Angiotensin II stimulates superoxide production by nitric oxide synthase in thick ascending limbs

Abstract

Angiotensin II (Ang II) causes nitric oxide synthase (NOS) to become a source of superoxide (O2−) via a protein kinase C (PKC)‐dependent process in endothelial cells. Ang II stimulates both NO and O2− production in thick ascending limbs. We hypothesized that Ang II causes O2− production by NOS in thick ascending limbs via a PKC‐dependent mechanism. NO production was measured in isolated rat thick ascending limbs using DAF‐FM, whereas O2− was measured in thick ascending limb suspensions using the lucigenin assay. Consistent stimulation of NO was observed with 1 nmol/L Ang II (P < 0.001; n = 9). This concentration of Ang II‐stimulated O2− production by 50% (1.77 ± 0.26 vs. 2.62 ± 0.36 relative lights units (RLU)/s/μg protein; P < 0.04; n = 5). In the presence of the NOS inhibitor L‐NAME, Ang II‐stimulated O2− decreased from 2.02 ± 0.29 to 1.10 ± 0.11 RLU/s/μg protein (P < 0.01; n = 8). L‐arginine alone did not change Ang II‐stimulated O2− (2.34 ± 0.22 vs. 2.29 ± 0.29 RLU/s/μg protein; n = 5). In the presence of Ang II plus the PKC α/β1 inhibitor Gö 6976, L‐NAME had no effect on O2− production (0.78 ± 0.23 vs. 0.62 ± 0.11 RLU/s/μg protein; n = 7). In the presence of Ang II plus apocynin, a NADPH oxidase inhibitor, L‐NAME did not change O2− (0.59 ± 0.04 vs. 0.61 ± ×0.08 RLU/s/μg protein; n = 5). We conclude that: (1) Ang II causes NOS to produce O2− in thick ascending limbs via a PKC‐ and NADPH oxidase‐dependent process; and (2) the effect of Ang II is not due to limited substrate.