McNeish, Alister J
(2002)
Inhibition of endothelium-derived hyperpolarizing factor by ascorbate in the bovine eye.
PhD thesis, University of Glasgow.
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Abstract
The aim of this study was to characterise vasodilator responses in the perfused ciliary vascular bed of the bovine eye. When bovine eyes were perfused at a constant rate of 2.5 ml min-1, infusion of the powerful vasodilator, papaverine (150 muM), produced a very small reduction in perfusion pressure. Under the same conditions, the nitric oxide synthase inhibitor, L- NAME (100 muM), had no effect but the inhibitor of soluble guanylate cyclase, ODQ (10 muM), produced a small vasoconstrictor response. These results indicate that there is a small component of intrinsic (myogenic) tone that may be suppressed by a basal release of nitric oxide. In the bovine eye, vasodilatation to acetylcholine or bradykinin was unaffected by L- NAME (100 muM), or the cyclo-oxygenase inhibitor, flurbiprofen (30 muM), but was significantly attenuated following treatment with a high concentration of KC1 (30 muM), or by damaging the endothelium with the detergent, CHAPS (0.3%, 2 min). Thus agonist-induced vasodilatation is not mediated by nitric oxide or prostacyclin but involves a K+ conductance and is endothelium-dependent. Acetylcholine-induced vasodilatation in the bovine eye was unaffected by glibenclamide (10 muM), an inhibitor of ATP-sensitive K+ channels (KATP), but was significantly attenuated by TEA (10 mM), a non-selective inhibitor of K+ channels. The blockade of vasodilatation by TEA but not glibenclamide could indicate that a calcium- sensitive K+ channel is involved in the response. The small conductance calcium-sensitive K+ channel (SKCa) inhibitor, apamin (100 nM), and the large conductance calcium-sensitive K+ channel (BKCa) inhibitor, iberiotoxin (50 nM), had no significant effect on acetylcholine-induced vasodilatation. In contrast, the intermediate (IKCa)/large conductance calcium-sensitive K+ channel inhibitor, charybdotoxin (50 nM), powerfully blocked these vasodilator responses, and uncovered a vasoconstrictor response. Thus, vasodilator responses appear to involve the opening of IKCa channels. The combination of apamin (100 nM) with a sub-threshold concentration of charybdotoxin (10 nM) significantly attenuated acetylcholine-induced vasodilatation, but the combination of apamin (100 nM) with iberiotoxin (50 nM) had no effect. This profile of blockade is consistent with the vasodilator responses being mediated by endothelium-derived hyperpolarizing factor (EDHF). Ascorbate is known to protect nitric oxide dependent vasodilatation under conditions of oxidant stress, however, EDHF-mediated vasodilator responses induced by acetylcholine or bradykinin were powerfully blocked when ascorbate (50 muM, 120 min) was included in the perfusion medium; with acetylcholine a normally masked muscarinic vasoconstrictor response was also uncovered. These results indicate that, ascorbate at a physiologically relevant concentration, can inhibit EDHF-mediated vasodilatations. The blockade of EDHF-mediated vasodilatation by ascorbate was time-dependent (maximum blockade at 120 min) and concentration-dependent (10-150 muM). Thus, the blocking action of ascorbate has a slow onset and occurs concentrations across the normal plasma concentration range (10 -150 muM). The ability of ascorbate to block EDHF-mediated vasodilatation in the bovine eye is likely to result from its reducing properties, since this action was mimicked by two other reducing agents, namely, N-acetyl-L-cysteine (1 mM) and dithiothreitol (100 muM), but not by the redox-inactive analogue, dehydroascorbate (50 muM). In the bovine eye, vasodilatations induced by the KATP opener, levcromakalim (100 pmol-30 nmol), or the nitric oxide donor, glyceryl trinitrate (10 nmol), were completely unaffected by the infusion of ascorbate (50 muM). Furthermore, the L-NAME induced vasoconstrictor response in the presence of U46619 (~200 nM) was unaffected by infusion of ascorbate (50 muM). Thus, the blockade of EDHF-mediated vasodilatation by ascorbate is highly selective and does not result from non-selective damage of the endothelium as basal release of nitric oxide is unaffected. (Abstract shortened by ProQuest.).
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