DL-methionine methyl ester hydrochloride

Objective: This study was designed to investigate the effects of Astragalus membranaceus (AM) and its main components, astragalus saponin (ASP), astragalus polysaccharide (APS) and aminobutyric acid (GABA), on homocysteine (Hcy) induced acute impairment of vascular tone and to explore whether the antioxidant mechanism was involved in AM protective effect. Methods: Inhibitory effects of Hcy and protective effects of AM and its main components on endothelium-dependent relaxation of aortic rings were determined by isometric tension recordings and nitric oxide signaling was assayed with 125I-cGMP RIA Kit. Furthermore, generation of reactive oxygen species (ROS) in endothelial cells was detected using 5-(6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCF-DA). Results: Hcy significantly inhibited endothelium-dependent relaxation to acetylcholine (ACh) in a dose-dependent manner, and decreased cGMP levels increased by ACh in aorta. Furthermore, superoxide dismutase (SOD), AM, and ASP markedly attenuated inhibition of vasorelaxation and downregulation of cGMP level by Hcy, and APS exerted a tendency to reverse both of the depressive responses, while GABA had no similar effects. Additionally, partially impaired relaxation by Hcy was completely blocked due to the presence of N(omega)-nitro-L-arginine-methyl ester (L-NAME), which could not be further altered by treatment with AM, ASP, APS or GABA. Finally, Hcy significantly increased intracellular ROS levels in endothelial cells as measured by CM-H2DCF-DA fluorescence. SOD, AM, ASP, and APS, but not GABA, inhibited Hcy-stimulated ROS generation. Conclusion: This study demonstrated that AM and ASP, potently protected endothelium-dependent relaxation against the acute injury from Hcy through nitric oxide regulatory pathways, in which antioxidation played a key role.

Purpose: Homocysteine (Hcy) has been shown to induce oxidative stress and apoptosis of endothelial progenitor cells (EPCs). AMP-activated protein kinase (AMPK) has been reported to have protective effects on endothelial function. However, effects of AMPK activation on Hcy-induced EPCs injury remain to be determined. In this study, we examined the effect of AMPK phosphorylation on Hcy-induced NO bioavailability impairment and NADPH oxidase 4 (Nox4) derived reactive oxygen species (ROS) accumulation in EPCs. Materials and methods: EPCs were pre-treated with various concentrations of 5-amino-4-imidazolecarboxamide riboside-l-beta-D-ribofuranoside (AICAR), a pharmacological agonist of AMPK, and then incubated with Hcy for 24 h. Furthermore, we challenged EPCs with Hcy in the presence or absence of atorvastatin and AMPK-DN which expressed a dominant-negative mutant of AMPK. Migration, proliferation and apoptosis were assayed to evaluate EPCs function. NO production, expression of endothelial nitric oxide synthase (eNOS), intracellular ROS levels and Nox4 activation were determined to explore the potential mechanisms of Hcy-induced EPCs dysfunction. Results: We observed that AICAR attenuated the inhibition effects of Hcy on EPCs migration and proliferation. The apoptosis rates of EPCs were down-regulated by AICAR compared with the group treated with Hcy only [(0.25 mmol/L AICAR: 10.48 ± 1.6%; 0.5 mmol/L AICAR: 8.70 ± 1.0%; 1 mmol/L AICAR: 5.83 ± 1.3%) vs. (500 μmol/L Hcy only: 12.60 ± 1.9%)]. We also found that NO production and eNOS expression were up-regulated by AICAR compared with the group treated with Hcy only, while ROS accumulation and Nox4 activation were inhibited. Furthermore, atorvastatin suppressed Hcy-induced dysfunction of EPCs, increased NO production and eNOS expression, and down-regulated ROS accumulation and Nox4 activation. And these effects of atorvastatin could be blunted by AMPK-DN. Conclusion: AMPK activation inhibits eNOS down-regulation and Nox4-derived ROS accumulation induced by Hcy in EPCs, and may contribute to the protective effects of atorvastatin on endothelial function.

Homocysteine (HCY) is a pro-inflammatory sulphur-containing redox active endogenous amino acid, which concentration increases in neurodegenerative disorders including amyotrophic lateral sclerosis (ALS). A widely held view suggests that HCY could contribute to neurodegeneration via promotion of oxidative stress. However, the action of HCY on motor nerve terminals has not been investigated so far. We previously reported that oxidative stress inhibited synaptic transmission at the neuromuscular junction, targeting primarily the motor nerve terminals. In the current study, we investigated the effect of HCY on oxidative stress-induced impairment of transmitter release at the mouse diaphragm muscle. The mild oxidant H2O2 decreased the intensity of spontaneous quantum release from nerve terminals (measured as the frequency of miniature endplate potentials, MEPPs) without changes in the amplitude of MEPPs, indicating a presynaptic effect. Pre-treatment with HCY for 2 h only slightly affected both amplitude and frequency of MEPPs but increased the inhibitory potency of H2O2 almost two fold. As HCY can activate certain subtypes of glutamate N-methyl D-aspartate (NMDA) receptors we tested the role of NMDA receptors in the sensitizing action of HCY. Remarkably, the selective blocker of NMDA receptors, AP-5 completely removed the sensitizing effect of HCY on the H2O2-induced presynaptic depressant effect. Thus, at the mammalian neuromuscular junction HCY largely increases the inhibitory effect of oxidative stress on transmitter release, via NMDA receptors activation. This combined effect of HCY and local oxidative stress can specifically contribute to the damage of presynaptic terminals in neurodegenerative motoneuron diseases, including ALS.