Arginine metabolism in keratinocytes and macrophages during nitric oxide biosynthesis: multiple modes of action of nitric oxide synthase inhibitors.

Nitric oxide is an important cellular mediator produced in keratinocytes and macrophages from arginine by the enzyme nitric oxide synthase during inflammatory reactions in the skin. We found that gamma-interferon stimulated nitric oxide production and the expression of inducible nitric oxide synthase in both cell types. However, macrophages produced more nitric oxide and nitric oxide synthase protein, and at earlier times than keratinocytes. Keratinocytes treated with gamma-interferon took up more arginine than macrophages; however, they were less efficient in metabolizing this amino acid and exhibited reduced nitric oxide synthase enzyme activity. In both cell types, the nitric oxide synthase inhibitors, N(G)-monomethyl-L-arginine (NMMA), L-N5-(iminoethyl)ornithine, L-canavanine, and N(omega)-nitro-L-arginine, as well as lysine, ornithine, and homoarginine markedly reduced arginine uptake. In contrast, N(omega)-nitro-L-arginine methyl ester and N(omega)-nitro-L-arginine benzyl ester were poor inhibitors of arginine uptake, while aminoguanidine had no effect on uptake of arginine by the cells. Moreover, NMMA was found to inhibit simultaneously arginine uptake and nitric oxide synthase enzyme activity in both cell types, whereas aminoguanidine only affected nitric oxide synthase activity. No major differences were observed between keratinocytes and macrophages. Taken together, these data demonstrate that, although keratinocytes and macrophages both synthesize nitric oxide, its production is regulated distinctly in these two cell types. Furthermore, in these cells, nitric oxide synthase inhibitors such as NMMA exhibit at least two sites of action: inhibition of nitric oxide synthase and cellular uptake of arginine.