Using subtractive cloning, we identified a 1.4 kb mRNA that was ubiquitously expressed in various tissues; this mRNA was highly up-regulated in amygdala nuclei in mice when morphine was repeatedly administered but not when an opiate-receptor antagonist was co-administered. The mRNA encodes a 23 kDa protein, designated 'addicsin'. This contains two putative PKC-phosphorylation motifs and several hydrophobic regions, and was recovered in a soluble protein fraction of brain lysate. Its primary structure showed 98% identity with that of rat glutamate-transporter-associated protein 3-18 (GTRAP3-18), a putative modulator of neural glutamate-transporter EAAC1. Up-regulation of addicsin expression by morphine may affect glutamate uptake in the amygdala, causing mice to develop morphine tolerance and dependence.

Addicsin (Arl6ip5) is a murine homologue of rat glutamate transporter-associated protein 3-18 (GTRAP3-18), a putative negative modulator of Na ؉ -dependent neural glutamate transporter-excitatory amino acid carrier 1 (EAAC1). Here we report that ADP-ribosylation factor-like 6 interacting protein 1 (Arl6ip1) is a novel addicsin-associated partner that indirectly promotes EAAC1-mediated glutamate transport activity in a protein kinase C activity-dependent manner. Like addicsin, Arl6ip1 is expressed in numerous tissues and proved likely to be co-localized with addicsin in certain neurons in the matured brain. Arl6ip1 was not translocated from the subcellular compartments under any of the test conditions and had no association with any molecules on the plasma membrane. Immunoprecipitation assay demonstrated that Arl6ip1 bound directly to addicsin and that the hydrophobic region located at amino acids 103-117 of addicsin was crucial to the formation of the Arl6ip1-addicsin heterodimer and addicsin homodimer. Glutamate transport assay revealed that increasing the expression of Arl6ip1 in C6BU-1 cells markedly enhanced Na ؉ -dependent EAAC1-mediated glutamate transport activity in the presence of 100 nM phorbol 12-myristate 13-acetate. Under these conditions, kinetic analyses demonstrated that EAAC1 altered glutamate transport activity by increasing its glutamate affinity but not its maximal velocity. Meanwhile, increasing expression of addicsin Y110A/L112A mutant lacking binding ability for Arl6ip1 showed no enhancement of EAAC1-mediated glutamate transport activity, regardless of phorbol 12-myristate 13-acetate activation, suggesting that association between addicsin and Arl6ip1 causes altered EAAC1-mediated glutamate transport activity. Our findings suggest that Arl6ip1 is a novel addicsin-associated partner that promotes EAAC1-mediated glutamate transport activity by decreasing the number of addicsin molecules available for interaction with EAAC1.

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In a previous study, we confirmed that orally administered L-ornithine can be transported into the brain of mice. In addition, orally administered L-ornithine, within a limited dose range, had an anxiolytic-like effect in the elevated plus-maze test. However, the mechanism by which orally administered L-ornithine reduced the stress response in mice is still unclear. Experiment 1 determined whether orally administered L-ornithine could reduce the stress-induced activation of hypothalamic-pituitary-adrenal axis. Mice were orally administered L-ornithine (0, 0.75, 1.5 and 3 mmol/10 ml/kg, p.o.), and restrained for 30 min from 30 min post administration. There was a significant decrease in the corticosterone levels in the group receiving 0.75 mmol of L-ornithine compared to the control group. In Experiment 2, the effect of orally administered L-ornithine (0 and 0.75 mmol/10 ml/kg, p.o.) on endogenous monoamine release was investigated using in vivo microdialysis. Only the monoamines metabolites 5-hydroxyindoleacetic acid (5-HIAA), dihydroxyphenylacetic acid (DOPAC) and homovallinic acids (HVA) were detected in the present study. Dialysate concentrations of 5-HIAA, DOPAC and HVA were not significantly changed immediately after administration of L-ornithine and restraint stress. In conclusion, changes of corticosterone concentrations by orally administered L-ornithine were not related to alterations in brain monoamine metabolisms.

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