Critical role of the β isoform of protein kinase C ( PKCβ ) in angiotensin II –induced oxidative stress in vascular smooth muscle cells

Figures

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  FIGURE 1. Effect of angiotensin II (Ang II) on reactive oxygen species (ROS) production and phosphorylation of protein kinase C (PKC)β in rat mesenteric vascular smooth muscle cells (VSMCs). Dihydroethidium (DHE) staining was used to detect ROS production in rat mesenteric artery primary VSMCs treated with Ang II (100 nM) for 30 min (a, n = 5) or 24 h (d, n = 7). Lucigenin assay was used to detect NADPH oxidase (NOX)‐derived superoxide production in VSMCs exposed to Ang II (100 nM) for 30 min (b, n = 4–5) or 24 h (e, n = 5–7) following pretreatment with apocynin (100 μM). Mitochondrial‐targeted fluorescent O2 − indicator was used to detect mitochondrial superoxide production in VSMCs exposed to Ang II (100 nM) for 30 min (c, n = 8) or 24 h (f, n = 8) following pretreatment with mitotempol (100 μM) as a negative control. Representative (g) and quantitative (h) Western blotting analysis of phosphorylation of PKCβ (S660), PKCβ, and GAPDH in VSMCs exposed to Ang II (0, 1, 10, 100, or 1000 nM) for 15 min (n = 7). Values are mean ± SEM. Exact p‐values are shown on the graphs.

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  FIGURE 2. Effect of PKCβ deletion on ROS production in rat mesenteric VSMCs. Expression of PKC isoforms in primary mesenteric VSMCs from PKCβ‐knockout (KO) rats (a–d). Quantitative PCR analysis of PKCβ (a), PKCα (b), PKCδ (c), and PKCε (d) expression in primary mesenteric VSMCs of non‐transgenic (NT) and PKCβ‐KO rats (n = 6–7). DHE staining was used to detect ROS production in NT‐ or PKCβ‐KO–VSMCs treated with Ang II (100 nM) for 30 min (e, n = 5) or 24 h (g, n = 7). Lucigenin assay was used to detect NOX‐derived superoxide production in NT‐ or PKCβ‐KO–VSMCs treated with Ang II (100 nM) for 30 min (f, n = 6) or 24 h (h, n = 8). Values are mean ± SEM. Exact p‐values are shown on the graphs. U.D., undetectable.

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  FIGURE 3. Effect of PKCβ inhibitor treatment on ROS production in rat mesenteric VSMCs. DHE staining was used to detect ROS production in VSMCs treated with Ang II (100 nM) for 30 min (a) (n = 14) or 24 h (c) (n = 11) following pretreatment with the PKCβ inhibitors LY333531 (100 nM, 1 h) or CGP53353 (100 nM, 1 h), or treatment with these PKCβ inhibitors alone. Lucigenin assay was used to detect NOX‐derived superoxide production in VSMCs exposed to Ang II (100 nM) for 30 min (b) (n = 6) or 24 h (d) (n = 6–10) following pretreatment with the PKCβ inhibitors LY333531 (100 nM, 1 h) or CGP53353 (100 nM, 1 h), or treatment with these PKCβ inhibitors alone. Values are mean ± SEM. Exact p‐values are shown on the graphs.

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  FIGURE 4. Effect of PKCβ inhibition on expression of NOX1‐related genes in rat mesenteric VSMCs. Expression of NOX1, p22phox, and NOX activator (NOXA)1 in primary mesenteric VSMCs from NT or PKCβ‐KO rats. Quantitative PCR analysis of NOX1 expression in NT‐VSMCs (a, c) or PKCβ‐KO–VSMCs (b) treated with Ang II (100 nM) for 6, 12, or 24 h without or with tempol (c) (n = 4). Quantitative PCR analysis of p22phox expression in NT‐VSMCs (d, f) or PKCβ‐KO–VSMCs (e) treated with Ang II (100 nM) for 6, 12, or 24 h without or with tempol (f) (n = 4). Quantitative PCR analysis of NOXA1 expression in NT‐VSMCs (g, i) or PKCβ‐KO–VSMCs (h) treated with Ang II (100 nM) for 6, 12, or 24 h without or with tempol (i) (n = 4). Representative (j) and quantitative (k) Western blotting analysis of the expression of NOX1 in NT‐VSMCs or PKCβ‐KO–VSMCs treated with Ang II (100 nM) for 24 h without or with tempol (n = 4). Values are mean ± SEM. Exact p‐values are shown on the graphs.

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  FIGURE 5. Effect of PKCβ inhibition on phosphorylation of p65, Akt, and ERK in rat mesenteric VSMCs. Representative (a) and quantitative (b) Western blotting analysis of the phosphorylation of p65 (p‐p65), Akt (p‐Akt), and ERK (p‐ERK) and expression of p65, Akt, and ERK in NT‐VSMCs or PKCβ‐KO–VSMCs treated with Ang II (100 nM) for 30 or 60 min without or with tempol (n = 4). Quantitative PCR analysis of NOX1 (c) and p22phox (d) expression in NT‐VSMCs treated with Ang II (100 nM, 6 h) without or with the NF‐κB inhibitor SC75741 (10 μM) (n = 4). DHE staining to detect ROS production in NT‐VSMCs treated with Ang II (100 nM, 6 h) without or with the NF‐κB inhibitor SC75741 (10 μM) (e) (n = 4). Values are mean ± SEM. Exact p‐values are shown on the graphs.

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  FIGURE 6. In vivo effect of PKCβ inhibition on ROS production. DHE staining was used to detect ROS production and representative (a, e) and quantified (b, f) ROS production in mesenteric artery from male (a, b) and female (e, f) NT and PKCβ‐KO rats without or with Ang II infusion (n = 4–6). Quantitative PCR analysis of NOX1 (c, g) and p22phox (d, h) expression in mesenteric artery from male (c, d) and female (g, h) NT and PKCβ‐KO rats without or with Ang II infusion (n = 4–6). Schematic diagram (i). Values are mean ± SEM. Exact p‐values are shown on the graphs. Scale bar: 100 μm.