Endothelin mediates the endothelium-dependent, flow-dependent constriction induced by ascorbate in the bovine ciliary artery

William Martin, John Craig
University of Glasgow, Glasgow, United Kingdom

In the bovine perfused ciliary artery and rat perfused mesentery, ascorbate blocks agonist-induced endothelium-derived hyperpolarising factor (EDHF) activity and produces constriction (McNeish et al., 2002; Stirrat et al., 2006; Stirrat et al., 2006). The magnitude of both effects of ascorbate is dependent upon the presence of flow and both are absent at zero or low flow. As the flow-dependent constrictor action of ascorbate requires the presence of the endothelium, we previously hypothesised that it might arise as a consequence of inhibition of flow-induced EDHF activity. The aim of this study was to test this hypothesis by making use of a pressure myograph to directly measure flow-induced dilatation.

Segments of bovine extraocular ciliary artery were cannulated at both ends and pressurised to 100 mm Hg in a pressure myograph, initially at zero flow. In some experiments, vessels were perfused continuously at a single flow rate of 2.5, 5 or 10 ml min-1 using a peristaltic pump. In others, flow-induced dilatation was elicited by perfusing at these flow rates for 2 min periods. Pressure was held constant at 100 mm Hg by adjusting the height of the outflow tube. External vessel diameter was monitored throughout. In all experiments nitric oxide synthase and cyclooxygenase were blocked using L-NAME (100 μM) and indomethacin (10 μM), respectively. In all experiments, n>5 and differences were determined by ANOVA followed by the Bonferroni post hoc test.

When pressurised to 100 mm Hg the external diameter of ciliary artery segments was 1,459 ± 53 μm. Infusion of ascorbate (50 μM) had no constrictor effect at zero flow but produced constrictions of 146 ± 55, 135 ± 33 and 457 ± 25 μm at flow rates of 2.5, 5 and 10 ml min-1, respectively. Perfusion for periods of 2 min at a range of flow rates (2.5 - 10 ml min-1) produced only modest flow-induced dilatation (18.4 ± 2.4 % at 10 ml min-1). It was therefore unlikely, that inhibition of this small dilatation could account for the profound, endothelium-dependent constrictor action of ascorbate. We therefore considered whether it might instead be mediated by endothelin. Treatment with the ETA receptor antagonist, BQ-123 (1 μM), reduced the constrictor action of ascorbate to 32.6 ± 60.5 μm at 10 ml min-1 (P<0.001). This action appeared to be selective, since BQ-123 also reduced endothelin-1 (25 pmol)-induced constriction from 247.7 ± 23.8 to 15.7 ± 11.9 μm (P<0.001), but had no effect on constriction to phenylephrine (50 nmol): 275.1 ± 34.0 and 289.0 ± 45.2 μm before and after BQ-123, respectively.

In conclusion, these data suggest that the profound endothelium-dependent, flow-dependent constrictor action of ascorbate is mediated by the release of endothelin.

McNeish, A. et al., (2002). Br. J. Pharmacol., 135, 1801-1809.
Stirrat, A. et al., (2006). Eur. J. Pharmacol., 534, 152-158.
Stirrat, A, et al., (2007). Br. J. Pharmacol., in press.

Supported by the Wellcome Trust