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Protein Disulfide Isomerase (PDI) Regulation of NADPH Oxidase Activity: Effects on Angiotensin II Redox Signaling in Hypertension
HNE is mediated by a specific amino acid residue of the LKB1 protein. Methods and Results: Cultured adult rat ventricular myocytes (ARVMs) treated with HNE (10 μM, 1 hr) were immunoblotted to detect changes indicative of hypertrophic signaling in the LKB1AMPK-mTOR-p70S6K protein synthesis pathway. Treatment with HNE resulted in a 40% decrease in P-LKB1 and P-AMPK, a 50% increase in P-mTOR, and a 40% increase in P-p70S6K protein levels. Furthermore, HNE treatment significantly increased the formation of HNE-LKB1 adducts (40% vs. control) as detected by immunoprecipitation of LKB1 followed by immunoblotting for HNE. LKB1 activity was assessed using an in vitro kinase assay in which HEK293T cells were transiently transfected with plasmids encoding wildtype GST-tagged LKB1 (WT-LKB1) or LKB1 with a double lysine to arginine mutation (K96-97R LKB1). HNE treatment (10 μM, 1 hr) increased the formation of HNE adducts with WT-LKB1 and this effect was attenuated with the K96-97R LKB1 mutant. Consistent with this observation, HNE decreased WT-LKB1 activity by 35% compared to untreated controls (n=9, p<0.01 student t-test). With the K96-97R mutant, the inhibitory effect of HNE on LKB1 activity was eliminated (n=4, p<0.001 ANOVA). Conclusions: In ARVMs, HNE treatment results in phosphorylation changes in the LKB1-AMPK-mTOR-p70S6K pathway consistent with increased protein synthesis. Our data suggests that the formation of HNE adducts on lysine residues 96 and/or 97 of LKB1 is a potential mechanism by which HNE inhibits LKB1 kinase activity. doi: 10.1016/j.freeradbiomed.2010.10.008
8 Protein Disulfide Isomerase (PDI) Regulation of NADPH Oxidase Activity: Effects on Angiotensin II Redox Signaling in Hypertension Livia de Lucca Camargo1, Aline Cristianne Depoli Androwiki1, Graziela Scalianti Ceravolo1, Alexandre Denadai-Souza1, Marcelo Nicolas Muscara1, Maria Helena Catelli de Carvalho1, Zuleica Bruno Fortes1, Mariano Janiszewski1, and Lucia Rossetti Lopes1 1 Department of Pharmacology, Institute of Biomedical Sciences, University of São Paulo Previous studies showed that overexpression of PDI in VSMC increases NOX 1 expression and activity. Since NOX 1 contributes to oxidative stress, blood pressure elevation and end organ damage in hypertension, we investigated the effect of PDI on Ang II redox signaling in spontaneously hypertensive rats (SHR). The expression of PDI and NOX isoforms was analysed by WB and immunohistochemistry in mesenteric arteries, aorta, heart, kidneys and lungs from SHR and Wistar rats. Ang II-7 induced (10 M) ROS production, c-Src activation and intracellular 2+ free calcium concentration ([Ca ]i) were measured by DHE derived fluorescence (5 µM), WB and fluo-3AM fluorescence, respectively, in VSMCs isolated from mesenteric arteries of SHR and Wistar rats. To evaluate the biological effect of PDI inhibition, vascular reactivity to Ang II was assessed in isolated mesenteric arteriolar bed from SHR and Wistar. Experiments were performed in the presence of the PDI inhibitor, bacitracin (0.5 mM), PDI inhibitory antibody (anti-PDI, 1:1000), apocynin (30µM), or the c-5 Src inhibitor, PP2 (10 M). PDI and NOX1 expression was increased in the heart (plus 38% increase; P<0.05, n=6), lungs (plus 58% increase; P<0.01, n=6) and mesenteric arteries (plus 50% increase; P<0.05, n=7) from SHR as compared to Wistar rats. Concomitantly ROS generation was increased in these tissues. VSMCs from SHR presented a higher expression of PDI (plus 50% increase, P< 0.05, n=3) as compared to Wistar rats. PDI inhibition significantly reduced Ang II dependent ROS generation and c-Src phosphorylation in Wistar (45%, P<0.01, n=4) and SHR cells (72%, P<0.001, n=3). Furthermore, PDI
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inhibition resulted in a reduction of Ang II- induced [Ca ]i responses in Wistar cells treated with bacitracin (33%, P<0.001) or anti-PDI antibody (31%, P<0.001). This effect was substantially higher in SHR cells, where bacitracin and anti-PDI reduced the 2+ [Ca ]i responses in 62% and 58% (P<0.001). PP2, reduced the 2+ Ang II elicited [Ca ]i responses by 20% in Wistar cells (P<0.01) and by 57% in SHR VSMCs (P<0.001). As a result bacitracin suppressed vasoconstrictor responses to Ang II by approximately 44 % in Wistar (P<0.01, n=6) and 62% in SHR (P<0.001, n=5). This effect could be related to a decrease in ROS generation, since apocynin reduced Ang II induced contraction by 30% in Wistar (P<0.05; n=5) and by 41% in SHR (P<0.00, n=6). Altogether, our results indicate that PDI could be a new player in oxidative stress and vascular dysfunction in hypertension. doi: 10.1016/j.freeradbiomed.2010.10.009
9 Dietary Inorganic Nitrate Attenuates Oxidative Stress and Hypertension, and Prevents Cardio renal injury in a Model of Renal and Cardio vascular Disease Mattias Carlstrom1,2, Erik G Persson2, Erik Larsson2, Michael Hezel1, Peter Scheffer3, Tom Teerlink3, Eddie Weitzberg1, and Jon O Lundberg1 1 2 3 Karolinska Institutet, Uppsala University, VU University Medical Centre Background: Reduced bioavailability of endogenous nitric oxide (NO) is a central pathophysiological event in hypertension and other cardiovascular diseases. Recently it has been demonstrated that inorganic nitrate from dietary sources is converted in vivo to form nitrite, NO and other bioactive nitrogen oxides. Aim: We tested the hypothesis that dietary inorganic nitrate supplementation may have therapeutic effects in a model of renal and cardiovascular disease. Methods and Results: Sprague Dawley rats subjected to unilateral nephrectomy and chronic high-salt diet from 3-weeks of age developed hypertension, cardiac hypertrophy and fibrosis, proteinuria and histological as well as biochemical signs of renal damage and oxidative stress. Simultaneous nitrate treatment (0.1 -1 -1 mmol or 1 mmol nitrate kg day ) for 8-10 weeks, with the lower dose resembling the nitrate content of a diet rich in vegetables, attenuated hypertension dose-dependently with no signs of tolerance. Nitrate treatment almost completely prevented proteinuria and histological signs of renal injury, and the cardiac hypertrophy and fibrosis were attenuated. Mechanistically, dietary nitrate restored the tissue levels of bioactive nitrogen oxides and reduced the levels of oxidative stress markers in plasma (MDA) and urine (iPF2α-VI and 8-OHdG). In addition, the increased circulating and urinary levels of dimethylarginines (ADMA and SDMA) in the hypertensive rats were normalized by nitrate supplementation. Conclusions: Dietary inorganic nitrate has therapeutical effects in this model of renal and cardiovascular disease. Future studies will elucidate if inorganic nitrate contributes to the cardioprotective effects associated with diets rich in vegetables. doi:10.1016/j.freeradbiomed.2010.10.010
SFRBM/SFRRI 2010
8 Protein Disulfide Isomerase (PDI) Regulation of NADPH Oxidase Activity: Effects on Angiotensin II Redox Signaling in Hypertension Livia de Lucca Camargo1, Aline Cristianne Depoli Androwiki1, Graziela Scalianti Ceravolo1, Alexandre Denadai-Souza1, Marcelo Nicolas Muscara1, Maria Helena Catelli de Carvalho1, Zuleica Bruno Fortes1, Mariano Janiszewski1, and Lucia Rossetti Lopes1 1 Department of Pharmacology, Institute of Biomedical Sciences, University of São Paulo Previous studies showed that overexpression of PDI in VSMC increases NOX 1 expression and activity. Since NOX 1 contributes to oxidative stress, blood pressure elevation and end organ damage in hypertension, we investigated the effect of PDI on Ang II redox signaling in spontaneously hypertensive rats (SHR). The expression of PDI and NOX isoforms was analysed by WB and immunohistochemistry in mesenteric arteries, aorta, heart, kidneys and lungs from SHR and Wistar rats. Ang II-7 induced (10 M) ROS production, c-Src activation and intracellular 2+ free calcium concentration ([Ca ]i) were measured by DHE derived fluorescence (5 µM), WB and fluo-3AM fluorescence, respectively, in VSMCs isolated from mesenteric arteries of SHR and Wistar rats. To evaluate the biological effect of PDI inhibition, vascular reactivity to Ang II was assessed in isolated mesenteric arteriolar bed from SHR and Wistar. Experiments were performed in the presence of the PDI inhibitor, bacitracin (0.5 mM), PDI inhibitory antibody (anti-PDI, 1:1000), apocynin (30µM), or the c-5 Src inhibitor, PP2 (10 M). PDI and NOX1 expression was increased in the heart (plus 38% increase; P<0.05, n=6), lungs (plus 58% increase; P<0.01, n=6) and mesenteric arteries (plus 50% increase; P<0.05, n=7) from SHR as compared to Wistar rats. Concomitantly ROS generation was increased in these tissues. VSMCs from SHR presented a higher expression of PDI (plus 50% increase, P< 0.05, n=3) as compared to Wistar rats. PDI inhibition significantly reduced Ang II dependent ROS generation and c-Src phosphorylation in Wistar (45%, P<0.01, n=4) and SHR cells (72%, P<0.001, n=3). Furthermore, PDI
S16
2+
inhibition resulted in a reduction of Ang II- induced [Ca ]i responses in Wistar cells treated with bacitracin (33%, P<0.001) or anti-PDI antibody (31%, P<0.001). This effect was substantially higher in SHR cells, where bacitracin and anti-PDI reduced the 2+ [Ca ]i responses in 62% and 58% (P<0.001). PP2, reduced the 2+ Ang II elicited [Ca ]i responses by 20% in Wistar cells (P<0.01) and by 57% in SHR VSMCs (P<0.001). As a result bacitracin suppressed vasoconstrictor responses to Ang II by approximately 44 % in Wistar (P<0.01, n=6) and 62% in SHR (P<0.001, n=5). This effect could be related to a decrease in ROS generation, since apocynin reduced Ang II induced contraction by 30% in Wistar (P<0.05; n=5) and by 41% in SHR (P<0.00, n=6). Altogether, our results indicate that PDI could be a new player in oxidative stress and vascular dysfunction in hypertension. doi: 10.1016/j.freeradbiomed.2010.10.009
9 Dietary Inorganic Nitrate Attenuates Oxidative Stress and Hypertension, and Prevents Cardio renal injury in a Model of Renal and Cardio vascular Disease Mattias Carlstrom1,2, Erik G Persson2, Erik Larsson2, Michael Hezel1, Peter Scheffer3, Tom Teerlink3, Eddie Weitzberg1, and Jon O Lundberg1 1 2 3 Karolinska Institutet, Uppsala University, VU University Medical Centre Background: Reduced bioavailability of endogenous nitric oxide (NO) is a central pathophysiological event in hypertension and other cardiovascular diseases. Recently it has been demonstrated that inorganic nitrate from dietary sources is converted in vivo to form nitrite, NO and other bioactive nitrogen oxides. Aim: We tested the hypothesis that dietary inorganic nitrate supplementation may have therapeutic effects in a model of renal and cardiovascular disease. Methods and Results: Sprague Dawley rats subjected to unilateral nephrectomy and chronic high-salt diet from 3-weeks of age developed hypertension, cardiac hypertrophy and fibrosis, proteinuria and histological as well as biochemical signs of renal damage and oxidative stress. Simultaneous nitrate treatment (0.1 -1 -1 mmol or 1 mmol nitrate kg day ) for 8-10 weeks, with the lower dose resembling the nitrate content of a diet rich in vegetables, attenuated hypertension dose-dependently with no signs of tolerance. Nitrate treatment almost completely prevented proteinuria and histological signs of renal injury, and the cardiac hypertrophy and fibrosis were attenuated. Mechanistically, dietary nitrate restored the tissue levels of bioactive nitrogen oxides and reduced the levels of oxidative stress markers in plasma (MDA) and urine (iPF2α-VI and 8-OHdG). In addition, the increased circulating and urinary levels of dimethylarginines (ADMA and SDMA) in the hypertensive rats were normalized by nitrate supplementation. Conclusions: Dietary inorganic nitrate has therapeutical effects in this model of renal and cardiovascular disease. Future studies will elucidate if inorganic nitrate contributes to the cardioprotective effects associated with diets rich in vegetables. doi:10.1016/j.freeradbiomed.2010.10.010
SFRBM/SFRRI 2010