We used laser scanning confocal microscopy in combination with the nitric oxide (NO)-sensitive fluorescent dye DAF-2 and the reactive oxygen species (ROS)-sensitive dyes CM-H(2)DCF and MitoSOX Red to characterize NO and ROS production by mitochondrial NO synthase (mtNOS) in permeabilized cat ventricular myocytes. Stimulation of mitochondrial Ca(2+) uptake by exposure to different cytoplasmic Ca(2+) concentrations ([Ca(2+)](i) = 1, 2 and 5 microm) resulted in a dose-dependent increase of NO production by mitochondria when L-arginine, a substrate for mtNOS, was present. Collapsing the mitochondrial membrane potential with the protonophore FCCP or blocking the mitochondrial Ca(2+) uniporter with Ru360 as well as blocking the respiratory chain with rotenone or antimycin A in combination with oligomycin inhibited mitochondrial NO production. In the absence of L-arginine, mitochondrial NO production during stimulation of Ca(2+) uptake was significantly decreased, but accompanied by increase in mitochondrial ROS production. Inhibition of mitochondrial arginase to limit L-arginine availability resulted in 50% inhibition of Ca(2+)-induced ROS production. Both mitochondrial NO and ROS production were blocked by the nNOS inhibitor (4S)-N-(4-amino-5[aminoethyl]aminopentyl)-N'-nitroguanidine and the calmodulin antagonist W-7, while the eNOS inhibitor L-N(5)-(1-iminoethyl)ornithine (L-NIO) or iNOS inhibitor N-(3-aminomethyl)benzylacetamidine, 2HCl (1400W) had no effect. The superoxide dismutase mimetic and peroxynitrite scavenger MnTBAP abolished Ca(2+)-induced ROS generation and increased NO production threefold, suggesting that in the absence of MnTBAP either formation of superoxide radicals suppressed NO production or part of the formed NO was transformed quickly to peroxynitrite. In the absence of L-arginine, mitochondrial Ca(2+) uptake induced opening of the mitochondrial permeability transition pore (PTP), which was blocked by the PTP inhibitor cyclosporin A and MnTBAP, and reversed by L-arginine supplementation. In the presence of the mtNOS cofactor (6R)-5,6,7,8,-tetrahydrobiopterin (BH(4); 100 microm) mitochondrial ROS generation and PTP opening decreased while mitochondrial NO generation slightly increased. These data demonstrate that mitochondrial Ca(2+) uptake activates mtNOS and leads to NO-mediated protection against opening of the mitochondrial PTP, provided sufficient availability of l-arginine and BH(4). In conclusion, our data show the importance of L-arginine and BH(4) for cardioprotection via regulation of mitochondrial oxidative stress and modulation of PTP opening by mtNOS.