Angiotensin II Stimulates Thick Ascending Limb Superoxide Production via Protein Kinase Cα-dependent NADPH Oxidase Activation

Angiotensin II (Ang II) stimulates thick ascending limb (TAL) O(2)radical anion. production, but the receptor(s) and signaling mechanism(s) involved are unknown. The effect of Ang II on O(2)radical anion is generally attributed to the AT(1) receptor. In some cells, Ang II stimulates protein kinase C (PKC), whose alpha isoform (PKC alpha) can activate NADPH oxidase. We hypothesized that in TALs, Ang II stimulates O(2)radical anion via AT(1) and PKC alpha-dependent NADPH oxidase activation. In rat TALs, 1 nM Ang II stimulated O(2)radical anion from 0.76 +/- 0.17 to 1.97 +/- 0.21 nmol/min/mg (p < 0.001). An AT(1) antagonist blocked the stimulatory effect of Ang II on O(2)radical anion (0.87 +/- 0.25 nmol/min/mg; p < 0.006), whereas an AT(2) 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(2)radical anion by 90% (p < 0.01). Ang II failed to stimulate O(2)radical anion 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(2)radical anion (1.47 +/- 0.21 versus 2.72 +/- 0.47 nmol/min/mg with Ang II alone; p < 0.03). A PKC alpha- and beta-selective inhibitor, Go6976, also blocked the stimulatory effect of Ang II on O(2)radical anion (0.59 +/- 0.15 versus 2.05 +/- 0.28 nmol/min/mg with Ang II alone; p < 0.001). To distinguish between PKC alpha and PKC beta, we used tubules expressing dominant-negative PKC alpha or -beta. In control TALs, Ang II stimulated O(2)radical anion by 2.17 +/- 0.44 nmol/min/mg (p < 0.011). In tubules expressing dominant-negative PKC alpha, Ang II failed to stimulate O(2)radical anion (change: -0.30 +/- 0.27 nmol/min/mg). In tubules expressing dominant-negative PKC beta 1, Ang II stimulated O(2)radical anion by 2.08 +/- 0.69 nmol/min/mg (p< 0.002). We conclude that Ang II stimulates TAL O(2)radical anion production via activation of AT1 receptors and PKC alpha-dependent NADPH oxidase.