Herrera Marcela, Silva Guillermo B, Garvin Jeffrey L
Hypertension and Vascular Research Division, Department of Internal Medicine, Henry Ford Hospital, Detroit, Michigan 48202, USA.
J Biol Chem. 2010 Jul 9;285(28):21323-8. doi: 10.1074/jbc.M110.109157. Epub 2010 May 6.
Angiotensin II (Ang II) stimulates thick ascending limb (TAL) O₂ production, but the receptor(s) and signaling mechanism(s)involved are unknown. The effect of Ang II on O₂. is generally attributed to the AT₁receptor. In some cells, Ang II stimulates protein kinase C (PKC), whose α isoform (PKCα) can activate NADPH oxidase. We hypothesized that in TALs, Ang II stimulates O₂. via AT₁and PKC α-dependent NADPH oxidase activation.In rat TALs, 1 nM Ang II stimulated O₂. from 0.760.17 to 1.97 0.21 nmol/min/mg (p < 0.001). An AT₁antagonist blocked the stimulatory effect of Ang II on O₂. (0.87 0.25 nmol/min/mg; p < 0.006), whereas an AT₂ antagonist had no effect (2.16 0.133 nmol/min/mg; p < 0.05 versus vehicle). Apocynin, an NADPH oxidase inhibitor, blocked Ang II-stimulated O₂by 90% (p <0.01). Ang II failed to stimulate O₂. in TALs from p47(phox) -/- mice (p < 0.02). Monitored by fluorescence resonance energy transfer, Ang II increased PKC activity from 0.02 0.03 to 0.13 0.02 arbitrary units (p < 0.03). A general PKC inhibitor, GF109203X, blocked the effect of Ang II on O₂(1.47 +/- .21 versus 2.72 +/- .47 nmol/min/mg with Ang II alone; p < 0.03). A PKCα- and ß-selective inhibitor, Gö6976, also blocked the stimulatory effect of Ang II on O₂. (0.59 +/- 0.15 versus 2.05 +/- 0.28 nmol/min/mg with Ang II alone; p < 0.001). To distinguish between PKC α and PKC ß, we used tubules expressing dominant-negative PKC α or -ß. In control TALs, Ang II stimulated O2. by 2.17 0.44 nmol/min/mg (p < 0.011). In tubules expressing dominant-negative PKC α, Ang II failed to stimulate O2. (change: -0.30 +/- 0.27 nmol/min/mg). In tubules expressing dominant-negative PKC ß1, Ang II stimulated O2. by 2.080.69 nmol/min/mg (p < 0.002). We conclude that Ang II stimulates TAL O₂production via activation of AT₁receptors and PKC α-dependent NADPH oxidase.
血管紧张素II（Ang II）刺激髓袢升支粗段（TAL）的氧生成，但相关的受体和信号传导机制尚不清楚。Ang II对氧的作用通常归因于AT₁受体。在某些细胞中，Ang II刺激蛋白激酶C（PKC），其α亚型（PKCα）可激活NADPH氧化酶。我们推测，在TAL中，Ang II通过AT₁和PKCα依赖性NADPH氧化酶激活来刺激氧生成。在大鼠TAL中，1 nM Ang II将氧生成速率从0.76±0.17提高至1.97±0.21 nmol/分钟/毫克（p<0.001）。一种AT₁拮抗剂可阻断Ang II对氧生成的刺激作用（0.87±0.25 nmol/分钟/毫克；p<0.006），而一种AT₂拮抗剂则无作用（2.16±0.133 nmol/分钟/毫克；与溶剂对照相比p<0.05）。NADPH氧化酶抑制剂阿朴吗啡可阻断Ang II刺激的氧生成的90%（p<0.01）。Ang II未能刺激p47（phox）-/-小鼠的TAL中的氧生成（p<0.02）。通过荧光共振能量转移监测，Ang II使PKC活性从0.02±0.03增加至0.13±0.02任意单位（p<0.03）。一种通用的PKC抑制剂GF109203X可阻断Ang II对氧生成的作用（1.47±0.21对比单独使用Ang II时的2.72±0.47 nmol/分钟/毫克；p<0.03）。一种PKCα和β选择性抑制剂Gö6976也可阻断Ang II对氧生成的刺激作用（0.59±0.15对比单独使用Ang II时的2.05±0.28 nmol/分钟/毫克；p<0.001）。为区分PKCα和PKCβ，我们使用了表达显性负性PKCα或-β的肾小管。在对照TAL中，Ang II刺激氧生成速率为2.17±0.44 nmol/分钟/毫克（p<0.011）。在表达显性负性PKCα的肾小管中，Ang II未能刺激氧生成（变化：-0.30±0.27 nmol/分钟/毫克）。在表达显性负性PKCβ1的肾小管中，Ang II刺激氧生成速率为2.08±0.69 nmol/分钟/毫克（p<0.002）。我们得出结论，Ang II通过激活AT₁受体和PKCα依赖性NADPH氧化酶来刺激TAL的氧生成。
