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 expres sion in rat cortical astrocyte differentiation. Astrocyte differentiation w as induced by dibutyryl cAMP (DBcAMP, 0.25 mM) for 7 days, and cells change d from a polygonal to process-bearing morphology. Basal activity of the cot ransporter was significantly increased in DBcAMP-treated astrocytes (P < 0. 05). Expression of an <similar to>161-kDa cotransporter protein was increas ed by 91% in the DBcAMP-treated astrocytes. Moreover, the specific [H-3] bu metanide binding was increased by 67% in the DBcAMP-treated astrocytes. Inh ibition of protein synthesis by cyclohexamide (2-3 mug/ml) significantly at tenuated 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 c otransporter-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+-i nduced stimulation of the cotransporter is attributed to membrane depolariz ation and Ca2+ influx, the role of the L-type voltage-dependent Ca2+ channe l was investigated. The high-K+-mediated stimulation of the cotransporter a ctivity was abolished in the presence of either 0.5 or 1.0 <mu>M of the L-t ype channel blocker nifedipine or Ca2+-free HEPES buffer. A rise in intrace llular free Ca2+ in astrocytes was observed in high K+. These results provi de 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 hi gh K+ in a Ca2+-dependent manner.