title: Induction of nitric oxide synthase in vascular smooth muscle
Creators: name: McKendrick, Joyce Dickson
Publisher: name: University of Glasgow
Year: 1996
Subjects: subject: Physiology.

description: The first aim of this study was to examine and assess the sensitivity and utility of a number of different measuring techniques for the detection of nitric oxide (NO). The systems examined did not measure NO directly. Instead, some of the systems employed measured nitrite, the major in vitro breakdown product of NO: these were the Griess reaction, chemlluminescence detection coupled to two reducing systems, i.e. 1,1'-dimethylferrocene/acetonitrile reflux and sodium iodide/glacial acetic acid reflux and lastly fluorometric detection. The Griess reaction was the least sensitive with a threshold detection limit of 300 pmol, but a large number of samples could be assayed each day. The 1,1'-dimethylferrocene/acetonitrile system produced chemiluminescence signals that were difficult to discern from background noise and was abandoned. The sodium iodide/glacial acetic acid reflux system did, however, produce good chemiluminescence signals, giving a threshold sensitivity of 100 pmol. This technique was slow, however, permitting only small numbers of samples to be assayed each day. The fluorometric detection system for nitrite had a similar sensitivity of around 120 pmol but as each sample was stable for around 10 min only small numbers of samples could be assayed each day. Thus, if samples contained large amounts of nitrite, the less sensitive Griess reaction (300 pmol) was by far the easiest system to use. A stronger reducing system, involving vanadium chloride/hydrochloric acid was tested which when combined with chemiluminescence could measure both of the breakdown products of NO, i.e. nitrite and nitrate. This system had a high sensitivity of around 200 pmol but in addition to being able to handle only small numbers of samples, it was much less selective since many 'false' positive signals were generated by constituents of tissue culture medium. Having assessed the suitability of the above detection systems, the second aim of this study was to use them in conjunction with other techniques to examine the expression of iNOS in different preparations. Expression of iNOS in J774.2 murine macrophages was seen after incubation with lipopolysaccharide (LPS; 1 - 1000 ng ml-1) or interferon-gamma (IFN-gamma; 1 - 100 u ml-1). In contrast, LPS (1 - 1000 ng ml-1) failed to stimulate expression of iNOS in bovine aortic endothelial cells (BAEC) in culture. Following an overnight incubation at 37°C in Krebs solution, phenylephrine (PE; 1 nM - 10 muM)-induced contraction of endothelium-denuded rings of rat aorta was depressed. This depression was prevented by including polymixin B (30 mug ml-1) during the incubation suggesting it resulted from LPS contaminating the Krebs solution. This depression was likely to have resulted from production of NO by iNOS as it could be reversed by the addition of L-NAME (1 mM), an inhibitor of NOS, methylene blue (10 muM), an inhibitor of soluble guanylate cyclase or haemoglobin (10 muM), which binds and inactivates NO. The effects of generators of superoxide anion, which destroy NO, were also examined. The intracellular generator of superoxide anion, LY 83583 (0.1 - 1 muM), reversed the depression but the extracellular generator, hypoxanthine (100 pM)/xanthine oxidase(16 mu ml-1), had no effect. These data suggest that NO is generated and acts within the same smooth muscle cells. In contrast to rat aorta, rat aortic smooth muscle cells (RASMC) in culture did not express iNOS, as assessed by measuring nitrite accumulation and cyclic GMP content, when stimulated by LPS (1 - 100 mug ml-1) alone except at exceptionally high concentrations (300 and 1000 mug ml-1). Certain cytokines, i.e. IFN-gamma (1 - 100 u ml-1') and interleukin-1 a (1 - 10 u ml-1), did however, lead to expression of iNOS. The presence of serum appeared to inhibit the expression of iNOS, probably because of the presence of platelet-derived growth factor and transforming growth factor. The production of nitrite and elevated levels of cyclic GMP were blocked following incubation with inhibitors of the expression of iNOS, dexamethasone (1 muM) and cycloheximide (30 u ml-1) and by the inhibitors of NOS, L-NAME (100 muM) and L-NMMA (300 muM). Thus, it appeared that LPS alone was able to stimulate expression of iNOS in isolated rings of rat aorta but not in cultured RASMC. Whether this apparent difference resulted from differences in smooth muscle phenotype in the vessel wall and in culture or from any other differences has yet to be resolved.