Swelling-induced activation of Na+,K+,2Cl− cotransport in C6 glioma cells: kinetic properties and intracellular signalling mechanisms

AbstractSwelling of C6 glioma cells in hypotonic medium (180 mOsm) results in two- to three-fold activation of K− (86Rb+) influx suppressed by 10 μM bumetanide. Bumetanide-sensitive transport of 86Rb+ is dependent on extracellular K−, Na− and Cl− both in iso-osmotic conditions and under hypo-osmotic shock, supporting the notion that it is mediated by Na+,K+,2Cl− cotransport. Inhibitors of protein kinase C (10 μM polymyxin B and 1 μM staurosporine) had no significant effect on basal cotransport but reduced its hypotonic stimulation by 70–80%. Similar results were obtained with calmodulin antagonist R24571 (10 μM), indicating Ca2+/calmodulin-dependence of the process. Influence of polymyxin B and R24571 was not additive. Swelling-activated Na+,K+,2Cl− cotransport was also suppressed by protein kinase C activator PMA (1 μM). By contrast, preincubation of cells with inhibitors of protein phosphatases (100 μM vanadate, 5 mM fluoride and 0.5 μM okadaic acid) activated greatly the bumetamide-sensitive 86Rb+ uptake in isotonic conditions, while a subsequent hypotonic swelling led to smaller or no increment. These results indicate the involvement of Ca2+/calmodulin-dependent staurosporine/polymyxin B-sensitive protein kinase other than protein kinase C in swelling-induced activation of Na+,K+,2Cl− cotransport in glial cells