![Figure 3. RBC P50 regulates nitrite-dependent vasodilation. (A) Representative traces of simultaneously measured vessel tension and oxygen tension as a function of time with RBCs alone (gray trace) or RBCs + nitrite (black trace). (B) Nitrite-dependent dilation starts at higher oxygen tensions in response to human RBCs (hRBCs), HBOC-201, or HbA and IHP. Final concentrations: nitrite (2 μM), human RBCs (0.3% HCT), HBOC-201 (25 μM), HbA (25 μM), and IHP (625 μM). †P < .03 relative to nitrite and P < .01 relative to hRBCs alone (n = 3). *P < .001 relative to nitrite alone. #P < .01 relative to Hb + IHP. Data represent mean ± SEM (n = 3-5). (C) Relationship of Hb P50 and the oxygen tension at the initiation of vessel dilation in the presence of nitrite (2 μM). Data from rat RBCs, hRBCs, HBOC-201, and cell-free HbA (in which P50 was modulated by the addition of varying concentrations of IHP, resulting in heme/IHP ratios ranging from 1:0.25 to 1:25) are shown. A linear relationship (y = 19.6 + 0.62x; r = 0.89; note intercept is not zero because of basal dilatory responses of vessels to hypoxia) between P50 and oxygen tension at onset of relaxation was observed. (inset) Calculated values for Hb fractional saturation (Y) at the oxygen tension at which nitrite-dependent vasodilation was initiated. (D) Using the calculated rate constants for nitrite reactions with RBC deoxyHb, the predicted velocity or initial rate of nitrite reaction with deoxyHb under physiologic conditions (total Hb = 20 mM in heme; circulating nitrite concentrations = 500 nM19) and as a function of Hb fractional saturation are shown and indicate a maximal rate of reaction around the Hb P50. Data were collected using RBCs from 3 different preparations. (inset) Representative traces showing NO formation (measured by NO chemiluminescence) in the head-space of the reaction between RBC (0.3% hc) and nitrite (500 μM) after 2 minutes in the open-flow respirometer (head-space volume and reaction solution volume 5 mL each with 100 mL/min gas flow). Traces show NO production after injection of 200 μL of [A] air alone or head-space from [B] nitrite in PBS at 28 mmHg oxygen or nitrite + RBC at [C] 0 mmHg (0% fractional saturation), [D] 28 mmHg oxygen (50% fractional saturation), and [E] 155 mmHg (100% fractional saturation).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/107/2/10.1182_blood-2005-07-2668/4/zh80020689370003.jpeg?Expires=1735496865&Signature=H63kN~zrhiYGMRkHNzDSQ3y26sUjg0nG-7RNGM9Pw1hdarTZXnON~hXvISY8LWXt-ne17VmAaXnEUHT3z2QNY~t-6O9j9ZXV1jzxUb6edXR-bnJkT4l0ot5k5~fovUqrHal6XscvVzYYDIpbB0yxGG-k5PZe7YZiH5jPwXc4ZNaI6henKWzO2DvNRk6f9CUHyI-ISuKFWmjma0iyIHeY8yJ5-GdtWJ4jwSrphuWTt6NJ2PO6kLtH-e1XbanSPVbvqooSfpea-g48hd6A9WREim2YaNbYNh7PtA9xcE44UYnxFD6JcNGcdVOO8GaYESCtUTU0nhv9oRvjTrik5a10eA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
RBC P50regulates nitrite-dependent vasodilation. (A) Representative traces of simultaneously measured vessel tension and oxygen tension as a function of time with RBCs alone (gray trace) or RBCs + nitrite (black trace). (B) Nitrite-dependent dilation starts at higher oxygen tensions in response to human RBCs (hRBCs), HBOC-201, or HbA and IHP. Final concentrations: nitrite (2 μM), human RBCs (0.3% HCT), HBOC-201 (25 μM), HbA (25 μM), and IHP (625 μM). †P < .03 relative to nitrite and P < .01 relative to hRBCs alone (n = 3). *P < .001 relative to nitrite alone. #P < .01 relative to Hb + IHP. Data represent mean ± SEM (n = 3-5). (C) Relationship of Hb P50 and the oxygen tension at the initiation of vessel dilation in the presence of nitrite (2 μM). Data from rat RBCs, hRBCs, HBOC-201, and cell-free HbA (in which P50 was modulated by the addition of varying concentrations of IHP, resulting in heme/IHP ratios ranging from 1:0.25 to 1:25) are shown. A linear relationship (y = 19.6 + 0.62x; r = 0.89; note intercept is not zero because of basal dilatory responses of vessels to hypoxia) between P50 and oxygen tension at onset of relaxation was observed. (inset) Calculated values for Hb fractional saturation (Y) at the oxygen tension at which nitrite-dependent vasodilation was initiated. (D) Using the calculated rate constants for nitrite reactions with RBC deoxyHb, the predicted velocity or initial rate of nitrite reaction with deoxyHb under physiologic conditions (total Hb = 20 mM in heme; circulating nitrite concentrations = 500 nM19 ) and as a function of Hb fractional saturation are shown and indicate a maximal rate of reaction around the Hb P50. Data were collected using RBCs from 3 different preparations. (inset) Representative traces showing NO formation (measured by NO chemiluminescence) in the head-space of the reaction between RBC (0.3% hc) and nitrite (500 μM) after 2 minutes in the open-flow respirometer (head-space volume and reaction solution volume 5 mL each with 100 mL/min gas flow). Traces show NO production after injection of 200 μL of [A] air alone or head-space from [B] nitrite in PBS at 28 mmHg oxygen or nitrite + RBC at [C] 0 mmHg (0% fractional saturation), [D] 28 mmHg oxygen (50% fractional saturation), and [E] 155 mmHg (100% fractional saturation).
