Differential role of angiotensin II receptor subtypes on endothelial superoxide formation

1 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. 2 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 are homology 2 domain containing phosphatases (SHP-1) was studied. 3 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 nM,) which was due to an activation of a NAD(P)H oxidase. This was abolished by candesartan (1 muM) 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. 4 It is concluded that AT2 functionally antagonizes the ATI induced endothelial superoxide formation by a pathway involving tyrosine phosphatases.