Angiotensin II stimulates thick ascending limb superoxide production via protein kinase C(α)-dependent NADPH oxidase activation

Abstract

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.

FIGURE 1.

Effect of 1 nm angiotensin II for 10 min on rat thick ascending limb O production in the absence and presence of 1 μm losartan (an AT₁ receptor blocker) or 1 μm PD123319 (an AT₂ receptor blocker). Ang II, angiotensin II; LOS, losartan; PD, PD123319. n = 11 for Ang II, 5 for vehicle and 6 for Ang II plus LOS and Ang II plus PD.

FIGURE 2.

Top, effect of 10 μm apocynin (an NADPH oxidase inhibitor) on the stimulatory effect of angiotensin II on O production by rat thick ascending limbs. Rat thick ascending limbs were incubated with 1 nm angiotensin II for 10 min in the presence or absence of apocynin and superoxide production measured. n = 5 per group. Bottom, effect of 1 nm angiotensin II for 10 min on O production by thick ascending limbs isolated from wild-type and p47^phox knock-out mice (p47^phox−/−). n = 4. Ang II, angiotensin II.

FIGURE 3.

Acute effect of 1 nm Ang II on total PKC activity by FRET. Rat thick ascending limbs expressing a PKC activity reporter were used. n = 6.

FIGURE 4.

Effect of 100 nm GF109203X (a general PKC inhibitor) on the stimulatory effect of 1 nm Ang II for 10 min on O production by rat thick ascending limbs. n = 5–6.

FIGURE 5.

Effect of 100 nm Gö6976 (a selective PKCα and β1 inhibitor) on the stimulatory effect of 1 nm Ang II for 10 min on O production by rat thick ascending limbs. n = 6.

FIGURE 6.

Effect of vehicle or 1 nm Ang II for 10 min on O production by rat thick ascending limbs expressing control, dn-PKCα, or dn-PKCβ1. n = 5–6.