RESUMEN
AIM: To analyze the neurotoxic potential of synthesized magnetite nanoparticles coated by dextran, hydroxyethyl starch, oxidized hydroxyethyl starch, and chitosan, and magnetic nanoparticles combined with ferritin as a native protein. METHODS: The size of nanoparticles was analyzed using photon correlation spectroscopy, their effects on the conductance of planar lipid membrane by planar lipid bilayer technique, membrane potential and acidification of synaptic vesicles by spectrofluorimetry, and glutamate uptake and ambient level of glutamate in isolated rat brain nerve terminals (synaptosomes) by radiolabeled assay. RESULTS: Uncoated synthesized magnetite nanoparticles and nanoparticles coated by different polysaccharides had no significant effect on synaptic vesicle acidification, the initial velocity of L-[(14)C]glutamate uptake, ambient level of L-[(14)C]glutamate and the potential of the plasma membrane of synaptosomes, and conductance of planar lipid membrane. Native ferritin-based magnetic nanoparticles had no effect on the membrane potential but significantly reduced L-[(14)C]glutamate transport in synaptosomes and acidification of synaptic vesicles. CONCLUSIONS: Our study indicates that synthesized magnetite nanoparticles in contrast to ferritin have no effects on the functional state and glutamate transport of nerve terminals, and so ferritin cannot be used as a prototype, analogue, or model of polysaccharide-coated magnetic nanoparticle in toxicity risk assessment and manipulation of nerve terminals by external magnetic fields. Still, the ability of ferritin to change the functional state of nerve terminals in combination with its magnetic properties suggests its biotechnological potential.
Asunto(s)
Materiales Biocompatibles Revestidos/toxicidad , Ferritinas/toxicidad , Ácido Glutámico/metabolismo , Nanopartículas de Magnetita/toxicidad , Polisacáridos/toxicidad , Vesículas Sinápticas/efectos de los fármacos , Sinaptosomas/efectos de los fármacos , Animales , Transporte Biológico , Quitosano/síntesis química , Quitosano/toxicidad , Materiales Biocompatibles Revestidos/síntesis química , Dextranos/síntesis química , Dextranos/toxicidad , Derivados de Hidroxietil Almidón/síntesis química , Derivados de Hidroxietil Almidón/toxicidad , Masculino , Potenciales de la Membrana/efectos de los fármacos , Ratas , Ratas Wistar , Vesículas Sinápticas/metabolismo , Sinaptosomas/metabolismoRESUMEN
The harmful effects of lunar dust (LD) on directly exposed tissues are documented in the literature, whereas researchers are only recently beginning to consider its effects on indirectly exposed tissues. During inhalation, nano-/microsized particles are efficiently deposited in nasal, tracheobronchial, and alveolar regions and transported to the central nervous system. The neurotoxic potential of LD and martian dust (MD) has not yet been assessed. Glutamate is the main excitatory neurotransmitter involved in most aspects of normal brain function, whereas disturbances in glutamate homeostasis contribute to the pathogenesis of major neurological disorders. The research was focused on the analysis of the effects of LD/MD simulants (JSC-1a/JSC, derived from volcanic ash) on the key characteristics of glutamatergic neurotransmission. The average size of LD and MD particles (even minor fractions) before and after sonication was determined by dynamic light scattering. With the use of radiolabeled l-[(14)C]glutamate, it was shown that there is an increase in l-[(14)C]glutamate binding to isolated rat brain nerve terminals (synaptosomes) in low [Na(+)] media and at low temperature in the presence of LD. MD caused significantly lesser changes under the same conditions, whereas nanoparticles of magnetite had no effect at all. Fluorimetric experiments with potential-sensitive dye rhodamine 6G and pH-sensitive dye acridine orange showed that the potential of the plasma membrane of the nerve terminals and acidification of synaptic vesicles were not altered by LD/MD (and nanoparticles of magnetite). Thus, the unique effect of LD to increase glutamate binding to the nerve terminals was shown. This can have deleterious effects on extracellular glutamate homeostasis in the central nervous system and cause alterations in the ambient level of glutamate, which is extremely important for proper synaptic transmission. During a long-term mission, a combination of constant irritation due to dust particles, inflammation, stress, low gravity and microgravity, radiation, UV, and so on may consequently change the effects of the dust and aggravate neurological consequences.
Asunto(s)
Encéfalo/efectos de los fármacos , Polvo , Marte , Luna , Neurotoxinas/toxicidad , Sinapsis/efectos de los fármacos , Naranja de Acridina , Animales , Encéfalo/metabolismo , Radioisótopos de Carbono , Colorantes Fluorescentes , Ácido Glutámico/metabolismo , Técnicas In Vitro , Masculino , Potenciales de la Membrana/efectos de los fármacos , Ratas , Ratas Wistar , Rodaminas , Pruebas de ToxicidadRESUMEN
Exposure to Cd(2+) and Pb(2+) has neurotoxic consequences for human health and may cause neurodegeneration. The study focused on the analysis of the presynaptic mechanisms underlying the neurotoxic effects of non-essential heavy metals Cd(2+) and Pb(2+). It was shown that the preincubation of rat brain nerve terminals with Cd(2+) (200 µM) or Pb(2+) (200 µM) resulted in the attenuation of synaptic vesicles acidification, which was assessed by the steady state level of the fluorescence of pH-sensitive dye acridine orange. A decrease in L-[(14)C]glutamate accumulation in digitonin-permeabilized synaptosomes after the addition of the metals, which reflected lowered L-[(14)C]glutamate accumulation by synaptic vesicles inside of synaptosomes, may be considered in the support of the above data. Using isolated rat brain synaptic vesicles, it was found that 50 µM Cd(2+) or Pb(2+) caused dissipation of their proton gradient, whereas the application of essential heavy metal Mn(2+) did not do it within the range of the concentration of 50-500 µM. Thus, synaptic malfunction associated with the influence of Cd(2+) and Pb(2+) may result from partial dissipation of the synaptic vesicle proton gradient that leads to: (1) a decrease in stimulated exocytosis, which is associated not only with the blockage of voltage-gated Ca(2+) channels, but also with incomplete filling of synaptic vesicles; (2) an attenuation of Na(+)-dependent glutamate uptake.
Asunto(s)
Cadmio/farmacología , Ácido Glutámico/metabolismo , Plomo/farmacología , Terminales Presinápticos/efectos de los fármacos , Vesículas Sinápticas/efectos de los fármacos , Animales , Transporte Biológico , Masculino , Terminales Presinápticos/metabolismo , Ratas , Ratas WistarRESUMEN
The effect of the cholesterol-depleting agent methyl-ß-cyclodextrin (MßCD) on exocytotic, transporter-mediated, tonic release, the ambient level and uptake of L-[(14)C]glutamate was assessed in rat brain synaptosomes using different methodological approaches of MßCD application. The addition of 15 mM MßCD to synaptosomes (the acute treatment, AT) immediately resulted in the extraction of cholesterol and in a two times increase in the extracellular L-[(14)C]glutamate level. When 15 mM MßCD was applied to synaptosomes for 35 min followed by washing of the acceptor (the long-term pretreatment, LP), this level was only one-third higher than in the control. The opposite effects of MßCD on tonic L-[(14)C]glutamate release and glutamate transporter reversal were found in AT and LP. Tonic release was dramatically enlarged in AT, but decreased after LP. Transporter-mediated release was increased several times in AT, but attenuated in LP. Depolarization-evoked exocytotic release of L-[(14)C]glutamate was completely lost in AT, whereas after LP, it was decreased by half in comparison with the control. Na(+)-dependent L-[(14)C]glutamate uptake was decreased by ~60% in AT, whereas in LP, it was lowered by ~40% only. The presence of MßCD in the incubation media during AT caused dramatic dissipation of the proton gradient of synaptic vesicles that was shown with the pH-sensitive dye acridine orange, whereas after LP, no statistically significant changes were registered in synaptic vesicle acidification. It was concluded that the diverse changes in glutamate transport in AT and LP were associated with the difference in the functional state of synaptic vesicles.
Asunto(s)
Ácido Glutámico/metabolismo , Terminales Presinápticos , Sinaptosomas , beta-Ciclodextrinas/farmacología , Naranja de Acridina/metabolismo , Animales , Colesterol/metabolismo , Exocitosis/efectos de los fármacos , Colorantes Fluorescentes/metabolismo , Concentración de Iones de Hidrógeno , Masculino , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/metabolismo , Terminales Presinápticos/ultraestructura , Ratas , Ratas Wistar , Vesículas Sinápticas/química , Vesículas Sinápticas/metabolismo , Sinaptosomas/química , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismoRESUMEN
The low level of ambient glutamate is important for the brain's spontaneous activity and proper synaptic transmission. Cholesterol deficiency has been implicated in the pathogenesis of several neurodegenerative disorders. It was examined whether membrane cholesterol modulated the extracellular glutamate level in nerve terminals and the processes responsible for its maintenance. The ambient L-[(14)C]glutamate level, being an equilibrium between Na(+)-dependent uptake and tonic release, was increased from 0.193+/-0.013 nmol/mg protein to 0.282+/-0.013 (extracellular endogenous glutamate-from 6.9+/-2.0 to 16.6+/-2.0, respectively) in rat brain synaptosomes treated with a cholesterol acceptor methyl-beta-cyclodextrin (MbetaCD). This alteration was not due to the change in the activity of glutamine synthetase that was shown with the specific blocker L-methionine sulfoximine. In the presence of DL-threo-beta-benzyloxyaspartate, which significantly reduced the contribution of glutamate transporters, net tonic release of L-[(14)C]glutamate was decreased by 38% and release in low-Na(+) medium was attenuated by 41% after cholesterol extraction. Also, cholesterol-deficient synaptosomes showed a reduced content of cytosolic L-[(14)C]glutamate and a lower initial velocity of L-[(14)C]glutamate uptake. We suggested that cholesterol deficiency altered the intra-to-extracellular glutamate ratio by the reduction of the cytosolic level of the neurotransmitter and the augmentation of the ambient glutamate level, thereby favoring a decrease in tonic glutamate release. Thus, increased extracellular glutamate in cholesterol-deficient nerve terminals was not a result of the changes in tonic release and/or glutamine synthetase activity, but was set by lack function of glutamate transporters.