Monoamine transporter inhibitors and norepinephrine reduce dopamine-dependent iron toxicity in cells derived from the substantia nigra.

The role of dopamine in iron uptake into catecholaminergic neurons, and dopamine oxidation to aminochrome and its one-electron reduction in iron-mediated neurotoxicity, was studied in RCSN-3 cells, which express both tyrosine hydroxylase and monoamine transporters. The mean +/- SD uptake of 100 microm 59FeCl3 in RCSN-3 cells was 25 +/- 4 pmol per min per mg, which increased to 28 +/- 8 pmol per min per mg when complexed with dopamine (Fe(III)-dopamine). This uptake was inhibited by 2 microm nomifensine (43%p < 0.05), 100 microm imipramine (62%p < 0.01), 30 microm reboxetine (71%p < 0.01) and 2 mm dopamine (84%p < 0.01). The uptake of 59Fe-dopamine complex was Na+, Cl- and temperature dependent. No toxic effects in RCSN-3 cells were observed when the cells were incubated with 100 microm FeCl3 alone or complexed with dopamine. However, 100 microm Fe(III)-dopamine in the presence of 100 microm dicoumarol, an inhibitor of DT-diaphorase, induced toxicity (44% cell death; p < 0.001), which was inhibited by 2 microm nomifensine, 30 microm reboxetine and 2 mm norepinephrine. The neuroprotective action of norepinephrine can be explained by (1) its ability to form complexes with Fe3+, (2) the uptake of Fe-norepinephrine complex via the norepinephrine transporter and (3) lack of toxicity of the Fe-norepinephrine complex even when DT-diaphorase is inhibited. These results support the proposed neuroprotective role of DT-diaphorase and norepinephrine.

Characterization of serotonin transporter in blood lymphocytes of rats. Modulation by in vivo administration of mitogens.

Serotonin transporter sites were characterized in blood lymphocytes of rats. Pharmacological characteristics of drug interactions were in concordance with recent studies in nervous and human immune cells. The potency order of inhibition of [(3)H]paroxetine binding was imipramine>citalopram>alaproclate>serotonin. Selective inhibitors of dopamine or noradrenaline transporters did not inhibit it. The specific binding of [(3)H]paroxetine was higher at intermediate than at low concentrations, and the plot of free vs. specific binding had a sigmoid shape. The affinity constant or K(d), 1.77 nM, was in close agreement with data obtained from kinetic studies (K(d)=1.33 nM), which evidences that the equilibrium was reached. In addition, serotonin transporter was evaluated by lipopolysaccharide or concanavalin A administration in vivo (0.1 mg/kg, i.p., 18 h). After the treatment with lipopolysaccharide, no changes were observed in the numbers of sites or B(max) or in the affinity, K(d). The treatment with concanavalin A showed a significant reduction in B(max) and reduction in K(d). Additionally, serotonin and 5-hydroxyindoleacetic acid levels were determined in plasma and lymphocytes by high-performance liquid chromatography. Treatment with lipopolysaccharide produced a significant increased of serotonin levels in lymphocytes without changes in 5-hydroxyindoleacetic acid level; in plasma, it produced an increase in serotonin and 5-hydroxyindolacetic acid levels. In addition, serotonin synthesis was evaluated by adding 300 microM of tryptophan in the medium, which significantly increased serotonin levels in control lymphocytes. Moreover, the concentrations of 5-hydroxyindoleacetic acid was enhanced significantly, both in plasma and lymphocytes in the presence of tryptophan after treatment with lipopolysaccharide. The administration of concanavalin A significantly decreased plasma levels of serotonin, as well as the concentrations of serotonin and 5-hydroxyindoleacetic acid in lymphocytes. These results demonstrate the presence of serotonin transporter in lymphocytes of rat blood, the capacity for serotonin synthesis in lymphocytes, and the modulation of these parameters by systemic administration of mitogens. The findings of this work contribute to understanding the immunological role of serotonin and the communication of immune and nervous systems.

Characterization of urea transport in Bufo arenarum oocytes.

Xenopus laevis oocytes have been extensively used for expression cloning, structure/function relationships, and regulation analysis of transporter proteins. Urea transporters have been expressed in Xenopus oocytes and their properties have been described. In order to establish an alternative system in which urea transporters could be efficiently expressed and studied, we determined the urea transport properties of ovarian oocytes from Bufo arenarum, a toad species common in Argentina. Bufo oocytes presented a high urea permeability of 22.3 x 10(-6) cm/s, which was significantly inhibited by the incubation with phloretin. The urea uptake in these oocytes was also inhibited by mercurial reagents, and high-affinity urea analogues. The urea uptake was not sodium dependent. The activation energy was 3.2 Kcal/mol, suggesting that urea movement across membrane oocytes may be through a facilitated urea transporter. In contrast, Bufo oocytes showed a low permeability for mannitol and glycerol. From these results, we propose that one or several specific urea transporters are present in ovarian oocytes from Bufo arenarum. Therefore, these oocytes cannot be used in expression studies of foreign urea transporters. The importance of Bufo urea transporter is not known but could be implicated in osmotic regulation during the laying of eggs in water.

[Effect of ammonium ions on the uptake of L-leucine in Saccharomyces cerevisiae. Repression and inhibition of transport systems]. / Efecto de iones amonio sobre la entrada de L-leucina en Saccharomyces cerevisiae. Represión e inhibición de los sistemas transportadores.

L-leucine entrance into Saccharomyces cerevisiae is mediated by the general amino acid permease, GAP and two transport systems, S1 and S2, kinetically characterized. S1 is a high-affinity, low-velocity transport system, operating at lower L-leucine external concentration (0.05-0.1 mM), while S2 is a low-affinity, high-velocity transport system, operating at higher L-leucine external concentration (1.0 mM). In cells grown in minimal medium containing ammonium as sole nitrogen source the values of L-leucine entrance and uptake are smaller than those in cells grown in L-proline containing medium. When GAP is repressed by ammonium, L-leucine entrance is mediate by systems S1 and S2. Both systems are inhibited by ammonium. When GAP is derepressed, in cells grown in L-proline medium, L-leucine is transported by systems S1 and GAP (lower L-leucine external concentration), and mainly by S2 (higher L-leucine external concentration). GAP is the largest system inhibited by ammonium.

Efecto de iones amonio sobre la entrada de L-leucina en Saccharomyces cerevisiae: represíon e inhibicíon of transport systems / Effect of ammoniun ions on the uptake of L-leucine: repression and inhibition of transport systems

En Saccharomyces cerevisiae la entrada de L-leucina es mediada por la permeasa general de aminoácidos, GAP, y dos sistemas cinéticamente caracterizados, uno de alta afinidad y baja velocidad, S1, para concentraciones externas de L-leucina 0,05-01mM y otro de baja afinidad, alta velocidad, S2, para concentraciones externas 1.0mM. En células crecidas en medios suplementados con amonio, como única fuente de nitrógeno, los valores de entrada e incorporación son menores que en células crecidas en medios suplementados con L-prolina. En condiciones de represión de la GAP por iones amonio, la entrada de L-leucina es mediada por los sistemas S1 y S2. Los dos sistemas son parcialmente inhibidos por efecto de iones amonio. En condiciones de depresion de la GAP, por crecimiento en L-prolina, la entrada de L-leucina es mediada por los sistemas S1 y GAP, bajas concentraciones externas de L-leucina mediada por centraciones externas. El amonio inhibe en mayor extensión la entrada de L-leucina mediada por La GAP

Efecto de iones amonio sobre la entrada de L-leucina en Saccharomyces cerevisiae: represíon e inhibicíon of transport systems / Effect of ammoniun ions on the uptake of L-leucine: repression and inhibition of transport systems

En Saccharomyces cerevisiae la entrada de L-leucina es mediada por la permeasa general de aminoácidos, GAP, y dos sistemas cinéticamente caracterizados, uno de alta afinidad y baja velocidad, S1, para concentraciones externas de L-leucina 0,05-01mM y otro de baja afinidad, alta velocidad, S2, para concentraciones externas 1.0mM. En células crecidas en medios suplementados con amonio, como única fuente de nitrógeno, los valores de entrada e incorporación son menores que en células crecidas en medios suplementados con L-prolina. En condiciones de represión de la GAP por iones amonio, la entrada de L-leucina es mediada por los sistemas S1 y S2. Los dos sistemas son parcialmente inhibidos por efecto de iones amonio. En condiciones de depresion de la GAP, por crecimiento en L-prolina, la entrada de L-leucina es mediada por los sistemas S1 y GAP, bajas concentraciones externas de L-leucina mediada por centraciones externas. El amonio inhibe en mayor extensión la entrada de L-leucina mediada por La GAP (AU)