OSMOSIGNALLING IN C6 GLIOMA-CELLS

The influence of aniso-osmolarity on the activity of the MAP kinases E rk-1 and Erk-2 was studied in C6 glioma cells, Hypo-osmotic treatment (205 mosmol/l) led to an increased activity of Erk-1 and Erk-2 within 3 min, which became maximal at 10 min and returned to basal level with in 120 min. In contrast, Erk activity was reduced under hyper-osmotic conditions (405 mosmol/l), compared to the normo-osmotic control (305 mosmol/l), Erk activation was accompanied by a mobility shift of Raf-1 , Hypo-osmotic exposure increased the cytosolic Ca2+ concentration ([C a2+](i)). Absence of extracellular Ca2+ largely abolished the [Ca2+](i ) response to hypo-osmolarity, whereas Erk activation following hypo-o smotic stimulation remained unaffected, suggesting a Ca2+ independence of the osmosignalling pathway to the MAP kinases, Both the Ca2+ respo nse as web as the Erk activation following hypo-osmotic exposure were maintained in the presence of the phospholipase C inhibitor U73122. Ap plication of 8-CPT cAMP, forskolin/isobutylmethylxanthine or isoproter enol blocked Erk activation following hypo-osmotic treatment of the ce lls, suggesting a role of the Ras/Raf pathway upstream from Erk-1 and Erk-2. Protein kinase C (PKC) is unlikely to play a role in the hypo-o smolarity-induced signalling towards MAP kinases, as revealed by inhib ition of PRC with Go6850. Inhibition of pertussis- or cholera toxin-se nsitive G-proteins as well as inhibition of tyrosine kinases with geni stein and of PI3 kinase by wortmannin had no effect on the Erk respons e to hypo-osmolarity. It is concluded that osmosignalling in C6 glioma cells differs upstream of the MAP kinases from that observed in prima ry rat astrocytes, H4IIE rat hepatoma cells and isolated rat hepatocyt es.