Regulation of Na(+)-K(+)-Cl(-) cotransporter in primary astrocytes by dibutyryl cAMP and high [K(+)](o).
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 approximately 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 microgram/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 microM 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.
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Department of Neurological Surgery, School of Medicine, University of Wisconsin, Madison, Wisconsin 53792, USA.
Haworth
R A
RA
Dempsey
R J
RJ
Sun
D
D
D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D003994:Bucladesine / Q000494:pharmacology*; D002034:Bumetanide / Q000378:metabolism / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020746:Calcium Channels, L-Type / Q000378:metabolism; D002352:Carrier Proteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D004232:Diuretics / Q000378:metabolism / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D009543:Nifedipine / Q000494:pharmacology; D011188:Potassium / Q000493:pharmacokinetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D028021:Sodium-Potassium-Chloride Symporters; D014316:Tritium
D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; D002352:Carrier Proteins; D004232:Diuretics; D028021:Sodium-Potassium-Chloride Symporters; D002034:Bumetanide; D014316:Tritium; D003994:Bucladesine; D009543:Nifedipine; D011188:Potassium; D002118:Calcium