Biomedical subjects

E Rodríguez-Farré

Publications and source records attributed to E Rodríguez-Farré.

At least 19 recordsLinked to original sources

PubMed2006

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.

Amyloid beta-Protein Precursor↗

PubMed2003

The organochlorine pesticides gamma-hexachlorocyclohexane (lindane), alpha-endosulfan and dieldrin differentially interact with GABA(A) and glycine-gated chloride channels in primary cultures of cerebellar granule cells.

The neurotoxic organochlorine pesticides gamma-hexachlorocyclohexane, alpha-endosulfan and dieldrin induce in mammals a hyperexcitability syndrome accompanied by convulsions. They reduce the GABA-induced Cl(-) flux. The strychnine-sensitive glycine receptor also regulates Cl(-)-flux inhibitory responses. We studied the effects of these compounds on Cl(-) channels associated with glycine receptors in cultured cerebellar granule cells in comparison to the GABA(A) receptor. Both GABA (EC(50): 5 microM) and glycine (EC(50): 68 microM) increased (36)Cl(-) influx. This increase was antagonized by bicuculline and strychnine, respectively. Lindane inhibited with similar potency both GABA(A) (IC(50): 6.1 microM) and glycine (5.0 microM) receptors. alpha-Endosulfan and dieldrin inhibited the GABA(A) receptor (IC(50) values: 0.4 microM and 0.2 microM, respectively) more potently than the glycine receptor (IC(50) values: 3.5 microM and 3 microM, respectively). Picrotoxinin also inhibited the glycine receptor, although with low potency (IC(50)>100 microM). A 3D pharmacophore model, consisting of five hydrophobic regions and one hydrogen bond acceptor site in a specific three-dimensional arrangement, was developed for these compounds by computational modelling. We propose that the hydrogen bond acceptor moiety and the hydrophobic region were responsible for the affinity of these compounds at the GABA(A) receptor whereas only the hydrophobic region of the molecules was responsible for their interaction with the glycine receptors. In summary, these compounds could produce neuronal hyperexcitability by blocking glycine receptors besides the GABA(A) receptor. We propose that two zones of the polychlorocycloalkane pesticide molecules (a lipophilic zone and a polar zone) differentially contribute to their binding to GABA(A) and glycine receptors.

Animals↗

PubMed2001

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.

Amiloride↗

PubMed2001

Mercury interaction with the GABA(A) receptor modulates the benzodiazepine binding site in primary cultures of mouse cerebellar granule cells.

Mercury compounds are neurotoxic compounds with a great specificity for cerebellar granule cells. The interaction of mercury compounds with proteins in the central nervous system may underlie some of their effects on neurotransmission. In this work we study the interaction of mercuric chloride (HgCl2) and methylmercury (MeHg) with the GABA(A) receptor in primary cultures of cerebellar granule cells. Both compounds increased, dose dependently, the binding of [3H]flunitrazepam to the benzodiazepine recognition site. EC50 values for this effect were 3.56 and 15.24 microM for HgCl2 and MeHg, respectively, after 30 min exposure of intact cultured cerebellar granule cells. The increase of [3H]flunitrazepam binding by mercury compounds was completely inhibited by the GABA(A) receptor antagonists bicuculline and picrotoxinin, and by the organochlorine pesticide alpha-endosulfan. It was also partially inhibited by the anion transporter blocker DIDS, however this effect could be due to a possible chelation of mercury by DIDS. Intracellular events, like intracellular calcium, kinase activation/inactivation or antioxidant conditions did not affect [3H]flunitrazepam binding or its increase induced by mercury compounds. The sulfhydryl alkylating agent N-ethylmaleimide mimicked the effect of mercury compounds on [3H]flunitrazepam binding suggesting a common mechanism. We conclude that mercury compounds interact with the GABA(A) receptor by the way of alkylation of SH groups of cysteinyl residues found in GABA(A) receptor subunit sequences.

Alkylation↗

PubMed2000

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.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2000

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.

Adrenergic Uptake Inhibitors↗

PubMed1999

Induction of cyclooxygenase-2 in the rat brain after a mild episode of focal ischemia without tissue inflammation or neural cell damage.

Cyclooxygenase-2, a key enzyme in prostanoid synthesis, is induced by inflammatory stimuli and it is associated with cell death after cerebral ischemia. Here we evaluated whether cyclooxygenase-2 was induced after a short (10-min) episode of focal ischemia, mild enough not to cause inflammation or cell death. One-hour ischemia leading to brain infarct was studied for comparative purposes. Induction of cyclooxygenase-2 mRNA and protein was detected after both 10-min and 1-h ischemia. However, signs of edema were only apparent after 1-h, but not 10-min ischemia, and only rats subjected to 1-h ischemia had developed brain infarct at 4 days. Therefore, cyclooxygenase-2, not linked with neural cell death or inflammation, is induced after focal ischemia.

Animals↗

PubMed1999

Degeneration and gliosis in rat retina and central nervous system following 3,3'-iminodipropionitrile exposure.

3,3'-Iminodipropionitrile (IDPN) exposure causes a neurofilamentous axonopathy and olfactory, audiovestibular and visual toxicity. Many events relevant to these effects and the neurotoxic properties of nitriles as a class remain to be elucidated. We characterized the gliosis associated with the IDPN-induced retinal degeneration in comparison to other effects on the visual and central nervous systems. Gliosis was quantified using an ELISA for the intermediate filament protein, glial fibrillary acidic protein (GFAP). IDPN (0-400 mg kg-1 day-1x3 days, i.p.) caused corneal opacity and dose- and time-dependent increases in retinal GFAP, up to 26-28 fold of control values at 4 weeks post-exposure; a second peak occurred at 16 weeks. In contrast, GFAP peaked at 1 week in olfactory bulbs (OB), cingulate cortex and hippocampus. Cerebellum and striatum showed no gliosis. Retinal dopamine decreased within 2 weeks. Delayed GFAP increases occurred in superior and inferior colliculi. Retina and superior colliculi also showed increased [3H]PK-11195 binding. Histological analysis demonstrated progressive degeneration and gliosis in retina and colliculi. Taken together, the data indicate that primary and secondary degenerative events occur in the retina, and that this retinal degeneration induces GFAP increases in retina and superior colliculus. In addition, GFAP assays demonstrated that the retinal toxicity of IDPN is enhanced by CCl4 hepatotoxicity and blocked by methimazole inhibition of flavin-mono-oxygenases, similarly to its ototoxicity. GFAP assays also indicated that neither vestibulotoxic doses of crotononitrile nor olfatotoxic doses of dichlobenil damage the retina. The data support the use of GFAP assays for assessing the retinal toxicity of IDPN and other nitriles.

Animals↗

PubMed1999

Effects of the conformationally restricted GABA analogues, cis- and trans-4-aminocrotonic acid, on GABA neurotransmission in primary neuronal cultures.

The effects of the GABA analogues, cis- and trans-4-aminocrotonic acid (ACA) on GABA(A) receptor function and GABA uptake, together with the presence of p-1 subunit mRNA and putative GABAc receptors, were studied in primary cultures of neocortical neurons and cerebellar granule cells. Both isomers induced a Cl- influx, which was inhibited by bicuculline, t-butylbicyclophosphorothionate (TBPS), picrotoxinin (PTX), and gamma-hexachlorocyclohexane (gamma-HCH or lindane). [3H]-flunitrazepam binding was also increased by both isomers and this increase was inhibited by bicuculline. In neocortical neurons, the transisomer completely inhibited the [3H]GABA uptake, whereas the cis-isomer produced only a 25% inhibition at the highest concentration used. The possible presence of GABAc receptors was investigated only in neocortical cultures by using RT-PCR in order to detect the presence of the mRNA encoding the p-1 subunit which assembles to form homooligomeric Cl-channels. The results presented here show that p-1 subunits, and thus GABAc receptors, may represent a very minor population of GABA receptors in these neuronal preparations. We conclude that both GABA analogues may act as agonists at the GABA(A) receptors, although with very different potencies.

Animals↗

PubMed1998

Cytotoxic action of lindane in neocortical GABAergic neurons is primarily mediated by interaction with flunitrazepam-sensitive GABA(A) receptors.

The cytotoxic action of the gamma-isomer of hexachlorocyclohexane (y-HCH; lindane) was studied in cultured mouse neocortical neurons by measurements of the reduction in mitochondrial function using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) test. The cells were exposed to 30-300 microM lindane in the culture medium for different periods of time and lindane cytotoxicity was found to be time- and concentration-dependent. Lindane cytotoxicity could be ameliorated by addition of gamma aminobutyric acid (GABA) in a concentration-dependent manner but this effect of GABA was not blocked by bicuculline or picrotoxinin (PTX). Lindane induced cytotoxicity was also reduced by the GABA(A) receptor agonists muscimol and THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol). This effect was enhanced by the simultaneous presence of flunitrazepam but only at the highest lindane concentrations studied (200 and 300 microM). Flunitrazepam by itself had no effect on lindane-induced cytotoxicity. The protective effect of GABA plus flunitrazepam was blocked by the benzodiazepine receptor antagonist flumazenil and by the GABA(A) antagonist bicuculline, suggesting the involvement of central benzodiazepine receptors allosterically coupled to the GABA recognition site at the GABA(A) receptor. When 100 microM PTX was used to suppress the protective effect of GABA and flunitrazepam, a significant effect of PTX was observed only at 300 microM lindane. The GABA(B) receptor agonist, baclophen, only marginally reduced the cytotoxic effect induced by the highest lindane concentrations. It is concluded that the cytotoxic action of lindane in neocortical neurons in culture is mediated primarily through an interaction with allosterically coupled GABA-benzodiazepine recognition sites at the GABA(A) receptor.

2-Amino-5-phosphonovalerate↗

PubMed1998

Cytotoxic action of lindane in cerebellar granule neurons is mediated by interaction with inducible GABA(B) receptors.

The cytotoxic action of the gamma-isomer of hexachlorocyclohexane (gamma-HCH, lindane) was studied in cultured mouse cerebellar granule neurons maintained in the presence or absence of the GABA(A) receptor agonist THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol). The cells were exposed for 24 hr to lindane (30-300 microM) in the culture medium. Changes in mitochondrial function were investigated by using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) test. The results showed that lindane-induced cytotoxicity was concentration-dependent. In cerebellar granule cells not treated with THIP, lindane-induced cytotoxicity did not appear to be related to GABA(A) or GABA(B) receptors. However, in THIP-treated cultures, lindane-induced cytotoxicity was found to be mediated by an action of the insecticide on GABA receptors. In the latter case, GABA reduced the lindane-induced cytotoxicity, but the protective effect was not potentiated by flunitrazepam. The GABA(A) receptor agonist muscimol (50 microM) also protected the THIP-treated cultures against lindane-induced cytotoxicity. In addition, the GABA(B) receptor agonist R(+)baclofen protected the cells from lindane-induced cytotoxicity and the effect of baclofen was blocked by GABA(B) receptor antagonists. Pertussis toxin was found to reverse the protective effect of baclofen only at the highest lindane concentration (300 microM). The lindane-induced cytotoxicity could be partly explained as being secondary to excitotoxicity as a mixture of the excitatory amino acid receptor antagonists APV (D-(-)-2-amino-5-phosphonopentanoate) and CNQX (6-cyano-7-nitro-quinoxaline-2,3-dione) shifted the concentration-response curve for lindane-induced cytotoxicity to the right. It is suggested that the cytotoxic effects of lindane in THIP-treated cerebellar granule neurons are primarily related to an action of lindane on GABA(B) receptors and to a lesser extent on inducible low-affinity, benzodiazepine insensitive GABA(A) receptors.

2-Amino-5-phosphonovalerate↗

PubMed1998

Behavioral disturbances and vestibular pathology following crotonitrile exposure in rats.

The dinitrile compound 3,3'-iminodipropionitrile causes a number of toxic effects in sensory systems, including degeneration of the vestibular sensory hair cells, as well as a neurofilamentous pathology in motor and sensory neurons. The chemical also causes permanent changes in behavior. These were initially attributed to the effect on neurofilaments, but have been recently linked to the vestibular toxicity. The present work studied the behavioral and pathological effects of the mononitrile compound crotonitrile. Adult male Long-Evans rats (n = 8/group) were exposed to crotonitrile (0, 100, 125, 150 mg/kg/day, for 3 days, i.p., in 1 ml/kg corn oil) and assessed for changes in rearing activity, locomotor activity, and rating scores in tests of vestibular function. Surface preparations of the vestibular sensory epithelia were observed for hair cell loss by scanning electron microscopy (n = 3/group). Control (n = 2) and 3 x 150 mg/kg crotonitrile (n = 3) rats were assessed for neurofilament accumulation in vestibular and dorsal root ganglion neurons by light microscopy observation of semi-thin sections from plastic-embedded ganglia. Crotonitrile dose-dependently increased locomotor activity and rating scores for vestibular dysfunction. A dose-dependent loss of vestibular hair cells was observed in the same animals. In contrast, no obvious neurofilament accumulations were observed in vestibular and dorsal root neurons. We conclude that vestibular toxicity is a property of the nitrile group, and that behavioral disturbances ensue from this toxic action.

Animals↗

PubMed1997

Allosteric interactions between gamma-aminobutyric acid, benzodiazepine and picrotoxinin binding sites in primary cultures of cerebellar granule cells. Differential effects induced by gamma- and delta-hexachlorocyclohexane.

Allosterism between gamma-aminobutyric acid (GABA), benzodiazepine and picrotoxinin recognition sites on the GABAA receptor was studied in primary cultures of cerebellar granule cells. The increase in [3H]flunitrazepam binding induced by GABA was inhibited by bicuculline and picrotoxinin and the decrease in [35S]t-butylbicyclophosphorothionate ([35S]TBPS) binding mediated by GABA was reverted by bicuculline. The effects of hexachlorocyclohexanes (the convulsant gamma- and the depressant delta-isomers, both acting at the picrotoxinin recognition site) on GABA and benzodiazepine sites were studied. delta-Hexachlorocyclohexane, but not the gamma-isomer (lindane), increased [3H]flunitrazepam binding in a concentration-dependent manner (EC50: 8.3 microM). This increase in [3H]flunitrazepam binding was reduced by bicuculline and picrotoxinin. The gamma-isomer reduced the increase in [3H]flunitrazepam binding induced by GABA or delta-hexachlorocyclohexane. Neither delta- nor gamma-hexachlorocyclohexane inhibited [3H]GABA binding. Moreover, the inhibition of [35S]TBPS binding induced by delta-hexachlorocyclohexane was not reverted by bicuculline. The results obtained in this study in vitro agree with the pharmacological properties and the effects of gamma- and delta-hexachlorocyclohexane in vivo. It is concluded that delta-hexachlorocyclohexane acts as a positive allosteric modulator and gamma-hexachlorocyclohexane acts as a non-competitive antagonist of the GABAA receptor.

Animals↗

PubMed1997

Properties of ryanodine receptors in cultured cerebellar granule neurons: effects of hexachlorocyclohexane isomers and calcium.

The binding of [3H]ryanodine was determined in microsomal membrane preparations obtained from cultured cerebellar granule cells. A KD of 1 nM and a Bmax of 64 fmol/mg protein were calculated from saturation experiments. This binding was calcium dependent and maximum values were obtained at 100-300 microM Ca+2. Caffeine increased [3H]ryanodine binding only at Ca+2 concentrations lower than optimum. The binding of [3H]ryanodine was inhibited by ruthenium red, procaine and the delta-isomer of hexachlorocyclohexane (delta-HCH). Dantrolene, a ryanodine receptor antagonist in skeletal sarcoplasmic reticulum, and the pesticide gamma-HCH (lindane) had no effect on [3H]ryanodine binding. The obtained binding parameters, the Ca+2 dependence and the effects of the agents tested agree with previous reports using brain microsomal membranes, further indicating a neuronal localization of [3H]ryanodine binding sites. When the interaction between dantrolene and gamma- and delta-HCH was tested, no changes were detected on the effects of HCH isomers on [3H]ryanodine binding. Dantrolene, which inhibits Ca+2 release from sarcoplasmic reticulum and from unidentified internal Ca+2 stores in neurons, also inhibits the intracellular Ca+2 mobilization induced by gamma-HCH but only marginally that induced by delta-HCH in the same preparation of cerebellar granule cells (Rosa et al.; Toxicol Appl Pharmacol, in press). Thus, the results obtained in this work verify the presence of different intracellular sites of action for the two HCH isomers: the ryanodine Ca+2 channel for delta-HCH and an unidentified dantrolene-sensitive Ca+2 channel for the gamma-HCH isomer.

Anesthetics, Local↗

PubMed1997

Striatal infarction in the rat causes a transient reduction of tyrosine hydroxylase immunoreactivity in the ipsilateral substantia nigra.

Dopaminergic neurons of the substantia nigra pars compacta were examined in the rat brain following striatal infarction subsequent to transient focal cerebral ischemia. Rats had the middle cerebral artery occluded for 2 h or were sham-operated, and tyrosine hydroxylase immunoreactivity was evaluated by Western blot and immunohistochemistry at different times ranging from 1 to 60 days after ischemia. The number of tyrosine hydroxylase-immunoreactive cells in the substantia nigra pars compacta was counted under the light microscope and compared to that in the contralateral side and controls. No changes of tyrosine hydroxylase immunoreactivity were detected in the ipsilateral versus the contralateral substantia nigra of sham-operated rats or 1 day after ischemia. However, a statistically significant reduction of tyrosine hydroxylase-immunoreactive cells became apparent in the ipsilateral compared with the contralateral substantia nigra at 7 and 14 days after ischemia. This reduction showed a clear recovery at 30 days after ischemia, and no signs of difference between the ipsilateral and the contralateral side were apparent by 60 days. Therefore, the reduction of tyrosine hydroxylase immunoreactivity in the ipsilateral substantia nigra was only transiently seen from 1 to 2 weeks following ischemia. The observed loss of tyrosine hydroxylase was not accompanied by signs of cell death or gliosis in the ipsilateral pars compacta. The present results show a transitory reduction of tyrosine hydroxylase immunoreactivity in the ipsilateral substantia nigra pars compacta after focal ischemia and suggest that striatal infarction causes a transient deficit of dopaminergic function.

Animals↗

PubMed1997

The mechanism for hexachlorocyclohexane-induced cytotoxicity and changes in intracellular Ca2+ homeostasis in cultured cerebellar granule neurons is different for the gamma- and delta-isomers.

The cytotoxic action of the delta- and gamma-isomers of hexachlorocyclohexane (HCH) as well as their ability to induce changes in intracellular Ca2+ homeostasis was studied in cultured rat cerebellar granule neurons. Changes in the free intracellular Ca2+ concentration ([Ca2+]i) related to Ca2+ influx and release from intracellular stores were investigated using, in addition to the physiological incubation medium, media without added Ca2+ (nominally Ca2+ free) or containing verapamil or dantrolene, drugs which block influx through voltage-gated Ca2+ channels and release from intracellular stores, respectively. Cytotoxicity was monitored using leakage of lactate dehydrogenase (LDH) and staining of damaged cells with either trypan blue or propidium iodide. In the latter case, when fluorescence microscopy was employed, undamaged cells were visualized using the vital stain calcein acetoxymethyl ester (calcein-AM). The delta-isomer of HCH was found to be more potent and active as a cytotoxic agent than the gamma-isomer (lindane) and it was demonstrated that the moderate cytotoxic action of lindane could be ameliorated by dantrolene which on the other hand had no effect on cytotoxicity induced by delta-HCH. It was noticed that pronounced (50% increase in fluorescence) staining with propidium iodide was associated with only a marginal (20%) LDH leakage and staining of only a few (<25%) cells with trypan blue. Both delta- and gamma-HCH induced an increase in [Ca2+]i which was most pronounced in case of delta-HCH. Analysis of the different mechanisms governing the increase in [Ca2+li using dantrolene, verapamil, and Ca2+-free medium revealed distinct differences between the two isomers with regard to the Ca2+ pools affected. Both isomers stimulated Ca2+ influx through voltage-gated Ca2+ channels but only the gamma-isomer affected a Ca2+-dependent, dantrolene-sensitive pool. On the other hand, delta-HCH affected mainly a Ca2+-independent dantrolene-insensitive pool. The finding that delta-HCH was more toxic than lindane may be correlated to the differences between the isomers with regard to the action on the different Ca2+ pools. Thus, delta-HCH affected primarily the dantrolene-insensitive Ca2+ pools which partly reflect influx. On the contrary, lindane had little effect on these Ca2+ pools but affected primarily dantrolene-sensitive intracellular Ca2+ stores. This may suggest that delta-HCH may exert its cytotoxic action by stimulating a large influx of Ca2+ possibly leading to release of Ca2+ from dantrolene-insensitive stores. In contrast, the toxic action of lindane may be primarily related to release of Ca2+ from the dantrolene-sensitive stores.

Animals↗

PubMed1997

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.

2-Amino-5-phosphonovalerate↗

PubMed1996

Apoptosis and c-Jun in the thalamus of the rat following cortical infarction.

Cortical infarction produces secondary neuronal damage in the thalamus. In this study we examined the thalamus of the rat following 2 h occlusion of the middle cerebral artery (MCA), and found degeneration and gliosis in the ipsilateral ventropostero-medial thalamic nucleus in those rats that showed cortical infarction 7 and 14 days after occlusion. This was accompanied by isolated cells with fragmented DNA, as revealed by in situ labelling of nuclear DNA fragmentation, and showing morphological features of apoptosis, i.e. chromatin condensation, extreme nuclear shrinkage and apoptotic bodies. In addition, cells immunoreactive for c-Jun showing morphological signs of apoptosis were observed. These results provide evidence of apoptosis in the ipsilateral thalamus following cortical infarction, and suggest that c-Jun is involved in this process.

Animals↗