Sohn H Y, Raff U, Hoffmann A, Gloe T, Heermeier K, Galle J, Pohl U
Institute of Physiology, Ludwig-Maximilians-University Munich, Schillerstrasse 44, 80336 Munich, Germany.
Br J Pharmacol. 2000 Oct;131(4):667-72. doi: 10.1038/sj.bjp.0703566.
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.
血管紧张素II AT2受体亚型的生理作用尚未完全明确。我们研究了AT2受体是否能拮抗AT1介导的内皮细胞超氧化物生成。在静息的人脐静脉内皮细胞（HUVEC）中，使用细胞色素c分析法，在存在或不存在其受体阻滞剂坎地沙坦（AT1）或PD123319（AT2）的情况下，用血管紧张素II进行长期孵育（6小时）后测量超氧化物生成。在单独的实验中，研究了AT2介导的对包括含src同源2结构域的磷酸酶（SHP-1）在内的细胞磷酸盐活性的影响。暴露于血管紧张素II（100 nM）后，HUVEC中的基础超氧化物生成（0.19±0.03 nmol超氧化物mg蛋白⁻¹分钟⁻¹）增加了37.1%，这是由于NAD(P)H氧化酶的激活。这被坎地沙坦（1 μM）以及酪氨酸激酶抑制剂染料木黄酮所消除。相比之下，PD123319对AT2受体的阻断使完整细胞中的超氧化物生成增加了73.7%。AT2的刺激伴随着总细胞裂解物中酪氨酸磷酸酶活性的增加（29.8%），特别是含src同源2结构域的磷酸酶（SHP-1，增加了293.4%）。酪氨酸磷酸酶抑制剂钒酸盐反过来阻止了AT2介导的对超氧化物生成的影响。通过RT-PCR分析证实了血管紧张素II受体亚型AT1和AT2的表达。结论是，AT2通过涉及酪氨酸磷酸酶的途径在功能上拮抗AT1诱导的内皮细胞超氧化物生成。
