Red blood cell ATP release correlates with red blood cell hemolysis

The precise matching of blood flow to skeletal muscle during exercise remains an important area of investigation. Release of adenosine triphosphate (ATP) from red blood cells (RBCs) is postulated as a mediator of peripheral vascular tone in response to shear stress, hypoxia, and mechanical deformation. We tested the following hypotheses: 1) RBCs of different densities contain different quantities of ATP; 2) hypoxia is a stimulus for ATP release from RBCs; and 3) hypoxic ATP release from RBCs is related to RBC lysis. Human blood was drawn from male and female volunteers (n = 11); the RBCs were isolated and washed. A Percoll gradient was used to separate RBCs based on cellular density. Density groups were then resuspended to 4% hematocrit and exposed to normoxia or hypoxia in a tonometer. Equilibrated samples were drawn and centrifuged; paired analyses of ATP (luminescence via a luciferase-catalyzed reaction) and hemolysis (Harboe spectrophotometric absorbance assay) were measured in the supernatant. ATP release was not different among low-density cells versus middle-density versus high-density cells. Similarly, hemoglobin (Hb) release was not different among the red blood cell subsets. No difference was found for either ATP release or Hb release following matched exposure to normoxic or hypoxic gas. The concentrations of ATP and Hb for all subsets combined were linearly correlated (r = 0.59, P ≤ 0.001). With simultaneous probing for Hb and ATP in the supernatant of each sample, we conclude that ATP release from RBCs can be explained by hemolysis and that hypoxia per se does not stimulate either ATP release or Hb release from RBCs.