Requirement for flow in the blockade of endothelium-derived hyperpolarizing factor (EDHF) by ascorbate in the bovine ciliary artery

Abstract

We previously reported that ascorbate inhibits endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilatation in the bovine perfused ciliary circulation and rat perfused mesentery, but not in rings of bovine or porcine coronary artery. In this study, we have compared the ability of ascorbate to inhibit EDHF-mediated vasodilatation in a single vessel, the bovine long posterior ciliary artery, when perfused and when mounted as rings in a myograph. Both in segments perfused at a flow rate of 2.5 ml min(-1) and in rings mounted in a myograph, bradykinin and acetylcholine each induced vasodilator responses that were mediated jointly by EDHF and nitric oxide, as revealed by their respective blocking agents, apamin/charybdotoxin, and L-NAME. Ascorbate (50 and 150 microm) induced a time (max at 2-3 h)-dependent inhibition of the EDHF-mediated component of vasodilatation to bradykinin or acetylcholine in perfused segments, but not in rings. Ascorbate (50 microm) failed to inhibit bradykinin-induced vasodilatation at a flow rate of 1.25 ml min(-1) or below, but produced graded blockade at the higher flow rates of 2.5 and 5 ml min(-1). Furthermore, using a pressure myograph where pressure and flow were independently controlled, it was confirmed that the inhibitory action of ascorbate (150 microm) was directly related to flow per se and not any associated changes in pressure. Thus, we have shown in the bovine ciliary artery that ascorbate inhibits EDHF-mediated vasodilatation under conditions of flow but not in a static myograph. The mechanism by which flow renders EDHF susceptible to inhibition by ascorbate remains to be determined.

Figure 1

Bradykinin induced dose-dependent vasodilatation of bovine perfused ciliary artery segments. (a) The EDHF-dependent component was inhibited following combined treatment with apamin and charybdotoxin (Ap+ChTx, both 100 nM) and the EDHF-independent component was blocked by L-NAME (L-N, 100 μM) and indomethacin (INDO, 3 μM). (b) Vasodilatation was also inhibited by ascorbate (ASC, 50 or 150 μM, >120 min) alone or in combination with L-NAME and indomethacin. Data are mean±s.e.m.; n⩾6; ^*P<0.05, ^**P<0.01, and ^***P<0.001 indicate significant differences in maximal vasodilatation from time-matched control or between groups joined by a bracket.

Figure 2

Acetylcholine induced dose-dependent vasodilatation of bovine perfused ciliary artery segments. (a) Vasodilatation was reversed to vasoconstriction following treatment with the EDHF blockers apamin/charybdotoxin (Ap+ChTx, both 100 nM) or with L-NAME (L-N, 100 μM) and indomethacin (INDO, 3 μM). (b) Vasodilatation was also reversed to constriction by ascorbate (ASC, 50 μM, >120 min) alone or in combination with L-NAME and indomethacin. Data are mean±s.e.m.; n⩾8; ^***P<0.001 indicates differences from time-matched control.

Figure 3

Bradykinin induced concentration-dependent relaxation of bovine ciliary artery rings in a wire myograph. (a) The EDHF-dependent component was inhibited following combined treatment with apamin and charybdotoxin (Ap+ChTx, both 100 nM) and the EDHF-independent component was blocked by L-NAME (L-N, 100 μM) and indomethacin (INDO, 3 μM). (b) Vasodilatation was unaffected by ascorbate (ASC, 150 μM, >120 min) alone or in combination with L-NAME and indomethacin. Data are mean±s.e.m.; n⩾6; ^**P<0.01 and ^***P<0.001 indicate differences in maximal relaxation from time-matched control or between groups joined by a bracket.

Figure 4

Acetylcholine induced concentration-dependent relaxation of bovine ciliary artery rings in a wire myograph. (a) The EDHF-dependent component was inhibited following combined treatment with apamin and charybdotoxin (Ap+ChTx, both 100 nM) and the EDHF-independent component was blocked by L-NAME (L-N, 100 μM) and indomethacin (INDO, 3 μM). Following endothelial denudation (-EC), acetylcholine-induced relaxation was reversed to constriction. (b) Vasodilatation was unaffected by ascorbate (ASC, 150 μM, >120 min) alone and the antioxidant did not enhance the inhibition induced by L-NAME and indomethacin. Data are mean±s.e.m.; n⩾6; ^***P<0.001 indicates differences from time-matched control.

Figure 5

The effect of flow rate on the ability of ascorbate to inhibit bradykinin (1 nmol)-induced vasodilatation in bovine perfused ciliary artery segments. (a) In control preparations, bradykinin-induced vasodilatation was similar at different rates of flow. Ascorbate (ASC, 50 μM, 3 h) inhibited vasodilatation at high but not low rates of flow. (b) In control preparations, sequentially lowering the flow rate from 5 to 0.3 ml min⁻¹ had no effect on bradykinin-induced vasodilatation. When inhibition of vasodilatation was established with ascorbate (ASC, 50 μM, 3 h) at 5 ml min⁻¹, sequentially reducing the flow rate to 2.5, 1.25, 0.6 and 0.3 ml min⁻¹had no effect on the level of inhibition observed. Data are mean±s.e.m.; n⩾6; ^*P<0.05, ^**P<0.01 and ^***P<0.001 indicate differences from time-matched control.

Figure 6

Experiments conducted in a pressure myograph examining the separate effects of pressure and flow on the ability of ascorbate to inhibit bradykinin-induced vasodilatation in bovine ciliary segments. (a) When pressurized to ∼100 mmHg, vessels developed myogenic tone, as revealed by a fall in outer diameter between the initial measurement and the end of a 2 h incubation period. This myogenic tone was relaxed powerfully by bradykinin (BK, 1 nmol) and completely by papaverine (PAP, 300 μM). (b) Bradykinin-induced dilatation remained constant for 3 h when pressurized in the absence or presence of flow at 5 ml min⁻¹, or in the presence of ascorbate (ASC, 150 μM) in the absence of flow, but was inhibited significantly by ascorbate in the presence of flow. Data are mean±s.e.m.; n⩾4; ^*P<0.05 indicates differences from control.