ABSTRACT
During cerebral ischemia and trauma, a complex cascade of events is set in motion that contributes to cellular injury. When cerebral blood flow falls to levels of approximately 20% of control, glucose and oxygen supply become limiting and bioenergetic failure occurs.1 As high-energy phosphate stores become depleted, the tissue depolarizes, leading to a loss of ion homeostasis. One of the consequences of a depletion of ATP is that the excitatory neurotransmitter glutamate, which is normally taken up from the extracellular space by glutamate transporter proteins (requiring the expenditure of energy), now begins to accumulate in the extracellular space and soon reaches neurotoxic levels.2 Calcium enters the cell through the activated neuronal Nmethyl-D-aspartate (NMDA) receptors, as well as through voltage-sensitive calcium channels and via reversal of 2Na+/Ca2+ exchange.3,4 This increase in cytosolic calcium is deleterious to the cell by way of a number of mechanisms including lipolysis, proteolysis, disaggregation of microtubuli, and activation of a number of enzymes, principal among them, nitric oxide synthase.5,6
When cytosolic Ca2+ increases precipitously during ischemia, two isoforms of nitric oxide synthase, NOS1 (nNOS) and NOS3 (eNOS), bind calmodulin in a Ca2+-dependent manner7 and nitric oxide (NO) synthesis increases by more than 20-fold.8 Although NOS2 (iNOS) is not Ca2+-dependent, the induction of this enzyme is stimulated by cytokines released during ischemia.9 The activation of NOS1 and NOS3 occurs within minutes after the start of ischemia, whereas the induction of NOS2 mRNA takes several hours, but persists over several days. In ischemia, and in particular during recirculation, the production of NO is accompanied by the production of superoxide anions and hydrogen peroxide. These can combine to form hydroxyl free radicals, which are highly reactive and can lead to lipid peroxidation. Of greater importance, however, is that NO combines with superoxide to form peroxynitrite (ONOO-).10,11 Although itself not a free radical, peroxynitrite is an anion that is extremely toxic to many biological molecules and to DNA. It is extremely reactive with moities that are involved with signal transduction,12 attacks iron/sulfer centers important for cell respiration,13 produces lipid peroxidation and causes calcium to be released from the intracellular stores in the mitochondria.14
