title: Quantitative assessment of tyrosine nitration of manganese superoxide dismutase in angiotensin II-infused rat kidney.
abstract: 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.
authors.affiliation: Vascular Biology Unit, Whitaker Cardiovascular Institute, Boston University Medical Center, Boston, MA 02118, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Guo
authors.forename: Takeshi
authors.initials: T
authors.lastname: Adachi
authors.forename: Reiko
authors.initials: R
authors.lastname: Matsui
authors.forename: Shanqin
authors.initials: S
authors.lastname: Xu
authors.forename: Bingbing
authors.initials: B
authors.lastname: Jiang
authors.forename: Ming-Hui
authors.initials: MH
authors.lastname: Zou
authors.forename: Michael
authors.initials: M
authors.lastname: Kirber
authors.forename: Wilfred
authors.initials: W
authors.lastname: Lieberthal
authors.forename: Richard A
authors.initials: RA
authors.lastname: Cohen
mesh_terms: 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*
chemical_list: D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D000804:Angiotensin II; C002744:3-nitrotyrosine; D014443:Tyrosine; D013482:Superoxide Dismutase