Abstract
Angiotensin II (Ang II) stimulates thick ascending limb (TAL) O&sub2; production, but the receptor(s) and signaling mechanism(s)involved are unknown. The effect of Ang II on O&sub2;. is generally attributed to the AT&sub1;receptor. 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 O&sub2;. via AT&sub1;and PKC α-dependent NADPH oxidase activation.In rat TALs, 1 nM Ang II stimulated O&sub2;. from 0.760.17 to 1.97 0.21 nmol/min/mg (p < 0.001). An AT&sub1;antagonist blocked the stimulatory effect of Ang II on O&sub2;. (0.87 0.25 nmol/min/mg; p < 0.006), whereas an AT&sub2; 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 O&sub2;by 90% (p <0.01). Ang II failed to stimulate O&sub2;. 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 O&sub2;(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 O&sub2;. (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 O&sub2;production via activation of AT&sub1;receptors and PKC α-dependent NADPH oxidase.