Regulation of RBC eNOS activity. eNOS activity in RBCs was measured depending on the availability of substrate (A) and calcium (B) and the level of phosphorylation of eNOS protein (C-D). (A) Changes in accumulated plasma nitrite were determined in blood samples after incubation with increasing concentrations of l-arginine, d-arginine, and l-NNA. Changes were compared with total depletion of l-arginine (achieved by arginase). indicates physiologic range of l-arginine concentration in human plasma. (B) Effects of Ca-Ionophore or Ca-Ionophore plus EDTA (n = 6) on plasma nitrite levels were determined after incubation of blood samples and compared with control (buffer). Changes in plasma nitrite reflect the sum of the release (due to NOS-dependent NO formation) and the reuptake of nitrite by RBCs. Thus complete inhibition of RBC-NOS by EDTA significantly reduced plasma nitrite compared with control buffer, unmasking the continuous uptake of plasma nitrite by RBCs during incubation period. (C) Challenging RBCs with insulin increased plasma nitrite whereas addition of the PI3K inhibitor wortmannin prevented this increase (n = 6). ^* indicates significant (P < .05) difference; and ^**, highly significant (P < .001) differences from control. (D) Phosphorylation of eNOS at Ser1177 was used to examine the phosphorylation-dependent activation status of the eNOS. RBCs incubated with insulin showed a significant rise of eNOS phosphorylated at Ser1177 compared with control (P < .05; n = 8). Bars, 10 μm.