FIGURE 7.
Respiratory chain activity was reduced at the level of COX after TBI and sphingosine inhibited the COX activity in baseline mitochondria. A, mitochondria were purified from the ipsilateral hemisphere of WT mouse brain at day 7 post-TBI. Sham-injured animal brain was used as a control (Con). Mitochondrial respiration was measured by recording oxygen consumption with a Clark-type oxygen electrode in the presence of Complex I substrate 5 mm glutamate plus 5 mm malate (glutamate + malate), Complex II substrate 10 mm succinate (succinate), and Complex IV (COX) substrate 1 mm ascorbate plus 250 μm TMPD (ascorbate + TMPD) in the presence of 100 μm ADP (state 3). Data are means ± S.E. (error bars); *, p < 0.05, n = 8. B, mitochondrial COX activity was measured by recording oxygen consumption of baseline mitochondria in the presence of COX substrate (1 mm ascorbate plus 250 μm TMPD), 1 μm antimycin, and 50 μm 2,4-DNP. Sphingosine (Sph) and dihydrosphingosine (DHSph) were delivered in ethanol. C16:0-ceramide and C18:0-ceramide were delivered in ethanol/dodecane (98:2, v/v). Data are means ± S.E.; *, p < 0.05, n = 8.