Francisco Alvarez (Committee Member), David Cool (Committee Member), Peter K. Lauf (Committee Member), James Olson (Advisor), Dawn Wooley (Committee Member)
Doctor of Philosophy (PhD)
Background. In osmotic edema, net efflux of taurine from neurons and accumulation by astrocytes contributes to neuronal volume regulation and astrocytic swelling. Taurine is accumulated in both cell types by a sodium- and chloride-dependent 72-75 kDa protein transporter, TauT. TauT functional activity decreases in osmotically swollen neurons but is unaltered in swollen astrocytes, in vitro. This swelling-induced downregulation of neuronal TauT activity is blocked with the tyrosine kinase (TK) inhibitor, genistein. In contrast, PKC activation has no effect on neuronal TauT, but inhibits astrocytic TauT. Thus, we hypothesize that during osmotic swelling, neuronal TauT activity is regulated by a TK signaling pathway whereas astroglial TauT activity is regulated by serine/threonine kinases. This differential regulation contributes to neuronal volume regulation and astrocytic swelling via taurine redistribution during osmotic brain edema. Methods. Primary neuronal and astrocytic cultures from rat hippocampus were incubated under iso- or hypo-osmotic conditions in the presence or absence of activators or inhibitors of TK or PKC. Subcellular TauT localization was measured after 30 min using cell fractionation, cell surface biotinylation, and western blot analyses. Phosphorylation was measured after 30 min using immunoprecipitation and western blot analyses with phosphoprotein-specific antibodies. Results. We found neuronal and astroglial TauT primarily localized to cytosolic and membrane/particulate fractions in isoosmotic conditions. However, cell surface biotinylation of TauT decreased in swollen neurons while phosphorylation of tyrosine residues increased. In contrast, phosphorylation of serine and threonine on neuronal TauT was unchanged. Surface biotinylation decreased and phosphorylation of serine and threonine residues on astrocytic TauT increased upon treatment with 1 µM PMA. Cell surface biotinylation and phosphorylation of TauT in astrocytes were unaffected by cell swelling. Conclusions. The results suggest the signal for neuronal TauT translocation from the cell membrane during hypoosmotic cell swelling involves tyrosine phosphorylation. Membrane localization of astroglial TauT remains unchanged during hypoosmotic cell swelling. These changes may account for the observed reduction in functional TauT activity in swollen neurons and may contribute to neuronal volume regulation during osmotic edema.
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