Diagram depicting the proposed pathways for the development of nitrate tolerance. Both endothelial and smooth muscle cells are involved in these processes. Superoxide anion is produced by membrane oxidases and, possibly, during nitroglycerin (glyceryl trinitrate; GTN) biotransformation (dashed arrow). Increased angiotensin II (AT II) production and responsiveness and increased oxidative stress may result in uncoupling of nitric oxide synthase (NOS) and further production of reactive oxygen species and peroxynitrite. In turn, these oxidant free radicals may cause many of the abnormalities observed in nitrate tolerance. Isoprostane formation and increased capillary permeability might be responsible for plasma volume expansion, and tyrosine nitrosylation and thiol group oxidation might be responsible for inactivation of multiple enzymes, including those involved in the biotransformation of nitroglycerin. Tetrahydrobiopterin (BH₄) oxidation may cause NOS uncoupling. Figure modified with permission from Circulation (2). PKC, protein kinase C; O₂^–, superoxide anion; GDN, glyceryl dinitrate; NO₂^–, nitrite.