Search

In the present work, we investigated the role of pre- and post-synaptic neuropeptide Y1 (NPY1) and Y2 receptors on the calcium responses and on glutamate release in the rat hippocampus. In cultured hippocampal neurones, we observed that only NPY1 receptors are involved in the modulation of intracellular free calcium concentration ([Ca(2+)](i)). In 88% of the neurones analysed, the increase in the [Ca(2+)](i), in response to depolarization with 50 mM KCl, was inhibited by 1 microM [Leu31,Pro34]NPY, whereas 300 nM NPY13-36 was without effect. However, studies with hippocampal synaptosomes showed that both NPY1 and Y2 receptors can modulate the [Ca(2+)](i) and glutamate release. The pharmacological characterization of the NPY-induced inhibition of glutamate release indicated that Y2 receptors play a predominant role, both in the modulation of Ca(2+)-dependent and -independent glutamate release. However, we could distinguish between Y1 and Y2 receptors by using [Leu31,Pro34]NPY and NPY13-36. Active pre-synaptic Y1 receptors are present in the dentate gyrus (DG) as well as in the CA3 subregion, but its activity was not revealed by using the endogenous agonist, NPY. Concerning the Y2 receptors, they are present in the three subregions (CA1, CA3 and DG) and were activated by either NPY13-36 or NPY. The present data support a predominant role for NPY2 receptors in mediating NPY-induced inhibition of glutamate release in the hippocampus, but the physiological relevance of the presently described DG and CA3 pre-synaptic NPY1 receptors remains to be clarified.

Photodynamic therapy (PDT) of cancer is a promising technique based on the formation of singlet oxygen following irradiation of a sensitizer with visible light. In the present work we investigated the role of calpains in PDT, using the human lymphoblastoid CCRF-CEM cells and bisulfonated aluminum phthalocyanine (AlPcS2) as a sensitizer. Photosensitization induced apoptotic cell death and a time-dependent activation of calpains, as determined using the fluorogenic substrate succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin (SLLVY-AMC). However, inhibition of calpains with calpain inhibitor II or with PD 150606 did not affect the demise process. The results indicate that although calpains are activated in PDT, they do not play a major role in tumor cell death. http://www.sciencedirect.com/science/article/B6T54-4D7K70T-1/1/6ab9c47e98e82c782e8b8eebda86004d

In this work, we investigated the involvement of calpains in the neurotoxicity induced by short-term exposure to kainate (KA) in non-desensitizing conditions of AMPA receptor activation (cyclothiazide present, CTZ), in cultured rat hippocampal neurons. The calpain inhibitor MDL28170 had a protective effect in cultures treated with KA plus CTZ (p

In photodynamic therapy (PDT) a sensitizer, light and oxygen are used to induce death of tumor cells and in the treatment of certain noncancerous conditions. Cell death in PDT may occur by apoptosis or by necrosis, depending on the sensitizer, on the PDT dose and on the cell genotype. Some sensitizers that have been used in PDT are accumulated in the mitochondria, and this may explain their efficiency in inducing apoptotic cell death, both in vitro and in vivo. In this review we will focus on the events that characterize apoptotic death in PDT and on the intracellular signaling events that are set in motion in photosensitized cells. Activation of phospholipases, changes in ceramide metabolism, a rise in the cytosolic free Ca2+ concentration, stimulation of nitric oxide synthase (NOS), changes in protein phosphorylation and alterations in the activity of transcription factors and on gene expression have all been observed in PDT-treated cells. Although many of these metabolic reactions contribute to the demise process, some of them may antagonize cell death. Understanding the signaling mechanisms in PDT may provide means to modulate the PDT effects at the molecular level and potentiate its antitumor effectiveness. http://www.sciencedirect.com/science/article/B6T23-4CNXHDF-1/1/3985caebee735db34936ae02462172be

In this work, we investigated the role of nitric oxide (NO) in neurotoxicity triggered by α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor activation in cultured hippocampal neurons. In the presence of cyclothiazide (CTZ), short-term exposures to kainate (KA; 5 and 15 min, followed by 24-h recovery) decreased cell viability. Both NBQX and d-AP-5 decreased the neurotoxicity caused by KA plus CTZ. Long-term exposures to KA plus CTZ (24 h) resulted in increased toxicity. In short-, but not in long-term exposures, the presence of NO synthase (NOS) inhibitors (l-NAME and 7-NI) decreased the toxicity induced by KA plus CTZ. We also found that KA plus CTZ (15-min exposure) significantly increased cGMP levels. Furthermore, short-term exposures lead to decreased intracellular ATP levels, which was prevented by NBQX, d-AP-5 and NOS inhibitors. Immunoblot analysis revealed that KA induced neuronal NOS (nNOS) proteolysis, gradually lowering the levels of nNOS according to the time of exposure. Calpain, but not caspase-3 inhibitors, prevented this effect. Overall, these results show that NO is involved in the neurotoxicity caused by activation of non-desensitizing AMPA receptors, although to a limited extent, since AMPA receptor activation triggers mechanisms that lead to nNOS proteolysis by calpains, preventing a further contribution of NO to the neurotoxic process.

The expression and localization of syntaxin isoforms 1A and 1B in adrenergic and noradrenergic chromaffin cells were examined by both immunoblot analysis and confocal immunofluorescence microscopy. Syntaxin 1A was found in higher levels in noradrenergic cells, whereas syntaxin 1B was similarly expressed in most noradrenergic and adrenergic cells. However, some heterogeneity was observed within each catecholaminergic phenotype. Although the majority of adrenergic cells appeared to express low levels of syntaxin 1A, about 7% was strongly stained for syntaxin 1A. A subpopulation of noradrenergic cells, about 17%, expressed greater levels of syntaxin 1B. Syntaxin 1B labeling showed a punctate appearance in the cytoplasm, whereas syntaxin 1A appeared predominantly localized to the plasma membrane. These data show differences in the exocytotic machinery of the two subtypes of chromaffin cells that may underlie some of the distinct characteristics of adrenaline and noradrenaline secretion.

Diacerhein and rhein are anthraquinone compounds that ameliorate the course of osteoarthritis. Recent reports also suggest that these compounds may have antiinflammatory properties, but the cellular mechanisms by which they exert antiosteoarthritic and possibly antiinflammatory effects are still incompletely understood. The purpose of this study was to investigate the ability of diacerhein and rhein to inhibit the activation of the transcription factor nuclear factor kappaB, induced by the proinflammatory cytokine interleukin-1beta, in primary monolayer cultures of bovine articular chondrocytes. We also studied the ability of diacerhein and rhein to prevent the expression of the inducible nitric oxide synthase gene, which is driven by nuclear factor-kappaB. We observed that interleukin-1beta induced the degradation of the inhibitor kappaB-alpha protein and the translocation of the protein p65 (a member of the nuclear factor-kappaB family) to the nucleus, which were inhibited by diacerhein and rhein, in a dose-dependent manner. Interleukin-1beta-induced nuclear factor-kappaB binding to a specific (gamma-(32)P)-labelled oligonucleotide probe was also inhibited by treatment of chondrocytes with diacerhein or rhein, as revealed by electrophoretic mobility shift assay. Inducible nitric oxide synthase mRNA and protein synthesis and nitric oxide production were also inhibited by diacerhein and rhein, in a dose-dependent manner. The half-maximal inhibitory concentrations of diacerhein and rhein, relative to nitric oxide production, were 8.2 microM ;and 7.7 microM, respectively. These results suggest that diacerhein and rhein inhibit nuclear factor-kappaB activation and, consequently, the expression of nuclear factor-kappaB-dependent genes, such as the inducible nitric oxide synthase gene, which can explain their antiosteoarthritic and antiinflammatory effects.

Carbamazepine (CBZ) has been extensively used in the treatment of epilepsy, as well as in the treatment of neuropathic pain and affective disorders. However, the mechanisms of action of this drug are not completely elucidated and are still a matter of debate. Since CBZ is not very effective in some epileptic patients and may cause several adverse effects, several antiepileptic drugs have been developed by structural variation of CBZ, such as oxcarbazepine (OXC), which is used in the treatment of epilepsy since 1990. (S)-(−)-10-acetoxy-10,11-dihydro-5H-dibenz[b,f]azepine-5-carboxamide (BIA 2-093) and 10,11-dihydro-10-hydroxyimino-5H-dibenz[b,f]azepine-5-carboxamide (BIA 2-024), which were recently developed by BIAL, are new putative antiepileptic drugs, with some improved properties. In this review, we will focus on the mechanisms of action of CBZ and its derivatives, OXC, BIA 2-093 and BIA 2-024. The available data indicate that the anticonvulsant efficacy of these AEDs is mainly due to the inhibition of sodium channel activity.

We investigated the role of kainate (KA) receptor activation and desensitization in inducing the increase in the intracellular free Ca2+ concentration ([Ca2+]i) in individual cultured rat hippocampal neurons. The rat hippocampal neurons in the cultures were shown to express kainate receptor subunits, KA2 and GluR6/7, either by immunocytochemistry or by immunoblot analysis. The effect of LY303070, an α-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) receptor antagonist, on the alterations in the [Ca2+]i caused by kainate showed cell-to-cell variability. The [Ca2+]i increase caused by kainate was mostly mediated by the activation of AMPA receptors because LY303070 inhibited the response to kainate in a high percentage of neurons. The response to kainate was potentiated by concanavalin A (Con A), which inhibits kainate receptor desensitization, in 82.1% of the neurons, and this potentiation was not reversed by LY303070 in about 38% of the neurons. Also, upon stimulation of the cells with 4-methylglutamate (MGA), a selective kainate receptor agonist, in the presence of Con A, it was possible to observe [Ca2+]i changes induced by kainate receptor activation, because LY303070 did not inhibit the response in all neurons analyzed. In toxicity studies, cultured rat hippocampal neurons were exposed to the drugs for 30 min, and the cell viability was evaluated at 24 hr using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The selective activation of kainate receptors with MGA, in the presence of Con A, induced a toxic effect, which was not prevented by LY303070, revealing a contribution of a small subpopulation of neurons expressing kainate receptors that independently mediate cytotoxicity. Taken together, these results indicate that cultured hippocampal neurons express not only AMPA receptors, but also kainate receptors, which can modulate the [Ca2+]i and toxicity. J. Neurosci. Res. 65:378–386, 2001. © 2001 Wiley-Liss, Inc.

We investigated the mechanism(s) of action of two new putative antiepileptic drugs (AEDs), (S)-(-)-10-acetoxy-10,11-dihydro-5H-dibenz[b,f]azepine-5-carboxamide (BIA 2-093) and 10,11-dihydro-10-hydroxyimino-5H-dibenz[b,f]azepine-5-carboxamide (BIA 2-024), by comparing their effects on the release of endogenous glutamate in hippocampal synaptosomes, with those of carbamazepine (CBZ) and oxcarbazepine (OXC). The AEDs inhibited the release of glutamate evoked by 4-aminopyridine (4-AP) or veratridine in a concentration-dependent manner, being CBZ more potent than the other AEDs. Using conditions of stimulation (30 mM KCl), where Na(+) channels are inactivated, the AEDs did not inhibit either the Ca(2+)-dependent or -independent release of glutamate. The results indicate that BIA 2-093 and BIA 2-024 have sodium channel-blocking properties, but CBZ and OXC are more potent than the new AEDs. Moreover, the present data also indicate that Ca(2+) channels coupled to the exocytotic release of glutamate and the activity of the glutamate transporter were not affected by the AEDs.

We used botulinum neurotoxins (BoNT) to examine whether differences in the secretory activity of noradrenergic and adrenergic chromaffin cells are related to differences in the exocytotic machinery of these two types of bovine adrenal medulla cells. Cleavage of syntaxin and SNAP-25 by BoNT/C1 decreased in a dose-dependent way the release of both noradrenaline and adrenaline, but noradrenaline release was more sensitive to BoNT/C1. Cleavage of SNAP-25 by BoNT/A also had a larger inhibitory effect on noradrenaline release than on adrenaline release. Neither BoNT/C1 nor BoNT/A affected the intracellular Ca2+ responses induced by K+-depolarisation, and the extent of the inhibition of K+-evoked catecholamine release by selective blockers of voltage-gated Ca2+ channels was not affected by BoNT/C1. Therefore, our data do not support the hypothesis of a regulatory effect of syntaxin or SNAP-25 on the activity of Ca2+ channels. The lower sensitivity of adrenaline release to BoNT was not due to a reduced ability of the toxins to enter or to cleave their protein targets in adrenergic cells, since immunoblot analysis showed the cleavage of a larger fraction of syntaxin 1A in adrenergic cells, as compared to the cleavage in noradrenergic cells. The immunoblot analysis also showed larger amounts of syntaxin 1A in noradrenergic chromaffin cells than in adrenergic cells. Thus, in spite of a greater cleavage of syntaxin 1A in adrenergic cells by BoNT/C1, adrenaline release was less sensitive to BoNT/C1, suggesting that the release process in noradrenergic cells might be more dependent on syntaxin 1A and SNAP-25, as compared to adrenergic cells.

The AMPA receptors for glutamate are oligomeric structures that mediate fast excitatory responses in the central nervous system. Phosphorylation of AMPA receptors is an important mechanism for short-term modulation of their function, and is thought to play an important role in synaptic plasticity in different brain regions. Recent studies have shown that phosphorylation of AMPA receptors by cAMP-dependent protein kinase (PKA) and Ca2+ - and calmodulin-dependent protein kinase II (CaMKII) potentiates their activity, but phosphorylation of the receptor subunits may also affect their interaction with intracellular proteins, and their expression at the plasma membrane. Phosphorylation of AMPA receptor subunits has also been investigated in relation to processes of synaptic plasticity. This review focuses on recent advances in understanding the molecular mechanisms of regulation of AMPA receptors, and their implications in synaptic plasticity.

Several evidences suggest that the pro-inflammatory cytokines IL-1b and the radical NO are implicated as effectors molecules in the pancreatic b-cells dysfunction; an event preceding the pathogenesis of diabetes. IL-1b induces the expression of the inducible isoform of NO synthase (iNOS), which use L-arginine as substrate to overproduce NO. However, it is not known whether these events may participate in the development of diabetic retinopathy, which is the main cause of blindness. In this work, we found an increased level of IL-1b in retinas from streptozotocin-induced (STZ) diabetic rats. We also observed that the activity of the NO synthase (NOS) and the L-arginine uptake are enhanced in retinas from STZ-induced diabetic rats as compared to retinas from control rats. We found that the uptake of L-arginine in retinas from control and diabetic rats occurs through a transporter resembling the Y system, i.e. it is saturable, not affected over the pH range 6.5 to 7.4, and is independent of the extracellular Na. Nevertheless, the L-arginine transport in retinas from diabetic rats occurs through a carrier with lower affinity (Km25 mM) and higher capacity (Vmax295922.4 pmol L-arginine:mg protein) than in retinas from control rats (Km 5 mM and Vmax158912.8 pmol L-arginine:mg protein) which is correlated with the increased NOS activity and consequent depletion of the intracellular pool of L-arginine. © 1999 Elsevier Science Ltd. All rights reserved.

The Dale's law postulates that a neuron releases the same neurotransmitter from all its branches. In the case of mulitple neurotransmitters it would require all transmitters to be released from all branches. The retinal cholinergic amacrine cells contain and release &ggr;-aminobutyric (GABA) and, therefore, if GABA and acetylcholine (ACh) are released at the same sites, this could mean that amacrine cells simultaneously excite and inhibit postsynaptic cells. Conversely, if the two neurotransmitters are released at different synapses, or if their release is regulated in a distinct manner, they may play different physiological roles. Recent studies carried out in cultured cholinergic amacrine-like neurons showed that Ca2+-dependent release of ACh and GABA have a different sensitivity to membrane depolarization, to the effect of blockers of voltage gated Ca2+ channels (VGCC) and to the effect of presynaptic A1 adenosine receptors. Therefore, it is proposed that in retinal amacrine cells the Ca2+-dependent release of ACh and GABA occurs at distinct cellular locations. The possible nature of these release sites and the physiological significance of this model are discussed in this review.J. Neurosci. Res. 58: 475-479, 1999. © 1999 Wiley-Liss, Inc.

Organophosphate and carbamate insecticides are inhibitors of cholinesterases (ChE). The depression of blood ChE activity is frequently used as indicative of exposure to these chemicals. However, it is not known whether the inhibition of blood ChE activity reflects the inhibition of ChE in target tissues (e.g. brain and muscle). In this study we investigated the possibility of using whole blood ChE activity to predict frontal cerebral cortex ChE activity in rats treated with parathion. Twenty four hours after the intraperitoneal administration of several doses of parathion, the activity of ChE in whole blood and the activity of ChE in frontal cerebral cortex were determined in each animal. A high correlation between the two parameters was found (r = 0.96, p < 0.05) and the model of linear regression fitted to the data accounted for 93% of its variability. Thus, these results seem to indicate that 24 hours after the treatment with parathion the effects induced on whole blood ChE activity may be used to predict the effects caused on frontal cerebral cortex ChE activity.

The changes in the intracellular free Ca2+ concentration, [Ca2+]i, mediated by glutamate and D-aspartate into rat hippocampal synaptosomes was studied. Glutamate increased the [Ca2+]i in a dose-dependent manner with an EC50 of 1.87 [mu]M and a maximal increase of 31.5±0.9 nM. We also observed that stimulation of the synaptosomes with 100 [mu]M [alpha]-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), 100 [mu]M kainate, or 100 [mu]M D-aspartate increased the synaptosomal [Ca2+]i. The effect of either of these non-NMDA receptor agonists and of D-aspartate was additive, suggesting the activation of two different components (the ionotropic non-NMDA receptors or the glutamate transporters). Stimulation of synaptosomes with 100 [mu]M glutamate increased the [Ca2+]i and prevented the effect of either non-NMDA receptor agonists and the effect of D-aspartate. We also observed that incubation of the synaptosomes with D-aspartate induced the Ca2+-independent release of glutamate, possibly through the reversal of the glutamate carrier. The aim of incubating the synaptosomes with D-aspartate was to avoid undesirable secondary activation of glutamate receptors. After incubating the synaptosomes with 100 [mu]M D-aspartate (10 min at 37°C), the subsequent stimulation with D-aspartate increased the [Ca2+]i due to glutamate transport. this increase in [Ca2+]i induced by 100 [mu]M D-aspartate was insensitive to 1 [mu]M nitrendipine, but was inhibited by about 50% by the presence of both 500 nM [omega]-CgTx GVIA and 100 nM [omega]-Aga IVA or by 500 nM [omega]-CgTx MVIIC. We clearly identified two different processes by which glutamate increased the [Ca2+]i in rat hippocampal synaptosomes: activation of non-NMDA receptors and activation of the glutamate transporters. We also characterized the voltage sensitive Ca2+ channels (VSCC) activated as a consequence of the glutamate transport, and determined that class B (N-type) and class A (P or Q-type) Ca2+ channels were responsible for about 50% of the signal. http://www.sciencedirect.com/science/article/B6T0B-3TDPXHV-1W/1/c4f513cb79dab43fc097b11df729bf79

A continuous fluorometric assay was adapted to measure the release of endogenous glutamate from cultured chick retinospheroids. The results obtained with this technique are compared with the release of [3H]d-aspartate from monolayer cultures of chick retina cells. It is shown that although excitatory amino acids may be released in a Ca2+-dependent manner, most of the neurotransmitter release from cultured retina cells occurs by reversal of the glutamate transporter. The presence of extracellular Ca2+ may actually inhibit glutamate release by the cells present in the retinospheroids, or the [3H]d-aspartate release by cells in monolayers, when veratridine is the depolarizing agent. Copyright © 1996 Elsevier Science Ltd.

Background and Purpose It is known that the extracellular accumulation of glutamate during anoxia/ischemia is responsible for initiating neuronal injury. However, little information is available on the release of monoamines and whether the mechanism of its release resembles that of glutamate, which may itself influence the release of monoamines by activating presynaptic receptors. This study was designed to characterize the release of both amino acids and monoamines under chemical conditions that mimic anoxia, hypoglycemia, and ischemia. Methods The contents of synaptosomes in adenine nucleotides (ATP, ADP, and AMP), amino acids (aspartate, glutamate, taurine, and γ-aminobutyric acid), and monoamines (dopamine, noradrenaline, and 5-hydroxytryptamine) were measured by high-performance liquid chromatography, after the synaptosomes were subjected to anoxia (KCN+oligomycin), hypoglycemia (2 mmol/L 2-deoxyglucose in glucose-free medium), and ischemia (anoxia plus hypoglycemia). Results The anoxia- and ischemia-induced release of noradrenaline, dopamine, 5-hydroxytryptamine, and glutamate correlated well with ATP depletion. The correlation observed between glutamate levels and the release of dopamine and 5-hydroxytryptamine in ischemic conditions suggests a functional linkage between the two transmitter systems. However, the antagonists of presynaptic glutamate receptors failed to alter the amount of monoamines released. The inhibition of Na + ,K + -ATPase by ouabain had an effect similar to that produced by ischemia. Conclusions The decrease in Na + and K + gradients resulting from the energy depletion of the synaptosomes under ischemic conditions or resulting from the inhibition of Na + ,K + -ATPase by ouabain promotes the reversal of the neurotransmitter transporters. The decrease in uptake of neurotransmitters may also contribute to the rise in the extracellular concentration of different transmitters observed during brain ischemia.

The effect of glutamate receptor agonists on the intracellular free calcium concentration ([Ca2+]i), measured with Indo-1, was studied in populations of cultured chick embryonic retina cells. The agonists of the ionotropic glutamate receptors,N-methyl-d-aspartate (NMDA), kainate, and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) increased the [Ca2+]i through a composite effect, comprising Ca2+ permeating the receptor-associated channels, and Ca2+ entering through voltage-gated Ca2+ channels. Furthermore, the [Ca2+i responses to NMDA and AMPA also involved Ca2+ release from intracellular stores, which could not be mobilized by stimulation of the metabotropic receptor.

In this work we investigated the possibility of using the enzyme acetylcholinesterase (AChE) activity in Daphnia magna homogenates, both in vivo and in vitro conditions, as a specific method for rapid toxicity evaluations. The results from in vivo and in vitro AChE inhibition tests were compared with 48 hours EC50 values obtained in conventional acute bioassays. EC50 values from in vivo AChE inhibition tests were: 2.4 micrograms/l for parathion, 0.2 microgram/l for paraoxon; DCA and cadmium at the concentrations tested had no effects on enzyme activity. I50 values were 764 micrograms/l for parathion, 0.08 micrograms/l for paraoxon and 3367 micrograms/l for cadmium; DCA did not affect AChE activity measured in in vitro conditions. EC50 values from conventional acute tests were: 2.2 micrograms/l for parathion, 0.2 microgram/l for paraoxon, 163 micrograms/l for DCA and 9.5 micrograms/l for cadmium. Our results indicated that the in vivo AChE inhibition test is selective, being very sensitive to detect toxicity of the organophosphates tested. The in vitro AChE inhibition assay is less time consuming, requires less human effort and produces less toxic waste than conventional acute bioassays and the in vivo AChE inhibition test. However, it does not take into account the effect of the metabolization of the toxicants inside live organisms; since the organophosphate metabolism may be activative or degradative, the toxic potential of the parent compound may be under or over evaluated in in vitro conditions.

Glutamate and NPY have been implicated in hippocampal neuropathology in temporal lobe epilepsy. Thus, we investigated the involvement of NPY receptors in mediating neuroprotection against excitotoxic insults in organotypic cultures of rat hippocampal slices. Exposure of hippocampal slice cultures to 2 μM AMPA (α-amino-3-hydroxy-5-methyl-isoxazole-4- propionate) induced neuronal degeneration, monitored by propidium iodide uptake, of granule cells and CA1 pyramidal cells. For dentate granule cells, selective activation of Y1, Y2, or Y5 receptors with 1 μM [Leu31,Pro34]NPY, 300 nM NPY13–36 or 1 μM 500 nM NPY(19–23)- 1 3 4 6 31 32 34 (Gly ,Ser ,Gln ,Thr ,Ala ,Aib ,Gln )-PP, respectively, had a neuroprotective effect against AMPA, whereas only the activation of Y2 receptors was effective for CA1 pyramidal cells. When the slice cultures were exposed to 6 μM kainate, the CA3 pyramidal cells displayed significant degeneration, and in this case the activation of Y1, Y2, and Y5 receptors was neuroprotective. For the kainic acid-induced degeneration of CA1 pyramidal cells, it was again found that only the Y2 receptor activation was effective. Based on the present findings, it was concluded that Y1, Y2, and Y5 receptors effectively can modify glutamate receptor-mediated neurodegeneration in the hippocampus

Rat thymic lymphocytes were used to determine the cytotoxic effect of the two toxicants, 3,4-dichloroaniline (DCA) and sodium bromide (NaBr), at concentrations that have effects in the 21-day reproduction test with Daphnia magna. To evaluate the effect of the chemical compounds on cell membrane integrity, we measured the permeability of the thymocytes to trypan blue and the leakage of lactate dehydrogenase (LDH) from the cells. The viability and metabolic activity of the thymocytes were quantified by the MTT assay. The DCA concentrations tested (from 31 to 310 nm) did not affect significantly the viability of thymocytes, as determined by trypan blue exclusion or by LDH leakage; however, with the MTT assay we could detect cytotoxicity at 62 nm. Both the trypan blue assay and the LDH assay indicated that concentrations of NaBr above 50 mm significantly affected the viability of the thymocytes. However, the MTT assay revealed a more pronounced toxic effect of NaBr, with significant cell damage being detected at 1 mm.

We investigated the contribution of L-, N- and P/Q-type Ca(2+) channels to the [Ca(2+)](i) changes, evoked by kainate, in the cell bodies of hippocampal neurons, using a pharmacological approach and Ca(2+) imaging. Selective Ca(2+) channel blockers, namely nitrendipine, omega-Conotoxin GVIA (omega-GVIA) and omega-Agatoxin IVA (omega-AgaIVA) were used. The [Ca(2+)](i) changes evoked by kainate presented a high variability, and were abolished by NBQX, a AMPA/kainate receptor antagonist, but the N-methyl-D-aspartate (NMDA) receptor antagonist, D-AP5, was without effect. Each Ca(2+) channel blocker caused differential inhibitory effects on [Ca(2+)](i) responses evoked by kainate. We grouped the neurons for each blocker in three subpopulations: (1) neurons with responses below 60% of the control; (2) neurons with responses between 60% and 90% of the control, and (3) neurons with responses above 90% of the control. The inhibition caused by nitrendipine was higher than the inhibition caused by omega-GVIA or omega-AgaIVA. Thus, in the presence of nitrendipine, the percentage of cells with responses below 60% of the control was 41%, whereas in the case of omega-GVIA or omega-AgaIVA the values were 9 or 17%, respectively. The results indicate that hippocampal neurons differ in what concerns their L-, N- and P/Q-type Ca(2+) channels activated by stimulation of the AMPA/kainate receptors.

Nuclear factor kappa B (NF-kB) and activating protein-1 (AP-1) transcription factors are ubiquitously expressed signaling molecules known to regulate the transcription of a large number of genes involved in immune responses, namely the inducible isoform of nitric oxide synthase (iNOS). In this study, we demonstrate that a fetal skin-derived dendritic cell line (FSDC) produces nitric oxide (NO) in response to the contact sensitizer nickel sulfate (NiSO(4)) and increases the expression of the iNOS protein, as determined by immunofluorescence and Western blot analysis. The sensitizer NiSO(4) increased cytoplasmic iNOS expression by 31.9 +/- 10.3% and nitrite production, as assayed by the Griess reaction, by 27.6 +/- 9.5%. Electrophoretic mobility shift assay (EMSA), showed that 30 min of FSDC exposure to NiSO(4) activates the transcription factor NF-kB by 58.2 +/- 7.0% and 2 h of FSDC exposure to NiSO(4) activates the transcription factor AP-1 by 26.0 +/- 1.4%. Together, these results indicate that NiSO(4) activates the NF-kB and AP-1 pathways and induces iNOS expression in skin dendritic cells.

The pro-inflammatory cytokine interleukin-1beta (IL-1) induces articular chondrocytes to produce reactive oxygen species (ROS), including hydrogen peroxide (H2O2), which mediate some IL-1-induced responses. This study aimed at elucidating the role of ROS, particularly H2O2, in mediating IL-1-induced activation of the transcription factor activator protein-1 (AP-1) in primary cultures of articular chondrocytes. AP-1 may function either as an inducer or as a repressor of the inducible nitric oxide synthase (iNOS) gene promoter. Since we observed that AP-1 is not required for iNOS expression in chondrocytes, we also investigated whether it is a repressor of this gene. The results of electrophoretic mobility shift assays showed that both IL-1 and H2O2 activated AP-1 and that inhibition of IL-1-induced ROS production abrogated AP-1 activation. The AP-1 complexes, induced by either IL-1 or H2O2, contained c-Fos/c-Jun and c-Fos/JunD heterodimers, but IL-1 activated AP-1 with a kinetics slower than that observed with H2O2. Pre-activation of AP-1, before stimulation of the cells with IL-1, did not inhibit iNOS mRNA and protein synthesis, relative to cells treated with IL-1 alone. These results indicate that H2O2 is a major mediator of IL-1-induced AP-1 activation in articular chondrocytes and that inhibition of ROS production is an effective strategy to block this IL-1-induced response. This study also identifies c-Fos/c-Jun and c-Fos/JunD heterodimers as the AP-1 transcription factors induced by IL-1, which, although not involved in the transcriptional regulation of the iNOS gene, may be important for the regulation of other genes also relevant in arthritic diseases, namely the collagenase-1 and IL-8 genes.

We investigated the functional interaction between neuropeptide Y (NPY) receptors using nerve terminals and cultured rat hippocampal neurons, and we evaluated the involvement of voltage-gated Ca2+ channels (VGCCs) in NPY receptors-induced inhibition of Ca2+ influx and glutamate release. The KCl-evoked release of glutamate from hippocampal synaptosomes was inhibited by 1 [mu]M NPY and this effect was insensitive to either BIBP3226 (Y1 receptor antagonist) or L-152,804 (Y5 receptor antagonist), but was sensitive to BIIE0246 (Y2 receptor antagonist). We could also pharmacologically dissect the NPY receptors activity by using Y1, Y2 and Y5 receptor agonists ([Leu31,Pro34]NPY, NPY13-36, NPY (19-23)-(Gly1,Ser3,Gln4,Thr6,Ala31,Aib32,Gln34)-pancreatic polypeptide (PP), respectively), and in all the cases we observed that these agonists could inhibited the KCl-induced release of glutamate. However, the selective and specific co-activation of both Y1 and Y2 or Y2 and Y5 receptors resulted in non-additive inhibition, and this effect was prevented in the presence of the Y2 antagonist, but was insensitive to the Y1 or Y5 receptor antagonist. Moreover, as we previously showed for Y1 receptors, we also observed that the activation of Y5 receptors inhibited the glutamate release in the dentate gyrus and CA3 subregion, without significant effect in the CA1 subregion of the hippocampus. The same qualitative results were obtained when we investigated the role of NPY Y1 and Y2 receptors in modulating the changes in [Ca2+]i due to KCl depolarisation in cultured hippocampal neurons. The inhibitory effect of nitrendipine (L-type VGCC blocker) or [omega]-conotoxin GVIA ([omega]-CgTx; N-type VGCC blocker) was not potentiated by the simultaneous activation of Y1 or Y2 receptors. Moreover, the exocytotic release of glutamate was inhibited by [omega]-agatoxin IVA ([omega]-Aga; P-/Q-type VGCC blocker), and this VGCC blocker did not potentiate Y1, Y2 or Y5 receptor-mediated inhibition of glutamate release. Also, the effect of ionomycin in inducing the exocytotic release of glutamate from hippocampal synaptosomes was insensitive to the activation of NPY receptors. In the present paper, we identified a role for NPY Y1, Y2 and Y5 receptors in modulating the exocytotic release of glutamate and the [Ca2+]i changes in the rat hippocampus. In conditions of co-activation, there appears to exist a physiological cross-talk between Y1 and Y2 and also between Y2 and Y5 receptors, in which Y2 receptors play a predominant role. Moreover, we also show that Y1 and Y2 receptors exert their inhibitory action by directly modulating L-, N-, and P-/Q-type VGCCs, whereas the inhibition of glutamate release mediated by the Y5 receptors seems to involve P-/Q-type VGCCs. http://www.sciencedirect.com/science/article/B6T0C-47RRRP0-C/1/70e1c3a1dbd6c37bea36062e48bf4a01

In this work, a dendritic cell line derived from mouse skin (FSDC) was used, as an in vitro experimental model, to evaluate the cytotoxic effect of two chemical sensitizers, a strong sensitizer (2,4-dinitrofluorobenzene, DNFB) and a weak sensitizer (2,4-dichloronitrobenzene, DCNB). The results indicated that DNFB reduces the cellular metabolism of FSDC, as evaluated by the reduction of the tetrazolium salt, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). All the DNFB concentrations tested, ranging from 5.2 micro M to 26 micro M, significantly inhibited the MTT reduction after 1 hour of cell exposure to the sensitizer. In contrast, incubation of FSDC with the weak sensitizer DCNB had no significant effect on the MTT reduction assay. When the cells were incubated with DNFB (13 micro M), for 3 and 6 hours, morphological changes characteristics of cell death by apoptosis were observed, as assessed by propidium iodide (PI) DNA staining and annexin-V externalization analysis. These results correlate well with an increase of caspase-3-like activity after FSDC exposure to DNFB (13 micro M) for 6 hours. Together, these results indicate that apoptotic death of skin dendritic cells occurs after exposure to the sensitizer DNFB, although necrotic cell death was also observed when the cells were incubated with high concentrations of DNFB (26 micro M), or after long periods of cell exposure to the chemical DNFB (13 micro M, for 6 hours).

The role of protein tyrosine kinases on glutamate release was investigated by determining the effect of broad range inhibitors of tyrosine kinases on the release of glutamate from rat hippocampal synaptosomes. We found that lavendustin A and herbimycin A did not inhibit glutamate release stimulated by 15 mM KCl, but genistein, also a broad range inhibitor of tyrosine kinases did inhibit the intracellular Ca(2+) concentration response to KCl and, concomitantly, decreased glutamate release evoked by the same stimulus, in a dose-dependent manner. These effects were not observed with the inactive analogue genistin. Therefore, we investigated the mechanism whereby genistein modulates Ca(2+) influx and glutamate release. Studies with voltage-gated Ca(2+) channel inhibitors showed that omega-conotoxin GVIA did not further inhibit glutamate release or the Ca(2+) influx stimulated by KCl in the presence of genistein. This tyrosine kinase inhibitor and omega-agatoxin IVA had a partially additive effect on those events. Nitrendipine did not reduce significantly the KCl-induced responses. Genistein further reduced Ca(2+) influx in response to KCl in the presence of nitrendipine, omega-conotoxin GVIA and omega-agatoxin IVA, simultaneously. The effect of tyrosine phosphatase inhibitors was also tested on the influx of Ca(2+) and on glutamate release stimulated by KCl-depolarization. We found that the broad range inhibitors sodium orthovanadate and dephostatin did not significantly affect these KCl-evoked events. Our results suggest that genistein inhibits glutamate release and Ca(2+) influx in response to KCl independently of tyrosine kinase inhibition, and that tyrosine kinases and phosphatases are not key regulators of glutamate release in hippocampal nerve terminals.

Aims:Annexin I (ANXA1), a 37 kDa member of the annexin family of Ca2+-binding and phospholipidbinding proteins, is particularly abundant in various populations of peripheral blood leukocytes. Since this protein modulates the anti-inflammatory actions of the steroid hormones, the purpose of this study was to investigate the effects of the female sex steroid hormone, 17β-estradiol (E2β), on the synthesis and secretion of ANXA1 in the human CCRF-CEM acute lymphoblastic leukemia cell line.Methods:Complementary reverse transcription-polymerase chain reaction and Western blot assays were performed to study the effect of E2β on the expression of mRNA and protein ANXA1, respectively.Results and discussion:Treatment of CCRF-CEM cells with E2β, for 30 min, stimulated the synthesis of ANXA1 mRNA molecules, and increased the cellular level of ANXA1 protein. Moreover, when the cells were incubated with E2β, under the same experimental conditions, a significant increase in the amount of ANXA1 secreted from the cells was also detected. ICI 182,780, a selective inhibitor of the intracellular estrogen receptor, had no effect on the E2β-stimulated expression and externalisation of ANXA1. Taken together, these results indicate that E2β inducesde novosynthesis of ANXA1 and stimulates its secretion in the CCRF-CEM cell line, apparently through a mechanism independent of the intracellular estrogen receptor.

Nitric oxide (NO) produced by skin dendritic cells and keratinocytes plays an important role in skin physiology, growth and remodelling. Nitric oxide is also involved in skin inflammatory processes and in modulating antigen presentation (either enhancing or suppressing it). In this study, we found that GM-CSF stimulates the expression of the inducible isoform of nitric oxide synthase (iNOS) in a fetal-skin-derived dendritic cell line (FSDC) and, consequently, increases the nitrite production from 11.9 +/- 3.2 micromol/L (basal level) to 26.9 +/- 4.2 micromol/L. Pyrrolidinedithiocarbamate (PDTC) inhibits nitrite production, with a half maximal inhibitory concentration (IC50) of 19.3 micromol/L and the iNOS protein expression in FSDC. In addition, western blot assays revealed that exposure of FSDC to GM-CSF induces the phosphorylation and degradation of the inhibitor of NF-kappaB (IkB), with subsequent translocation of the p50, p52 and RelB subunits of the transcription nuclear factor kappa B (NF-kappaB) from the cytosol to the nucleus. Electrophoretic mobility shift assays (EMSA) showed that FSDC exposure to GM-CSF activates the transcription factor NF-kappaB. Together, these results show that GM-CSF induces iNOS expression in skin dendritic cells by a mechanism involving activation of the NF-kappaB pathway.

The aim of this work was to investigate whether excitotoxicity induced by overstimulation of different ionotropic glutamate receptors could trigger different intracellular signaling cascades. Cultured chick neuronal retina cells, essentially amacrine-like, were particularly sensitive to the toxicity induced by non-NMDA glutamate receptor agonists. One hour stimulation with 100 muM kainate induced a reduction of cell viability of about 44%, as assessed by the MTT test 24 hr after stimulation. Kainate-induced toxicity was mediated through AMPA receptors. Glutamate (100 muM, 1 hr) reduced cell viability by 26%, essentially acting through N-methyl-D-aspartate receptors. Five hours after stimulation, neuronal retina cells had an apoptotic-like nuclear morphology. In retinal neurons, the excitotoxic stimulation, with either glutamate or kainate, induced a calcium-dependent enhancement of the DNA-binding activity of the activating protein-1 (AP-1) transcription factor, which was maximal 2 hr after stimulation. Glutamate induced a greater increase in the AP-1 DNA-binding activity than did kainate. Supershift assays using antibodies directed against different members of the Fos and Jun protein families showed that the AP-1 complex in retinal neurons includes proteins of the Fos family, namely, Fra-2, c-Jun, and Jun D. The DNA-binding activity of the nuclear factor-kappaB transcription factor was not significantly changed upon excitotoxic stimulation with any agonist. Stimulation of glutamate receptors with 100 muM kainate or 100 muM glutamate for 2 min was sufficient to induce the activation of the extracellular signal-regulated kinase (ERK). Inhibition of the ERK activation with the MEK inhibitors U 0126 and PD 98059 increased the toxicity induced by kainate but was without effect on the toxicity induced by glutamate. These results indicate that, although stimulation with both glutamate receptor agonists increased ERK phosphorylation, only kainate-induced ERK activation correlates with the activation of a survival signaling pathway. Our results suggest that, in chick embryo retinal neurons, the signaling pathways that mediate excitotoxic cell death and neuroprotection are stimulus specific. J. Neurosci. Res. 66:643-655, 2001. © 2001 Wiley-Liss, Inc.

We investigated and compared the toxicity profile, as well as possible neuroprotective effects, of some antiepileptic drugs in cultured rat hippocampal neurons. We used two novel carbamazepine derivatives, (S)-(-)-10-acetoxy-10,11-dihydro-5H-dibenz[b,f]azepine-5-carboxamide (BIA 2-093) and 10,11-dihydro-10-hydroxyimino-5H-dibenz[b,f]azepine-5-carboxamide (BIA 2-024), and compared their effects with the established compounds carbamazepine and oxcarbazepine. The assessment of neuronal injury was made by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl (MTT) assay, as well as by analysing morphology and nuclear chromatin condensation (propidium iodide staining), after hippocampal neurons were exposed to the drugs for 24 h. The putative antiepileptic drugs, BIA 2-093 or BIA 2-024 (at 300 [mu]M), only slightly decreased MTT reduction, whereas carbamazepine or oxcarbazepine were much more toxic at lower concentrations. Treatment with the antiepileptic drugs caused nuclear chromatin condensation in some neurons, which is characteristic of apoptosis, and increased the activity of caspase-3-like enzymes, mainly in neurons treated with carbamazepine and oxcarbazepine. The toxic effect caused by carbamazepine was not mediated by N-methyl--aspartate (NMDA) or by [alpha]-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) receptors. Moreover, the antiepileptic drugs failed to protect hippocampal neurons from the toxicity caused by kainate, veratridine, or ischaemia-like conditions. http://www.sciencedirect.com/science/article/B6T1J-41B7099-4/1/bea648de55f3f3e30bc78c53e37f394d

We investigated the effect of adenosine A1 receptors on the release of acetylcholine (ACh) and GABA, and on the intracellular calcium concentration ([Ca2+]i) response in cultured chick amacrine-like neurons, stimulated by KCl depolarization. The KCl-induced release of [3H]ACh, but not the release of [14C]GABA, was potentiated when adenosine A1 receptor activation was prevented by perfusing the cells with adenosine deaminase (ADA) or with 1,3-dipropyl-8-cycloentylxanthine (DPCPX). The changes in the [Ca2+]i induced by KCl depolarization, measured in neurite segments of single cultured cells, were also modulated by endogenous adenosine, acting on adenosine A1 receptors. Our results show that adenosine A1 receptors inhibit Ca2+ entry coupled to ACh release, but not to the release of GABA, suggesting that the synaptic vesicles containing each neurotransmitter are located in different zones of the neurites, containing different VSCC and/or different densities of adenosine A1 receptors. http://www.sciencedirect.com/science/article/B6SYR-3Y6Y47X-2/1/84f62dfae41c6a6377492d716b4449b8

Adenosine triphosphate (ATP) has been proposed to play a role as a neurotransmitter in the retina, but not much attention has been given to the regulation of ATP release from retinal neurons. In this work, we investigated the release of ATP from cultures enriched in amacrine-like neurons. Depolarization of the cells with KCl, or activation of alpha-amino-3-hydroxy- 5-methyl-4-isoxazole-propionate (AMPA) receptors, evoked the release of ATP, as determined by the luciferin/luciferase luminescent method. The ATP release was found to be largely Ca2+ dependent and sensitive to the botulinum neurotoxin A, which indicates that the ATP released by cultured retinal neurons originated from an exocytotic pool. Nitrendipine and omega-Agatoxin IVA, but not by omega-Conotoxin GVIA, partially blocked the release of ATP, indicating that in these cells, the Ca2+ influx necessary to trigger the release of ATP occurs in part through the L- and the P/Q types of voltage-sensitive Ca2+ channels (VSCC), but not through N-type VSCC. The release of ATP increased in the presence of adenosine deaminase, or in the presence of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), an adenosine A1 receptor antagonist, showing that the release is tonically inhibited by the adenosine A1 receptors. To our knowledge, this is the first report showing the release of endogenous ATP from a retinal preparation. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 340-348, 1999

We compared the effects of sodium nitroprusside (SNP), and of 8-bromo guanosine 3',5'-cyclic monophosphate (8-BrcGMP), on the 4-aminopyridine (4-AP)-evoked Ca2+-dependent release of glutamate from hippocampal nerve terminals and further investigated the role of protein kinase G (PKG) in this mechanism. SNP and 8-BrcGMP dose-dependently inhibited glutamate release, however SNP concentrations ([SNP])>500 [mu]M abolished the 4-AP evoked release, whereas 8-BrcGMP maximally inhibited the release by about 30%. The inhibition of glutamate release at low concentrations of SNP (50 [mu]M strongly inhibited glutamate release, and this was not reversed by either inhibitor. Furthermore, [SNP]

In order to better understand the mechanism(s) of action of carbamazepine (CBZ), we studied its effects on the increase in [Ca2+]i and [Na+]i stimulated by glutamate ionotropic receptor agonists, in cultured rat hippocampal neurons, as followed by indo-1 or SBFI fluorescence, respectively. CBZ inhibited the increase in [Ca2+]i stimulated either by glutamate, kainate, [alpha]-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA), or N-methyl--aspartate (NMDA), in a concentration-dependent manner. In order to discriminate the effects of CBZ on the activation of glutamate receptors from possible effects on Ca2+ channels, we determined the inhibitory effects of Ca2+ channel blockers on [Ca2+]i changes in the absence or in the presence of CBZ. The presence of 1 [mu]M nitrendipine, 0.5 [mu]M [omega]-conotoxin GVIA ([omega]-CgTx GVIA), or of both blockers, inhibited the kainate-stimulated increase in [Ca2+]i by 51.6, 32.9 or 68.7%, respectively. In the presence of both 100 [mu]M CBZ and nitrendipine, the inhibition was similar (54.1%) to that obtained with nitrendipine alone, but in the presence of both CBZ and [omega]-CgTx GVIA, the inhibition was greater (54%) than that caused by [omega]-CgTx GVIA alone. However, CBZ did not inhibit the increase in [Na+]i stimulated by the glutamate receptor agonists, but inhibited the increase in [Na+]i due to veratridine. Tetrodotoxin, or MK-801, did not inhibit the influx of Na+ stimulated by kainate, indicating that Na+ influx occurs mainly through the glutamate ionotropic non-NMDA receptors. Moreover, LY 303070, a specific AMPA receptor antagonist, inhibited the [Na+]i response to kainate or AMPA by about 70 or 80%, respectively, suggesting that AMPA receptors are mainly involved. Taken together, the results suggest that CBZ inhibits L-type Ca2+ channels and Na+ channels, but does not inhibit activation of glutamate ionotropic receptors. http://www.sciencedirect.com/science/article/B6T0C-3X1W5Y4-D/1/65c32835840a01052a606b944eb4cabd

We have investigated the effect of endogenous adenosine on the release of [ 3 H]acetylcholine ([ 3 H]ACh) in cultured chick amacrine-like neurons. The release of [ 3 H]ACh evoked by 50 mM KCI was mostly Ca 2+ dependent, and it was increased in the presence of adenosine deaminase and in the presence of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), an adenosine A 1 receptor antagonist. The effect of adenosine on [ 3 H]ACh release was sensitive to pertussis toxin (PTX) and was due to a selective inhibition of N-type Ca 2+ channels. Ligand binding studies using [ 3 H]DPCPX confirmed the presence of adenosine A 1 receptors in the preparation. Using specific inhibitors of the plasma membrane adenosine carriers and of the ectonucleotidases, we found that the extracellular accumulation of adenosine in response to KCI depolarization was due to the release of endogenous adenosine per se and to the extracellular conversion of released nucleotides into adenosine. Activation of adenosine A 1 receptors was without effect on the intracellular levels of cyclic AMP under depolarizing conditions, but it inhibited the accumulation of inositol phosphates. Our results indicate that in cultured amacrine-like neurons, the Ca 2+ -dependent release of [ 3 H]ACh evoked by KCI is under tonic inhibition by adenosine, which activates A 1 receptors. The effect of adenosine on the [ 3 H]ACh release may be due to a direct inhibition of N-type Ca 2+ channels and/ or secondary to the inhibition of phospholipase C and involves the activation of PTX-sensitive G proteins.

In this work we investigated the acute effects of 3,4-dichloroaniline (DCA) on the changes of specific biochemical and cellular blood parameters using male Wistar rats, 24 hours after the intraperitoneal administration of five different doses of DCA (0, 81, 162, 324, 486, and 568 mg/Kg of body weight). We also evaluated the dose-dependent toxicity of DCA on the ratio “organ weight: total body weight” for kidney, liver, spleen and thymus, and on the changes of morphological characteristics of the spleen. The LOEL (lowest observed effect level) value for spleen and thymus injury was 324 mg DCA/Kg. The cellular blood parameters affected by DCA include: methemoglobin concentration, the number of circulating leukocytes and the number and size of platelets. The relative sensitivity of all the analysis used to assess the acute toxicity of DCA was evaluated by comparing their ED50 values, which ranged from 224 to 837 mg DCA/Kg. We concluded that the most sensitive endpoint to measure the acute toxicity of DCA was the methemoglobin formation. Platelet counts, urea and creatinine showed ED50 values slightly higher than methemoglobin (329 – 367 mg/Kg).

We examined the conversion of L-[3H]arginine to L-[3H]citrulline in lysate from rat thymocytes, which was dependent on Ca2+and cofactors (FAD, BH4, NADPH). Removal of Ca2+of the medium, reduced the total L-[3H]citrulline formation by about 97%. The L-[3H]citrulline formation was completely inhibited by the NO synthase inhibitors, NG-nitro-L-arginine and NG-monomethyl-L-arginine, with values for IC50of 1.2 [mu]M and 19.4 [mu]M, respectively. In intact thymocytes, the L-[3H]citrulline formation was dependent on the intracellular Ca2+([Ca2+]i) concentration. Increasing the extracellular free-Ca2+concentration up to 1.5 mM, was accompanied by an increase in [Ca2+]iinside the thymocytes and there was a parallel increase in the intracellular L-[3H]citrulline formation, which reached a maximal value of 371.2 nM of [Ca2+]i. Addition of NG-nitro-L-arginine to the medium, completely inhibited the formation of L-[3H]citrulline. The immunolabeling study revealed that 15% of the thymocytes isolated from rat thymus constitutively expressed the endothelial isoform of NO synthase. http://www.sciencedirect.com/science/article/B6WBK-45KN875-KN/1/6f9bc800d346384564098806d560df12

The functional modulation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors by protein kinase C (PKC) was investigated in cultures enriched in retinal amacrine-like cells. The kainate-evoked [Ca2+]i increase is due to Ca2+ entry through open AMPA receptor channels, because it was blocked by the active isomer of a 2,3-benzodiazepine (LY 303070), an AMPA receptor antagonist. The AMPA receptor response to kainate was potentiated by phorbol 12-myristate 13-acetate, which specifically stimulates PKC, and it was decreased by bisindolylmaleimide I, a selective inhibitor of PKC, as well as by PKC down-regulation. The results indicate not only that the AMPA receptor activation has a PKC requirement, but also that PKC amplifies maximal receptor activation by 100 µM kainate. The effect of PKC activation or inhibition on voltage-gated Ca2+-channel activity was also investigated. Activation of PKC caused inhibition of Ca2+ channels, and the same effect was produced by inhibition of PKC, whereas the inactive analogue of the phorbol ester did not affect channel activity. Our results show an important role for PKC in regulating the function of both AMPA receptors and Ca2+ channels in cultured retina cells.

The hippocampal CA3 subregion of the rat is characteristically enriched in kainate receptors. At the synaptic level, the subcellular localization of these receptors is still a matter of debate. The CA3 pyramidal cells are particularly sensitive to excitotoxicity induced by kainate, which is in agreement with the high levels of kainate receptors in the stratum lucidum of the hippocampal CA3 subregion. Immunocytochemical studies, using antibodies against kainate receptor subunits, clearly demonstrated the presence of postsynaptic kainate receptors. However, it was not possible at the time to identify the activity of postsynaptic kainate receptors as mediators of the synaptic transmission. There are also reports showing the labeling of unmyelinated axons and nerve terminals with antibodies against kainate receptor subunits. The evidence for the presence of presynaptic kainate receptors in the hippocampus is further substantiated by the demonstration that stimulation of kainate receptors in synaptosomes isolated from the rat hippocampal CA3 subregion increases the intracellular free Ca2+ concentration ([Ca2+]i) coupled to the release of glutamate. These results support the model proposed by Coyle (1983), in which the excitotoxicity induced by kainate involves the activation of presynaptic kainate receptors, causing the release of glutamate. According to this model, the neurotoxic effect of kainate in the rat hippocampal CA3 subregion involves a direct effect on presynaptic kainate receptors and an indirect effect on postsynaptic glutamate receptors due to the enhanced release of glutamate. http://www.sciencedirect.com/science/article/B6T0B-3TDPXHV-1V/1/2426990c6a815f0f527290618c066fe1

1. Glutamate is the neurotransmitter released by bipolar cells at their synapses with amacrine cells. The amacrine cells express ionotropic (NMDA, AMPA and kainate) and metabotropic (mGluR1, mGluR2, mGluR4 and mGluR7) glutamate receptors and may take up glutamate from the synaptic cleft. 2. Activation of the ionotropic glutamate receptors increases the intracellular free calcium concentration ([Ca2+]i), owing to Ca2+ entry through the receptor-associated channels as well as through voltage-gated Ca2+ channels. The [Ca2+]i response to glutamate may be amplified by Ca2+-induced Ca2+ release from intracellular sources. 3. Activation of NMDA and non-NMDA glutamate receptors stimulates the release of GABA and acetylcholine from amacrine cells. GABA is released by a Ca2+-dependent mechanism and by reversal of the neurotransmitter transporter. 4. Excessive activation of glutamate receptors during ischemia leads to amacrine cell death. An increase in [Ca2+]i due to Ca2+ influx through NMDA and AMPA/kainate receptor channels is related to cell death in studies in vitro. In other studies, it was shown that nitric oxide may also take part in the process of cell damage during ischemia.

In adrenal chromaffin cells, depolarization-evoked Ca2+ influx and catecholamine release are partially blocked by blockers of L-type voltage-sensitive Ca2+ channels. We have now evaluated the sensitivity of the dihydropyridine-resistant components of Ca2+ influx and catecholamine release to a toxin fraction (FTX) from the funnel-web spider poison, which is known to block P-type channels in mammalian neurons. FTX (1:4,000 dilution, with respect to the original fraction) inhibited K(+)-depolarization-induced Ca2+ influx by 50%, as monitored with fura-2, whereas nitrendipine (0.1-1 microM) and FTX (3:3), a synthetic FTX analogue (1 mM), blocked the [Ca2+]i transients by 35 and 30%, respectively. When tested together, FTX and nitrendipine reduced the [Ca2+]i transients by 70%. FTX or nitrendipine reduced adrenaline and noradrenaline release by approximately 80 and 70%, respectively, but both substances together abolished the K(+)-evoked catecholamine release, as measured by HPLC. The omega-conotoxin GVIA (0.5 microM) was without effect on K(+)-stimulated 45Ca2+ uptake. Our results indicate that FTX blocks dihydropyridine- and omega-conotoxin-insensitive Ca2+ channels that, together with L-type voltage-sensitive Ca2+ channels, are coupled to catecholamine release.

We have studied the fusion activity of Sendai virus, a lipid-enveloped paramyxovirus, towards a line of adherent cells designated PC-12. Fusion was monitored by the dequenching of octadecyl-rhodamine, a fluorescent non-exchangeable probe. The results were analysed with a mass action kinetic model which could explain and predict the kinetics of virus-cell fusion. When the temperature was lowered from 37 degrees C to 25 degrees C, a sharp inhibition of the fusion process was observed, probably reflecting a constraint in the movement of viral glycoproteins at low temperatures. The rate constants of adhesion and fusion were reduced 3.5-fold and 7-fold, respectively, as the temperature was lowered from 37 degrees C to 25 degrees C. The fusion process seemed essentially pH-independent, unlike the case of liposomes and erythrocyte ghosts. Preincubation of the virus in the absence of target cell membranes at neutral and alkaline pH (37 degrees C, 30 min) did not affect the fusion process. However, a similar preincubation of the virus at pH = 5.0 resulted in marked, though slow, inhibition in fusion with the fusion rate constant being reduced 8-fold. Viral preincubation for 5 min in the same acidic conditions yielded a mild inhibition of fusogenic activity, while preincubation in the cold (4 degrees C, 30 min) did not alter viral fusion activity. These acid-induced inhibitory effects could not be fully reversed by further viral preincubation at pH = 7.4 (37 degrees C, 30 min). Changes in internal pH as well as endocytic activity of PC-12 cells had small effect on the fusion process, thus indicating that Sendai virus fuses primarily with the plasma membranes.

The Ca2+ actively accumulated by sarcoplasmic reticulum isolated from skeletal muscle is composed of two fractions; one represented by intravesicular free Ca2+ and another represented by Ca2+ selectively bound to the membranes. Both of these Ca2+ fractions depend on ATP, although it is not clear whether ATP hydrolysis is essential for accumulation of the second Ca2+ fraction. The existence of the membrane-bound Ca2+ induced by ATP is clearly shown in experiments in which the Ca2+ retention by sarcoplasmic reticulum is measured in the presence and in the absence of X-537A, a Ca2+ ionophore, which makes the membrane permeable to Ca2+. Thus, in the presence of X-537A all Ca2+ accumulated due to ATP is bound to the membranes. This membrane-bound Ca2+ represents about 30 nmol/mg protein in the range of external pCa values of 7 to 3.5. The magnitude of this Ca2+ fraction is slightly higher whether or not the experiments are performed in the presence of oxalate, which greatly increased the intravesicular Ca2+ accumulation. Furthermore, taking advantage of the impermeability of sarcoplasmic reticulum to EGTA, it is possible to show the existence of the membrane-bound Ca2+ as a distinct fraction from that which exists intravesicularly.

The Ca 2+ ionophore X-537A is employed as a tool to distinguish between intravesicular Ca 2+ and surface membrane-bound Ca 2+ in sarcoplasmic reticulum isolated from rabbit skeletal muscle. When sarcoplasmic reticulum is incubated in 20 mM Ca 2+ in the absence of ATP, 10–12 h are necessary for measurable amounts of Ca 2+ to penetrate into the vesicular space, as determined by the fact that X-537A releases Ca 2+ from ‘loaded’ vesicles only after this period of incubation. A fraction of Ca 2+ of 50–60nmol/mg protein, rapidly taken up by sarcoplasmic reticulum, exchanges with Mg 2+ and K + in the medium and is readily released by ethyleneglycol-bis-( β -aminoethyl ether)- N , N ′-tetraacetic acid, but it is not released by X-537A. The slow-penetrating fraction of Ca 2+ (30–40 nmol/mg protein) is rapidly released by X-537A. The results indicate that most of the Ca 2+ retained by sarcoplasmic reticulum under conditions of passive uptake is bound to the external side of the membrane. The fraction of Ca 2+ that slowly penetrates the vesicles remains essentially free inside the vesicles and only a small part is bound to the internal side of the membrane.

The mechanism of Ca2+ transport by rat liver mitochondria was investigated with respect to the possible involvement of calmodulin in this process. We studied the action of exogenous calmodulin isolated from brain tissue on the Ca2+-transport system, as well as the effect of two types of calmodulin antagonists; the phenothiazine drugs trifluoperazine and chlorpromazine and the more specific substance compound 48/80. Our results show that Ca2+ transport by mitochondria and mitochondrial ATPase activity are insensitive to exogenous calmodulin, although they can be inhibited by the phenothiazines. Since no effect of compound 48/80 was observed, we believe that the phenothiazines act through a mechanism that does not involve calmodulin. This is in accord with our inability to locate significant quantities of calmodulin in mitochondria by radioimmunoassay analysis. Our results further show that trifluoperazine and chlorpromazine also inhibit the electron-carrier system of the respiratory chain, and this effect may mediate their inhibitory action on Ca2+ transport when it is energized by respiration instead of ATP hydrolysis.