Arsélio P. Carvalho

Neuroprotection by BDNF against glutamate-induced apoptotic cell death is mediated by ERK and PI3-kinase pathways.

Neurotrophins protect neurons against glutamate excitotoxicity, but the signaling mechanisms have not been fully elucidated. We studied the role of the phosphatidylinositol 3-kinase (PI3-K) and Ras/mitogen-activated protein kinase (MAPK) pathways in the protection of cultured hippocampal neurons from glutamate induced apoptotic cell death, characterized by nuclear condensation and activation of caspase-3-like enzymes. Pre-incubation with the neurotrophin brain-derived neurotrophic factor (BDNF), for 24 h, reduced glutamate-evoked apoptotic morphology and caspase-3-like activity, and transiently increased the activity of the PI3-K and of the Ras/MAPK pathways. Inhibition of the PI3-K and of the Ras/MAPK signaling pathways abrogated the protective effect of BDNF against glutamate-induced neuronal death and similar effects were observed upon inhibition of protein synthesis. Moreover, incubation of hippocampal neurons with BDNF, for 24 h, increased Bcl-2 protein levels. The results indicate that the protective effect of BDNF in hippocampal neurons against glutamate toxicity is mediated by the PI3-K and the Ras/MAPK signaling pathways, and involves a long-term change in protein synthesis.

Inhibition of acetylcholinesterase activity as effect criterion in acute tests with juvenile Daphnia Magna

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.

Mechanisms of Action of Carbamazepine and Its Derivatives, Oxcarbazepine, BIA 2-093, and BIA 2-024*

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.

Relationships Between ATP Depletion, Membrane Potential, and the Release of Neurotransmitters in Rat Nerve Terminals: An In Vitro Study Under Conditions That Mimic Anoxia, Hypoglycemia, and Ischemia

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 gamma-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 or 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.

Activation energies of the ATPase activity of sarcoplasmic reticulum

Summary Arrhenius plots of Ca ++ -stimulated ATP hydrolysis by sarcoplasmic reticulum (SR) show breaks (T t ) at 16.7°C and 11.5°C for rabbit and lobster preparations, respectively. The energies of activation (Eact) are about 10 and 19.5 Kcal/mole above and below T t , respectively, and are similar for both lobster and rabbit SR. The antibiotic filipin increases T t by about 7°C for both preparations, but the Eact above and below the new T t values remain similar to those of the controls. Desintegrated membranes do not show breaks in Arrhenius plots and the Eact assume relatively high values. The Ca ++ ionophore X-537A does not affect either the T t values nor the Eact.

Activation of neuropeptide Y receptors is neuroprotective against excitotoxicity in organotypic hippocampal slice cultures

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)‐(Gly1,Ser3,Gln4,Thr6,Ala31,Aib32,Gln34)‐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.

l-Arginine transport in retinas from streptozotocin diabetic rats: correlation with the level of IL-1β and NO synthase activity

Several evidences suggest that the pro-inflammatory cytokines IL-1 beta and the radical NO are implicated as effectors molecules in the pancreatic beta-cells dysfunction; an event preceding the pathogenesis of diabetes. IL-1 beta 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-1 beta 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 (K(m) = 25 microM) and higher capacity (Vmax = 295 +/- 22.4 pmol L-arginine/mg protein) than in retinas from control rats (K(m) = 5 microM and Vmax = 158 +/- 12.8 pmol L-arginine/mg protein) which is correlated with the increased NOS activity and consequent depletion of the intracellular pool of L-arginine.

Regulation of AMPA receptors by phosphorylation.

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.

Carbamazepine inhibits L-type Ca2+ channels in cultured rat hippocampal neurons stimulated with glutamate receptor agonists

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- 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-D-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 microM nitrendipine, 0.5 microM 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 microM 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.

Ca2+ influx through glutamate receptor-associated channels in retina cells correlates with neuronal cell death

We studied the effect of glutamate, N-methyl-D-aspartate (NMDA), kainate or K+ depolarization, on neurotoxicity in cultured chick retinal cells, under conditions in which we could discriminate between Ca2+ entering through ionotropic glutamate receptors and voltage-sensitive Ca2+ channels (VSCCs). When neurons were challenged with NMDA, kainate or glutamate, in Na(+)-containing medium, a decrease in cell survival was observed, whereas K+ depolarization did not affect the viability of the cells. The Mg2+ ion completely prevented the toxic effect mediated by the NMDA receptor, and had a small but significant protective effect at the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate (AMPA/kainate) receptor-induced cell death. We observed that, in a Na(+)-free N-methyl-D-glucamine (NMG) medium, to avoid the activation of VSCCs indirectly by the glutamate receptor agonists, stimulation of the glutamate receptors causes Ca2+ influx only through NMDA and AMPA/kainate receptor-associated channels, and that Ca2+ entry correlates well with subsequent cell death. These results show that the activation of NMDA or AMPA/kainate receptors can cause excitotoxicity in retinal neurons by mechanisms not involving Na+ influx, but rather depending on the permeation of Ca2+ through glutamate receptor-associated channels. For small Ca2+ loads the entry of Ca2+ through the NMDA receptor-associated channel was more efficient in triggering cell death than the influx of Ca2+ through the AMPA/kainate receptor.