Regulation of the mouse Na+-dependent glutamate/aspartate transporter GLAST: putative role of an AP-1 DNA binding site.

Appropriate removal of L: -glutamate from the synaptic cleft is important for prevention of the excitotoxic effects of this neurotransmitter. The Na+-dependent glutamate/aspartate transporter GLAST is regulated in the short term, by a transporter-dependent decrease in uptake activity while in the long term, a receptor's-dependent decrease in GLAST protein levels leads to a severe reduction in glutamate uptake. The promoter region of the mouse glast gene harbors an Activator Protein-1 site (AP-1). To gain insight into the molecular mechanisms triggered by Glu-receptors activation involved in GLAST regulation, we took advantage of the neonatal mouse cerebellar prisms model. We characterized the glutamate uptake activity; the glutamate-dependent effect on GLAST protein levels and over the interaction of nuclear proteins with a mouse glast promoter AP-1 probe. A time and dose dependent decrease in transporter activity matching with a decrease in GLAST levels was recorded upon glutamate treatment. Moreover, a significant increase in glast AP-1 DNA binding was found. Pharmacological experiments established that both effects are mediated through alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors, favoring the notion of the critical involvement of glutamate in the regulation of its binding partners: receptors and transporters.

Signal transducers and activators of transcription 1 and 3 in prostate: effect of sexual activity.

The signal transducers and activators of transcription (Stat) are effector molecules downstream of cytokine receptors. Ligand occupancy of these receptors results in the tyrosine phosphorylation, dimerization and nuclear translocation of the Stat family of transcription factors and by these means regulate gene expression. Prolactin receptors as members of the cytokine-hematopoietin receptor superfamily, are linked to Stat activation. Sexual stimulation leads to an increase in prolactin secretion that might be involved in long-term changes in the protein repertoire associated to prostate hyperplasia. In order to gain insight into this phenomenon, we analyzed the tyrosine phosphorylation and DNA binding activity of two members of the Stat family in the prostate of sexual experienced rats after different number of ejaculations. A significant increase in Stat-1 and Stat-3 tyrosine phosphorylation was found after three ejaculations. Concomitantly an increase in Stat-1 and Stat-3 DNA-binding activity is detected after two and three ejaculation series. These results, favor the notion that ejaculation-induced prolactin secretion activates its prostate receptors resulting in Stat-1 and Stat-3 nuclear translocation, event likely to be associated to the so-called benign prostate hyperplasia.

Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors signaling complexes in Bergmann glia.

Glutamate, the major excitatory neurotransmitter, induces a wide array of signals from the membrane to the nucleus regulating gene expression. In Bergmann glia, Ca2+ -permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazole- propionic acid (AMPA) receptors are involved in the short- and long-term interactions between these cells and the neurons that they surround. After activation, AMPA receptors become tyrosine phosphorylated and by these means form multiprotein signaling complexes. To characterize these events, cultured chick Bergmann glia cells as well as chick cerebellar slices were exposed to glutamate, and, by using a combination of immunoprecipitation assays coupled to Western blot analysis, we identified several signaling proteins that become associated with these receptors. A dose- and time-dependent association among AMPA receptors, the focal adhesion kinase pp125FAK, the phosphatidylinositol-3 kinase and paxillin was found. These results extend the concept of the transducisome to AMPA receptors and provide a framework in which a plausible control of the cytoskeletal network by glutamate is taking place, most possibly through AMPA receptors.

Regulation of the Na+-dependent glutamate/aspartate transporter in rodent cerebellar astrocytes.

The regulation of the Na+-dependent glutamate/aspartate transporter system GLAST expressed in rat and mouse cerebellar and cortical astrocytic cultures was examined. Pretreatment of the cerebellar cells with L-glutamate and 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a known Ca2+/diacylglicerol-dependent protein kinase (PKC) activator, produced a decrease in [3H]-D-aspartate uptake. This reduction was dose- and time-dependent and sensitive to PKC inhibitors. Furthermore, the L-glutamate-dependent [3H]-D-aspartate uptake decrease is a non-receptor dependent process, because neither of the agonists or antagonists were effective in mimicking or reverting the effect. Interestingly, transportable substrates could reproduce the L-glutamate effect. In sharp contrast, in cortical astrocytes, both L-glutamate and TPA pre-exposure result in an augmentation of the [3H]-D-aspartate uptake. These findings suggest that the Na+-dependent glutamate uptake GLAST undergoes a region-specific regulation.