Abbreviations COX, cytochrome c oxidase; CRC, calcium retention capacity; CsA, cyclosporin A; Δ Ψ m, mitochondrial membrane potential; GC, nitric oxide-sensitive guanylyl cyclase; KT, KT5823; MPTP, mitochondrial permeability transition pore; mtNOS, mitochondrial NO synthase; mtNOS-SS , mtNOS/ GC/PKG-signaling system; 7-NI, 7-nitroindasole; NO, nitric oxide; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; PKG, cGMP-dependent kinase G; PC, D,Lpalmitoylcarnitine; ROS, reactive oxygen species; SNAP, S-nitrosoacetylpenacillamine; SNP, sodium nitroprusside; VO 2 ss , steady state respiration rate. Keywords nitric oxide; mitochondrial respiration; permeability transition pore; mitochondrial nitric oxide synthase, guanylate cyclase and protein kinase G AbstractThe role of mitochondrial calcium-dependent NO synthase in the control of respiration and mitochondrial permeability transition pore (MPTP) opening, as well as possible involvement of mitochondrial NO synthase/guanylate cyclase/kinase G-signaling system (mtNOS-SS) in the regulation of these processes are not sufficiently studied. In this work, using rat liver mitochondria, we applied specific inhibitors of the enzymes of this signaling system to evaluate its role in the control of respiration and MPTP. The respiration was supported by pyruvate and glutamate or succinate in the presence of hexokinase, glucose and ADP. The results indicate that L-arginine and NO donors SNP and SNAP produce bidirectional concentration-dependent effects on the respiration and MPTP opening evoked by calcium ions or D,L-palmitoylcarnitine. Maximal activation of respiration was observed at 20 µM of L-arginine or SNP. At low concentrations, L-arginine (to 500 µM) and NO donors (to 50 µM) increased the threshold concentrations of calcium and D,L-palmitoylcarnitine required for the dissipation of the mitochondrial membrane potential and pore opening. The application of the inhibitors of NO synthase, guanylate cyclase, and kinase G eliminated both effects. These data indicate the involvement of mtNOS-SS in the activation of respiration and deceleration of MPTP opening. At high concentrations, L-arginine and NO donors inhibited the respiration and promoted pore opening, indicating that the inhibition induced by NO excess dominates over the protection caused by mtNOS-SS. These results demonstrate that the functioning of mtNOS-SS might provide a feedforward activation of respiration and a lowering of MPTP sensitivity to calcium and palmitoylcarnitine overload.