Oxyhemoglobin stimulation of endothelin production in cultured endothelial cells

Oxyhemoglobin and endothelin have both been linked to the development of the severe and sustained cerebral vasospasm associated with subarachnoid hemorrhage. The effects of oxyhemoglobin on endothelin biosynthesis in cultured endothelial cells were evaluated. Oxyhemoglobin (0.01 to 100 microM) produced concentration-dependent increases in immunoreactive endothelin levels in bovine pulmonary artery endothelial cell-conditioned medium. The median effective concentration for oxyhemoglobin-induced increases in immunoreactive endothelin levels was approximately 0.5 microM, and the maximum stimulation of immunoreactive endothelin levels was approximately 5.5-fold over basal conditions. In addition to directly stimulating basal production of immunoreactive endothelin, oxyhemoglobin significantly augmented immunoreactive endothelin production following platelet-mediated stimulation of endothelin production. An l-arginine analog inhibitor of nitric oxide synthase, L-NG-monomethyl arginine (L-NMMA, 200 microM), did not significantly affect basal immunoreactive endothelin levels. However, L-NMMA significantly augmented platelet-induced immunoreactive endothelin production. Methylene blue (10 microM), an inhibitor of soluble guanylate cyclase, did not significantly affect basal immunoreactive endothelin levels, nor did it significantly affect the platelet-mediated stimulation of immunoreactive endothelin production in cultured endothelial cells. The present results reveal that oxyhemoglobin can directly stimulate endothelin biosynthesis in cultured endothelial cells. This newly identified property of oxyhemoglobin suggests a potential mechanism for the sustained and severe cerebral vasospasm associated with subarachnoid hemorrhage.