Anthie Ellis

Differential actions of L‐cysteine on responses to nitric oxide, nitroxyl anions and EDRF in the rat aorta

The effects of L‐cysteine were tested in rat aortic rings on responses to nitric oxide free radical (NO•), nitroxyl (NO−) derived from Angelis salt and endothelium‐derived relaxing factor (EDRF) activated by acetylcholine, ATP and the calcium ionophore A23187. Concentrations of 300 μM or less of L‐cysteine had no effect on responses. Relaxations produced by exogenous NO• (0.25–2.5 μM) were markedly prolonged and relaxations produced by sodium nitroprusside (0.001–0.3 μM) were enhanced by 1 and 3 mM L‐cysteine. The enhancements by L‐cysteine of responses to NO• and sodium nitroprusside may be attributed to the formation of S‐nitrosocysteine. Relaxations mediated by the nitroxyl anion (0.3 μM) donated from Angelis salt were more prolonged than those produced by NO•, and nitroxyl‐induced relaxations were reduced by L‐cysteine (1 and 3 mM). EDRF‐mediated relaxations produced by acetylcholine (0.01–10 μM), ATP (3–100 μM) and the calcium ionophore A23187 (0.1 μM) were significantly reduced by 3 mM L‐cysteine. The similarity between the inhibitory effects of L‐cysteine on responses to EDRF and on those to nitroxyl suggests that a component of the response to EDRF may be mediated by nitroxyl anion.

Catalase has negligible inhibitory effects on endothelium-dependent relaxations in mouse isolated aorta and small mesenteric artery.

The current study examined the hypothesis that endothelial production of hydrogen peroxide (H2O2) mediates relaxations to acetylcholine (ACh) in aorta and small mesenteric arteries (SMA) from mice. Relaxations to ACh (0.01–10 μM) and H2O2 (0.1–1000 μM) were produced in aorta and SMA isolated from wild‐type C57BL/6 mice and type II diabetic mice (db/db). In SMA, relaxations to ACh were produced in the presence of Nω‐nitro‐L‐arginine methyl ester (100 μM) and indomethacin (Indo, 10 μM). 1‐H[1,2,4]oxadiazolo[4,3‐]quinoxalin‐1‐one (10 μM) significantly reduced ACh‐induced relaxations in SMA, abolished responses in aorta, but had no effect on relaxations induced by H2O2. Catalase (2500 U ml−1) abolished responses to H2O2, but did not alter relaxations to ACh in the SMA and only caused a small rightward shift in responses to ACh in the aorta. ACh‐, but not H2O2‐, mediated relaxations were significantly reduced by tetraethylammonium (10 mM), the combination of apamin (1 μM) and charybdotoxin (100 nM), and 25 mM potassium chloride (KCl). Higher KCl (60 mM) abolished relaxations to both ACh and H2O2. Polyethylene glycolated superoxide dismutase (100 U ml−1), the catalase inhibitor 3‐amino‐1,2,4‐triazole (3‐AT, 50 mM) and treatment with the copper chelator diethyldithiolcarbamate (3 mM) did not affect relaxations to ACh. H2O2‐induced relaxations were endothelium‐independent and were not affected by ethylene diamine tetraacetic acid (EDTA 0.067 mM), 4‐aminopyridine (1 mM), ouabain (100 μM) and barium (30 μM), 3‐AT or Indo. Although the data from this study show that H2O2 dilates vessels, they do not support the notion that H2O2 mediates endothelium‐dependent relaxations to ACh in either aorta or SMA from mice.

Effects of agents that inactivate free radical NO (NO•) on nitroxyl anion‐mediated relaxations, and on the detection of NO• released from the nitroxyl anion donor Angeli's salt

The effects of agents that inactivate free radical nitric oxide (carboxy‐PTIO, hydroxocobalamin and pyrogallol) were tested on relaxations produced by the nitroxyl anion (NO−) donor Angelis salt in rat aortic rings and anococcygeus muscles. The amount of NO• generated from Angelis salt in the presence of these agents was measured using a NO•‐selective electrode sensor. Carboxy‐PTIO (100, 300 μM), hydroxocobalamin (30, 100 μM) and pyrogallol (10, 30 μM) significantly reduced relaxations produced by Angelis salt (0.3 μM) in aortic rings but not in anococcygeus muscles. NO• generated from Angelis salt (0.1 – 10 μM), as detected by the sensor electrode, was less than 0.5% of the amount of Angelis salt added. Carboxy‐PTIO (100 μM) and hydroxocobalamin (30 μM), but not pyrogallol significantly increased the amount of NO• detected. In the presence of an oxidizing agent copper [II] (as CuSO4 100 μM), the amount of NO• detected from 0.3 μM of Angelis salt increased from an undetectable level of 142.7±15.7 nM (equivalent to 47.6% of Angelis salt added). Under these conditions, carboxy‐PTIO, hydroxocobalamin and pyrogallol significantly reduced the amount of NO• detected from Angelis salt as well as the signal generated by an equivalent amount of authentic NO (0.33 μM). The difference in effects of these agents on relaxations to Angelis salt in the aorta and the anococcygeus muscle may be explained by the ready conversion of NO− to NO• in the aorta through an unidentified mechanism, which makes NO− susceptible to inactivation by these agents. Furthermore, in addition to inactivating NO•, carboxy‐PTIO and hydroxocobalamin may themselves oxidize NO− to NO•, albeit slightly.

Effects of a Western diet versus high glucose on endothelium-dependent relaxation in murine micro- and macro-vasculature

Vascular contractility and endothelium-dependent vasodilatation were studied in mesenteric, aorta and coronary vasculature from male and female LDL receptor deficient (LDLR(-/-)) and wild type C57BL/6 mice fed either a high-fat Western Diet (WD) or regular animal chow (RD). Endothelium-dependent vasodilatation was also studied in small mesenteric arteries and aorta from C57BL/6 mice following a 20 h exposure in vitro to 30 mM glucose. Compared with RD-fed animals, WD-fed LDLR-/- animals had increased body weights, elevated triglycerides and total cholesterol, but not glucose. Control C57BL6 animals had elevated body weight without increased cholesterol, triglyceride or glucose levels. The contractile sensitivity to cirazoline (pD(2)) of small mesenteric arteries was the same for RD-fed LDLR-/- and RD-fed C57BL6 mice, but was reduced in WD-fed male LDLR-/- and WD-fed female C57BL/6 mice. Maximum mesenteric contractile values for cirazoline (Emax) were unchanged; however, the Emax for phenylephrine in the aorta from WD-fed male C57BL/6 (but not LDLR-/- or female C57BL/6) mice was reduced. The Emax for acetylcholine-mediated endothelium-dependent vasodilatation in micro- and macro vessels (small mesenteric artery, coronary artery and aorta) from WD-fed LDLR-/- and C57BL/6 mice was unaltered, in contrast to the reduction in Emax for glucose-exposed tissues. Furthermore, the component of acetylcholine-mediated vasodilatation resistant to the combination of inhibitors of nitric oxide synthase, cyclooxygenase and guanylyl cyclase (nitro L-arginine methyl ester - 100 microM; indomethacin 10 microM and 1H-[1,2,4]-oxadiazolo[4,3,-a]quinoxalin-1-one, ODQ - 10 microM, respectively) was generally greater in WD-fed mice. Thus, vasculature from WD-fed mice with short-term dyslipidaemia do not exhibit reduced endothelium-dependent vasodilatation, but the WD is associated with changes in the overall endothelial-dependent relaxation and contractile responses thus suggesting an impact of diet rather than dyslipidaemia on cellular signalling pathways in vascular tissue. In contrast, acute hyperglycaemia resulted in endothelial dysfunction in both small mesenteric arteries and thoracic aorta.

Effect of xanthine oxidase inhibition on endothelium-dependent and nitrergic relaxations

The effects of inhibition of xanthine oxidase on responses mediated by nitric oxide (NO) were examined using the selective xanthine oxidase inhibitors allopurinol and 4-amino-6-hydroxypyrazolo[3,4-d]pyrimidine (AHPP). In rat aortic rings precontracted with phenylephrine (1 microM), allopurinol (300 microM) and AHPP (100, 300 microM) significantly reduced tone, an effect not seen after inhibition of NO synthase with Nomega-nitro-L-arginine (NOLA 100 microM). Relaxations produced by acetylcholine (0.01-10 microM) were significantly enhanced by AHPP (100, 300 microM) but not by allopurinol. Nitrergic relaxations in the rat anococcygeus muscle (field stimulation 1 ms pulses; 1 Hz: 10 s) were not affected by either allopurinol or AHPP. However, relaxations produced by exogenous NO (0.25 microM) were significantly enhanced by AHPP, allopurinol (100 microM) and superoxide dismutase (100 U/ml). Xanthine (500 microM) partially, but significantly, reversed the enhancement produced by AHPP. These findings suggest that superoxide generated by xanthine oxidase modulates the activity of basal and stimulated NO derived from the rat aortic endothelium, but does not affect the activity of the nitrergic transmitter in the rat anococcygeus muscle, despite its ability to modulate responses to exogenous NO.