In addition to the generation from specific
nitric-oxide (NO) synthases, NO formation from
nitrite occurs in ischemic tissues, such as the heart. Although NO binding to
heme-centers is the basis for NO-mediated signaling as occurs through
guanylate cyclase, it is not known if this process is triggered with physiologically relevant periods of sublethal
ischemia and if
nitrite serves as a critical substrate. Therefore electron paramagnetic resonance studies were performed to measure
nitrosylheme formation during the time course of
myocardial ischemia and reperfusion and the role of
nitrite in this process. Rat hearts were either partially
nitrite-depleted by
nitrite-free
buffer perfusion or
nitrite-enriched by preinfusion with 50 microm
nitrite. Ischemic hearts loaded with
nitrite showed prominent spectra of six-coordinate nitrosyl-
heme complexes, primarily NO-
myoglobin, that increased as a function of ischemic duration, whereas in nonischemic-controls these signals were not seen. Total nitrosyl-
heme concentrations within the heart were 6.6 +/- 0.7 microm after 30 min of
ischemia.
Nitrite-depleted hearts also gave rise to NO-
heme signals during
ischemia, but levels were 8-fold lower.
Nitrite-mediated NO-
heme complex formation during
ischemia was associated with activation of
guanylate cyclase. Upon reperfusion, the levels of NO-
heme complexes decreased 3-fold by the first 15 min but remained elevated for over 45 min. The decrease in NO-
heme complex levels was paralleled by the formation of
nitrate, suggesting the oxidation of
heme-bound NO upon reperfusion. Thus,
nitrite-mediated NO-
heme formation occurs progressively during
ischemia, with these complexes serving as a store of NO with concordant activation of NO signaling pathways.