Hydroxyurea stimulates the release of ATP from rabbit erythrocytes through an increase in calcium and nitric oxide production

Abstract

Hydroxyurea, a proven therapy for sickle cell disease, is known to improve blood flow and reduce vaso-occlusive crises, although its exact mechanism of action is not clear. The objective of this study was to determine if hydroxyurea results in an increase of ATP release from the red blood cell (RBC) via the drug's ability to stimulate nitric oxide (NO) production in these cells. A system enabling the flow of RBCs through microbore tubing was used to investigate ATP release from the RBC. Incubation of rabbit RBCs (7% hct) with 50 μM hydroxyurea resulted in a significant increase in the release of ATP from these cells. This level of ATP release was not detected in the absence of flow. Studies also showed that increments in hydroxyurea and NO (from spermine NONOate) resulted in an initial increase in ATP release, followed by a decrease in this release at higher concentrations of hydroxyurea and the NO donor. Incubation with L-NAME abolished the effect of the hydroxyurea, suggesting that NO production by the RBC was involved. Indeed, in the presence of 50 μM hydroxyurea, the amount of total Ca 2+ measured (by atomic absorption spectroscopy) in a 7% solution of RBCs increased from 363 ± 47 ng/ml and 530 ± 52 ng/ml. Finally, EPR studies suggest that an increase in nitrosylated Hb in the RBC is only measured for those studies involving hydroxyurea and a Ca 2+-containing buffer.