A possible role of the non-GAT1 GABA transporters in transfer of GABA from GABAergic to glutamatergic neurons in mouse cerebellar neuronal cultures.

Cultures of dissociated cerebellum from 7-day-old mice were used to investigate the mechanism involved in synthesis and cellular redistribution of GABA in these cultures consisting primarily of glutamatergic granule neurons and a smaller population of GABAergic Golgi and stellate neurons. The distribution of GAD, GABA and the vesicular glutamate transporter VGlut-1 was assessed using specific antibodies combined with immunofluorescence microscopy. Additionally, tiagabine, SKF 89976-A, betaine, beta-alanine, nipecotic acid and guvacine were used to inhibit the GAT1, betaine/GABA (BGT1), GAT2 and GAT3 transporters. Only a small population of cells were immuno-stained for GAD while many cells exhibited VGlut-1 like immuno-reactivity which, however, never co-localized with GAD positive neurons. This likely reflects the small number of GABAergic neurons compared to the glutamatergic granule neurons constituting the majority of the cells. GABA uptake exhibited the kinetics of high affinity transport and could be partly (20%) inhibited by betaine (IC(50) 142 microM), beta-alanine (30%) and almost fully (90%) inhibited by SKF 89976-A (IC(50) 0.8 microM) or nipecotic acid and guvacine at 1 mM concentrations (95%). Essentially all neurons showed GABA like immunostaining albeit with differences in intensity. The results indicate that GABA which is synthesized in a small population of GAD-positive neurons is redistributed to essentially all neurons including the glutamatergic granule cells. GAT1 is not likely involved in this redistribution since addition of 15 microM tiagabine (GAT1 inhibitor) to the culture medium had no effect on the overall GABA content of the cells. Likewise the BGT1 transporter cannot alone account for the redistribution since inclusion of 3 mM betaine in the culture medium had no effect on the overall GABA content. The inhibitory action of beta-alanine and high concentrations of nipecotic acid and guvacine on GABA transport strongly suggests that also GAT2 or GAT3 (HUGO nomenclature) could play a role.

Modulation of noradrenaline release from hippocampal slices by hexachlorocyclohexane isomers. Effects of GABAergic compounds.

The effects of hexachlorocyclohexane (HCH) isomers and some GABAergic compounds on [3H]noradrenaline (NA) release from rat hippocampal slices prelabelled with 80 nM [3H]NA were determined. The convulsant gamma-HCH isomer facilitated (EC50 = 21 microM) and the depressant delta-HCH isomer reduced (EC50 = 48 microM) the Ca(2+)-dependent K(+)-evoked release of [3H]NA, whereas alpha- and beta-HCH isomers did not show any effect. Moreover, alpha- and delta-HCH isomers antagonized the facilitation of evoked [3H]NA release induced by the gamma-HCH isomer. The GABAergic convulsant drugs, bicuculline, picrotoxin and pentylenetetrazol, did not cause any modification of the evoked [3H]NA release even at high concentrations. Neither bicuculline nor picrotoxin blocked the effects of HCH isomers on K(+)-evoked release of [3H]NA. Exposure of slices to diazepam reduced the K(+)-evoked release of [3H]NA (EC50 = 33 microM) in a manner similar to that of the delta-HCH isomer. In addition, diazepam (50 microM) blocked the gamma-HCH effect and caused an additive inhibitory response with the delta-HCH isomer. On the other hand, diazepam and delta-HCH induced a time-dependent Ca(2+)-independent enhancement of basal [3H]NA release. The results suggest that modulation of [3H]NA release in the hippocampus by HCH isomers may be involved in the central actions of these compounds, and that sites other than the classic GABAA receptor may underlie their presynaptic mechanisms of action.

Hippocampal noradrenaline release is modulated by gamma- and delta-hexachlorocyclohexane isomers: which mechanisms are involved?

The differential effects of gamma- and delta-hexachlorocyclohexane isomers on 25 mM K(+)-evoked release of [3H]noradrenaline were studied in hippocampal slices treated with selected agents to activate or block L- and N-type Ca2+ and Na+ voltage-sensitive ion channels, Cl- transport and Ca(2+)-dependent protein activity. At maximally effective concentrations, the L- and N-type Ca2+ channel blockers nifedipine and omega-conotoxin, respectively, and the Na+ channel antagonist tetrodotoxin did not modify the enhancement of K(+)-evoked [3H]noradrenaline release induced by gamma-hexachlorocyclohexane. Likewise, under activation of protein kinase C by phorbol 12,13-dibutyrate (PDB) or inhibition of calmodulin by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), the stimulatory effect of gamma-hexachlorocyclohexane remained almost unchanged. The Cl- transport blocker 4,4-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) significantly reduced the effect of gamma-hexachlorocyclohexane on [3H]noradrenaline release. The enhanced release in the presence of Bay K 8644, the L-type Ca2+ channel activator, was significantly inhibited by nifedipine but not by delta-hexachlorocyclohexane. The combination of omega-conotoxin and tetrodotoxin with delta-hexachlorocyclohexane did not alter the [3H]noradrenaline release effects of each agent alone. Activation of protein kinase C in the presence of delta-hexachlorocyclohexane resulted in a reduction of the delta isomer effect and in a potentiation of the PDB effect. W-7 did not further facilitate the inhibition induced by delta-hexachlorocyclohexane alone. These data suggest that hexachlorocyclohexane isomers may modify K(+)-evoked [3H]noradrenaline release by interacting with presynaptic molecular processes involving changes in Cl- membrane permeability and intracellular Ca2+ homeostasis.

Differential presynaptic effects of hexachlorocyclohexane isomers on noradrenaline release in cerebral cortex.

To investigate presynaptic effects of hexachlorocyclohexane (HCH) isomers, the release of noradrenaline (NA) in brain tissue was analyzed using rat cerebral cortical slices preloaded with [3H]-NA. gamma-HCH (lindane) 50 microM significantly enhanced the [3H]-NA release evoked by 15-25 mM K+. alpha- and beta-HCH (50 microM) did not produce any significant effect on K(+)-evoked [3H]-NA release. delta-HCH (50 microM) induced a significant decrease of the 25 mM K(+)-evoked release of [3H]-NA. The effect of the gamma- and delta-HCH isomers on the presynaptic action of the alpha 2-agonist clonidine and the alpha 2-antagonist yohimbine was also studied. The presynaptic inhibitory effect of clonidine and the stimulatory effect of yohimbine on [3H]-NA release was attenuated by lindane and delta-HCH, respectively. These results are consistent with a presynaptic action of the HCH isomers on noradrenergic release processes.

Effects of gamma and delta hexachlorocyclohexane isomers on inositol phosphate formation in cerebral cortex and hippocampus slices from developing and adult rat.

Hexachlorocyclohexane (HCH) isomers, mainly gamma-HCH (insecticide with stimulant and convulsant effects in man) and delta-HCH (neurodepressant agent), are known to inhibit the synthesis of phosphatidylinositol (PI) in diverse cell systems, but action on phosphoinositide hydrolysis has been scarcely studied. The present work examines the effects of gamma-HCH and delta-HCH on the accumulation of [3H]inositol phosphates (InsP) from the hydrolysis of prelabelled phosphoinositides, in cerebral cortical and hippocampal slices from developing (8-day-old) and adult rats. In developing and adult animals both isomers increased InsP formation in a concentration-related manner. delta-HCH was statistically more potent in developing than in adult animals (maximum effects obtained were 280% and 200% of basal InsP accumulation at 300 microM, respectively). gamma-HCH showed lower stimulation (maximal effect was 170% at 300 microM, at both ages) than delta isomer. No differences between brain regions were observed after treatment with HCH isomers. Effects of HCH isomers (200 microM) on phosphoinositide hydrolysis stimulation by glutamate, carbachol and noradrenaline were selective for the transmitter receptor agonist and age studied. delta-HCH inhibited glutamate and carbachol stimulation in cerebral cortex from developing animals and did not modify it in adults. Combination of delta-HCH and noradrenaline increased the effects of this neurotransmitter in immature rats, while only noradrenaline stimulation was observed in adult rats. A noticeable effect of gamma-HCH was that it did not increase glutamate and carbachol stimulation in cerebral cortex of developing rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of glucose and oxygen deprivation on phosphoinositide hydrolysis in cerebral cortex slices from neonatal rats.

The effects of glucose deprivation, hypoxia and glucose-free hypoxia conditions on phosphoinositide (PI) hydrolysis were studied in cortical slices from 8-day-old rats. Only glucose-free hypoxia induced a significant increase of inositol phosphate formation. The inositol phosphate formation induced by noradrenaline, carbachol and several excitatory amino acid receptor agonists, but not the Ca2+ ionophore A23187-induced stimulation, was blocked by glucose-free hypoxia and differentially reduced by glucose and oxygen deprivation depending on the neurotransmitter receptor agonist. The stimulatory effect of glucose-free hypoxia was not reduced by the muscarinic receptor antagonist atropine or by the inhibitors of the excitatory amino acid-stimulated PI hydrolysis DL-2-amino-3-phosphono-propionic acid and L-aspartate-beta-hydroxamate, and neither by the voltage-sensitive Na+ channel tetrodotoxin. The effect of glucose-free hypoxia was partially dependent on extracellular Ca2+ and it was blocked by verapamil and amiloride, but not by nifedipine, Co2+ and neomycin. These results suggest that Ca2+ influx through the Na(+)-Ca2+ exchanger underlies the PI hydrolysis stimulation induced by combined glucose and oxygen deprivation in neonatal cerebral cortical slices.

Protein binding and stability of norepinephrine in human blood plasma. Involvement of prealbumin, alpha 1-acid glycoprotein and albumin.

The binding of norepinephrine (NE) to plasma proteins of fresh human blood obtained from healthy volunteers was studied by ultrafiltration at different NE concentrations and incubation times at 37 degrees C. At 1.7 nM L-[3H]-NE binding was approximately 25%. The binding was rapid and was not influenced by the incubation time. [3H]-NE could be dissociated from its binding sites by acid precipitation and, after HPLC, showed to be unchanged NE. No difference in NE binding was found between plasma collected in EGTA-GSH or heparin solution. There was no degradation of NE when incubated in plasma at 37 degrees C for 10 h, even without the addition of antioxidants. Therefore, in the present study, binding represented interaction of unchanged NE with plasma proteins. The whole plasma binding was saturable over the range of 0.66 nM to 0.59 mM of NE. Scatchard plot of specific binding revealed high-affinity sites with a Kd of 5.4 nM and a Bmax of 3.9 fmoles.mg-1 protein, and low-affinity sites with a Kd of 2.7 microM and a Bmax of 3.3 pmoles.mg-1 protein. Electrophoretic characterization of NE-binding proteins showed that about 60% of bound NE was associated to albumin, and 20% to prealbumin. NE binding to pure human plasma proteins was also studied using ultrafiltration. Scatchard analyses revealed a single class of very high-affinity binding sites for prealbumin (Kd 4.9 nM), a single class of binding sites for alpha 1-acid glycoprotein (Kd 54 microM) and two classes of binding sites for albumin with high (Kd 1.7 microM) and low (Kd 0.8 mM) affinities respectively. The main results obtained in this study - a) reversibility of NE binding, b) stability of free and bound NE in plasma, c) involvement of the prealbumin as a specific binding protein - point out to a specific transport for NE in human blood plasma.

Pharmacological characterization of the effects of methylmercury and mercuric chloride on spontaneous noradrenaline release from rat hippocampal slices.

The environmental contaminants methylmercury (MeHg) and mercuric chloride (HgCl2) stimulated the spontaneous release of [3H]noradrenaline ([3H]NA) from hippocampal slices in a time- and concentration-dependent manner. Both MeHg and HgCl2 were similarly potent, with an EC50 of 88.4 microM and 75.9 microM, respectively. The releasing effects of MeHg and HgCl2 increased in the presence of desipramine, showing that the mechanism does not involve reversal of the transmitter transporter, and were completely blocked by reserpine preincubation, indicating a vesicular origin of [3H]NA release. The voltage-gated Na+ channel blocker tetrodotoxin (TTX) did not affect the response to mercury compounds. [3H]NA release elicited by MeHg was partially dependent on extracellular Ca2+, since it decreased significantly in a Ca2+-free EGTA-containing medium whereas HgCl2 induced a release of [3H]NA independent of extracellular Ca2+. Neither Ca2+-channels blockers, cobalt chloride (CoCl2) and (omega-conotoxin-GVIA, nor the Na+/Ca2+-exchanger inhibitor benzamil reduced MeHg-evoked [3H]NA release. Moreover, thapsigargin or caffeine, endoplasmic reticulum Ca2+-depletors, did not modify metal-evoked [3H]NA release, whereas ruthenium red, which inhibits the mitochondrial Ca2+ transport, decreased the effect of both MeHg and HgCl2. All these data indicate that, in hippocampal slices, mercury compounds release [3H]NA from the vesicular pool by a mechanism involving Ca2+ mobilization from mitochondrial stores.

Use of human central nervous system cell cultures in neurotoxicity testing.

The nervous system is highly sensitive to toxic damage. Many environmental contaminants can produce acute or chronic neurological effects, and contribute to neural damage and cell death in neurodegenerative diseases. The utilization of primary cultures of neurons and glial cells is an essential step in investigating the specificity of the effects and mechanisms of action of the test chemical. If we take into account interspecies differences, cultures of human central nervous system (CNS) cells would be the best-suited test models for in vitro neurotoxicity testing. For practical and ethical reasons, human neuronal and glial cultures cannot be used for routine neurotoxicity testing, but they may be very useful for validating results from murine cultures and to address specific toxicity questions. For instance, we are investigating the action of agents producing oxygen radical damage in CNS cells. Oxidative stress is known to trigger apoptotic death of neurons and lead to neurodegeneration. A useful model in which to study these processes could be neuronal cultures obtained from CNS tissue with trisomy 21, since these cells suffer oxidative stress and apoptotic cell death in vitro. Besides primary cultures, human-derived clonal cell lines such as neuroblastoma SH-SY5Y can offer a first-step approach in neurotoxicity testing.

Expression of GDNF transgene in astrocytes improves cognitive deficits in aged rats.

Glial cell line-derived neurotrophic factor (GDNF) was assayed for its neurotrophic effects against the neuronal atrophy that causes cognitive deficits in old age. Aged Fisher 344 rats with impairment in the Morris water maze received intrahippocampal injections at the dorsal CA1 area of either a lentiviral vector encoding human GDNF or the same vector encoding human green fluorescent protein as a control. Recombinant lentiviral vectors constructed with human cytomegalovirus promotor and pseudotyped with lyssavirus Mokola glycoprotein specifically transduced the astrocytes in vivo. Astrocyte-secreted GDNF enhanced neuron function as shown by local increases in synthesis of the neurotransmitters acetylcholine, dopamine and serotonin. This neurotrophic effect led to cognitive improvement of the rats as early as 2 weeks after gene transduction. Spatial learning and memory testing showed a significant gain in cognitive abilities due to GDNF exposure, whereas control-transduced rats kept their performance at the chance level. These results confirm the broad spectrum of the neurotrophic action of GDNF and open new gene therapy possibilities for reducing age-related neurodegeneration.

Astrocytes aged in vitro show a decreased neuroprotective capacity.

Alterations in astrocyte function that may affect neuronal viability occur with brain aging. In this study, we evaluate the neuroprotective capacity of astrocytes in an experimental model of in vitro aging. Changes in oxidative stress, glutamate uptake and protein expression were evaluated in rat cortical astrocytes cultured for 10 and 90 days in vitro (DIV). Levels of glial fibrillary acidic protein and S100beta increased at 90 days when cells were positive for the senescence beta-galactosidase marker. In long-term astrocyte cultures, the generation of reactive oxygen species was enhanced and mitochondrial activity decreased. Simultaneously, there was an increase in proteins that stained positively for nitrotyrosine. The expression of Cu/Zn-superoxide dismutase (SOD-1) and haeme oxygenase-1 (HO-1) proteins and inducible nitric oxide synthase (iNOS) increased in aged astrocytes. Glutamate uptake in 90-DIV astrocytes was higher than in 10 DIV ones, and was more vulnerable to inhibition by H2O2 exposure. Enhanced glutamate uptake was probably because of up-regulation of the glutamate/aspartate transporter protein. Aged astrocytes had a reduced ability to maintain neuronal survival. These findings indicate that astrocytes may partially loose their neuroprotective ability during aging. The results also suggest that aged astrocytes may contribute to exacerbating neuronal injury in age-related neurodegenerative processes.

Carboxyl-terminal fragment of amyloid precursor protein and hydrogen peroxide induce neuronal cell death through different pathways.

Carboxyl-terminal fragments (CTs) of the amyloid precursor protein have been shown to be highly neurotoxic and are though to contribute to the neuropathology of Alzheimer's disease. We compared the effects of expressing CT99 in the human neuroblastoma MC65 with the effects of hydrogen peroxide on the parental SK-N-MC cells. CT99 and hydrogen peroxide generated a different pattern of free radicals and their toxic effects were differentially protected by a battery of antioxidants. Hydrogen peroxide caused a cell cycle arrest at phase S and apoptosis mediated through caspase-3 activation in a pattern similar to that described for amyloid-beta neurotoxicity. However, CT99 apoptosis appeared to be mediated through an unidentified mitochondrial pathway. Both oxidative injury types induced heme oxygenase-1 expression as a neuroprotective response. Overall we found a coincidence in the nonespecific stress oxidative effects of CT99 and hydrogen peroxide, but clear differences on their respective potencies and pathways of neurotoxicity.

Sulphur-containing amino acids modulate noradrenaline release from hippocampal slices.

The L- and D-enantiomers of the sulphur-containing amino acids (SAAs)-homocysteate, homocysteine sulphinate, cysteate, cysteine sulphinate, and S-sulphocysteine-stimulated [3H] noradrenaline release from rat hippocampal slices in a concentration-dependent manner. The relative potencies of the L-isomers (EC50 values of 1.05-1.96 mM) were of similar order to that of glutamate (1.56 mM), which was 10-fold lower than that of NMDA (0.15 mM), whereas the D-isomers exhibited a wider range of potencies (0.75 to > 5 mM). All stimulatory effects of the SAAs were significantly inhibited by the voltage-sensitive Na+ channel blocker tetrodotoxin (55-71%) and completely blocked by addition of Mg2+ or Co2- to the incubation medium. All SAA-evoked responses were concentration-dependently antagonized by the selective NMDA receptor antagonist D-(-)-2-amino-5-phosphonopentanoic acid (IC50 values of 3.2 - 49.5 microM). 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA receptor antagonist, at 100 microM inhibited the [3H]noradrenaline release induced by glutamate and NMDA (65 and 76%, respectively) and by all SAAs studied (65-85%), whereas 10 microM CNQX only inhibited the effects of S-sulpho-L-cysteine and L- and D-homocysteate (33, 32, and 44%, respectively). However, the more selective AMPA/kainic acid receptor antagonist 6-nitro-7-sulphamoylbenzo (f) quinoxaline-2,3-dione (100 microM), which did not antagonize the [3H]noradrenaline release induced by glutamate and NMDA, reduced only the S-sulpho-L-cysteine-evoked response (25%). Thus, the stimulation of Ca2(+)-dependent [3H]noradrenaline release from hippocampal slices elicited by the majority of the SAAs appears to be mediated by the NMDA receptor.

Antioxidant compounds and Ca(2+) pathway blockers differentially protect against methylmercury and mercuric chloride neurotoxicity.

The effects of the environmental contaminants methylmercury (MeHg) and inorganic mercury (HgCl(2)) on cell viability, intracellular calcium concentration ([Ca(2+)](i)), and reactive oxygen species (ROS) generation were studied in rat cerebellar granule neuron cultures using fluorescent methods. MeHg exhibited an LC(50) (2.47 microM) tenfold lower than that of HgCl(2) (26.40 microM). To study the involvement of oxidative stress and Ca(2+) homeostasis disruption in mercury-induced cytotoxicity, we tested the neuroprotective effects of several agents that selectively interfere with these mechanisms. After a 24 hr exposure, the cytotoxic effect of both mercury compounds was reduced by thapsigargin, an inhibitor of endoplasmic reticulum Ca(2+)-ATPase; the Ca(2+) channel blocker flunarizine; and the Na(+)/Ca(2+) exchanger blocker benzamil. All these compounds decreased the mercury-mediated [Ca(2+)](i) rise. These results indicate that Ca(2+) influx through Ca(2+) channels and the Na(+)/Ca(2+) exchanger and Ca(2+) mobilization from the endoplasmic reticulum are involved in mercury-mediated cytotoxicity. The antioxidants probucol and propyl gallate reduced the HgCl(2) toxicity. Probucol and vitamin E partially inhibited the MeHg toxicity after a 24 hr period, whereas propyl gallate completely prevented this effect. Probucol slightly reduced ROS generation in methylmercury-exposed cultures and decreased mercury-mediated rise of [Ca(2+)](i). Propyl gallate abolished ROS generation and partially inhibited the increase of [Ca(2+)](i) induced by both mercury compounds. Propyl gallate also protected human cerebral cortical neuron cultures from the MeHg effect even after 72 hr of MeHg exposure, thus showing a long-lasting effect. Our data suggest that disruption of redox equilibrium and Ca(2+) homeostasis contribute equally to HgCl(2)-mediated toxicity, whereas oxidative stress is the main cause of MeHg neurotoxicity.

Trimethyltin and triethyltin differentially induce spontaneous noradrenaline release from rat hippocampal slices.

The environmental contaminants trimethyltin (TMT) and triethyltin (TET) stimulated the spontaneous release of [(3)H]noradrenaline ([(3)H]NA) from hippocampal slices in a time- and concentration-dependent manner. TMT was the most potent compound, exhibiting an EC50 value 10-fold lower (3.8 microM) than that of TET (39.5 microM). Metal-evoked [(3)H]NA release did not increase in the absence of desipramine and was completely blocked by reserpine preincubation, indicating a vesicular origin of [(3)H]NA release but not a mechanism involving reversal of the transmitter transporter. The voltage-gated Na(+) channel blocker tetrodotoxin (TTX) did not affect metal-evoked [(3)H]NA release. [(3)H]NA release elicited by TMT was partially extracellular Ca(2+)-dependent, since it was significantly decreased in a Ca(2+)-free EGTA-containing medium, whereas TET induced an extracellular Ca(2+)-independent release of [(3)H]NA. Neither inhibitors of Ca(2+)-entry through Na(+)/Ca(2+)exchanger and voltage-gated calcium channels, nor agents that interfere with Ca(2+)-mobilization from intracellular stores affected [(3)H]NA release induced by TMT. TET-evoked [(3)H]NA release was reduced by ruthenium red, which depletes mitochondrial Ca(2+)stores, but was not modified by caffeine and thapsigargin, which interfere with Ca(2+)mobilization from endoplasmic reticulum. The fact that TET effect was also attenuated by DIDS, an inhibitor of anion exchange, indicates that the effect of TET on spontaneous [(3)H]NA release may be mediated by intracellular mobilization of Ca(2+) from mitochondrial stores through a Cl(-) dependent mechanism.

Peroxiredoxin 2 (PRDX2), an antioxidant enzyme, is under-expressed in Down syndrome fetal brains.

Suppression subtractive hybridization performed on Down syndrome (DS) versus control fetal brains revealed differential expression of peroxiredoxin 2 (PRDX2), mapped at 13q12. Peroxiredoxins are antioxidant enzymes involved in protein and lipid protection against oxidative injury and in cellular signalling pathways regulating apoptosis. The under-expression of PRDX2 observed in DS samples was confirmed by real-time PCR (0.73-fold). To test whether decreased expression is associated with enhanced sensitivity of DS neurons to reactive oxygen species, we down-regulated PRDX2 through stable transfections of SH-SY5Y neuroblastoma cells with antisense contructs of the complete PRDX2 coding sequence. In addition, we over-expressed SOD1 and compared the effects of the two genes on cell viability. Cells transfected with either construct showed similar sensitivity to oxidative stress in addition to increased apoptosis under basal conditions and after treatment with oxidative cytotoxic agents. This suggests that the decreased expression of PRDX2 may contribute to the altered redox state in DS at levels comparable to that of the increased expression of SOD1.