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Regulation of Na + -K + -Cl − cotransporter in primary astrocytes by dibutyryl cAMP and high [K + ] o

2000; American Physical Society; Volume: 279; Issue: 6 Linguagem: Inglês

10.1152/ajpcell.2000.279.6.c1710

1522-1563

Gui Su, Robert A. Haworth, Robert J. Dempsey, Dandan Sun,

Neuroscience and Neuropharmacology Research

In this study, we examined the Na + -K + -Cl − cotransporter activity and expression in rat cortical astrocyte differentiation. Astrocyte differentiation was induced by dibutyryl cAMP (DBcAMP, 0.25 mM) for 7 days, and cells changed from a polygonal to process-bearing morphology. Basal activity of the cotransporter was significantly increased in DBcAMP-treated astrocytes ( P < 0.05). Expression of an ∼161-kDa cotransporter protein was increased by 91% in the DBcAMP-treated astrocytes. Moreover, the specific [ 3 H]bumetanide binding was increased by 67% in the DBcAMP-treated astrocytes. Inhibition of protein synthesis by cyclohexamide (2–3 μg/ml) significantly attenuated the DBcAMP-mediated upregulation of the cotransporter activity and expression. The Na + -K + -Cl − cotransporter in astrocytes has been suggested to play a role in K + uptake. In 75 mM extracellular K + concentration, the cotransporter-mediated K + influx was stimulated by 147% in nontreated cells and 79% in DBcAMP-treated cells ( P < 0.05). To study whether this high K + -induced stimulation of the cotransporter is attributed to membrane depolarization and Ca 2+ influx, the role of the L-type voltage-dependent Ca 2+ channel was investigated. The high-K + -mediated stimulation of the cotransporter activity was abolished in the presence of either 0.5 or 1.0 μM of the L-type channel blocker nifedipine or Ca 2+ -free HEPES buffer. A rise in intracellular free Ca 2+ in astrocytes was observed in high K + . These results provide the first evidence that the Na + -K + -Cl − cotransporter protein expression can be regulated selectively when intracellular cAMP is elevated. The study also demonstrates that the cotransporter in astrocytes is stimulated by high K + in a Ca 2+ -dependent manner.