármacos , Sustancia Negra/metabolismoRESUMEN
Since ascorbic acid (AA) reportedly suppresses tolerance to and dependence on morphine in humans and rodents, levels of dopamine (DA), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 3-methoxytyramine (3-MT), 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), AA, dehydroascorbic acid (DHAA), uric acid, xanthine, hypoxanthine, glutamate and gamma-aminobutyric acid (GABA) were determined by high-pressure liquid chromatography (HPLC) in the striatum and in the limbic forebrain of the rat following morphine treatment (single or repeated) and withdrawal. Single morphine administration (20 mg/kg s.c.) increased DOPAC + HVA/DA, 5-HIAA/5-HT and DHAA/AA ratios, uric acid levels, and decreased xanthine, hypoxanthine, glutamate and GABA levels in both regions. 3-MT levels were decreased in the striatum and increased in the limbic forebrain. After 7 days of morphine treatment, striatal DOPAC + HVA/DA and DHAA/AA ratios and uric acid levels were still higher and striatal and limbic xanthine levels still lower than in controls, while all other parameters were in the range of control values in both regions. Morphine treatment also increased the glutamate/GABA ratio in the striatum. In all morphine-treated rats, individual striatal DOPAC + HVA/DA and DHAA/AA ratio values were directly correlated. After a 48 h withdrawal period, both striatal AA oxidation and glutamate/GABA ratio further increased; limbic 3-MT levels further decreased, while all other parameters did not differ from control values. We conclude that: (i) tolerance to morphine-induced increase in hypoxanthine, xanthine and AA oxidation develops in the limbic forebrain faster than in the striatum; (ii) the morphine-induced increase in striatal and limbic AA oxidation may be considered a consequence of increased formation of reactive oxygen species due to increased DA, hypoxanthine and xanthine oxidative metabolism; (iii) a striatal excitotoxic imbalance characterizes the withdrawal state and may be taken into account to explain the further increase in striatal AA oxidation.
Asunto(s)
Aminas/metabolismo , Ácido Ascórbico/metabolismo , Cuerpo Estriado/efectos de los fármacos , Morfina/farmacología , Prosencéfalo/efectos de los fármacos , Ácido 3,4-Dihidroxifenilacético/metabolismo , Animales , Dopamina/metabolismo , Masculino , Ratas , Ratas Wistar , Síndrome de Abstinencia a Sustancias , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Levels of uric acid, xanthine, hypoxanthine, ascorbic acid (AA), dehydroascorbic acid (DHAA), glutathione (GSH), noradrenaline (NA), dopamine (DA), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridinium ion (MPP+) were determined in the striatum and/or in the brainstem of 3-month-old male Wistar rats, given allopurinol (500 mg/kg day by gavage) for 3 days before a single MPTP 52 mg/kg dose i.p. Allopurinol alone decreased uric acid and hypoxanthine levels in the striatum and in the brainstem; moreover, allopurinol increased AA oxidation and decreased striatal DA metabolites. Allopurinol affected neither regional MPTP and MPP+ levels nor the MPTP-induced inhibition of striatal DA oxidative metabolism. On the contrary, the MPTP-induced increase in uric acid levels and decrease in xanthine, hypoxanthine and NA levels were fully antagonised. Such findings demonstrate that the claimed MPP(+)-induced oxidative stress mediated by xanthine oxidase may be involved at least in the NA depletion; moreover, uric acid may have a physiological role as an active component of the neuronal antioxidant pool.
Asunto(s)
1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/antagonistas & inhibidores , Alopurinol/farmacología , Tronco Encefálico/metabolismo , Neostriado/metabolismo , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/metabolismo , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , 1-Metil-4-fenilpiridinio/metabolismo , Animales , Ácido Ascórbico/metabolismo , Tronco Encefálico/efectos de los fármacos , Dopamina/metabolismo , Dopamina/fisiología , Glutatión/metabolismo , Hipoxantina , Hipoxantinas/metabolismo , Masculino , Neostriado/efectos de los fármacos , Norepinefrina/fisiología , Ratas , Ratas Wistar , Ácido Úrico/metabolismo , Xantinas/metabolismoRESUMEN
Oxidative stress is thought to play a key role both in the neurotoxin MPTP- and manganese (Mn)-induced neurotoxicity and in apoptotic cell death. In the present study, we report that Mn and the MPTP analogue 1-methyl-4-(2'-ethylphenyl)-1,2,3,6-tetrahydropyridine (2'Et-MPTP), which is metabolized by MAO-A to 1-methyl-4-(2'-ethylphenyl)-pyridinium ion (at concentrations of 0.5 and 1.0 mM), induced apoptosis in PC12 cells. Apoptosis was tested by terminal deoxynucleotidyl transferase-mediated 2'-deoxy-uridine-5'-triphosphate nick end labelling (TUNEL) technique, flow cytometry and fluorescence microscopy. Both Mn and 2'Et-MPTP induced also a time-dependent decrease in cell viability, as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Only Mn-induced apoptosis and decrease in cell viability were inhibited by the antioxidant ascorbic acid. We conclude that apoptosis may be an important mechanism of cell death in MPTP- and Mn-induced parkinsonism. However, an oxidative stress mechanism may be recognized only in the Mn-induced apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Manganeso/farmacología , Animales , Ácido Ascórbico/farmacología , Supervivencia Celular/efectos de los fármacos , Citometría de Flujo , Técnicas Genéticas , Manganeso/antagonistas & inhibidores , Microscopía Fluorescente , Estrés Oxidativo/efectos de los fármacos , Células PC12 , Ratas , Uridina Trifosfato/genética , Uridina Trifosfato/metabolismoRESUMEN
In 6-month-old male Wistar rats, levels of dopamine (DA), dihydroxyphenylacetic acid (DOPAC), ascorbic acid (AA), dehydroascorbic acid (DHAA), uric acid, glutathione (GSH) and 1-methyl-4-phenylpyridinium ion (MPP+) were determined by HPLC in the crude striatal synaptosomal fraction after single injections of MPTP 35 mg/kg i.p. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced a 32.5% death rate within 15 min to 10 h. Groups of surviving rats were sacrificed 1, 3, 8 and 24 h after MPTP. MPTP significantly increased levels of DHAA and uric acid and decreased levels of DOPAC and GSH. Individual synaptosomal levels of MPP+ were correlated inversely with DOPAC (r = -0.601, P < 0.002) and GSH levels (r = -0.496, P < 0.02) and directly with levels of uric acid (r = +0.627, P < 0.001); these latter, in turn, were correlated with DHAA (r = +0.418, P < 0.05) and GSH levels (r = -0.357, P = 0.07). In conclusion, the response of the endogenous antioxidant system (increase in AA oxidation, decrease in GSH levels) correlates well with the MPTP-induced increase in uric acid levels and provides further evidence for a mechanism of MPTP neurotoxicity involving oxidative stress produced by xanthine oxidase.
Asunto(s)
1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/análogos & derivados , Ácido Ascórbico/metabolismo , Cuerpo Estriado/metabolismo , Dopaminérgicos/farmacología , Glutatión/metabolismo , Compuestos de Piridinio/metabolismo , Sinaptosomas/metabolismo , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/metabolismo , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , Ácido 3,4-Dihidroxifenilacético/metabolismo , Animales , Conducta Animal/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Cuerpo Estriado/efectos de los fármacos , Dopamina/metabolismo , Dopaminérgicos/metabolismo , Dopaminérgicos/toxicidad , Intoxicación por MPTP , Masculino , Terminaciones Nerviosas/efectos de los fármacos , Terminaciones Nerviosas/metabolismo , Oxidación-Reducción , Ratas , Ratas Wistar , Sinaptosomas/efectos de los fármacos , Ácido Úrico/metabolismoRESUMEN
Levels of uric acid, xanthine, hypoxanthine, ascorbic acid (AA), dehydroascorbic acid, glutathione (GSH), noradrenaline (NA), dopamine (DA), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and and 3-methoxytyramine were determined in the striatum and/or in the brainstem of 3-month-old male Wistar rats given manganese (MnCl2, 200 mg/kg/day for 7 days by gavage) alone or associated with allopurinol. Allopurinol alone (300 mg/kg/day for 4 days by gavage) decreased uric acid and increased xanthine levels both in the striatum and in the brainstem; moreover, allopurinol decreased the striatal DOPAC + HVA/DA ratio. Allopurinol antagonised the Mn-induced: (a) increase in the DOPAC + HVA/DA ratio; (b) increase in uric acid levels and AA oxidation; and (c) decrease in GSH and NA levels. We conclude that allopurinol may protect against Mn-induced oxidative stress by inhibiting both DA oxidative metabolism and xanthine oxidase-mediated formation of reactive oxygen species.
Asunto(s)
Alopurinol/farmacología , Tronco Encefálico/metabolismo , Cuerpo Estriado/metabolismo , Manganeso/farmacología , Estrés Oxidativo/efectos de los fármacos , Animales , Tronco Encefálico/efectos de los fármacos , Cuerpo Estriado/efectos de los fármacos , Dopamina/metabolismo , Masculino , Norepinefrina/metabolismo , Ratas , Ratas Wistar , Ácido Úrico/metabolismo , Xantina , Xantinas/metabolismoRESUMEN
Reportedly, the generation of nitric oxide (NO) may lead to iron mobilization from ferritin disrupting intracellular iron homeostasis and increasing levels of reactive oxygen species. In the present study, we evaluated the role of endogenous iron in NO-induced apoptosis in PC12 cells. Apoptosis was tested by flow cytometry, fluorescence microscopy and terminal deoxynucleotidyl transferase-mediated 2'-deoxy-uridine 5'-triphosphate nick end labeling (TUNEL) technique. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. When incubated with 0.5-0.75 mM sodium nitroprusside (SNP, a chemical NO donor), PC12 cells were shown to undergo apoptosis. In addition, SNP induced a time-dependent decrease in cell viability. Since deferoxamine (0.05-0.1 mM), a powerful iron chelator, inhibited both SNP-induced apoptosis and the decrease in cell viability, we suggest that these NO effects may be dependent upon iron mobilization within the cell.
Asunto(s)
Apoptosis/efectos de los fármacos , Deferoxamina/farmacología , Nitroprusiato/farmacología , Células PC12/efectos de los fármacos , Animales , Células PC12/patología , RatasRESUMEN
In 3- and 20-month-old male Wistar rats, levels of noradrenaline (NA), dopamine (DA), 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), ascorbic acid (AA), dehydroascorbic acid (DHAA), uric acid and glutathione (GSH) were determined by HPLC in the brainstem after subchronic oral exposure to MnCl2 200 mg/kg (3-month-old) and 30-100-200 mg/kg (20-month-old). In aged rats, manganese (Mn) significantly decreased levels of NA, DA and GSH and increased 5-HIAA/5-HT ratio values and DHAA and uric acid levels. All these parameters were scarcely affected in young rats. In aged rats, individual total Mn doses/rat were inversely correlated with individual DA levels (r = -0.405) and GSH levels (r = -0.450). In conclusion, Mn induces changes in markers of monoaminergic systems activity in the brainstem of aged rats considerably greater than in young rats. The increase in AA oxidation and decrease in GSH levels are consistent with a Mn-induced increase in formation of reactive oxygen species. The increase in uric acid levels provides evidence that one of these species might arise from the activity of xanthine-oxidase on uric acid precursors.
Asunto(s)
Envejecimiento/fisiología , Monoaminas Biogénicas/metabolismo , Tronco Encefálico/efectos de los fármacos , Cloruros/toxicidad , Intoxicación por Manganeso , Animales , Biomarcadores , Tronco Encefálico/fisiología , Cloruros/administración & dosificación , Ácido Deshidroascórbico/metabolismo , Esquema de Medicación , Glutatión/análisis , Masculino , Compuestos de Manganeso/administración & dosificación , Oxidación-Reducción , Ratas , Ratas Wistar , Ácido Úrico/metabolismoRESUMEN
Rats whose frontoparietal cortex had been bilaterally ablated were allowed 21 days for recovery and then treated with apomorphine (APO), 1 mg/kg SC or scopolamine (SCOP), 0.6 mg/kg SC. Soon after a behavioral test, dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), ascorbic acid (AA), and dehydroascorbic acid (DHAA) levels were determined by HPLC/EC in striatal synaptosomes (left side) and whole striatum (right side). SCOP behavioural effects were attenuated by cortical ablation, while those of APO were affected to a lesser extent. In the striatum of unoperated and sham-operated rats DHAA contents and DHAA/AA ratio resulted increased after drugs administration. No change in AA oxidation was observed in the striatum of ablated rats. In the synaptosomes of unoperated and sham-operated rats both drugs led to a decrease in DHAA contents and DHAA/AA ratio. In unoperated and sham-operated rats APO and SCOP caused a decrease of the DOPAC/DA ratio in the whole striatum and striatal synaptosomes. In ablated rats APO caused a decrease of DOPAC/DA ratio in the whole striatum and synaptosomes, while SCOP effects on DA turnover resulted attenuated in the whole striatum and abolished in synaptosomes. We conclude that drug-induced AA oxidation is likely to occur in the extracellular space and requires intact corticostriatal glutamatergic pathways. The latter may play an enabling role in SCOP behavioral effects.
Asunto(s)
Ácido Ascórbico/metabolismo , Conducta Animal/efectos de los fármacos , Corteza Cerebral/fisiología , Neostriado/metabolismo , Ácido 3,4-Dihidroxifenilacético/metabolismo , Animales , Apomorfina/farmacología , Ácido Deshidroascórbico/metabolismo , Dopamina/metabolismo , Ácido Glutámico/farmacología , Masculino , Actividad Motora/efectos de los fármacos , Neostriado/efectos de los fármacos , Oxidación-Reducción , Ratas , Ratas Wistar , Escopolamina/farmacología , Conducta Estereotipada/efectos de los fármacos , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismoRESUMEN
Levels of ascorbic acid (AA), dehydroascorbic acid (DHAA), glutathione (GSH), uric acid, dopamine (DA), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 3-methoxytyramine (3-MT), noradrenaline (NA), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and 1-methyl-4-phenylpyridinium ion (MPP+) were determined in the striatum, striatal synaptosomes, and/or brain stem of 3- and 6-month-old male Wistar rats given MPTP 35-52 mg/kg IP. In older rats, MPTP 35 mg/kg caused a 38% death rate within 15 min-12 h. Levels of MPTP and MPP+ in the striatum, synaptosomes, and brain stem were directly correlated with the absolute MPTP dose/rat. MPTP decreased striatal DA metabolites and NA levels in the striatum and brain stem, and increased uric acid levels in all regions in all rats. All these changes were significantly correlated with MPP+ levels. GSH levels were increased in younger rats and decreased in older rats. AA oxidation was increased mainly in older rats. We conclude that acute lethality and regional brain MPTP and MPP+ levels depend upon the absolute dose of MPTP/rat rather than the relative dose/kg. In younger rats, the neuronal antioxidant GSH system is more efficient than in older rats, in which the response to MPP(+)-induced oxidative stress also involves AA oxidation. The increase in uric acid levels provides further evidence for a mechanism of MPTP neurotoxicity involving oxidative stress mediated by xanthine oxidase.
Asunto(s)
Antioxidantes/metabolismo , Tronco Encefálico/metabolismo , Intoxicación por MPTP , Neostriado/metabolismo , Neuronas/metabolismo , Estrés Oxidativo/fisiología , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/metabolismo , 1-Metil-4-fenilpiridinio/metabolismo , 1-Metil-4-fenilpiridinio/farmacología , Animales , Ácido Ascórbico/metabolismo , Conducta Animal/efectos de los fármacos , Tronco Encefálico/citología , Tronco Encefálico/efectos de los fármacos , Dopamina/metabolismo , Glutatión/metabolismo , Masculino , Neostriado/citología , Neostriado/efectos de los fármacos , Norepinefrina/metabolismo , Estrés Oxidativo/efectos de los fármacos , Células PC12 , Ratas , Ratas Wistar , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismo , Ácido Úrico/metabolismoRESUMEN
Levels of uric acid, xanthine, hypoxanthine, ascorbic acid (AA), dehydroascorbic acid (DHAA), glutathione (GSH), noradrenaline (NA), dopamine (DA), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridinium ion (MPP+) were determined in the striatum and/or in the brain stem of 3-month-old male Wistar rats given allopurinol (300 mg/kg day by gavage) for 3 days before a single MPTP 35 mg/kg dose IP. Allopurinol alone decreased uric acid and increased xanthine levels both in the striatum and in the brain stem; moreover, allopurinol decreased striatal DOPAC + HVA/DA ratio and increased 5-HIAA/5HT ratio in the brainstem. Allopurinol affected neither regional MPTP nor MPP+ disposition. Allopurinol potentiated the MPTP-induced decrease in the DOPAC+HVA/DA ratio and increase in striatal AA oxidation; in addition, allopurinol antagonised the MPTP-induced: (i) increase in uric acid levels; (ii) decrease in NA levels in both regions, in DA levels, and in the 5-HIAA/5-HT ratio in the brain stem: (iii) increase in AA oxidation in the brain stem. In conclusion, the MPP(+)-induced oxidative stress mediated by xanthine oxidase seems to be involved in DA depletion in the brainstem and in NA depletion in both regions; moreover, striatal uric acid may have an active role in the neuronal antioxidant pool.
Asunto(s)
1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , Alopurinol/farmacología , Antimetabolitos/farmacología , Tronco Encefálico/metabolismo , Dopaminérgicos/farmacología , Neostriado/metabolismo , Estrés Oxidativo/efectos de los fármacos , Animales , Ácido Ascórbico/metabolismo , Tronco Encefálico/efectos de los fármacos , Catecolaminas/metabolismo , Hipoxantina/metabolismo , Masculino , Neostriado/efectos de los fármacos , Purinas/metabolismo , Ratas , Ratas Wistar , Xantinas/metabolismoRESUMEN
In 6-month-old male Wistar rats, levels of dopamine (DA), dihydroxyphenylacetic acid (DOPAC), ascorbic acid (AA), dehydroascorbic acid (DHAA), uric acid and glutathione (GSH) were determined by HPLC in the striatum and striatal synaptosomes after subchronic oral exposure to MnCl2 50-100-150 mg/kg. Mn significantly decreased levels of DA and GSH and increased levels of DHAA and uric acid both in the striatum and synaptosomes. In synaptosomes, individual total Mn doses/rat were directly correlated with individual DOPAC/DA ratio values (r = +0.647), uric acid (r = +0.532) and DHAA levels (r = +0.889) and inversely correlated with DA (r = -0.757) and GSH levels (r = -0.608). In turn, GSH levels were inversely correlated with uric acid (r = -0.451) and DHAA levels (r = -0.460). In conclusion, the response of striatal cellular defense mechanisms (increase in AA oxidation, decrease in GSH levels) correlated well with changes in markers of dopaminergic system activity and increase in uric acid levels. The latter provides evidence of an Mn-induced oxidative stress mediated by xanthine oxidase.