Inhibition of endothelium-derived hyperpolarizing factor by ascorbate in the bovine eye

McNeish, Alister J (2002) Inhibition of endothelium-derived hyperpolarizing factor by ascorbate in the bovine eye. PhD thesis, University of Glasgow.

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.).

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: vasodilator responses, bovine eye.
• Subjects: Q Science > QH Natural history > QH345 Biochemistry
• Colleges/Schools: College of Medical Veterinary and Life Sciences
• Supervisor's Name: Martin, Prof. William and Wilson, Dr. William
• Date of Award: 2002
• Depositing User: Enlighten Team
• Unique ID: glathesis:2002-71245
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 10 May 2019 10:49
• Last Modified: 05 Aug 2022 16:35
• Thesis DOI: 10.5525/gla.thesis.71245
• URI: https://theses.gla.ac.uk/id/eprint/71245

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