Figure 2.
Figure 2. Nitrite metabolism by deoxygenated RBCs stimulates NO-dependent vasodilation and inhibition of mitochondrial respiration. (A) Representative vessel tension traces showing rat RBCs (0.3% HCT) and nitrite-dependent vasodilation at 25 mmHg oxygen in the presence (black) and absence (gray) of 200 μM C-PTIO. Arrows indicate the times and concentrations of nitrite added. (B) Changes in cGMP in rat thoracic aorta treated as indicated for 10 minutes at 25 mmHg oxygen (▪) or 600 mmHg oxygen (□). Final concentrations were nitrite 3 μM, rat RBCs 0.3% HCT, and C-PTIO 200 μM. *P < .01 relative to control and nitrite alone; *P < .05 relative to RBCs alone (n = 3). (inset) To estimate the amount of NO produced by RBC/nitrite, a calibration curve was determined for increased vessel cGMP in response to increasing concentrations of the NO donor, DeaNonoate, at 25 mmHg oxygen. In a concentration-dependent manner, NO increased cGMP, which was inhibited by C-PTIO (○). Specifically, 100 nM DeaNonoate increased cGMP from 526.3 ± 46 pmol/g to 5602.6 ± 1340 pmol/g in the absence of C-PTIO and to 653 ± 97.9 nM in the presence of C-PTIO (values represent mean ± SEM). Using this curve, an estimated 20 nM NO was produced by RBCs (0.3% HCT) and nitrite (3 μM) over 10 minutes. (C) Representative traces of oxygen concentration as a function of time for mitochondria in state 3 respiration (black line) in the presence of 20 μM nitrite (gray line), 0.3% HCT RBCs (dashed line), or RBCs and nitrite (dotted line) are shown. Respiratory substrates were added to oxygen electrode chamber containing mitochondria and RBCs. The arrow indicates the time of nitrite addition. (D) Respiration rate measured at 30 μM oxygen. Inhibition of respiration by RBCs + nitrite was completely reversed by C-PTIO, consistent with NO formation. C-PTIO had no effect alone (not shown), and neither did RBCs alone. *P < .001 relative to RBCs alone. #P < .002 relative to RBCs + nitrite + PTIO (n = 3).

Nitrite metabolism by deoxygenated RBCs stimulates NO-dependent vasodilation and inhibition of mitochondrial respiration. (A) Representative vessel tension traces showing rat RBCs (0.3% HCT) and nitrite-dependent vasodilation at 25 mmHg oxygen in the presence (black) and absence (gray) of 200 μM C-PTIO. Arrows indicate the times and concentrations of nitrite added. (B) Changes in cGMP in rat thoracic aorta treated as indicated for 10 minutes at 25 mmHg oxygen (▪) or 600 mmHg oxygen (□). Final concentrations were nitrite 3 μM, rat RBCs 0.3% HCT, and C-PTIO 200 μM. *P < .01 relative to control and nitrite alone; *P < .05 relative to RBCs alone (n = 3). (inset) To estimate the amount of NO produced by RBC/nitrite, a calibration curve was determined for increased vessel cGMP in response to increasing concentrations of the NO donor, DeaNonoate, at 25 mmHg oxygen. In a concentration-dependent manner, NO increased cGMP, which was inhibited by C-PTIO (○). Specifically, 100 nM DeaNonoate increased cGMP from 526.3 ± 46 pmol/g to 5602.6 ± 1340 pmol/g in the absence of C-PTIO and to 653 ± 97.9 nM in the presence of C-PTIO (values represent mean ± SEM). Using this curve, an estimated 20 nM NO was produced by RBCs (0.3% HCT) and nitrite (3 μM) over 10 minutes. (C) Representative traces of oxygen concentration as a function of time for mitochondria in state 3 respiration (black line) in the presence of 20 μM nitrite (gray line), 0.3% HCT RBCs (dashed line), or RBCs and nitrite (dotted line) are shown. Respiratory substrates were added to oxygen electrode chamber containing mitochondria and RBCs. The arrow indicates the time of nitrite addition. (D) Respiration rate measured at 30 μM oxygen. Inhibition of respiration by RBCs + nitrite was completely reversed by C-PTIO, consistent with NO formation. C-PTIO had no effect alone (not shown), and neither did RBCs alone. *P < .001 relative to RBCs alone. #P < .002 relative to RBCs + nitrite + PTIO (n = 3).

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