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.

NADPH Oxidase-Derived Reactive Oxygen Species Mediate the Cerebrovascular Dysfunction Induced by the Amyloid β Peptide

“…Peroxynitrite also leads to the nitration of other protein involved in vascular regulation. These include the enzyme prostacyclin synthase, which produces the vasodilator prostacyclin, and the free-radical scavenging enzyme superoxide dismutase (Zou et al, 1999;Guo et al, 2003;Ischiropoulos and Beckman, 2003). Furthermore, ROS inactivate the essential NOS cofactor tetrahydrobiopterin and attenuate NO production (Katusic, 2001).…”

Nitric oxide, oxidants, and protein tyrosine nitration

“…Interestingly, MnSOD nitration via the hemeperoxidase͞ • NO 2 pathway does not lead to significant inactivation (70), suggesting that nitration occurs at tyrosines that are more superficial than the metaladjacent Tyr-34, and that the association of nitration plus inactivation of MnSOD may serve to unravel the chemical nature of the nitrating species. A recent quantitation of the extent of MnSOD nitration in rat kidneys during angiotensin II infusion provided support for the hypothesis of nitration as a direct cause of enzyme inactivation (71). Similarly, the vascular enzyme prostacyclin synthase (PGI 2 ) synthase becomes site-specifically nitrated by peroxynitrite at heme-adjacent Tyr-430 in a process catalyzed by the active site heme-thiolate involving transient ferrylspecies (72,73).…”

Proteomic analysis of age dependent nitration of rat cardiac proteins by solution isoelectric focusing coupled to nanoHPLC tandem mass spectrometry

“…Here, biological aging leads to the selective nitration of the slow-twitch skeletal and cardiac muscle isoform SERCA2a nitrated at Tyr 294 and Tyr 295 , which can be linked to the loss of enzyme activity Viner et al, 1999;Knyushko et al, 2005;Xu et al, 2006). Other proteins studied to similar functional detail include manganese superoxide dismutase (MnSOD) (Guo et al, 2003;Quint et al, 2006), prostacyclin synthase (Zou et al, 1999), and actin (Aslan et al, 2003).…”