The Activity of a Low-Affinity L-Arginine Transporter Quenches Peroxynitrite-Induced Fluorescence in Ventricular Cardiomyocytes

Abstract

We discovered a low-affinity, high-capacity L-arginine (L-Arg) transport process in rat cardiomyocytes consistent with the activity of the CAT-2A member of the y(+) family of cationic amino acid transporters (Peluffo, J Physiol, 580:925-936, 2007), set to function in parallel with the previously described high-affinity, low-capacity CAT-1 (Lu et al., Biosci Rep, 29:271-281, 2009). In assessing the role of a low-affinity transporter in this setting, we propose that CAT-2A protects cardiac muscle cells by ensuring the availability of proper L-Arg levels for the synthesis of nitric oxide (NO) via NO synthase (NOS). To test this hypothesis, acutely-isolated cardiomyocytes were loaded with the dye coelenterazine that greatly increases its fluorescence quantum yield in the presence of peroxynitrite (ONOO-) and superoxide radicals. Cells were then exposed to 20 or 100 μM ONOO- and changes in fluorescence were followed with a spectrofluorometer. Addition of extracellular L-Arg reduced ONOO-induced fluorescence in a concentration-dependent manner, an effect that was not mimicked by D-arginine or L-lysine and was fully blocked by the NOS inhibitor L-NAME. L-Arg reduced fluorescence with Ki values of 0.84 ± 0.12 and 1.26 ± 0.16 mM at 20 and 100 μM ONOO-, respectively. L-Arg “zero effect” on ONOO-induced fluorescence was also dependent on ONOO- concentration, with values of 145 and 363 μM for 20 and 100 μM ONOO-, respectively. Below these values, decreasing concentrations of L-Arg progressively increased ONOO-induced fluorescence, an effect that was also blocked by L-NAME. All these effects can be explained by NOS-mediated NO synthesis, which may turn to ONOO- production at limiting L-Arg. Since ONOO- has detrimental effects on cardiac contractility, these results suggest a cardioprotective role for the low-affinity L-Arg transporter, ensuring proper supply of NOS substrate under a variety of physiological and pathological conditions.