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cotransporter in primary astrocytes by dibutyryl cAMP and high
[K+]o
Departments of 1 Neurological Surgery, 2 Physiology, and 3 Surgery, School of Medicine, University of Wisconsin, Madison, Wisconsin 53792
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
[3H]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 Ca2+ influx, the role of the
L-type voltage-dependent Ca2+ 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 Ca2+-free HEPES buffer. A
rise in intracellular free Ca2+ 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
Ca2+-dependent manner.
potassium uptake; bumetanide; L-type calcium channel; intracellular calcium; rat cortical astrocytes; dibutyryl-cAMP
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