Endothelin-1 is not involved in hemoglobin associated vasoactivities

This study tested the hypothesis: is hemoglobin (Hb) associated vasoconstriction mediated by endothelins (ET)? Two millimeter segments of the thoracic aorta with intact endothelium were obtained from Sprague-Dawley rats (200-350g) and suspended in oxygenated Krebs buffer (37C, pH 7.4). The vessels were equilibrated at 2g of imposed tension for 1 hour. After submaximal tone was induced with norepinephrine (50nM), the presence of functional endothelium was verified by an acetylcholine (33uM) dilation test. The vessel rings were then treated with either ET-1 (3nM) or human stroma-free Hb (SFH; 2.2uM) and vascular response characteristics observed. Subsequently, the vessel rings were treated with BQ-123 (15uM), an ET-1 receptor antagonist, to test whether the ET-1 pathway is involved in the Hb associated vasoconstriction. Both ET-1 and SFH elicited contractory responses in aortic rings; the maximal tension increases at the doses tested were 54.8 +/- 8.5% and 39.2 +/- 22.5%, respectively, over the pretreatment values. The contractory response characteristics were, however, distinct; ET-1 caused a slow (time to maximal response; TMR = 28.8 +/- 6.4 min, N = 6) but prolonged contraction (> 30 min) while SFH caused a faster contraction (TMR = 7.2 +/- 1.7 min, N = 6) with a shorter duration (< 30 min). The TMR of ET-1 treated rings were significantly longer than that of SFH (P < 0.0005, t-test). Treatment with BQ-123 caused an immediate reversal of the SFH induced contraction but had no significant effect on the SFH induced contraction. If the Hb associated vasoconstriction were mediated by ET-1, BQ-123 should have also reversed the contraction. These results suggest that ET-1 pathway is not involved in the Hb mediated vasoconstriction.