Figure 6.

Effect of selective inhibition of Trx or GSH scavenging systems on H₂O₂ emission from heart mitochondria during respiratory states 4 and 3. Freshly isolated mitochondria (∼100 μg mitochondrial protein) from guinea pig hearts were preincubated with the indicated concentrations of AF (A), or DNCB (C) in the presence of G/M (5 mM each). Monitoring of H₂O₂ was performed with the Amplex Red assay during state 4 (black solid square) and 3 (+1 mM ADP, gray solid square) respiration (see Materials and Methods). Shown are the specific fluxes of H₂O₂ emission obtained from two experiments with duplicates in each (24). For simulating AF inhibition with the ME-R model, the concentrations of Trx reductase from mitochondrial (E_trxm) and extra-matrix (E_trx) compartments were simultaneously lowered from a control concentration of 3.5 × 10⁻⁴ mM to 7 × 10⁻⁶ mM. The steady state values of H₂O₂ emission (V_H2O2dif expressed in the same units as the experimental plots) were computed at each inhibitory concentration (B). DNCB inhibition was simulated by simultaneously decreasing mitochondrial (Et_GRm) and extra-matrix (Et_GR) GR from a control concentration of 9 × 10⁻⁴ mM to 3 × 10⁻⁵ mM. In the simulations, the percent inhibition was calculated from dividing the control concentration by Et_GRm and the result multiplied by 100. In experiments and simulations, the kinetic parameters (V_max and K_0.5) that characterize the H₂O₂ emission fluxes as a function of the inhibitor concentrations, were determined by nonlinear regression of the data points with a hyperbolic Michaelis-Menten or Hill type equation (solid lines).