L-arginine and Tetrahydrobiopterin Inhibit Mitochondrial Permeability Transition Pore by Preventing ROS Formation by Mitochondrial Nitric Oxide Synthase

Abstract

Background: The functional role of mitochondrial nitric oxide synthase (mtNOS) in heart has remained a matter of debate. Methods: We used laser scanning confocal microscopy in combination with fluorescent dyes to characterize mitochondrial NO and ROS production and the permeability transition pore (PTP) activity in permeabilized cat ventricular myocytes. Results: Stimulation of mitochondrial calcium uptake resulted in a dose-dependent increase in mitochondrial NO production when L-arginine, a substrate for mtNOS, was present. The potential contribution of the caveolae-located eNOS and SR-targeted nNOS was ruled out based on the fact that disruption of caveolae with methyl-βeτα-cyclodextrin or prevention of SR uptake with thapsigargin did not affect calcium-induced NO production. Collapsing the mitochondrial membrane potential, blocking the mitochondrial calcium uniporter and respiratory chain abolished mitochondrial NO production. In the absence of L-arginine, calcium-induced NO production was significantly decreased; however an increased ROS production was observed. Inhibition of mitochondrial arginase (which limits L-arginine availiability) resulted in 50% inhibition of calcium-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-NIO or the iNOS inhibitor 1400W had no effect. The superoxide dismutase mimetic MnTBAP abolished calcium-induced ROS generation and increased NO production threefold. In the absence of L-arginine, mitochondrial calcium uptake induced opening of the mitochondrial PTP, which was blocked by cyclosporin A, MnTBAP and reversed by L-arginine. The essential mtNOS co-factor tetrahydrobiopterin also inhibited mitochondrial ROS generation and PTP opening at a concentration of 100 μM, while 10 μM tetrahydrobiopterin had no effect. Conclusion: Our data demonstrate the importance of L-arginine and tetrahydrobiopterin for the regulation of mitochondrial oxidative stress and modulation of PTP opening by mtNOS.