Abstract
We recently described a role for the circulating anion nitrite as a hypoxic vasodilator, mediated by reduction of nitrite to nitric oxide (NO) by a reaction with deoxyhemoglobin (deoxyHb) (Cosby et al, Nat Med, 2003). The rate of deoxyHb dependent reduction of nitrite is regulated by the structural transition of deoxyHb from T (deoxy) to R (oxy) state, with maximal NO production (and vasodilatory activity) occurring at the P50 of Hb (Huang et al, J Clin Invest, 2005). New studies from our laboratory suggest that this paradigm may extend beyond Hb to other heme proteins, and that hypoxia dependent production of NO from nitrite mediates hypoxic responses beyond vasodilation. Particularly, in a murine model of ischemia/reperfusion (I/R), low doses of nitrite (48nmol) have been shown to protect cardiac and hepatic tissue against reperfusion injury (Duranski et al, J Clin Invest, 2005). One major characteristic of I/R injury is mitochondrial damage, resulting in decreased respiratory rate, decreased ATP production and increased reactive oxygen species production. Since NO is a known reversible inhibitor of mitochondrial respiration and the rate of deoxymyoglobin (deoxyMb) dependent reduction of nitrite to NO is approximately 50-fold higher than the deoxyHb-nitrite reaction, we sought to determine whether the reaction between deoxyMb and nitrite in tissues, where concentrations of Mb and nitrite can reach micromolar levels, could produce bioavailable NO to regulate mitochondrial respiration, and whether this could protect mitochondria against I/R injury. Here we show evidence that nitrite has both acute and chronic effects on mitochondrial respiration. In experiments using respiring isolated rat liver mitochondria, the addition of Mb (25μM) and nitrite (100μM) results in the inhibition of respiratory rate only at oxygen tensions below 2.7mmHg (the P50of Mb), while nitrite or Mb alone have no significant effect on respiration. This acute effect is consistent with spectrophotometric data showing that the rate of reduction of nitrite (100μM) by deoxyMb (25μM) to NO is 9 nM/sec, suggesting that within 20 seconds enough NO would be produced to inhibit respiration (IC50=100nM). In an in vitro model of anoxia-induced mitochondrial injury, respiration rates of isolated rat liver mitochondria were measured before and after the organelles were subjected to anoxia for 30 minutes. In untreated control mitochondria, respiration rate decreased by 50% after the anoxia, consistent with ischemic injury, while mitochondria treated with nitrite (10μM) during anoxia had only a 25% decrease in respiratory rate. Preservation of post-anoxic respiratory rate was dependent on nitrite concentration (5–50μM,) with the maximal effect occurring at 10μM, a concentration coinciding with the tissue levels of nitrite at the nitrite dose which conferred maximal protection in previous animal models of I/R. Remarkably, administration of nitrite (480nmol) to rats 24 hours before the isolation of mitochondria was also shown to prevent the anoxia-induced decrease in respiration rate. In summary, these data suggest that nitrite bioactivation in tissue, both by Mb-dependent and independent mechanisms, modulates mitochondrial respiration and stress response. These data support a more global role for nitrite as a biochemical HIF-1 alpha with pleiotropic effects on both vascular and tissue response to hypoxia.
Stimulation of the respiration rate of rat liver mitochondria by sub-micromolar concentrations of extramitochondrial Ca2+
