Increased AP-1 DNA-binding activity and nuclear REF-1 accumulation in lead-exposed primary cultures of astrocytes.

Pb was shown to perturb neuronal and glial function either directly by interacting with protein thiol groups or indirectly by mimicking Ca(2+) and increasing oxidative stress. In view of the potential action of Pb on cellular redox homeostasis we studied the regulation of activator protein-1 (AP-1) DNA binding. A 1h incubation of astrocyte primary cultures with 10 microM Pb caused a 2.5 fold increase in AP-1 DNA binding. An assessment of how Pb elicited this increase revealed the involvement of 1. transcriptional and 2. posttranslational processes. The first one was documented by an increase of c-jun mRNA content after 15 to 30 min of 10 microM Pb exposure. The second one was suggested by an enhanced nuclear accumulation of redox factor-1 after 30 to 60 min of 10 microM Pb exposure. The Pb-elicited increase of the reduction/oxidation-sensitive AP-1 signal transduction may regulate target genes operative in cell survival or cell death.

Molecular markers of oxidative stress vulnerability.

In astrocyte primary cultures of trisomy 16 mice, an animal model for Down's syndrome, protein oxidation was 50% higher than in diploid littermates. Exposure to 10 microM H2O2 or 50 microM kainic acid incremented protein oxidation in trisomic but not in diploid cultures. Studies on stress response genes showed that metallothionein (MT) level was 2-3 times higher in trisomy 16 than in diploid cultures. Kainic acid or H2O2 exposure increased the MT protein level in diploid cultures but failed to increase it in trisomy 16 mouse beyond its elevated basal level. The reduced responsiveness of MT to simulated oxidative stress may result in insufficient removal of ROS, which could partially explain the further increase of protein oxidation in trisomy 16 cultures. In contrast, Pb exposure increased MT in trisomy 16 and diploid primary cultures to a similar extent. The similar metal responsiveness of MT in both phenotypes indicated that MT in trisomic glial cultures was not yet maximally stimulated. The flawed redox sensitivity in trisomy 16 mouse suggests possible alterations in the binding activity of ROS-sensitive transcription factors on the MT promoter.

Activator protein-1 DNA binding activation by hydrogen peroxide in neuronal and astrocytic primary cultures of trisomy-16 and diploid mice.

The effect of H(2)O(2) on DNA binding activity of activator protein-1 (AP-1) was studied by electrophoretic mobility shift assay (EMSA) in cortical primary cultures of trisomy-16 mice and their diploid littermates. Exposure to 10 microM H(2)O(2) for 15 min elicited a greater and earlier occurring increase of AP-1 DNA binding in neuronal primary cultures of trisomy-16 mice than of diploid mice. When astrocyte-rich primary cultures were exposed to 10 microM H(2)O(2) a two-fold increase of AP-1 DNA binding activity was found in trisomy-16 and diploid mice. Supershift EMSA analysis revealed that c-jun was a component of AP-1 in neuronal and glial cultures of diploid and trisomic mice. A 15-min exposure to 10 microM H(2)O(2) increased c-jun mRNA in cortical neuronal cultures by six-fold, compared with a two-fold increase in cultured astrocytes. The results documented that H(2)O(2)-elicited activation of AP-1 DNA binding in trisomy-16 primary cultures is transcriptionally regulated. Since oxidative stress also activates various stress-inducible protein kinases that may phosphorylate AP-1 dimers, the increase of AP-1 DNA binding may, in part, be triggered by phosphorylation.

Neuroprotection by nitric oxide against hydroxyl radical-induced nigral neurotoxicity.

We investigated the effects of nitric oxide on an in vitro and in vivo generation of hydroxyl radicals, and in vivo neurotoxicity caused by intranigral infusion of ferrous citrate in rats. The formation of hydroxyl radicals in vitro, without exogenous hydrogen peroxide, was dose-dependent. Some nitric oxide donors (e.g. sodium nitroprusside) stimulated, while others (nitroglycerin, diethylamine/nitric oxide, nitric oxide in Ringer's solution) suppressed hydroxyl radical generation in vitro. A significant increase in extra-cellular hydroxyl radicals was detected in a brain microdialysis study. Intranigral infusion of ferrous citrate caused long-lasting lipid peroxidation and dopamine depletion in the ipsilateral nigral region and striatum, respectively. Sub-acute dopamine depletion in the striatum was positively correlated with acute lipid peroxidation in substantia nigra. Intranigral administration of nitric oxide did not affect striatal dopamine. Interestingly, nitric oxide in Ringer's protected nigral neurones against the oxidative injury. The results demonstrate that a regional increase in the levels of iron can result in hydroxyl radical generation and lipid peroxidation leading to neurotoxicity. It also demonstrates that exogenous nitric oxide can act as hydroxyl radical scavenger and protect neurones from oxidative injury.

Effect of lead on cytoskeletal proteins expressed in E14 mesencephalic primary cultures.

Several lines of evidence indicated that Pb exposure in vivo and in vitro altered neurite morphology in central and peripheral neurons. The present report shows that neurite length in mesencephalic primary cultures, consisting of neurons and glia, was decreased by Pb exposure when serum factors, presumably essential for glial functions, were absent in the culture medium. We studied whether a serum factor might control the mechanisms involved in the uptake and accumulation of Pb and its effect on cytoskeleton proteins. The total amount of Pb taken up in cell cultures was measured by atomic absorption spectroscopy and appeared to be down-regulated by a non-albumin-like serum component. In presence of serum, Pb exposure failed to alter cytoskeletal proteins. Instead, in serum-free neurobasal medium, Pb uptake failed to reach saturation within 6 h. Western blot analysis showed that the tau, 280 kDa MAP-2b, 70 kDa MAP-2c and GAP-43 protein bands were decreased 24 h after a 3 h exposure to 3 or 6 microM Pb in absence of serum. However, if cultures were maintained in serum-containing media after a 3 h Pb exposure without serum, the immunoblots did not differ from those of controls. It can be inferred that a serum factor prevents cytoskeletal protein alterations by Pb. In serum free medium, Pb that is primarily scavenged by the metallothionein I/II isoforms present in glial cells, may bind to thiol residues of proteins involved in either oxidative stress response or transcriptional regulation of cytoskeletal proteins.

In cortical cultures of trisomy 16 mouse brain the upregulated metallothionein-I/II fails to respond to H2O2 exposure or glutamate receptor stimulation.

To assess whether a defective oxidative defense may contribute to Down's syndrome, we studied the regulation of the metallothionein(MT)-I/II isoforms in primary cultures of cerebral cortex from fetal trisomy 16 mice and their euploid littermates. Western blot analysis showed that MT-I/II was upregulated and the protein carbonyl content was higher in trisomy 16 compared with euploid cultures. Addition of N-acetyl-l-cysteine to the culture medium reduced the increment of MT-I/II in trisomy 16 cortical cells. In euploid, but not trisomic cortical cultures, kainic acid, trans-(+/-)-ACPD, or H2O2 exposure elicited a dose-dependent increase of the MT-I/II immunoblots. In trisomic cells, the MT-I/II immunoblot densities were not increased beyond their elevated basal levels. In contrast, 25 microM Pb induced MT-I/II, to a similar extent, in cortical cultures from euploid and trisomy 16 mice. This suggests that the antioxidant-but not the metal-response element of the MT-I/II promoter was altered by increased oxidative stress. Our data suggest that, in the trisomy 16 mouse, the effects of increased production of reactive oxygen species, due to the increased SOD-1, GluR5, or amyloid precursor protein gene dosage, is exacerbated by an insufficient or missing antioxidant response.

A method for the rapid analysis of neuronal proportions and neurite morphology in primary cultures.

This article provides basic guidelines for a rapid analysis of subpopulation proportions and neurite morphology in primary cultures. We describe, in E14 mesencephalic primary cultures, an immunohistochemical method for the simultaneous identification of multiple neuronal phenotypes and an estimation of the ratio of subpopulations. In addition, we describe the use of the Renaissance TSA-Direct kit (NEN, DuPont) to enhance the visualization of neurites when the antigen is in low abundance. Finally, a modified sholl analysis is used to rapidly and reliably estimate neurite number and length.

Evidence of increased oxidative stress in hippocampal primary cultures of trisomy 16 mouse. Studies on metallothionein-I/II.

In the trisomy 16 mouse the increased gene dosage of SOD-1 increases H2O2 production that results in increased oxidative stress. We report here that in hippocampal primary cultures, metallothionein (MT)-I/II immunoreactivity was present mainly in glial fibrillary acidic protein-immunolabeled cells. Western blot analysis showed a two-fold higher level of MT-I/II in trisomy 16 mice then in euploid littermates. In contrast, the immunoreactivity of glutamine synthetase, another glia-expressed protein, was similar in hippocampal cultures of trisomy 16 mouse and euploid littermates. Oxyblot analysis of hippocampal cultures showed that the carbonyl content in several protein bands was higher in trisomy 16 mice than in euploid littermates giving evidence for increased oxidative stress in trisomy 16 mouse cultures. To evaluate the responsiveness of MT-I/II to agents that increase the level of reactive oxygen species in cells we measured the effect of H2O2, kainic acid, (+/-) ACPD, and beta-amyloid peptide 1-42. Western blot analysis documented that in hippocampal cultures of euploid littermates MT-I/II was maximally increased by 50 micro M H2O2, 100 micro M kainic acid, 10 micro M (+/-)ACPD, or 1.0 mM beta-amyloid peptide 1-42, whereas in those of trisomy 16 mice no further increase above the elevated level was observed. Our data suggest that in the trisomy 16 mouse the production of reactive oxygen species may have shifted the intracellular redox environment that could have alerted the susceptibility of MT-I/II transcription. The possibility that transcription factors whose activation may be essential to initiate MT-I/II transcription get oxidized has yet to be examined.

Lead exposures increases oxidative stress in serum deprived E14 mesencephalic cultures. Role of metallothionein and glutathione.

E14 mesencephalic cultures grown 6 days in Neurobasal Medium containing 10% horse serum consist of differentiated neurons and astroglia. In these cultures, glutathione and metallothionein-I/II are enriched in astrocytes and play an important role in heavy metal scavenging and oxidative stress response. A 24 h exposure to 25 micro M Pb, in serum-containing medium, elevated the glutathione content by more than twofold and increased the metallothionein I/II-immunolabeled protein band. In contrast, exposure to 3 to 25 micro M Pb is serum-free medium increased Pb uptake by cells 2 to 4-times above the levels found in 10% serum-containing medium, reduced the glutathione level and obliterated the metallothionein-I/II protein band. The rapid decrease of metallothionein-I/II and glutathione levels in serum-free medium implies that their regulation may depend on a serum factor operative in inducing immediate early genes. Exposure to 6 micro M Pb in serum-free or in B27-supplemented medium increased the carbonyl content of several protein bands above control levels indicating that under conditions that curtail metallothionein induction Pb exposure causes increased oxidative stress.

The effect of lead exposure and serum deprivation on mesencephalic primary cultures.

The effect of Pb2+ was studied in embryonic mesencephalic primary cultures that contain neurons and glia. Pb2+ exposure in absence of serum, damaged more efficaciously the cultured cells than Pb2+ exposure in presence of serum. In serum-free medium, Pb2+ elicited mainly necrosis and apoptosis in maximally 13% of the cells in culture. The glial fibrillary acidic protein (GFAP) content was decreased by Pb2+ exposure in serum-containing medium. The abundance of GFAP was also decreased by serum deprivation that was augmented by the addition of 12.5 microM Pb2+ in serum-free medium. A 6h exposure to 6 microM Pb2+ in serum-free medium also lowered the low affinity 3H-D-aspartate uptake. A 6h exposure of mesencephalic cells to 3-25 microM Pb2+ in serum-free medium failed to alter the number of tyrosine hydroxylase- and calretinin-immunoreactive cells, whereas, 50 microM Pb2+ obliterated both cell types. A 6h exposure of cells to 3 microM Pb2+ in serum-free medium decreased 3H-dopamine uptake by 50 % and 12.5 microM Pb2+ obliterated it. Addition of albumin to serum-free medium failed to prevent the Pb2+ -elicited inhibition of [3H]-dopamine uptake suggesting that the serum-afforded delay of cell death may not be due to a removal of reactive Pb2+ by protein/chelate formation but rather to the Pb2+ -scavenging function of glial cells. Serum deprivation may exacerbate the Pb2+ -induced neurotoxicity presumably by impairing the metal scavenging function of astrocytes.

Role of antioxidants in the nitric oxide-elicited inhibition of dopamine uptake in cultured mesencephalic neurons. Insights into potential mechanisms of nitric oxide-mediated neurotoxicity.

Under aerobic conditions the addition of (C2N5)2N(N[O]NO)-.Na+(DEA/NO), S-nitroso-N-acetyl penicillamine and nitric oxide (NO)-saturated buffer, but not S-nitroso-L-glutathione, to dopamine solutions resulted in dopamine o-semiquinone formation that was dependent on the formation of a NO/oxygen intermediate. High pressure liquid chromatography (HPLC) electrochemical analysis of dopamine demonstrated that the DEA/NO-induced oxidation of dopamine was abrogated in the presence of the antioxidants, ascorbate and glutathione. NO spontaneously released from DEA/NO decreased [3H]dopamine accumulation in primary cultures of mesencephalic neurons in a dose-dependent fashion. In contrast, [3H] gamma-aminobutyric acid uptake by mesencephalic neurons tested under the same conditions was unchanged. When DEA/NO was added to incubation buffer that contained [3H]dopamine and the antioxidant, ascorbate or glutathione, [3H]dopamine uptake was also inhibited. These data excluded that oxidation of extracellular [3H]dopamine by the intermediates of the NO/O2 reaction could have caused this decrease. Instead, NO may have acted directly on a not yet identified target operative in the regulation of dopamine storage and release. Analysis of the rate constants for the NO reaction with ascorbate, glutathione and dopamine revealed that dopamine quinone formation was delayed by the presence of antioxidants. Since the formation of NO as well as neurotransmitter release are activated during ischemia reperfusion injury, it is possible that prolonged NO exposure could deplete antioxidants and facilitate the oxidation of dopamine and thereby cause neurotoxicity.

Differential effects of excitatory amino acids on mesencephalic neurons expressing either calretinin or tyrosine hydroxylase in primary cultures.

In mesencephalic primary cultures derived from E14 rat embryos, calretinin- and tyrosine hydroxylase-immunoreactive neurons comprised 2% and 5% of the total cell population, respectively, at 6-7 days in vitro. The number of calretinin-immunoreactive neurons was unchanged after a 12- or 24-h exposure to 500 microM kainic acid (KA), but a 50% cell loss was detected after a 48-h exposure to KA. Tyrosine hydroxylase-immunoreactive neurons demonstrated a 50% and 67% cell loss at 24- and 48-h exposures to 500 microM KA. A 500 microM N-methyl-D-aspartic acid (NMDA) incubation for 24 h had no effect on calretinin-immunoreactive cell number, but did significantly reduce tyrosine hydroxylase-immunoreactive cell numbers by 26%. In tyrosine hydroxylase-immunoreactive cells, exposure to KA appeared to stimulate the retraction of the neuritic tree and to cause somatic swelling. In contrast, calretinin-immunoreactive neurons developed larger and more complex neuritic trees after a 24-h exposure to 500 microM KA but not NMDA. Immunohistochemical colocalization studies revealed that all tyrosine hydroxylase-immunoreactive and the majority of calretinin-immunoreactive neurons expressed the glutamate receptor subunits GluR2-R3. Very low levels of NMDAR1 receptor subunits were detected on cells in this culture and GluR4 receptor subunits were not detectable. Our experiments showed that glutamate receptors present in both calretinin- and tyrosine hydroxylase-immunoreactive cells were functional, since phosphorylated cAMP/Ca2+ response element-binding protein levels were increased in both cell types after 10 or 30 min exposures to 500 microM KA. The present results indicate that in the mesencephalic cultures tyrosine hydroxylase-immunoreactive cells are more vulnerable to KA excitotoxicity than calretinin-immunoreactive neurons.

Rat mesencephalic neuronal cells cultured for different periods as a model of dopamine transporter ontogenesis.

Ventral mesencephalic neurons contained only low-affinity and sodium-independent binding sites of [3H]WIN 35,428 (marker of dopamine transporter) during the first 10 d in primary cultures. These sites were present in cytosol, and they are not very probably related to dopamine transporter. After 12 d in culture, membrane-bound, high-affinity, and sodium-dependent [3H]WIN 35,428 binding sites were detected. In membranes prepared from cells 14 d in culture, cocaine displaced [3H]WIN 35,428 binding with similar potency to that in striatal membranes of adult rat brain. The high-affinity [3H]WIN 35,428 binding sites in mesencephalic neuronal cell cultures are very probably related to dopamine transporter. The development of high-affinity [3H]WIN 35,428 binding sites in neurons cultured for different time periods could be a useful model of dopamine transporter ontogenesis.

Nitric oxide protects against alkyl peroxide-mediated cytotoxicity: further insights into the role nitric oxide plays in oxidative stress.

Endogenously formed nitric oxide (NO) possesses diverse properties such as regulating physiological functions, exerting specific toxic effects, and protecting against various toxic substances. Recent studies suggest that in the presence of reactive oxygen species, NO can serve as an antioxidant. We show here that NO delivered from the NO donor compound, PAPA/NO (NH2(C3H6)(N[N(O)NO](C3H7)), protects Chinese hamster V79 lung fibroblasts from the cytotoxicity of t-butyl hydroperoxide and cumene hydroperoxide. In contrast, the other end products of PAPA/NO degradation in aqueous solution, NH2(C3H6)NH(C3H7) and nitrite, did not protect. The NONOate DEA/NO releases NO six times faster than PAPA/NO, yet did not afford protection, which implies that NO must be present throughout the alkyl hydroperoxide exposure. Measurements of NO concentrations released from PAPA/NO suggest that micromolar levels protect against cytotoxicity induced by alkyl hydroperoxides. These findings demonstrate that the flux of NO sustained over the duration of the peroxide exposure determines protection and not the total of NO delivered. These results suggest that concentrations of NO produced in the microenvironment of endothelial cells are high enough to protect cells from Fenton-type-mediated toxicity and support the premise that NO may exert a salutary effect in certain diseases associated with membrane damage.

Reaction kinetics for nitrosation of cysteine and glutathione in aerobic nitric oxide solutions at neutral pH. Insights into the fate and physiological effects of intermediates generated in the NO/O2 reaction.

The critical regulatory function of nitric oxide (NO) in many physiologic processes is well established. However, in an aerobic aqueous environment NO is known to generate one or more reactive and potentially toxic nitrogen oxide (NOx) metabolites. This has led to the speculation that mechanisms must exist in vivo by which these reactive intermediates are detoxified, although the nature of these mechanisms has yet to be elucidated. This report demonstrates that among the primary bioorganic products of the reaction of cellular constituents with the intermediates of the NO/O2 reaction are S-nitrosothiol (S-NO) adducts. Anaerobic solutions of NO are not capable of nitrosating cysteine or glutathione, while S-NO adducts of these amino acids are readily formed in the presence of O2 and NO. Investigation of the kinetics for the formation of these S-NO adducts has revealed a rate equation of d[RSNO]/dt = kSNO[NO]2[O2], where kSNO = (6 +/- 2) x 10(6) M-2S-1, a value identical to that for the formation of reactive intermediates in the autoxidation of NO. Competition studies performed with a variety of amino acids, glutathione, and azide have shown that cysteine residues have an affinity for the NOx species that is 3 orders of magnitude greater than that of the nonsulfhydryl amino acids, and > 10(6) times greater than that of the exocyclic amino groups of DNA bases. The dipeptide alanyltyrosine reacts with the intermediates of the NO/O2 reaction with an affinity 150 times less than that of the sulfhydryl-containing compounds. Furthermore, Chinese hamster V79 lung fibroblasts depleted of glutathione display enhanced cytotoxicity on exposure to NO.(ABSTRACT TRUNCATED AT 250 WORDS)

Persistent AMPA receptor stimulation alters [Ca2+]i homeostasis in cultures of embryonic dopaminergic neurons.

The effect of the ionotropic glutamate receptor agonist, AMPA, on intracellular Ca2+ concentrations ([Ca2+]i) was studied in dopaminergic neurons present in primary cultures of ventral tegmental mesencephalon of 14 day rat embryos. Exposure of cells to 10 microM AMPA for 1 min increased [Ca2+]i by 2-3 fold in dopaminergic and other neurons and this response was obliterated within 5 min by superfusion with AMPA-free incubation buffer. In dopaminergic neurons, 1 min or 5 min exposure to 50 microM AMPA increased [Ca2+]i 3 to 5 times over control values. This rise in [Ca2+]i persisted even after a 20 min superfusion with AMPA-free media, whereas, [Ca2+]i in non-dopaminergic neurons was reversed to control values during this time. Preincubation (2 min) of cultured cells with NBQX or the L-type channel blocker, nifedipine, but not with MK-801 blunted the rise of [Ca2+]i in dopaminergic and other neurons. Pretreatment with 2 microM NBQX shifted the dose response curve for AMPA to the right without changing the basal [Ca2+]i. The presence of 10 microM dantrolene, a blocker of Ca2+ release from intracellular stores, did not alter the initial rise of [Ca2+]i elicited by 50 microM AMPA, but prevented the destabilization of Ca2+ homeostasis by facilitating the recovery to normal of basal [Ca2+]i. Exposure to 50 microM AMPA (5 min) caused an irreversible increase of [Ca2+]i in dopaminergic neurons and cell death was manifested by propidium iodide uptake 6-7 h after AMPA exposure.(ABSTRACT TRUNCATED AT 250 WORDS)