HEAT SHOCK-INDUCED AND ETHANOL-INDUCED IONIC CHANGES IN C6 RAT GLIOMA-CELLS DETERMINED BY NMR AND FLUORESCENCE SPECTROSCOPY

The effects of two different stressors, heat shock (HS; 44 degrees C, 20 min) and ethanol (1.2 M, 60 min), on ion content and membrane poten tial were investigated in C6 rat glioma cells. Both treatments were pr eviously shown to induce the HS response [26]. Intracellular pH (pH(i) ), sodium ion concentration ([Na+](i)), potassium ion concentration ([ K+](i)) and membrane potential were determined by means of continuous P-31 and Na-23 nuclear magnetic resonance (NMR), continuous fluorescen ce spectroscopy and Rb-86 uptake. Lactate extrusion was determined in addition with respect to pH(i) regulation. The aim of this study was a detailed picture of HS and ethanol-induced ion changes in a single ce ll type, because stress-induced changes in the intracellular ionic bal ance may be important factors for determining proliferation, stress re sponse and apoptosis. HS lowered the pH(i) from 7.38 +/- 0.04 to about 7.05. +/- 0.04. [Na+](i) decreased during HS to 50% of the control an d recovered to normal levels 95 min after HS treatment. During HS, [K](i) remained constant but increased after HS. The membrane potential hyperpolarized from -83 mV to -125 mV and returned to initial values d uring HS treatment. Lactate extrusion increased 3-fold after HS. Ethan ol (1.2 M) lowered the pH(i) from pH 7.38 +/- 0.04 to pH 7.0 +/- 0.04, but in contrast to heat strongly increased [Na+](i). it hyperpolarize d the membrane potential from -83 mV to -125 mV. Ethanol also increase d lactate extrusion similar to HS. Also in contrast to the effect of H S, the potassium concentration decreased during ethanol treatment. The Na+-H+ exchanger monensin was used to overcome the apparent inhibitio n of the cellular Na+-H+ exchanger by HS. At normal pH(e) (7.4) monens in increased [Na+]i (a)nd pH(i) considerably. A subsequent HS reduced [Na+](i) only minimally. Acidification of the cells by low pH(e) (6.2) prior to HS did not abolish the HS-induced drop of pH(i), indicating that the Na+-H+ exchanger was also inhibited at low pH(i). At low pH(e ), monensin transports H+ into the cell. A subsequent HS decreased pH( i) only little, showing the importance of inhibition of the Na+-H+ exc hanger for the HS-induced pH(i) decrease. 100 mu M amiloride reduced p H(i) and [Na+](i) in a similar way as HS, but did not change pH(i) and [Na+](i) much during a HS. These results indicate that some of the HS -induced ionic changes are mediated by inhibition of the Na+-H+ exchan ger, activation of Na+-K+-ATPase and changes of membrane conductance f or ions.