title: 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.
authors.affiliation: Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Ciudad Autónoma de Buenos Aires, Argentina agustin.gonzalezvicente@case.edu.
authors.forename: Agustin
authors.initials: A
authors.lastname: Gonzalez-Vicente
authors.affiliation: Department of Internal Medicine, Hypertension and Vascular Research Division, Henry Ford Hospital, Detroit, MI.
authors.forename: Jagannath H
authors.initials: JH
authors.lastname: Saikumar
authors.affiliation: Department of Internal Medicine, Hypertension and Vascular Research Division, Henry Ford Hospital, Detroit, MI.
authors.forename: Katherine J
authors.initials: KJ
authors.lastname: Massey
authors.affiliation: Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio.
authors.forename: Nancy J
authors.initials: NJ
authors.lastname: Hong
authors.affiliation: Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Ciudad Autónoma de Buenos Aires, Argentina Instituto de Química y Fisicoquímica Biológicas, CONICET, Ciudad Autónoma de Buenos Aires, Argentina.
authors.forename: Fernando P
authors.initials: FP
authors.lastname: Dominici
authors.affiliation: Department of Internal Medicine, Hypertension and Vascular Research Division, Henry Ford Hospital, Detroit, MI.
authors.forename: Oscar A
authors.initials: OA
authors.lastname: Carretero
authors.affiliation: Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio Department of Internal Medicine, Hypertension and Vascular Research Division, Henry Ford Hospital, Detroit, MI.
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Garvin
mesh_terms: D000804:Angiotensin II / Q000378:metabolism*; D000818:Animals; D008138:Loop of Henle / Q000378:metabolism*; D008163:Luminescent Measurements; D008297:Male; D008856:Microscopy, Fluorescence; D019255:NADPH Oxidases / Q000378:metabolism; D009399:Nephrons / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D009924:Organ Culture Techniques; D051381:Rats; D017207:Rats, Sprague-Dawley; D013481:Superoxides / Q000378:metabolism*
chemical_list: D013481:Superoxides; D000804:Angiotensin II; D019001:Nitric Oxide Synthase; D019255:NADPH Oxidases
keywords: Kidney; NADPH oxidase; protein kinase C; reactive oxygen species; uncoupling