Role of S-Nitrosothiols in Non-Adrenergic Non-Cholinergic Neurotransmission

Liu, Xiaorong (1993) Role of S-Nitrosothiols in Non-Adrenergic Non-Cholinergic Neurotransmission. PhD thesis, University of Glasgow.

Abstract

1. The aim of this study was to examine the possibility that S-nitrosocysteine or S- nitrosoglutathione rather than nitric oxide functions as the inhibitory non- adrenergic, non-cholinergic (NANC) neurotransmitter in the bovine retractor penis (BRP) muscle. This was investigated firstly, by examining whether free sulfhydryl groups are required for NANC relaxation to take place upon nerve stimulation in the BRP muscle and secondly, by examining the properties of the new relaxant produced when nitric oxide is reacted with a range of sulfhydryl compounds. 2. Treatment of BRP muscle with the sulfhydryl oxidising agent, diamide (1 mM), inhibited NANC relaxation induced by nerve stimulation. This effect was completely prevented and almost completely reversed by treating the tissue with the sulfhydryl compounds, L-cysteine (3 mM), L-glutathione (3 mM) or dithiothreitol (3 mM). The inhibition was not specific, however, since the oxidising agent also inhibited the relaxant actions of glyceryl trinitrate (0.01-0.1 muM) and isoprenaline (0.01-1 muM). 3. Treatment of BRP muscle with the sulfhydryl alkylating agent, N-ethylmaleimide (0.3 mM), inhibited NANC relaxation induced by nerve stimulation. This effect was completely prevented but not reversed by treating the tissue with the sulfhydryl compounds, L-cysteine (3 mM), L-glutathione (3 mM) or dithiothreitol (3 mM). As with diamide, inhibition was not specific, however, since the alkylating agent also inhibited the relaxant actions of glyceryl trinitrate (0.01-0.1 muM) and isoprenaline (0.01-1 muM). 4. The weak vasodilator activity of sodium nitrite (10 muM) was greatly enhanced by acidification. The optimal pH for enhancement of vasodilator activity was pH 1-2. Deoxygenation before acidification further enhanced and bubbling to saturation with oxygen for 10 minutes before acidification inhibited relaxant activity. Increases in relaxant activity upon acidification were associated with decreases in nitrite content. Neutralisation of acidified samples led to a rapid loss of relaxant activity. These results are consistent with formation of nitric oxide upon acidification of nitrite and this is destroyed by oxygen and protected in an oxygen-free environment. 5,The first means adopted to form an S-nitrosothiol was to acidify nitrite (10 mM) in the presence of L-cysteine (1.5 M). The relaxant activity, assessed both in magnitude and duration, greatly exceeded that of equivalent solutions of acidified nitrite. Again maximum generation of relaxant activity occurred at pH 2, but unlike solutions of acidified nitrite, these solutions retained their relaxant activity upon neutralisation. These solutions were pink in colour and their relaxant activity was destroyed by the nitric oxide-binding substance, haemoglobin, but was unaffected by the inhibitor of nitric oxide synthase, NG-nitro-L-arginine. 6, In the spectrophotometer at 190-900 nm, nitric oxide in the gas phase produced several narrow absorption bands at wavelengths less than 230 nm, whereas in aqueous solution it produced only a single peak at 190 nm. Upon admission of oxygen, nitrogen dioxide was rapidly formed in both the gas and liquid phases assessed by formation of its characteristic absorption peaks at 300-400 nm. 7, A second means adopted for the formation of S-nitrosothiols was by reacting nitric oxide gas with L-cysteine. In phenylephrine (0.3 muM)-contracted rabbit aortic rings denuded of endothelium and in BRP strips, nitric oxide (1-1,000 nM) alone induced transient relaxation in a concentration-dependent manner, whereas L-cysteine (0.15-4.5 mM) was without effect. When aqueous solutions of L-cysteine (15 mM) were reacted with nitric oxide (5 mM) in nominally oxygen-free conditions at pH 3 for 10 minutes followed by purging with oxygen-free nitrogen to remove free nitric oxide and neutralisation and added to achieve bath concentrations equal to 10 nM nitric oxide and 30 muM L-cysteine, however, more powerful and prolonged relaxation was produced than could be attributed to nitric oxide alone. 8. In the HPLC, aqueous solutions of L-cysteine (10 mM) at pH 3.0 produced two peaks corresponding to L-cystine (-0.85 %, peak 1) and L-cysteine C(-99.15 %, peak 2), respectively. After saturating this solution with nitric oxide gas for 10 minutes under nominal oxygen-free conditions, the solution produced three peaks in the HPLC corresponding to L-cystine (-0.85 %), L-cysteine (- 98 %) and a new substance (-1.15%, peak 3) which was entirely responsible for relaxant activity. Re-run of the peaks in the HPLC revealed that L-cystine was stable, whereas L-cysteine and the new relaxant decayed slowly to form L-cystine.9. Using a series of structural analogues of L-cysteine (all at 15 mM) it was found that removal of the carboxyl group (L-cysteamine), replacement of the carboxyl with an ester function (L-cysteine methyl ester) or substitution at the amino group (N-acetyl-L-cysteine) had no effect on the ability to generate new relaxant activity upon reaction with nitric oxide (0.1 mM). (Abstract shortened by ProQuest.).

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: J S Gillespie
• Keywords: Pharmacology, Neurosciences
• Date of Award: 1993
• Depositing User: Enlighten Team
• Unique ID: glathesis:1993-74990
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 27 Sep 2019 14:44
• Last Modified: 27 Sep 2019 14:44
• URI: https://theses.gla.ac.uk/id/eprint/74990