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1.
Anal Biochem ; 380(2): 323-30, 2008 Sep 15.
Article in English | MEDLINE | ID: mdl-18577368

ABSTRACT

A novel dual channel in vitro apparatus, derived from a previously described design, has been coupled with dopamine (DA) microsensors for the flow-through detection of DA secreted from PC12 cells. The device, including two independent microdialysis capillaries, was loaded with a solution containing PC12 cells while a constant phosphate-buffered saline (PBS) medium perfusion was carried out using a dual channel miniaturized peristaltic pump. One capillary was perfused with normal PBS, whereas extracellular calcium was removed from extracellular fluid of the second capillary. After a first period of stabilization and DA baseline recording, KCl (75 mM) was added to the perfusion fluid of both capillaries. In this manner, a simultaneous "treatment-control" experimental design was performed to detect K+-evoked calcium-dependent DA secretion. For this purpose, self-referencing DA microsensors were developed, and procedures for making, testing, and calibrating them are described in detail. The electronic circuitry was derived from previously published schematics and optimized for dual sensor constant potential amperometry applications. The microdialysis system was tested and validated in vitro under different experimental conditions, and DA secretion was confirmed by high-performance liquid chromatography with electrochemical detection (HPLC-EC). PC12 cell viability was quantified before and after each experiment. The proposed apparatus serves as a reliable model for studying the effects of different drugs on DA secretion through the direct comparison of extracellular DA increase in treatment-control experiments performed on the same initial PC12 cell population.


Subject(s)
Dopamine/analysis , Electrochemistry/methods , Microdialysis/methods , Animals , Calcium/pharmacology , Calibration , Cell Count , Cell Survival , Dopamine/metabolism , Electrochemistry/instrumentation , Microdialysis/instrumentation , PC12 Cells , Potassium Chloride/pharmacology , Rats , Reproducibility of Results , Secretory Rate/drug effects , Software
2.
Neuroscience ; 138(3): 869-78, 2006.
Article in English | MEDLINE | ID: mdl-16337092

ABSTRACT

Post-menopausal estrogen deficiency is recognized to play a pivotal role in the pathogenesis of a number of age-related diseases in women, such as osteoporosis, coronary heart disease and Alzheimer's disease. There are also sexual differences in the progression of diseases associated with the nigrostriatal dopaminergic system, such as Parkinson's disease, a chronic progressive degenerative disorder characterized by the selective degeneration of mesencephalic dopaminergic neurons in the substancia nigra pars compacta. The mechanism(s) responsible for dopaminergic neuron degeneration in Parkinson's disease are still unknown, but oxidative stress and neuroinflammation are believed to play a key role in nigrostriatal dopaminergic neuron demise. Estrogen neuroprotective effects have been widely reported in a number of neuronal cell systems including the nigrostriatal dopaminergic neurons, via both genomic and non-genomic effects, however, little is known on estrogen modulation of astrocyte and microglia function in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson's disease. We here highlight estrogen modulation of glial neuroinflammatory reaction in the protection of mesencephalic dopaminergic neurons and emphasize the cardinal role of glia-neuron crosstalk in directing neuroprotection vs neurodegeneration. In particular, the specific role of astroglia and its pro-/anti-inflammatory mechanisms in estrogen neuroprotection are presented. This study shows that astrocyte and microglia response to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine injury vary according to the estrogenic status with direct consequences for dopaminergic neuron survival, recovery and repair. These findings provide a new insight into the protective action of estrogen that may possibly contribute to the development of novel therapeutic treatment strategies for Parkinson's disease.


Subject(s)
Estrogens/physiology , Nerve Degeneration/physiopathology , Neuroglia/physiology , Parkinson Disease/physiopathology , 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine , Animals , Disease Models, Animal , Humans , Inflammation/physiopathology , Models, Neurological , Nerve Degeneration/prevention & control , Nervous System/physiopathology , Neurons/pathology , Neurons/physiology , Oxidative Stress
3.
Br J Pharmacol ; 134(2): 275-82, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11564645

ABSTRACT

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.


Subject(s)
Corpus Striatum/drug effects , Dopamine/metabolism , Iron/pharmacology , Molsidomine/analogs & derivatives , Molsidomine/pharmacology , Nitric Oxide Donors/pharmacology , Nitric Oxide/physiology , 3,4-Dihydroxyphenylacetic Acid/metabolism , Acetylcysteine/pharmacology , Animals , Ascorbic Acid/metabolism , Corpus Striatum/metabolism , Deferoxamine/pharmacology , Dialysis Solutions/chemistry , Dose-Response Relationship, Drug , Free Radical Scavengers/pharmacology , Homovanillic Acid/metabolism , Male , Metalloporphyrins/pharmacology , Movement , Rats , Rats, Wistar , Uric Acid/metabolism , Uric Acid/pharmacology
4.
Brain Res Mol Brain Res ; 92(1-2): 138-48, 2001 Aug 15.
Article in English | MEDLINE | ID: mdl-11483250

ABSTRACT

The effects of long-term exposure to, and subsequent withdrawal of, diazepam or imidazenil (full and partial agonists of the benzodiazepine receptor, respectively) on the abundance of GABA(A) receptor subunit mRNAs and peptides were investigated in rat cerebellar granule cells in culture. Exposure of cells to 10 microM diazepam for 5 days significantly reduced the amounts of alpha(1) and gamma(2) subunit mRNAs, and had no effect on the amount of alpha(4) mRNA. These effects were accompanied by a decrease in the levels of alpha(1) and gamma(2) protein and by a reduction in the efficacy of diazepam with regard to potentiation of GABA-evoked Cl- current. Similar long-term treatment with 10 microM imidazenil significantly reduced the abundance of only the gamma(2)S subunit mRNA and had no effect on GABA(A) receptor function. Withdrawal of diazepam or imidazenil induced a marked increase in the amount of alpha(4) mRNA; withdrawal of imidazenil also reduced the amounts of alpha(1) and gamma(2) mRNAs. In addition, withdrawal of diazepam or imidazenil was associated with a reduced ability of diazepam to potentiate GABA action. These data give new insights into the different molecular events related to GABA(A) receptor gene expression and function produced by chronic treatment and withdrawal of benzodiazepines with full or partial agonist properties.


Subject(s)
Anti-Anxiety Agents/pharmacology , Benzodiazepines/pharmacology , Diazepam/pharmacology , GABA Agonists/pharmacology , Imidazoles/pharmacology , Nerve Tissue Proteins/biosynthesis , Receptors, GABA-A/biosynthesis , Up-Regulation/drug effects , Animals , Anti-Anxiety Agents/administration & dosage , Benzodiazepines/administration & dosage , Cell Membrane/drug effects , Cells, Cultured , Cerebellum/cytology , Cerebellum/drug effects , Cerebellum/metabolism , Chloride Channels/drug effects , Chloride Channels/metabolism , Chlorides/metabolism , Diazepam/administration & dosage , Drug Tolerance/genetics , Drug Tolerance/physiology , Female , Flumazenil/administration & dosage , Flumazenil/pharmacology , GABA Agonists/administration & dosage , GABA Antagonists/pharmacology , GABA-A Receptor Agonists , GABA-A Receptor Antagonists , Imidazoles/administration & dosage , Ion Transport/drug effects , Membrane Potentials/drug effects , Microinjections , Nerve Tissue Proteins/agonists , Nerve Tissue Proteins/genetics , Neurons/drug effects , Neurons/metabolism , Oocytes , Protein Subunits , RNA, Messenger/biosynthesis , Rats , Receptors, GABA-A/genetics , Substance Withdrawal Syndrome/genetics , Substance Withdrawal Syndrome/metabolism , Substance-Related Disorders/genetics , Substance-Related Disorders/metabolism , Xenopus laevis
5.
Br J Pharmacol ; 132(4): 941-9, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11181436

ABSTRACT

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.


Subject(s)
Ascorbic Acid/physiology , Corpus Striatum/drug effects , Dopamine/metabolism , Nitric Oxide Donors/pharmacology , Oxidative Stress , Penicillamine/pharmacology , Acetylcysteine/pharmacology , Animals , Corpus Striatum/metabolism , Iron/metabolism , Male , Microdialysis , Penicillamine/analogs & derivatives , Rats , Rats, Wistar , S-Nitroso-N-Acetylpenicillamine
6.
Aging (Milano) ; 12(5): 393-7, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11126527

ABSTRACT

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 glutamate 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.


Subject(s)
Aging/metabolism , Brain Stem/metabolism , Corpus Striatum/metabolism , Energy Metabolism/drug effects , Glutamic Acid/metabolism , Manganese/pharmacology , Phosphates/metabolism , Animals , Brain Stem/drug effects , Corpus Striatum/drug effects , Hypoxanthine/metabolism , Male , Osmolar Concentration , Rats , Rats, Wistar , Time Factors , Uric Acid/metabolism , Xanthine/metabolism
7.
Br J Pharmacol ; 131(4): 836-42, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11030735

ABSTRACT

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.


Subject(s)
Ascorbic Acid/pharmacology , Corpus Striatum/metabolism , Dopamine/metabolism , Iron/physiology , Molsidomine/analogs & derivatives , Nitric Oxide Donors/pharmacology , Nitric Oxide/physiology , Nitroprusside/pharmacology , Animals , Deferoxamine/pharmacology , Male , Microdialysis , Molsidomine/pharmacology , Rats , Rats, Wistar
8.
Aging (Milano) ; 12(2): 77-84, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10902049

ABSTRACT

Gender accounts for important differences in the incidence and prevalence of a variety of age-related diseases. Considering people of far advanced age, demographic data document a clear-cut prevalence of females compared to males, suggesting that sex-specific mortality rates follow different trajectories during aging. In the present investigation, we report data from a nationwide study on Italian centenarians (a total of 1162 subjects), and from two studies on centenarians living in two distinct zones of Italy, i.e., the island of Sardinia (a total of 222 subjects) and the Mantova province (Northern Italy) (a total of 43 subjects). The female/male ratio was about 2:1 in Sardinia, 4:1 in the whole of Italy, and about 7:1 in the Mantova province. Thus, a complex interaction of environmental, historical and genetic factors, differently characterizing the various parts of Italy, likely plays an important role in determining the gender-specific probability of achieving longevity. Gender differences in the health status of centenarians are also reported, and an innovative score method to classify long-lived people in different health categories, according to clinical and functional parameters, is proposed. Our data indicate that not only is this selected group of people, as a whole, highly heterogeneous, but also that a marked gender difference exists, since male centenarians are less heterogeneous and more healthy than female centenarians. Immunological factors regarding the age-related increase in pro-inflammatory status, and the frequency of HLA ancestral haplotypes also show gender differences that likely contribute to the different strategies that men and women seem to follow to achieve longevity. Concerning the different impact of genetic factors on the probability of reaching the extreme limits of the human life-span, emerging evidence (regarding mtDNA haplogroups, Thyrosine Hydroxilase, and IL-6 genes) suggests that female longevity is less dependent on genetics than male longevity, and that female centenarians likely exploited a healthier life-style and more favorable environmental conditions, owing to gender-specific cultural and anthropological characteristics of the Italian society in the last 100 years.


Subject(s)
Longevity , Sex Characteristics , Aged , Aged, 80 and over , Female , Health Status , Humans , Immune System/physiology , Longevity/genetics , Male , Stress, Physiological/physiopathology
9.
Br J Pharmacol ; 130(4): 937-45, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10864903

ABSTRACT

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.


Subject(s)
Corpus Striatum/drug effects , Levodopa/metabolism , Manganese/pharmacology , 3,4-Dihydroxyphenylacetic Acid/metabolism , Acetylcysteine/pharmacology , Animals , Ascorbic Acid/metabolism , Chlorides/pharmacology , Chromatography, High Pressure Liquid , Corpus Striatum/metabolism , Dialysis Solutions/chemistry , Dopamine/metabolism , Homovanillic Acid/metabolism , Infusion Pumps , Levodopa/pharmacology , Levodopa/therapeutic use , Male , Manganese Compounds/pharmacology , Microdialysis , Movement , Oxidation-Reduction/drug effects , Parkinson Disease/drug therapy , Rats , Rats, Wistar , Time Factors , Uric Acid/metabolism
10.
Br J Pharmacol ; 129(3): 582-8, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10711358

ABSTRACT

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).


Subject(s)
Ascorbic Acid/metabolism , Dextroamphetamine/pharmacology , Dopamine Uptake Inhibitors/pharmacology , Glutamic Acid/metabolism , Neostriatum/metabolism , Uric Acid/metabolism , 3,4-Dihydroxyphenylacetic Acid/metabolism , Animals , Dextroamphetamine/administration & dosage , Dopamine/metabolism , Dopamine Antagonists/pharmacology , Dopamine Uptake Inhibitors/administration & dosage , Haloperidol/pharmacology , In Vitro Techniques , Injections , Male , Microdialysis , Motor Activity/drug effects , Neostriatum/drug effects , Oxidation-Reduction , Rats , Rats, Wistar , Stereotaxic Techniques , Stereotyped Behavior/drug effects
11.
Aging (Milano) ; 12(6): 470-7, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11211958

ABSTRACT

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.


Subject(s)
Aging/metabolism , Brain/metabolism , Energy Metabolism/drug effects , Glutathione/deficiency , Manganese/pharmacology , Phosphates/metabolism , Animals , Antimetabolites/pharmacology , Ascorbic Acid/metabolism , Buthionine Sulfoximine/pharmacology , Dehydroascorbic Acid/metabolism , Drug Synergism , Glutathione/antagonists & inhibitors , Glutathione/metabolism , Hypoxanthine/metabolism , Inosine/metabolism , Male , Rats , Rats, Wistar , Tissue Distribution , Uric Acid/metabolism , Xanthine/metabolism
12.
J Neurochem ; 73(3): 1155-63, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10461907

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 microM 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 microM) induced decreases in both cell viability and apoptosis. When 0.2 mM MnCl2 was associated with 10, 20, or 50 microM 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.


Subject(s)
Apoptosis/drug effects , Dopamine Agents/toxicity , Levodopa/toxicity , Manganese Poisoning , Oxidative Stress/physiology , Acetylcysteine/pharmacology , Animals , Antioxidants/pharmacology , Ascorbic Acid/pharmacology , Cell Survival/drug effects , Chromatography, High Pressure Liquid , Dopamine/biosynthesis , Drug Synergism , Flow Cytometry , Oxidative Stress/drug effects , PC12 Cells , Rats
13.
Differentiation ; 64(2): 91-101, 1999 Jan.
Article in English | MEDLINE | ID: mdl-10234806

ABSTRACT

It has previously been shown that mammary stromal cells possess the ability to maintain a fibroblast-like phenotype or differentiate in vitro into mature adipocytes in a hormone-dependent manner. This paper reports that rat mammary stromal cells can also differentiate into capillary-like structures in vitro when cultured on a reconstituted basement membrane (RBM). The differentiation potential of mammary stromal cells was compared with that of human umbilical vein endothelial cells (HUVEC) and 3T3-L1 preadipocytes. When cultured on plastic, mammary stromal cells, 3T3-L1 and HUVEC maintained a fibroblast-like phenotype. Mammary stromal cells and 3T3-L1, but not HUVEC, differentiated into mature adipocytes when cultured in adipogenic medium. When plated on reconstituted basement membrane, all three cell types began to migrate and organize themselves into an interconnected capillary network. By 18-20 h, mammary stromal cells organized into complex, highly branched capillary-like tubules whereas 3T3-L1 cells and HUVEC formed more simple structures. Cross-sectional analysis demonstrated the presence of an internal lumen. Mammary stromal cells were unique in their ability to progressively develop into a three-dimensional, highly branched network invading the RBM surface. The network formation was enhanced by the presence of vascular endothelial growth factor (VEGF) and was inhibited by the anti-angiogenic drug suramin. Western blotting analysis demonstrated the presence of the endothelial-specific marker flk-1, as well as the presence of the tight-junction-associated protein ZO-1. Mammary stromal cell differentiation into capillary structures was not a terminal state, since these cells were still able to differentiate into adipocytes when exposed to adipogenic medium. These findings suggest that mammary stromal cells differentiate into fibroblasts, adipocytes or vascular structures in a hormone- and substatum-dependent manner, and may explain the dramatic changes in stromal composition during both normal mammary gland development and tumorigenesis.


Subject(s)
Adipocytes/cytology , Endothelium, Vascular/cytology , Extracellular Matrix/metabolism , Mammary Glands, Animal/cytology , Mammary Glands, Animal/metabolism , 3T3 Cells/metabolism , Animals , Cell Culture Techniques/methods , Cell Differentiation/physiology , Cell Membrane , Cells, Cultured , Culture Media , Endothelial Growth Factors/pharmacology , Female , Fibroblasts/cytology , Hormones/metabolism , Humans , Lipid Metabolism , Lymphokines/pharmacology , Mammary Glands, Animal/drug effects , Membrane Proteins/metabolism , Mice , Phenotype , Phosphoproteins/metabolism , Rats , Rats, Sprague-Dawley , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Growth Factor/metabolism , Receptors, Vascular Endothelial Growth Factor , Stromal Cells/cytology , Stromal Cells/drug effects , Stromal Cells/metabolism , Suramin/pharmacology , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors , Zonula Occludens-1 Protein
14.
Neurosci Lett ; 247(1): 1-4, 1998 May 08.
Article in English | MEDLINE | ID: mdl-9637395

ABSTRACT

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.


Subject(s)
Apoptosis/drug effects , Deferoxamine/pharmacology , Nitroprusside/pharmacology , PC12 Cells/drug effects , Animals , PC12 Cells/pathology , Rats
15.
Brain Res ; 797(1): 94-102, 1998 Jun 22.
Article in English | MEDLINE | ID: mdl-9630540

ABSTRACT

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.


Subject(s)
Analgesics, Opioid/pharmacology , Ascorbic Acid/metabolism , Dopamine/metabolism , Glutamic Acid/metabolism , Morphine/pharmacology , Uric Acid/metabolism , 3,4-Dihydroxyphenylacetic Acid/metabolism , Animals , Corpus Striatum/drug effects , Corpus Striatum/metabolism , Homovanillic Acid/metabolism , Hydroxyindoleacetic Acid/metabolism , Locomotion/physiology , Male , Microdialysis , Naloxone/pharmacology , Narcotic Antagonists/pharmacology , Rats , Rats, Wistar , Substantia Nigra/drug effects , Substantia Nigra/metabolism
16.
Neurochem Int ; 31(2): 169-76, 1997 Aug.
Article in English | MEDLINE | ID: mdl-9220449

ABSTRACT

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.


Subject(s)
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine/analogs & derivatives , Apoptosis/physiology , Manganese/pharmacology , Oxidative Stress , PC12 Cells/drug effects , PC12 Cells/physiology , 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine/pharmacology , Acetylcysteine/pharmacology , Animals , Antioxidants/pharmacology , Apoptosis/drug effects , Ascorbic Acid/pharmacology , Cell Survival/drug effects , Rats
17.
Pharmacol Res ; 35(6): 577-85, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9356212

ABSTRACT

In the present study in vivo and ex vivo experiments were combined to evaluate the effects of allopurinol on the neurochemical changes induced by an acute morphine challenge (2 mg kg-1, s.c.). In samples from rat striatum, levels of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 3-methoxytyramine (3-MT), ascorbate (AA), dehydroascorbate (DHAA), hypoxanthine, xanthine and uric acid (UA) were measured. Brain microdialysis experiments were carried out in freely moving rats. Striatal dialysate levels were assayed for DA, DOPAC + HVA, AA and UA using liquid chromatography followed by electrochemical detection. Morphine administration increased the striatal levels of DA metabolites, UA and DHAA and the extracellular concentrations of DA, DOPAC + HVA, UA and AA. Allopurinol (50 mg kg-1 by gavage), an inhibitor of xanthine oxidase which catalyses oxidation of xanthine to UA, decreased basal UA and AA concentrations and the morphine-induced increase in DA metabolites and AA oxidation. Since oxidation of DA and xanthines generates reactive oxygen species (ROS) and AA and UA are the main cellular antioxidants, these findings suggest that: (a) single morphine administration increases DA and xanthine oxidative metabolism with a consequent increase in ROS production, which may account for changes in concentrations of extracellular AA and tissue DHAA; (b) allopurinol decreases morphine-induced DA and xanthine oxidation; (c) UA and AA may act in concert to regulate levels of ROS in the brain.


Subject(s)
Allopurinol/pharmacology , Ascorbic Acid/metabolism , Corpus Striatum/drug effects , Dopamine/metabolism , Enzyme Inhibitors/pharmacology , Morphine/pharmacology , Uric Acid/metabolism , Xanthine Oxidase/antagonists & inhibitors , Animals , Corpus Striatum/metabolism , Male , Microdialysis , Rats , Rats, Wistar , Reactive Oxygen Species
18.
Brain Res ; 745(1-2): 173-82, 1997 Jan 16.
Article in English | MEDLINE | ID: mdl-9037407

ABSTRACT

Recent ex vivo findings have shown that morphine increases dopamine (DA) and xanthine oxidative metabolism and ascorbic acid (AA) oxidation in the rat striatum. In the present study, we evaluated the effects of subcutaneous daily morphine (20 mg/kg) administration on DA, dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), AA and uric acid in the striatum of freely moving rats using microdialysis. Dialysates were assayed by high performance liquid chromatography with electrochemical detection. On the first day, morphine administration caused a significant increase in extracellular DA, DOPAC, HVA, AA and uric acid concentrations over a 3 h period after morphine. In all treated rats (n = 7), individual concentrations of DOPAC + HVA were directly correlated with individual AA and uric acid concentrations. Last morphine administration on the 4th day increased DOPAC, HVA, AA and uric acid concentrations but failed to increase those of DA. Individual DOPAC + HVA concentrations were still directly correlated with individual AA and uric acid concentrations. These results suggest that systemic morphine increases both striatal DA release and DA and xanthine oxidative metabolism. Only the former effect undergoes tolerance. The increase in DA oxidative metabolism is highly correlated with that of xanthine. The subsequent enhancement in reactive oxygen species production may account for the increase in extracellular AA.


Subject(s)
Ascorbic Acid/metabolism , Dopamine/metabolism , Morphine/pharmacology , Narcotics/pharmacology , Neostriatum/metabolism , Uric Acid/metabolism , 3,4-Dihydroxyphenylacetic Acid/metabolism , Animals , Homovanillic Acid/metabolism , Male , Microdialysis , Neostriatum/drug effects , Rats , Rats, Wistar
19.
Pharmacol Res ; 36(4): 285-92, 1997 Oct.
Article in English | MEDLINE | ID: mdl-9425617

ABSTRACT

Levels of dopamine (DA), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), noradrenaline (NA), glutathione (GSH), ascorbic acid (AA), dehydroascorbic acid (DHAA) and uric acid (UA) were determined in the striatum and/or in the brainstem of 3-month-old male Wistar rats after subchronic oral exposure to MnCl2 (20 mg kg-1 daily) alone or associated to buthionine (S,R)sulphoximine-ethyl ester (BSO-E), an inhibitor of GSH synthesis. The NA, DA, DOPAC, GSH and glutathione disulphide (GSSG) concentrations were also determined in PC12 cells incubated with Mn alone or associated with either BSO-E or AA. When PC12 cells were incubated with AA, cellular AA and DHAA concentrations were also determined. It was found that BSO-E: (a) decreased GSH levels in the striatum and in the brainstem; (b) potentiated the Mn-induced increase in AA oxidation and uric acid formation in both brain regions; and (c) potentiated the Mn-induced DA and NA depletion in the brainstem. Moreover, the changes in striatal DA metabolism induced by the BSO-E association with Mn (decrease in DA, DOPAC and HVA levels and in the DOPAC + HVA/DA ratio) are consistent with the hypothesis of a loss of dopaminergic neurons. In PC12 cells, BSO-E decreased GSH and GSSG levels and potentiated the Mn-induced decrease-in DA and NA concentrations. On the contrary, AA antagonised the Mn-induced DA and NA depletion. AA antagonised also the Mn- and MN+ BSO-induced decrease in PC12 cells viability. In conclusion, the impairment of neuronal antioxidant system activity plays a permissive role in the oxidative stress-mediated Mn neurotoxicity.


Subject(s)
Brain Stem/drug effects , Chlorides/toxicity , Dopamine/metabolism , Glutathione/deficiency , Manganese Compounds , Manganese Poisoning , Neostriatum/drug effects , Animals , Ascorbic Acid/pharmacology , Brain Stem/metabolism , Male , Methionine Sulfoximine/analogs & derivatives , Methionine Sulfoximine/pharmacology , Neostriatum/metabolism , PC12 Cells/drug effects , Rats , Rats, Wistar
20.
Brain Res ; 723(1-2): 154-61, 1996 Jun 03.
Article in English | MEDLINE | ID: mdl-8813393

ABSTRACT

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.


Subject(s)
Amines/metabolism , Ascorbic Acid/metabolism , Corpus Striatum/drug effects , Morphine/pharmacology , Prosencephalon/drug effects , 3,4-Dihydroxyphenylacetic Acid/metabolism , Animals , Dopamine/metabolism , Male , Rats , Rats, Wistar , Substance Withdrawal Syndrome , gamma-Aminobutyric Acid/metabolism
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