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Differential role of angiotensin II receptor subtypes on endothelial superoxide formation.

ABSTRACT: 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 nM,) which was due to an activation of a NAD(P)H oxidase. This was abolished by candesartan (1 microM) 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.

SUBMITTER: Sohn HY

PROVIDER: S-EPMC1572372 | biostudies-other | 2000 Oct

REPOSITORIES: biostudies-other

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Project description:AimsAngiotensin II (AngII)-induced superoxide (O2(•-)) production by the NADPH oxidases and mitochondria has been implicated in the pathogenesis of endothelial dysfunction and hypertension. In this work, we investigated the specific molecular mechanisms responsible for the stimulation of mitochondrial O2(•-) and its downstream targets using cultured human aortic endothelial cells and a mouse model of AngII-induced hypertension.ResultsWestern blot analysis showed that Nox2 and Nox4 were present in the cytoplasm but not in the mitochondria. Depletion of Nox2, but not Nox1, Nox4, or Nox5, using siRNA inhibits AngII-induced O2(•-) production in both mitochondria and cytoplasm. Nox2 depletion in gp91phox knockout mice inhibited AngII-induced cellular and mitochondrial O2(•-) and attenuated hypertension. Inhibition of mitochondrial reverse electron transfer with malonate, malate, or rotenone attenuated AngII-induced cytoplasmic and mitochondrial O2(•-) production. Inhibition of the mitochondrial ATP-sensitive potassium channel (mitoK(+)ATP) with 5-hydroxydecanoic acid or specific PKCɛ peptide antagonist (EAVSLKPT) reduced AngII-induced H2O2 in isolated mitochondria and diminished cytoplasmic O2(•-). The mitoK(+)ATP agonist diazoxide increased mitochondrial O2(•-), cytoplasmic c-Src phosphorylation and cytoplasmic O2(•-) suggesting feed-forward regulation of cellular O2(•-) by mitochondrial reactive oxygen species (ROS). Treatment of AngII-infused mice with malate reduced blood pressure and enhanced the antihypertensive effect of mitoTEMPO. Mitochondria-targeted H2O2 scavenger mitoEbselen attenuated redox-dependent c-Src and inhibited AngII-induced cellular O2(•-), diminished aortic H2O2, and reduced blood pressure in hypertensive mice.Innovation and conclusionsThese studies show that Nox2 stimulates mitochondrial ROS by activating reverse electron transfer and both mitochondrial O2(•-) and reverse electron transfer may represent new pharmacological targets for the treatment of hypertension.