Effects of angiotensin II on vascular endothelial cells: formation of receptor-mediated reactive nitrogen species.
Angiotensin II (ANG II) participates in many cardiovascular disease states, but the mechanisms involved are not completely defined. Doses of ANG II that do not affect blood pressure significantly can still cause early changes in vascular endothelial performance and cell-specific protein 3-nitrotyrosine formation (protein-3NT, marker of peroxynitrite formation) in vivo. Here, we have tested the hypothesis that ANG II induces endothelial cell peroxynitrite (ONOO-) formation in vitro, and investigated the mechanisms involved. Endothelial cells were incubated with ANG II (1nM-250 microM), and protein nitration was assessed by immunoblotting. ANG II caused concentration-dependent increases in protein-3NT above detectable basal control levels, at concentrations greater than 100nM. This response was inhibited significantly by co-incubation with losartan or diphenyleneiodonium chloride. Endothelial cell lysates incubated with nitrated protein standards demonstrated significant protein-3NT modification activity only in the presence of serum. However, endothelial cell lysates did not modify the free amino acid form of 3NT (free-3NT) in identical experimental conditions, assessed by capillary electrophoresis. Finally, free-3NT was cytotoxic to cultured endothelial cells (fitted LC(50)=98 microM). These data demonstrate that stimulation of angiotensin receptor subtype 1 by ANG II can cause increased endothelial cell protein nitration in vitro in the absence of other cell types or stimuli, at concentrations that are pathophysiologically relevant. Furthermore, endothelial cells selectively modified nitrated protein tyrosine residues only in the presence of a cofactor(s), and did not modify the free modified amino acid. Protein nitration may be a regulated endothelial signaling process, while free-3NT may be toxic to endothelial cells.
Mihm
Michael J
MJ
Center for Developmental Pharmacology & Toxicology, Columbus Children's Research Institute, 700 Children's Drive, Columbus, OH 43205, USA.
Wattanapitayakul
Suvara K
SK
Piao
Sheng-Fu
SF
Hoyt
Dale G
DG
Bauer
John Anthony
JA
D000804:Angiotensin II / Q000494:pharmacology*; D000818:Animals; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D026361:Reactive Nitrogen Species / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D026361:Reactive Nitrogen Species; D000804:Angiotensin II; C002744:3-nitrotyrosine; D014443:Tyrosine