GL-3 accumulation occurs primarily in endothelial cells with impairment of vessel reactivity

sociated with altered Cav-1 expression in the aortas of ApoE2/2 mice. In this study we investigated whether aortic vascular reactivity was altered in AngII-infused ApoE2/2 mice by measuring aortic contraction and relaxation in isolated thoracic aortas of AngII-infused ApoE2/2 mice. We also assessed eNOS activity and Cav-1 expression in this animal model. sia, topical application of local anaesthetic was provided as post-operative analgesia and the animals were allowed to recover under supplemental heat using a warming pad. Male ApoE2/2 mice were housed under a 12:12-h light-dark cycle and were given standard chow and water ad libitum. At 6 months of age mice were anaesthetised by intraperitoneal injection of ketamine and xylazine. Osmotic minipumps were placed into the subcutaneous space along the dorsal midline to deliver 1 mg/kg/min of AngII dissolved in distilled water over 14 days. Age-matched ApoE2/2 mice in which osmotic pumps delivered distilled water served as vehicle controls. Mice were maintained on a normal laboratory diet 23446639 throughout the infusion period. After the 14 day infusion, mice were sacrificed by CO2 asphyxiation, blood was collected by cardiac puncture and the aortas were isolated for further assessments. Non-invasive Tail-cuff Blood Pressure and Heart Rate Measurement Blood pressure and heart rate were measured at day 0, day 7 and day 14 of the experiment using a computerized, non-invasive, tail-cuff system. Animals were habituated to the device before measuring the pressures to R 115777 web ensure accurate measurements. Good reproducibility of this technique has been established previously: 14.827.5 and mean of absolute difference for mean blood pressure: 12.3 mmHg, 95% CI: 8.416.1). Isometric Tension Measurement Angiotensin II-Induced Endothelium Dysfunction Australia). Relaxation in response to individual agents was expressed as % of the phenylephrine contraction and 100% relaxation was considered when the active tone returned to the baseline level. was converted to nitrite using nitrate reductase. Subsequently addition of the Griess reagents converted nitrite into a deep purple azo compound and the absorbance was measured at 540 nm using an Omega plate reader. Plasma Nitrite/nitrate and Cholesterol Measurement Measurements of total plasma cholesterol, high density lipoprotein and low density/very low density lipoprotein were performed in plasma in duplicate using a commercial kit in accordance with the manufacturers’ instructions . Cholesterol oxidase specifically recognizes free cholesterol and produces products which react with a probe to generate fluorescence and were measured using an Omega plate reader. Total nitrate was measured in plasma samples by a nitrate/ nitrite colorimetric assay kit following manufacturer’s protocol . Briefly, nitrate Western Blotting Thoracic aortas were homogenized in RIPA buffer in the presence of protease inhibitors to obtain extracts of proteins. Protein concentrations were determined using the Bradford protein assay kit. Samples were loaded onto a 10% SDS-polyacrylamide gel electrophoresis gel. After electrophoresis, the separated proteins were transferred to polyvinylidene difluoride membranes. Non-specific sites were blocked with 5% non-fat dry milk in TBSt for 60 min, and the blots were then incubated with anti-phospho-eNOS antibody, 1:1000; 10422886 anti-eNOS, 1:1000 Concentration-response curve of endotheliumdependent relaxation to acetylcholine, with or without the present of L-NAME and endo