Abstract-Endothelial function is impaired in aging because of a decrease in NO bioavailability. This may be, in part, attributable to increased arginase activity, which reciprocally regulates NO synthase (NOS) by competing for the common substrate, L-arginine. However, the high K m of arginase (Ͼ1 mmol/L) compared with NOS (2 to 20 mol/L) seemingly makes direct competition for substrate unlikely. One of the mechanisms by which NO exerts its effects is by posttranslational modification through S-nitrosylation of protein cysteines. We tested the hypothesis that arginase1 activity is modulated by this mechanism, which serves to alter its substrate affinity, allowing competition with NOS for L-arginine. We demonstrate that arginase1 activity is altered by S-nitrosylation, both in vitro and ex vivo. Furthermore, using site-directed mutagenesis we demonstrate that 2 cysteine residues (C168 and C303) are able to undergo nitrosylation. S-Nitrosylation of C303 stabilizes the arginase1 trimer and reduces its K m value 6-fold. Finally, arginase1 nitrosylation is increased (and thus its K m decreased) in blood vessels from aging rats, likely contributing to impaired NO bioavailability and endothelial dysfunction. This is mediated by inducible NOS, which is expressed in the aging endothelium. These findings suggest that S-nitrosylated arginase1 can compete with NOS for L-arginine and contribute to endothelial dysfunction in the aging cardiovascular system. (Circ Res. 2007;101:692-702.)Key Words: arginase Ⅲ NO synthase Ⅲ S-nitrosylation Ⅲ aging A ging is accompanied by impaired endothelial function caused by reduced NO bioavailability. Arginase, the final enzyme of the urea cycle, uses L-arginine as a substrate 1-4 and reciprocally regulates NO synthase (NOS) by substrate depletion. [5][6][7][8] Increased arginase activity, therefore, leads to diminished production of NO. 6,[9][10][11][12] In vascular tissue, this further affects 2 important functions of NO: (1) cGMPdependent signaling and (2) modulation of protein function through S-nitrosylation. Several studies have demonstrated reciprocal regulation of arginase and NOS, where inhibition of arginase leads to increased NO activity. 7,8,13,14 Conversely, upregulation of arginase functionally inhibits NOS activity and contributes to the pathophysiology of several disease processes, 8,[15][16][17][18] including age-related vascular dysfunction. 5,6 The high K m value of human arginase for L-arginine (Ͼ1 mmol/L 19 ) is significantly higher than that of NOS (2 to 20 mol/L 20 ) and suggests that there should not be a direct competition for L-arginine. However, the role of arginase in regulating NOS activity through substrate depletion is now well detailed, as discussed above.Given the interaction between NOS and arginase signaling, we hypothesized that S-nitrosylation of arginase1 might be an important posttranslational modification mechanism that regulates its activity. The protein sequence for human arginase1 contains 3 cysteines, C45, C168, and C303, with C303 being very close...