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Protein Kinase C and PI3 Kinase Mediate Ang II‐Dependent Generation of Reactive Oxygen Species in Renal Nuclei
Author(s) -
Pendergrass Karl D,
Gwathmey TanYa M,
Grayson Jason M,
Chappell Mark C
Publication year - 2009
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.23.1_supplement.803.7
Subject(s) - nox4 , protein kinase c , reactive oxygen species , nadph oxidase , chemistry , ly294002 , angiotensin ii , microbiology and biotechnology , signal transduction , receptor , pi3k/akt/mtor pathway , medicine , biology , biochemistry
We previously reported that nuclei isolated from rat renal cortex contain a high density of AT 1 receptors (AT1R) that are functionally linked to reactive oxygen species (ROS) generation. Moreover, the protein kinase C (PKC) mimetic, PMA, stimulated ROS to a similar extent as Ang II. Therefore, the present studies elucidated the signaling pathways responsible for Ang II‐AT1R dependent generation of ROS in renal nuclei. Ang II (1 nM, 5 min) increased fluorescence of the ROS indicator dichlorofluorescein (DCF) [39±6% above control, P≤0.05, N=8], which was abolished by the oxidase inhibitor DPI [‐5±5%, P≤0.01 vs. Ang II, N=4]. The selective PKC inhibitor GF109203X also attenuated Ang II‐dependent ROS generation [‐8±8%, P≤0.01 vs. Ang II, N=3]. Immunoblot screening of five PKC isoforms revealed a predominant band for PKC‐δ (80 kDa) in renal nuclei consistent with the inhibitory spectrum of the PKC agent. As activation of PKC is typically downstream from PI3‐kinase (PI3K), addition of the highly selective PI3K inhibitor LY294002 abolished Ang II‐stimulated ROS [2±2%, P≤0.01 vs. Ang II, N=3] that was consistent with protein expression of the PI3K subunits p85 and p110 in isolated nuclei. Finally, immunoblots revealed NOX4 (65 kDa) in isolated nuclei and flow analysis localized NOX4 to 65±4% of nuclei [N=4]. We conclude that the nuclear Ang II‐AT1R‐ROS axis may involve NOX4, as well as PKC‐ and PI3K‐dependent pathways.