Effect of metabolic inhibitors on membrane potential and ion conductance of rat astrocytes

1. The aim of this study was to elucidate the effect of metabolic inhibition on the membrane potential and ion conductance of rat astrocytes, The metabolic inhibitors investigated were dinitrophenol (DNP), carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP), cyanide, and oligomycin. 2. Primary cultures of astroglial cells from newborn rat cerebral cortex were cultivated for 13-20 days on chamber slides. The effect of metabolic inhibitors on the cellular ATP concentration was estimated from the decrease in peak chemiluminescence from the luciferin/luciferase reaction. The membrane potential and ion conductances were measured from whole-cell recordings with the patch-clamp technique. 3. After 2.0 min of incubation ATP decreased from the control level to 43% with cyanide (2 mM), 58% with DNP (1 mM), 47% with FCCP (1 mu M), and 69% with oligomycin (10 mu M). 4. Under normal conditions V was -74.4 +/- 1.0 mV. DNP and FCCP both caused a rapid and reversible depolarization equivalent to a shift in the I/V curve of 8.2 +/- 1.3 and 19.7 +/- 3.8 mV, respectively. DNP decreased the slope conductance (g) by 22.1% but FCCP had no significant effect on g. In contrast, neither oligomycin nor cyanide had any significant effect on the I/V curve, 5. Tetraethylammonium (TEA; 10 mM) depolarized the cells by 7.1 +/- 2.0 mV but had no significant effect on g. In the presence of TEA, DNP caused a depolarization of 52.8 +/- 3.5 mV and increased g by 45.5 +/- 9.6%. The action of FCCP was not affected by the presence of TEA. 6. Perfusion of the astrocytes with a Cl- free solution inhibited the action of DNP and FCCP. Thus the depolarization was only 4.2 +/- 1.5 mV in DNP and 3.7 +/- 0.3 mV in FCCP, which were significantly smaller effects than in the presence of a high intracellular [Cl-]. 7. Block of tentative K-ATP channels with tolbutamide (1 mM) or Cl- channels with Zn2+ (1 mM) did not inhibit the depolarization caused by DNP or FCCP. 8. In conclusion, DNP and FCCP have specific effects on the plasmalemma in rat astrocytes which may be due to opening of Cl- channels: This effect was not seen with cyanide or oligomycin and should be considered as a possible complication when DNP and FCCP are used for metabolic inhibition.