ToxSci Advance Access originally published online on July 26, 2007
Toxicological Sciences 2007 99(2):566-571; doi:10.1093/toxsci/kfm187
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45Ca2+ Influx in Rat Brain: Effect of Diorganylchalcogenides Compounds
* Departamento de Análises Clínicas e Toxicológicas, Centro de Ciências da Saúde, Universidade Federal de Santa Maria, 97105-900—Santa Maria, RS, Brasil
Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brasil
Departamento de Química, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, Santa Maria, RS, Brasil
1 To whom correspondence should be addressed. Fax: +55-3220-8018. E-mail: Beatriz{at}smail.ufsm.br.
Received May 2, 2007; accepted June 26, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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In nervous tissue, the calcium (Ca2+) release induces neurotransmitter exocytosis and synaptic plasticity in neurons and is essential for Ca2+ waves and oscillations in astrocytes. In this work, we have investigated the effect of organocalchogens on calcium influx in synaptosomal preparations under basal and depolarizing conditions. Acute administration of ebselen caused a significant increase of 34% (p < 0.05) Ca2+ influx, when under basal conditions but showed no effect on potassium stimulated calcium conditions by brain synaptosomes. Diphenyl ditelluride (PhTe)2 increased 45Ca2+ influx by 40% (p < 0.05) under depolarizing conditions, while diphenyl diselenide (PhSe)2 had no effect on the brain synaptosomes studied. In addition, we characterized an "in vitro" model with the purpose of studying Ca2+ movements in slices. In this model, we examined the effect of diorganylchalcogenides using brain hippocampal slices, which showed the decrease of calcium influx with the three drugs studied. These findings showed that there are different effects of diorganylchalcogenides in the different models evaluated. It is possible that these differential effects result from the action of neural signal transduction pathways at different levels, possibly involving neurotransmitter release and channel targeting.
Key Words: calcium uptake; selenium compounds; tellurium compounds; slices; synaptosomes.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Calcium ions are critically important in many functions of the nervous system from neurotransmitter release to intracellular signal transduction. The large difference between intracellular and extracellular Ca2+ ion concentration [Ca2+] highlights the importance of the mechanisms controlling influx and efflux of this ion. Neurons control both intracellular Ca2+ levels and the location of Ca2+ ions through a complex interplay between Ca2+ influx, Ca2+ efflux, Ca2+, buffering, and internal Ca2+ storage. However, excessive Ca2+ or release from intracellular stores can elevate Ca2+ loads to levels that exceed the capacity of Ca2+ regulatory mechanisms. Numerous studies have indicated various disturbances of Ca2+ homeostasis on different levels, such as Ca2+ channel properties, 45Ca2+ uptake, or Ca2+-binding proteins (Arundine and Tymianski, 2003
).
Transient changes in the intracellular concentration of free calcium ([Ca2+])i act as a trigger or modulator for a large number of important neuronal processes. Such transients can originate from voltage- or ligand-gated fluxes of Ca2+ into the cytoplasm from the extracellular space or by ligand- or Ca2+-gated release from intracellular stores. Characterizing the sources and spatiotemporal patterns of [Ca2+]i transients is critical for understanding the role of different neuronal compartments in dendritic integration and synaptic plasticity. Thus, the increase in intracellular Ca2+ plays a well-established role in neurotransmitter release, synaptic plasticity, enzyme activation, and gene expression. Regarding astrocytes, neurotransmitter-activated [Ca2+]i changes also have substantial functional significance. Indeed, a transient rise in cytosolic Ca2+ can produce several immediate, intermediate, and long-term structural and functional changes in astrocytes (Carmignoto, 2000).
In this context, calcium channels play important and diverse roles in neurological function. It has been known for many years that calcium channels are likely targets of a variety of organic and inorganic toxicants. The activity of calcium channels is regulated by a wide variety of intracellular signaling pathways. These pathways not only modulate calcium channel activity in various ways by calcium influx through calcium channels but also interact with one another. Thus, complex feedback mechanisms among calcium channels and intracellular signaling exist and are likely targets for both environmental and genetic disorders (Audesirk et al., 2000; Kiryushko et al., 2006). In fact, pathological conditions (stroke, ischemia, AIDS, etc.) and toxicological insult (e.g., by methylmercury, lead) produce alterations in calcium homeostasis that can lead to neuronal death (Denny and Atchison, 1996; Nikonenko et al., 2005).
Organochalcogen compounds have been described to possess very interesting biological activities. Several reports have been published on glutathione peroxidase–mimetic activity of chalcogen compounds, which, like the native enzyme, relies on the redox cycling of selenium or tellurium moiety of the compounds (Parnham and Sies, 2000). The organoselenium compound ebselen has been demonstrated to play a protective role against brain ischemia and stroke (Yamaguchi et al., 1998). Besides, these effects were observed in experimental models against glutamate excitotoxicity (Porciuncula et al., 2001; Rossato et al., 2002) and exposure to methylmercury (Moretto et al., 2005a).
Recently, studies have showed diphenyl diselenide presents neuroprotective and anti-inflammatory activities (Ghisleni et al., 2003; Nogueira et al., 2004). In contrast to the selenium-containing compounds, diphenyl ditelluride, an analogous molecule of diphenyl diselenide, was extremely toxic to rodents and caused marked neurotoxic effects in mice after acute or prolonged exposure (Nogueira et al., 2004; Stangherlin et al., 2005). Moreover, data from our laboratory have shown that the selenium compounds ebselen and diselenide present protective actions toward the alterations of the phosphorylating system associated with the intermediate filament proteins induced by methylmercury and diphenyl ditelluride in slices of the cerebral cortex of young rats (Moretto et al., 2005a,b).
The main purpose of the present study was to determine whether the previous published effect of diphenyl ditelluride, diphenyl diselenide, and ebselen on 45Ca2+ influx into brain synaptosomal could be, at least, related to changes in the Ca2+ influx at presynaptic level under basal and depolarizing conditions in ex vivo treatment in adult rats. It is important to point out that a variety of agents that changes Ca2+ channel activity in isolated nerve terminal also changes neurotransmitter release with glutamate (Nogueira et al., 2001; Rao and Sikdar, 2006; Sitges et al., 2006). At the same time, taking into account the vast number of reports showing that Ca2+-dependent neurotoxicity, as in many physiological events, occurs through distinct intracellular signaling pathways, we have developed a method to investigate the transport of calcium ions through 45Ca2+ influx using brain slices of rats in different conditions. In addition, we investigated the effects of diphenyl ditelluride, diphenyl diselenide, and ebselen on calcium influx into brain hippocampal slices.
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MATERIALS AND METHODS |
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Chemicals.
Diphenyl diselenide (PhSe)2, diphenyl ditelluride (PhTe)2, and ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one] were synthesized according to literature methods (Engman, 1989
; Paulmier, 1986; Petragnani, 1994). These drugs were dissolved in dimethylsulfoxide (DMSO). Analysis of the 1H-NMR and 13C-NMR spectra showed that all the compounds obtained presented analytical and spectroscopic data in full agreement with their assigned structures. Final concentration of DMSO was 0.5%, which did not affect the glutamate uptake. 45Ca2+ was purchased from Amersham International. All other reagents were of analytical grade. All other chemicals were of analytical grade and obtained from standard commercial suppliers.
Animals.
Male Wistar rats 60 days old (P60) were obtained from our own breeding colony. Animals were kept in separate animal rooms, on a 12-h light:dark cycle, at a room temperature of 22°C and with free access to food and water. The animals were used according to the guidelines of the Committee on Care and Use of Experimental Animal Resources, School of Veterinary Medicine and Animal Science of the University of São Paulo, Brazil.
Exposure.
Male adult rats were treated with one ip injection of 25 µmol/kg diphenyl diselenide (PhSe)2, 25 µmol/kg ebselen (2-phenyl-1,2-benzisoselenazol-3[2H]-one), or 3 µmol/kg dyphenyl ditelluride (PhTe)2 (experimental groups) or vehicle (DMSO) (control group, 103 µl/kg). The doses of diorganylchalcogenides and the time of exposure were chosen based on a previous study (Nogueira et al., 2001). Animals were killed, 48 h after the injection, and synaptosomes were prepared as described below.
Preparation of synaptosomes.
Male Wistar rats were decapitated and their brains removed and processed as described previously by Rocha et al. (1990) with some modifications. In brief, the tissue was homogenized (12 strokes at 900 rpm) in 10 volumes of 8mM Tris-HCl, 0.32M sucrose, and buffered at pH 7.4. The homogenate was first centrifuged (20 min, 1000 x g), and synaptosomes were then isolated from the supernatant by centrifugation at 12,000 x g for 20 min. The synaptosomal pellet was then resuspended in the medium described above. All the above procedures were performed at 0°C–4°C. Protein concentrations were measured by the method of Bradford (1976) using bovine serum albumin as the standard.
Calcium uptake.
Ca2+ uptake was carried out essentially as described by Eason and Aronstam (1984) with little modifications. In short, two salt solutions were used in these studies: (1) Na+ (nondepolarized) buffer contained 130mM NaCl, 5mM KCl, 1.2mM Na2HPO4, 0.1mM CaCl2, 10mM glucose, and 20mM Tris-HCl, pH 7.4; (2) K+ (depolarizing) buffer, where the NaCl was omitted and the KCl concentration raised to 135mM. To measure calcium influx, 25 µl of synaptosomes were added to prewarmed Na+ or K+ buffer mixtures containing 2–3 µCi 45Ca2+to give a final protein concentration of 0.4–0.6 mg/ml in 0.5 ml final volume. After 30 s, the samples were filtered on Whatman GF/B glass fiber filters. The filters were washed three times with 3 ml of cold Na+ buffer containing 0.1mM La (NO3)2, and their radioactivity content was determined by liquid scintillation counting. All experiments were performed in triplicate.
Preparation of slices.
Rats (weighing 150–250 g) were killed by decapitation, and their brains were promptly removed and hippocampal or cortex slices (400 µm) were obtained using a McIlwain chopper. Hippocampal and cortex brain slices were carefully separated and placed one for each well of a plate with 1 ml of incubation medium at room temperature.
Calcium uptake assays.
45Ca2+ uptake was carried out as described by Ichida et al. (1981) with some modifications. Slices were preincubated at 35°C (incubation medium) containing (mM) 127 NaCl, 1.2 Na2HPO4 H2O, 5.0 KCl, 0.44 KH2PO4, 0.95 MgCl2, 1.25 CaCl2, 10 glucose, and 0.6 HEPES (pH 7.4). 45Ca2+ influx was started by adding 1.28 µCi 45Ca2+ for different times according with the region of brain (cortex or hippocampus). After incubation, the slices were washed two times with ice-cold incubation medium by CaCl2 replaced by 10mM La (NO3)2, homogenized with 0.5mM NaOH, and transferred to a counting vial. The radioactivity content was determined by liquid scintillation counting. All experiments were performed in triplicate. Protein was determined using the method of Peterson (1977). The nonspecific uptake was through correction for losses of 45Ca2+ from extracellular sites by subtracting the influx at 0°C incubated with ice-cold medium by CaCl2 replaced by 10mM La(NO3)2, as specific conditions above.
Statistical analysis.
Data were analyzed using one-way ANOVA followed by Duncan multiple range test. Values of p < 0.05 were considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Acute administration of 25 µmol/kg ebselen caused a significant increase of 34% (p
0.05) Ca2+ influx, when under basal conditions, but showed no effect on K+-stimulated calcium conditions by brain synaptosomes. Diphenyl ditelluride (3 µmol/kg) increased 40% (p < 0.05) Ca2+ influx under depolarizing conditions while 25 µmol/kg of diphenyl diselenide had no effect on rat brain synaptosomes (Fig. 1).
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In the present protocol, we used hippocampal slice preparations in a complex system that retains many of the neuronal-glial interactions in different conditions, which might be a useful tool to examine interactions closely related with movements Ca2+ and intracellular signaling pathways which regulate channel function. First, we investigated the effect of nonspecific antagonist channels calcium, CoCl2, which decreased the 45Ca2+ influx (Fig. 2A). Inversely, 35mM of KCl in incubation medium increased the 45Ca2+ influx in hippocampal slices of rat 113% (Fig. 2B).
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To examine the optimal time for assaying 45Ca2+ uptake, we carried out incubation time courses using slices of cerebral hippocampal and cortex (Figs. 3A and 3B). Next, we tested (0.1–3.0mM) CaCl2 and determined 1.25mM concentration of CaCl2 in all assays (Fig. 4). We also observed that cortical slices incubated with BAPTA, a calcium intracellular chelator caused no effect, indicating that the results reported reflected net influx of Ca2+ into the system (data not shown).
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Data reported here demonstrate that all the tested compounds inhibited significantly the calcium influx in cerebral hippocampal slices of rats. The following order was observed: ebselen, diphenyl diselenide (PhSe)2, and dyphenyl ditelluride (PhTe)2 (Fig. 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Chemical agents that potentially interfere with Ca2+ homeostasis are considered potential toxic agents. In recent work, we have demonstrated that selenium compounds inhibited the 45Ca2+ uptake (Moretto et al., 2003
) with a synaptosomal model using uptake of radiolabeled 45Ca2+ typically used in investigations in the Ca2+ channel function (Atchison, 2003). The present findings show that the effects of organochalcogenides were rather complex depending on the condition examined. The findings also showed that when exposures were done in vitro and ex vivo the results obtained changed. Indeed, (PhSe)2 and (PhTe)2 had no significant effect on basal calcium influx by brain synaptosomes after acute treatment (Fig. 1). Only ebselen caused a significant increase on Ca2+ influx when under basal conditions by brain synaptosomes. Nogueira et al. (2002) showed that the acute administration of ebselen caused significant increase in basal glutamate release by brain synaptosomes, suggesting that ebselen acts on glutamate release–dependent calcium in nerve terminals. As we know, calcium accomplishes the regulation of the exocytosis of the neurotransmitters in nerve terminals. Besides, according to Moretto et al. (2004), it possibly acts on the direct modulation of kinases/phosphatases associated with changes in the phosphorylation of neurofilaments and other cytoskeletal proteins.
Following ex vivo exposure, the effect of (PhSe)2 and (PhTe)2 varied under K+-stimulated conditions, that is, (PhSe)2 had no significant effect, but (PhTe)2 increased 40% when K+ stimulated was used, indicating that different populations of channels may be functional during basal or depolarization protocols. In the present work, (PhTe)2 increased 40% depolarization stimulated by K+ raising neuronal Ca2+ overload. This depolarization may result from an excessive glutamate-evoked membrane depolarization as well as from the Ca2+ influx and the activation of metabotropic receptors. The consecutive intracellular Ca2+ mobilization is known to have direct toxic effects on the cytoskeleton and the cell metabolism of neurons (Schubert et al., 1997). These findings may reflect the physiological response of the cell to the insult caused by the compound and corroborate with the tellurium toxicity. Previous data have demonstrated that organotellurium compounds can be potentially toxic to rodents (Nogueira et al., 2004; Stangherlin et al., 2005) and that ditelluride induced hyperphosphorylation of cytoskeletal proteins (Moretto et al., 2005b).
Changes in the intracellular free Ca2+ concentration, [Ca2+]i, mediated by glutamate were previously demonstrated (Malva et al., 1998; Tanaka et al., 2003). This difference depends on the state of the neuronal activity and accordingly when situations that cause excessive neuronal firing and glutamate release, such as convulsion state and ischemia (Armijo et al., 2000; Calabresi et al., 2003). In these situations, ebselen could have the neuroprotective effect preventing the calcium accumulation in the cytoplasm. Moreover, the neuroprotective action of ebselen can be related, in addition to its glutathione peroxidase-like and antilipoperoxidative activity, to a direct interaction with the glutamatergic system by reducing K+-evoked glutamate release. On the other hand, despite ebselen abolished the inhibitory effect of Hg2+ on Ca2+ influx in synaptosomes, it did not modify glutamate uptake inhibition caused by Hg2+ in synaptosomes (Moretto et al., 2004). Experimental evidences also show that ebselen causes cell death in several different cell types (Shi et al., 2006).
Additionally, in this work, we have evaluated the calcium influx in a system where the properties of neurons and glial cells are preserved (slices). We observed (Figs. 2–4) that this procedure may constitute a tool to study the transport of calcium in the maintenance of the cellular calcium homeostasis. Considering that calcium influx plays an important role for neuronal cell function and interneuronal cell to cell communication (Carmignoto, 2000), glial cells of the nervous system influence directly neuronal and synaptic activities in the transmitter release (Angulo et al., 2004). The present experiments have indicated that these protocols could be useful to study the events and to bring them into relation with CNS. Thus, we investigated the effects of organotellurium and organoselenium compounds on this system. It is well known that a Ca2+-dependent activation of glial cells along with the loss of physiologically required mature astrocyte functions and with the acquisition of potentially neurotoxic microglial properties has recently been recognized as an additive pathogenic factor (Carmignoto, 2000; Laming et al., 2000). This may provide an effective target for pharmacological interference, especially to organochalcogens that are important intermediates and useful reagents in organic synthesis. Interestingly, the calcium influx was markedly reduced by the three drugs studied (10–400µM) in hippocampal slices (Fig. 5). Glial cells are electrically inexcitable cells, which often employ Ca2+ signaling in response to chemical or mechanical stimuli (Deitmer et al., 1998). However, excessive Ca2+ or release from intracellular stores can elevate Ca2+ loads to levels that exceed the capacity of Ca2+ regulatory mechanisms. Thus, astrocytes can modulate synaptic transmission by the release of glutamate (Dani and Smith, 1995; Rao and Sikdar, 2006). In brain pathology, excessive release of glutamate triggers excitotoxic neural cell death through necrotic or apoptotic pathways (Verkhratsky and Kirchhoff, 2007). The impaired function of calcium channels may explain the deleterious disturbances observed after exposure of these compounds.
The underlying mechanisms which show the toxic or protective effect of organochalcogens are not completely understood. This way, this work comes to help the understanding not only of the complex nature of intercellular signaling mechanisms in the brain, which involve all types of neural cells, but also the factors and signaling systems which control the release of glutamate as well as of other neurotransmitters and compounds capable of interacting with neuronal membranes under normal and pathological conditions.
Since organochalcogenides, such as tellurium or selenium, have a rather complex effect on glutamate homeostasis depending on the compound and the schedule of exposition (Nogueira et al., 2001, 2002), they may operate an endogenous homeostatic regulator that has been a theme of study in this group. These findings reveal novel effects of organochalcogens on glutamatergic nerve terminals by hippocampal slices and demonstrate that the effects of compounds in vitro and ex vivo are not always identical.
Presently, the biochemistry and clinical significance of diorganylchalcogenides exposures are poorly understood. In fact, studies dealing with the distribution of Se and Te and their concomitant toxicology are scarce in the literature. Even though it is difficult to extrapolate our results to the human condition, if that is the case, we presume that interference with these systems here studied would probably lead to the deleterious action of (PhTe)2 on the brain, a fact that might explain at least in part its neurotoxicity. Thus, this study corroborates our continuous efforts in investigating the mechanisms involved in toxicity induced by organochalcogens.
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FUNDING |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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The Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES); the FINEP research grant "Rede Instituto Brasileiro de Neurociência (IBN-Net)" # 01.06.0842-00.
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