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 m ediated superoxide formation in endothelial cells. 2 In quiescent human umbilical vein endothelial cells (HUVEC) superoxide fo rmation was measured after long-term incubation (6 h) with angiotensin II i n the presence or absence of its receptor blocker candesartan (AT1) or PD12 3319 (AT2) using the cytochrome c assay. In separate experiments, the effec ts of AT2 mediated effects on activities of cellular phosphates including t he 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 g enistein. In contrast, blockade of AT2 receptors by PD123319 enhanced the s uperoxide 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 c ontaining phosphatases (SHP-1, by 293.4%). The tyrosine phosphatase inhibit or vanadate, in turn, prevented the AT2 mediated effects on superoxide form ation. 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 endothe lial superoxide formation by a pathway involving tyrosine phosphatases.