Open AccessAngiotensin II stimulates superoxide production by nitric oxide synthase in thick ascending limbs
Author(s) -
GonzalezVicente Agustin,
Saikumar Jagannath H.,
Massey Katherine J.,
Hong Nancy J.,
Dominici Fernando P.,
Carretero Oscar A.,
Garvin Jeffrey L.
Publication year - 2016
Publication title -
physiological reports
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.918
H-Index - 39
ISSN - 2051-817X
DOI - 10.14814/phy2.12697
Subject(s) - angiotensin ii , apocynin , superoxide , protein kinase c , nitric oxide synthase , nitric oxide , medicine , nadph oxidase , endocrinology , chemistry , microbiology and biotechnology , biology , kinase , receptor , biochemistry , oxidative stress , enzyme
Angiotensin II (Ang II ) causes nitric oxide synthase ( NOS ) to become a source of superoxide (O 2 − ) via a protein kinase C ( PKC )‐dependent process in endothelial cells. Ang II stimulates both NO and O 2 − production in thick ascending limbs. We hypothesized that Ang II causes O 2 − 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 O 2 − 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 O 2 − production by 50% (1.77 ± 0.26 vs. 2.62 ± 0.36 relative lights units ( RLU )/s/ μ g protein; P < 0.04; n = 5). In the presence of the NOS inhibitor L‐ NAME , Ang II ‐stimulated O 2 − decreased from 2.02 ± 0.29 to 1.10 ± 0.11 RLU /s/ μ g protein ( P < 0.01; n = 8). L‐arginine alone did not change Ang II ‐stimulated O 2 − (2.34 ± 0.22 vs. 2.29 ± 0.29 RLU /s/ μ g protein; n = 5). In the presence of Ang II plus the PKC α / β 1 inhibitor Gö 6976, L‐ NAME had no effect on O 2 − production (0.78 ± 0.23 vs. 0.62 ± 0.11 RLU /s/ μ g protein; n = 7). In the presence of Ang II plus apocynin, a NADPH oxidase inhibitor, L‐ NAME did not change O 2 − (0.59 ± 0.04 vs. 0.61 ± ×0.08 RLU /s/ μ g protein; n = 5). We conclude that: (1) Ang II causes NOS to produce O 2 − 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.