Quantitative assessment of tyrosine nitration of manganese superoxide dismutase in angiotensin II-infused rat kidney.
Hypertension caused by angiotensin II is characterized by an increase in tissue oxidant stress as evidenced by increased quantities of reactive oxygen and nitrogen species. Manganese superoxide dismutase (MnSOD) is a key mitochondrial antioxidant enzyme that is inactivated in conditions of oxidant stress by reacting with peroxynitrite to form 3-nitrotyrosine in its active site. The increase in 3-nitrotyrosine content in MnSOD in the kidney of angiotensin II-infused rats was assessed in this study by immunohistochemistry, Western blotting, immunoprecipitation, and HPLC with UV detection (HPLC-UV). MnSOD activity decreased approximately 50% in angiotensin II-infused rat kidneys (24 +/- 4.6 vs. 11 +/- 5.2 U/mg) without a change in protein expression. Immunohistochemical staining showed 3-nitrotyrosine predominantly in distal tubules and collecting duct cells in the angiotensin II-infused rat kidneys. By two-photon microscopy, 3-nitrotyrosine colocalized with MnSOD. Total 3-nitrotyrosine content in kidney homogenates was increased in angiotensin II-infused rat kidney [3.2 +/- 1.9 (sham treated) vs. 9.5 +/- 2.3 ng/mg protein by HPLC-UV detection]. With tracer amounts of tyrosine-nitrated recombinant MnSOD, the most sensitive technique to detect tyrosine nitration of MnSOD was immunoprecipitation from tissue with anti-MnSOD antibody, followed by detection of 3-nitrotyrosine by Western blotting or HPLC. By HPLC, 3-nitrotyrosine content of kidney MnSOD increased 13-fold after angiotensin II infusion, representing an increase from approximately one-twentieth to one-fifth of the total 3-nitrotyrosine content in sham-treated and angiotensin II-infused rat kidney, respectively. Angiotensin II-induced hypertension is accompanied by increased tyrosine nitration of MnSOD, which, because it inactivates the enzyme, may contribute to increased oxidant stress in the kidney.
Guo
Wei
W
Vascular Biology Unit, Whitaker Cardiovascular Institute, Boston University Medical Center, Boston, MA 02118, USA.
Adachi
Takeshi
T
Matsui
Reiko
R
Xu
Shanqin
S
Jiang
Bingbing
B
Zou
Ming-Hui
MH
Kirber
Michael
M
Lieberthal
Wilfred
W
Cohen
Richard A
RA
D000804:Angiotensin II / Q000494:pharmacology*; D000818:Animals; D000911:Antibodies, Monoclonal; D002851:Chromatography, High Pressure Liquid; D007150:Immunohistochemistry; D007668:Kidney / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D011233:Precipitin Tests; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000378:metabolism; D012680:Sensitivity and Specificity; D013482:Superoxide Dismutase / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D000804:Angiotensin II; C002744:3-nitrotyrosine; D014443:Tyrosine; D013482:Superoxide Dismutase