Ascorbate inhibits endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilatation induced by acetylcholine and bradykinin in the bovine perfused ciliary circulation (McNeish et al., 2002). The inhibition is not immediate, but becomes significant within ~60 min and maximal at ~120 min. Moreover, the magnitude of inhibition is dependent upon the flow rate, with none seen in rings of ciliary artery suspended in a static wire myograph (Nelli et al, 2004). We have recently observed that at low levels of vasoconstrictor tone, the inhibition of agonist-induced EDHF by ascorbate in ciliary artery segments appears to be associated with an elevation of perfusion pressure. The aim of this study was to determine if the ascorbate-induced elevation of perfusion pressure might arise through blockade of a tonic EDHF activity.

Terminal segments of extraocular long posterior ciliary artery (LPCA) were dissected from bovine eyes obtained at a local abattoir. These were then either cannulated and perfused at 2.5 ml min^-1 with Krebs solution, or cut into 2 mm rings and mounted in a wire myograph at a transmural pressure equivalent to ~100 mmHg. The thromboxane A 2-mimetic, U46619 (0.1-1 µM), was used to constrict the vessels before treatment with ascorbate or the cytochrome P₄₅₀ and intermediate conductance calcium-activated K⁺ channel inhibitor, clotrimazole. Data are expressed as mean ± s.e.m., n6, and differences determined using one-way ANOVA with Bonferroni’s post-test.

Ascorbate (10-150 µM) induced a further rise in perfusion pressure in LPCA segments constricted sub-maximally (17.9 ± 8.4 mmHg) with U46619. The rise was not immediate, but became apparent at ~60 min and maximal (50 µM: 74.3 ± 21.1 mmHg, p<0.01) at ~120 min. Ascorbate (50 µM) added in the absence of U46619 failed to raise perfusion pressure. The rise in U46619-induced pressure produced by L-NAME (100 µM: 42.2 ± 7.0 mmHg) through blockade of basal NO activity was unaffected by the presence of ascorbate (48.1 ± 19.3 mmHg). The EDHF blocker, clotrimazole (1-100 µM), also induced a concentration-dependent rise in U46619-induced perfusion pressure and this attained a similar maximum (at 30 µM: 70.1 ± 15.9 mmHg) to that induced by ascorbate. Ascorbate (150 µM) failed to raise sub-maximal U46619-induced tone (75.8 ± 12 mN) in rings of LPCA during 180 min incubation in a wire myograph.

Thus, the ability of ascorbate to raise U46619-induced perfusion pressure in the LPCA follows a time course (latency ~60 min, max at ~120 min) similar to that for blockade of agonist-induced EDHF (McNeish et al., 2002). The rise appears to occur through blockade of a tonic vasodilator influence not involving NO, rather than from a constrictor action per se, since ascorbate has no effect in the absence of U46619. Flow may be required for this rise, since it is absent in arterial rings in a static myograph. These data, together with the ability of clotrimazole to mimic the action of ascorbate, suggest that the rise in perfusion pressure may occur through blockade of tonic, perhaps flow-induced, EDHF activity.

McNeish, A.J. et al., (2002). Br. J. Pharmacol., 135, 1801-1809
Nelli, S. et al., (2004). Br. J. Pharmacol. in press

Supported by the BHF and Welcome Trust.