Abstract
Angiotensin II (Ang II) stimulates thick ascending limb (TAL) O
production, but the receptor(s) and signaling mechanism(s) involved are unknown. The effect of Ang II on O
is generally attributed to the AT
1
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
via AT
1
and PKCα-dependent NADPH oxidase activation. In rat TALs, 1 n
m
Ang II stimulated O
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
(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
by 90% (
p
< 0.01). Ang II failed to stimulate O
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
(1.47 ± 0.21
versus
2.72 ± 0.47 nmol/min/mg with Ang II alone;
p
< 0.03). A PKCα- and β-selective inhibitor, Gö6976, also blocked the stimulatory effect of Ang II on O
(0.59 ± 0.15
versus
2.05 ± 0.28 nmol/min/mg with Ang II alone;
p
< 0.001). To distinguish between PKCα and PKCβ, we used tubules expressing dominant-negative PKCα or -β. In control TALs, Ang II stimulated O
by 2.17 ± 0.44 nmol/min/mg (
p
< 0.011). In tubules expressing dominant-negative PKCα, Ang II failed to stimulate O
(change: −0.30 ± 0.27 nmol/min/mg). In tubules expressing dominant-negative PKCβ1, Ang II stimulated O
by 2.08 ± 0.69 nmol/min/mg (
p
< 0.002). We conclude that Ang II stimulates TAL O
production via activation of AT
1
receptors and PKCα-dependent NADPH oxidase.