title: Angiotensin II stimulates thick ascending limb superoxide production via protein kinase C(α)-dependent NADPH oxidase activation.
abstract: Angiotensin II (Ang II) stimulates thick ascending limb (TAL) O₂ production, but the receptor(s) and signaling mechanism(s)involved are unknown. The effect of Ang II on O₂. is generally attributed to the AT₁receptor. 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 O₂. via AT₁and PKC α-dependent NADPH oxidase activation.In rat TALs, 1 nM Ang II stimulated O₂. from 0.760.17 to 1.97 0.21 nmol/min/mg (p < 0.001). An AT₁antagonist blocked the stimulatory effect of Ang II on O₂. (0.87 0.25 nmol/min/mg; p < 0.006), whereas an AT₂ 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 O₂by 90% (p <0.01). Ang II failed to stimulate O₂. 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 O₂(1.47 +/- .21 versus 2.72 +/- .47 nmol/min/mg with Ang II alone; p < 0.03). A PKCα- and ß-selective inhibitor, Gö6976, also blocked the stimulatory effect of Ang II on O₂. (0.59 +/- 0.15 versus 2.05 +/- 0.28 nmol/min/mg with Ang II alone; p < 0.001). To distinguish between PKC α and PKC ß, we used tubules expressing dominant-negative PKC α or -ß. In control TALs, Ang II stimulated O2. by 2.17 0.44 nmol/min/mg (p < 0.011). In tubules expressing dominant-negative PKC α, Ang II failed to stimulate O2. (change: -0.30 +/- 0.27 nmol/min/mg). In tubules expressing dominant-negative PKC ß1, Ang II stimulated O2. by 2.080.69 nmol/min/mg (p < 0.002). We conclude that Ang II stimulates TAL O₂production via activation of AT₁receptors and PKC α-dependent NADPH oxidase.
authors.affiliation: Hypertension and Vascular Research Division, Department of Internal Medicine, Henry Ford Hospital, Detroit, Michigan 48202, USA.
authors.forename: Marcela
authors.initials: M
authors.lastname: Herrera
authors.forename: Guillermo B
authors.initials: GB
authors.lastname: Silva
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Garvin
mesh_terms: D000804:Angiotensin II / Q000378:metabolism*; D000809:Angiotensins / Q000737:chemistry; D000818:Animals; D004789:Enzyme Activation; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D015971:Gene Expression Regulation, Enzymologic; D005799:Genes, Dominant; D008297:Male; D008954:Models, Biological; D019255:NADPH Oxidases / Q000737:chemistry / Q000378:metabolism*; D018384:Oxidative Stress; D010100:Oxygen / Q000737:chemistry; D051571:Protein Kinase C-alpha / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013481:Superoxides / Q000378:metabolism*
chemical_list: D000809:Angiotensins; D013481:Superoxides; D000804:Angiotensin II; D019255:NADPH Oxidases; D051571:Protein Kinase C-alpha; D010100:Oxygen
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