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Angiotensin II Stimulates Thick Ascending Limb Superoxide Production via activation of AT1 Receptors and PKCα‐stimulated NADPH oxidase
Author(s) -
Herrera Marcela,
Silva Guillermo B,
Garvin Jeffrey L
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.812.16
Subject(s) - losartan , nadph oxidase , angiotensin ii , apocynin , protein kinase c , medicine , endocrinology , chemistry , receptor , superoxide , angiotensin ii receptor type 1 , receptor antagonist , oxidase test , antagonist , signal transduction , reactive oxygen species , biology , biochemistry , enzyme
Angiotensin II (Ang II) stimulates thick ascending limb (TAL) O 2 − production but the receptor involved and signaling cascade is unknown. In other cells, activation of AT 1 receptors (AT 1 R) results in PKC‐stimulated NADPH oxidase. We hypothesized that Ang II stimulates TAL O 2 − production via AT 1 R and PKC‐stimulated NADPH oxidase. In TAL suspensions, 1nM Ang II stimulated O 2 − production from 0.8 ± 0.2 to 2.0 ± 0.2 nmoles/min/mg ( p <0.003). Losartan, an AT 1 antagonist (1μM), blocked the effect of Ang II on O 2 − (0.9 ± 0.3 nmoles/min/mg; p < 0.02 vs. Ang II alone). PD123319, an AT 2 antagonist (1μM), had no effect. Apocynin, a NADPH oxidase inhibitor (10μM), blocked the Ang II‐stimulated O 2 − by 90% ( p <0.01). In isolated TALs, Ang II increased PKC activity, as measured by FRET, from 0.2 ± 0.3 to 1.3 ± 0.2 arbitrary units ( p <0.02). A PKC inhibitor, Gö6976 (100nM), blocked the Ang II‐stimulated O 2 − (0.6 ± 0.2 vs. 2.1 ± 0.3 nmoles/min/mg; p <0.008). To investigate which PKC isoform was involved, we measured O 2 − in tubules expressing dominant‐negative (dn) PKCα or β1. In control TALs, Ang II stimulated O 2 − from 1.4 ± 0.1 to 3.6 ± 0.5 nmoles/min/mg ( p <0.003). In TALs expressing the dn‐PKCα, Ang II failed to stimulate O 2 − . In tubules expressing the dn‐PKCβ1, Ang II stimulated O 2 − from 1.8 ± 0.4 to 3.9 ± 0.4 nmoles/min/mg ( p <0.002). We conclude that Ang II stimulates TAL O 2 − production via activation of AT 1 R and in PKCα‐stimulation of NADPH oxidase. Support: PO1 HL028982