Angiotensin II Infusion and Blood Pressure Measurement
Objective: To examine the role of p47phox, a cytosolic subunit of NADPH oxidase, in angiotensin II-induced hypertension and vascular oxidative stress by comparing blood pressure responses and superoxide production in wild-type versus p47phox-deficient mice
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Equipment2
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Protocol Steps
Angiotensin II infusion
Administer angiotensin II infusion to induce hypertension in mice
Note: Dose: 0.7 mg/kg per day. Performed in both wild-type and p47phox-deficient mice
View evidence from paper
“In vivo, angiotensin II infusion (0.7 mg/kg per day for 7 days) increased systolic blood pressure”
Systolic blood pressure measurement
Measure systolic blood pressure response in both wild-type and p47phox-deficient mice
Note: Baseline and post-infusion measurements recorded. Wild-type mice showed increase from 105±2 to 151±6 mm Hg; p47phox-/- mice showed blunted response at 122±4 mm Hg
View evidence from paper
“In vivo, angiotensin II infusion (0.7 mg/kg per day for 7 days) increased systolic blood pressure from 105±2 to 151±6 mm Hg”
In situ superoxide staining
Perform in situ staining for superoxide production using dihydroethidium in vascular tissue
Note: Staining reveals superoxide production in endothelial and vascular smooth muscle cells
View evidence from paper
“In situ staining for O2•- using dihydroethidium revealed a marked increase of O2•- production in both endothelial and vascular smooth muscle cells”
Endothelial cell culture
Culture endothelial cells from wild-type and p47phox-deficient mice
Note: Cells used for direct examination of NADPH oxidase role in superoxide production
View evidence from paper
“To directly examine the role of the NAD(P)H oxidase in endothelial production of O2•-, endothelial cells from WT and p47phox-/- mice were cultured”
Western blotting
Confirm absence of p47phox protein in p47phox-deficient mice
Note: Verification of genotype at protein level
View evidence from paper
“Western blotting confirmed the absence of p47phox in p47phox-/- mice”
Angiotensin II treatment of cultured cells
Treat cultured endothelial cells with angiotensin II
Note: Applied to both wild-type and p47phox-deficient endothelial cells
View evidence from paper
“Angiotensin II increased O2•- production in endothelial cells from WT mice, but not in those from p47phox-/- mice”
Superoxide measurement in cultured cells
Measure superoxide production in cultured endothelial cells using electron spin resonance spectroscopy
Note: Determines role of NADPH oxidase in endothelial superoxide production
View evidence from paper
“as determined by electron spin resonance spectroscopy”