The mechanism of protein S-nitrosation in cells is not fully understood. Using rat 3Y1 cells, we addressed this issue. Among S-nitrosothiols and NO donors tested, only S-nitrosocysteine (CysNO) induced S-nitrosation when exposed in Hanks' balanced salt solution (HBSS) and not in serum-containing general culture medium. In HBSS, NO release from CysNO was almost completely abolished by sequestering metal ions with a metal chelator without affecting cellular S-nitrosation. In contrast, L-leucine, a substrate of L-type amino acid transporters (LATs), significantly inhibited S-nitrosation. The absence of S-nitrosation with CysNO in general culture medium resulted not only from a competition with amino acids in the medium for LATs but also from transnitrosation of cysteine residues in serum albumin. Collectively, these results suggest that in simple buffered saline, CysNO-dependent S-nitrosation occurs through a cellular incorporation-dependent mechanism, but if it occurs in general culture media, it may be through an NO-dependent mechanism. 1. Introduction Nitric oxide (NO) plays diverse roles in physiological processes, such as vasodilatation, host defenses against infection, and neuromodulation, some of which are mediated by the activation of the guanylate cyclase (GS)/cGMP pathway [1, 2]. Accumulating evidence suggests that NO also acts as a signaling molecule through the formation of S-nitrosothiols in proteins. S-nitros(yl)ation is a post-translational modification of proteins and low molecular weight thiols in which the nitrosonium cation attaches to the thiolate anion of a cysteine residue in proteins in a net reaction [2, 3]. Protein S-nitrosation has been shown to regulate the functions of various proteins including caspases [3, 4]. However, excessive S-nitrosation of certain proteins has been proposed as a causative event for some diseases [5–7]. More than 100 proteins have been identified to undergo S-nitrosation [3]. Incubation of cysteine-containing proteins or low molecular weight thiols with NO donors in a simple aqueous solution under aerobic conditions yields S-nitrosothiols. The principal mechanism has been proposed to be mediated by dinitrogen trioxide (N2O3) formation as follows [8]: 2 N O + O 2 ？ 2 N O 2 ( 1 ) N O 2 + N O ？ N 2 O 3 N ( 2 ) 2 O 3 + P r o t e i n - C y s - S H ？ P r o t e i n - C y s - S N O + N O 2 ？ + H + ( 3 ) In case of S-nitrosation of intact cells by NO, similar mechanisms are believed to occur in the hydrophobic interior of proteins or the plasma membranes [9, 10]; however, recent studies have implicated the
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