Differential role of angiotensin II receptor subtypes on endothelial superoxide formation

The physiological role of the angiotensin II AT2 receptor subtype is not fully characterized. We studied whether AT2 receptor could antagonize AT1 mediated superoxide formation in endothelial cells. In quiescent human umbilical vein endothelial cells (HUVEC) superoxide formation was measured after long‐term incubation (6 h) with angiotensin II in the presence or absence of its receptor blocker candesartan (AT1) or PD123319 (AT2) using the cytochrome c assay. In separate experiments, the effects of AT2 mediated effects on activities of cellular phosphates including the src homology 2 domain containing phosphatases (SHP‐1) was studied. The basal superoxide formation (0.19±0.03 nmol superoxide mg protein −1 min −1 ) in HUVEC was increased by 37.1% after exposure to angiotensin II (100 n M ,) which was due to an activation of a NAD(P)H oxidase. This was abolished by candesartan (1 μ M ) as well as the tyrosine kinase inhibitor genistein. In contrast, blockade of AT2 receptors by PD123319 enhanced the superoxide formation by 73.7% in intact cells. Stimulation of AT2 went along with an increased activity of tyrosine phosphatases in total cell lysates (29.8%) and, in particular, a marked stimulation of src homology 2 domain containing phosphatases (SHP‐1, by 293.4%). The tyrosine phosphatase inhibitor vanadate, in turn, prevented the AT2 mediated effects on superoxide formation. The expression of both angiotensin II receptor subtypes AT1 and AT2 was confirmed by RT–PCR analysis. It is concluded that AT2 functionally antagonizes the AT1 induced endothelial superoxide formation by a pathway involving tyrosine phosphatases.British Journal of Pharmacology (2000) 131 , 667–672; doi: 10.1038/sj.bjp.0703566