Mechanism of oxyhemoglobin-induced release of endothelin-1 from cultured vascular endothelial cells and smooth-muscle cells

Release of endothelin-1 from cultured endothelial cells can be induced with oxyhemoglobin (oxyHb). The present study was conducted to explore whether oxyHb affects the release of endothelin-1 and the induction of endothelin-1 messenger ribonucleic acid (mRNA) and to examine the mechanism whereby oxyHb induces endothelin-1 production in cultured vascular smooth-muscle cells as well as in cultured endothelial cells. Oxyhemoglobin produces concentration-dependent (0.1 to 10 microM) and time-dependent (0 to 24 hours) increases in immunoreactive endothelin-1 in conditioned medium from bovine arterial endothelial cells. Oxyhemoglobin induces immunoreactive endothelin-1 in rat aortic smooth-muscle cells in the same fashion, although the rate is 30-fold less than that of endothelial cells. This promoting effect is much higher than that of other stimulators such as thrombin and phorbol 12-myristate 13-acetate. Northern blot analysis of total RNA from endothelial cells also showed endothelin-1 mRNA induction. Staurosporine, a protein kinase C (PKC) inhibitor, inhibited oxyHb-induced endothelin-1 production in both vascular endothelial and smooth-muscle cells, whereas an increase of intracellular cyclic adenosine monophosphate (cAMP) by forskolin or an addition of 8-bromo-cAMP only inhibited this effect in smooth-muscle cells. These findings suggest that oxyHb-induced endothelin-1 production in endothelial cells is regulated by PKC, and in smooth-muscle cells by both PKC and the cAMP-dependent pathway. The production of endothelin, the most potent vasoconstrictor, in both vascular endothelial and smooth-muscle cells by oxyHb may have significance in the pathogenesis of cerebral vasospasm.