SWELLING-INDUCED ACTIVATION OF NA-) COTRANSPORT IN C6 GLIOMA-CELLS - KINETIC-PROPERTIES AND INTRACELLULAR SIGNALING MECHANISMS(,K+,2CL()

Swelling of C6 glioma cells in hypotonic medium (180 mOsm) results in two- to three-fold activation of K+ (Rb-86(+)) influx suppressed by 10 mu M bumetanide. Bumetanide-sensitive transport of Rb-86(+) is, depen dent on extracellular K+, Na+ and Cl- both in iso-osmotic conditions a nd under hypo-osmotic shock, supporting, the notion that it is mediate d by Na+,K+,2Cl(-) cotransport. Inhibitors of protein kinase C (10 mu M polymyxin B and 1 mu M staurosporine) had no significant effect on b asal cotransport but reduced its hypotonic stimulation by 70-80%. Simi lar results were obtained with calmodulin antagonist R24571 (10 mu M), indicating Ca2+/calmodulin-dependence of the process. Influence of po lymyxin B and R24571 was not additive. Swelling-activated Na+,K+,2Cl(- ) cotransport was also suppressed by protein kinase C activator PMA(I mu M). By contrast, preincubation of cells with inhibitors of protein phosphatases (100 mu M vanadate, 5 mM fluoride and 0.5 mu M okadaic ac id) activated greatly the bumetanide-sensitive Rb-86(+) uptake in isot onic conditions, while a subsequent hypotonic swelling led to smaller or no increment. These results indicate the involvement of Ca2+/calmod ulin-dependent staurosporine/polymyxin B-sensitive protein kinase othe r than protein kinase C in swelling-induced activation of Na+,K+,2Cl(- ) cotransport in glial cells.