Endothelium-Derived Nitric Oxide (NO) Activates the NO-Epidermal Growth Factor Receptor-Mediated Signaling Pathway in Bradykinin-Mediated Angiogenesis

dependent vasodilation via endothelial nitric oxide synthase (eNOS) activity. Thus, we hypothesized that eNOS dysfunction consequential to continuous GTN exposure might underlie nitrate tolerance and endothelial dysfunction secondary to GTN. Caveolin-1 (CAV-1), a membrane protein located at the cholesterol-rich lipid rafts of caveolae, is known to interact with eNOS through its scaffolding domain and tightly regulate eNOS function, warranting temporal and quantitative NO synthesis by the enzyme. Loss of caveolin-1 is known to result in eNOS hyperactivation which evolves into eNOS dysfunction and converts the enzyme into a source of oxidative stress. In this study we investigated the roles of CAV-1 and eNOS in tolerance. We report that when endothelial cells are continuously exposed to GTN for extended periods of time, eNOS becomes a source of peroxynitrite secondary to CAV-1 depletion. We demonstrate that CAV-1 is degraded in both GTN treated endothelial cells and animal tissues which parallels a marked increase in the phosphorylation (Ser 1177) and accumulation of the monomeric form of eNOS. We also demonstrate in endothelial cells that although CAV-1 mRNA level is not changed, ubiquitination on the protein is increased with GTN, and that treatment of the proteasome inhibitor MG132 partially restores CAV-1 level, which indicates that GTN-induced CAV-1 loss is based on posttranslational modifications. In addition, we also confirmed that eNOS is dysfunctional in a caveolin-1 deficient mouse model, which is naturally tolerant to pharmacologic relevant doses of GTN. Moreover, we show that the elevated production of peroxynitrite from dysregulated eNOS is antagonized by either CAV-1 transfection or L-Arginine treatment, suggesting that eNOS may be uncoupled in the absence of CAV-1. Taken together, these results imply that GTN-induced CAV-1 depletion and sequential eNOS dysfunction contributes to tolerance.

doi:10.1016/j.freeradbiomed.2012.10.503

462 Visualization of Protein S-Nitrosylation Reveals Essential Roles of Nitric Oxide in Endothelial Signaling Homeostasis

Ming-Fo Hsu1, Yi-Yun Chen1, Geen-Dong Chang2, and Tzu-Ching 1 Meng 1 2 Academia Sinica, Taiwan, National Taiwan University, Taiwan Recent studies have suggested that nitric oxide (NO) acts as a modulator of diverse signaling pathways via protein Cys Snitrosylation. To date hundreds of proteins are considered being potentially S-nitrosylated. However, the spatiotemporal control of S-nitrosylation under physiological and pathological settings is unknown. Here we present two cases to demonstrate how endothelial NO regulates signaling homeostasis. We have developed a novel method using the combination of chemical labeling and subsequent detection by house-made specific antibodies that allows microscopic visualization of S-nitrosylated proteins in cells. The application of this method indicated that cytosolic proteins in aortic endothelia were constitutively Snitrosylated. Surprisingly, in response to hypoxic stress, protein Snitrosylaion levels decreased dramatically, concomitant with the onset of endothelial barrier dysfunction. We further identified caspase-3 in the denitrosylated form as a key player in hypoxiainduced endothelial injury. This method was further used in visualizing S-nitrosylated proteins in microvascular endothelia response to insulin stimulation. Our results showed that only a small fraction of cellular proteins at the plasma membrane proximity were targeted by NO, which was generated rapidly by insulin-activated NO synthase. Interestingly, plasma membraneassociated protein tyrosine phosphatase 1B (PTP1B), a negative regulator of insulin receptor, was S-nitrosylated and inactivated,

permitting a full activation of insulin signaling. Employing this novel imaging platform, we have demonstrated how diverse signaling events are controlled by NO-mediated dynamic regulation of protein S-nitrosylation.

doi:10.1016/j.freeradbiomed.2012.10.504

463 Competition between Arginase and Nitric Oxide Synthase for the Intracellular Pool of the Substrate LArginine is not Rate Limiting for NO Production 1

Tony Yoshiya Momma , Javier Ottaviani1,2, Carl Keen1, and Hagen Schroeter1,2 1 2 University of California, Davis, Mars, Inc. The concept that arginase (ARG) and nitric oxide synthase (NOS) compete for an intracellular pool of the common substrate Larginine is well accepted, and ARG inhibition has been advanced as a possible means of increasing nitric oxide (NO) production. 14 Based on high-sensitivity measurements of [1- C]-L-arginine and its cellular metabolites in NOS II- and ARG I-induced RAW246.7γNO(í) cells, we developed a well-defined and highly controlled model to re-examine the putative competition between NOS and ARG for the intracellular L-arginine pool. In NOS- and ARG-induced RAW cells, increasing extracellular L-arginine concentration (up to 200μM) led to a linear increase in intracellular L-arginine levels and increases in NOS and ARG activities. At a steady-state concentration of 100μM extracellular L-arginine, ARG inhibition by nor-NOHA (100μM) significantly increased intracellular L-arginine level (44% increase, p<0.001). However, such ARG inhibition-dependent increases in intracellular L-arginine did not result in an increase in NO production (p=0.99). To substantiate the above finding, we tested a wider range of extracellular L-arginine concentrations (01000μM) and measured nitrite accumulation in the cell media as an alternative readout for NO production. The inhibition of ARG did not alter nitrite accumulation in the media of RAW cells at all L-arginine concentrations tested. Similarly, in human umbilical artery endothelial cells co-expressing NOS III and ARG II, the inhibition of ARG did not increase NO production. The above results indicate that intracellular substrate competition between ARG and NOS is not rate limiting for NO production. Consequently, the results from our current study question the precise mechanisms-of-action that underlie the beneficial effects on NO-mediated biological functions, which have been described previously in the context of L-arginine supplementation in humans as well as ARG inhibition in animal models.

doi:10.1016/j.freeradbiomed.2012.10.505

464 Endothelium-Derived Nitric Oxide (NO) Activates the NO-Epidermal Growth Factor Receptor-Mediated Signaling Pathway in Bradykinin-Mediated Angiogenesis

Miriam S Moraes1, Paulo Eduardo da Costa1, Murched Omar Taha1, 1 Arnold Stern2, and Hugo Pequeno Monteiro 1 2 Universidade Federal de São Paulo, Brazil, New York University School of Medicine The intracellular generation of NO following stimulation of rabbit aortic endothelial cells (RAEC) and human umbilical vein endothelial cells (HUVEC) with 100 nM Bradykinin (BK) activates

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the endothelial isoform of NO synthase (eNOS) and causes tyrosine phosphorylation of the EGF receptor (EGF-R). The NO-EGF-R signaling pathway was studied in endothelial cells stimulated with BK. The signaling pathway was investigated using ® immunoprecipitation, western blot analysis, and a Matrigel -based in vitro assay for angiogenesis. Increase in NO production correlated with enhanced phosphorylation of tyrosine residues of the EGF-R as seen by immunoprecipitation and western blot analysis. The activation of the ERK1/2 MAP kinases downstream to EGF-R by BK was dependent on fully functional EGF-R. ERK1/2 MAP kinases in turn trans-activated the EGF-R by phosphorylating specific Ser/Thr residues. BK-induced proliferation in endothelial cells was partially inhibited by the NOS inhibitor (L-NAME) and by the MEK inhibitor (PD98059). A ® Matrigel -based in vitro assay for angiogenesis showed that BK induced the formation of capillary-like structures in HUVEC, but not in those cells expressing a mutant of the EGF-R lacking tyrosine kinase activity. Pre-treatment of BK-stimulated HUVEC with SU5416, a specific VEGF inhibitor resulted in inhibition of in vitro angiogenesis. Specific inhibition of the EGF-R prevented the pro-angiogenic effects of VEGF in BK-stimulated HUVEC. In addition, pre-treatment of HUVEC with L-NAME and PD98059 also resulted in inhibition of in vitro angiogenesis. These findings suggest that BK-mediated angiogenesis in endothelial cells involves the activation of the NO-EGFR signaling pathway. Financial Support: Provided by the Brazilian Institutions FAPESP and CNPq.

doi:10.1016/j.freeradbiomed.2012.10.506

465 NO Generation: Impact in Tumor Viability Under Nutrients Restriction 1

Graciele Almeida de Oliveira , Paulo Eduardo da Costa1, Tatiana Alvarez Rinaldi1, and Hugo Pequeno Monteiro1 1 Universidade Federal de São Paulo, Brazil Nitric oxide (NO) is a free radical permeable to cell membranes that functions as a messenger in intercellular communication and intracellular signaling. In mammalian cells, the inducible (iNOS) and constitutive (eNOS, nNOS) nitric oxide synthases are responsible for NO biosynthesis. Caloric restriction has been widely employed as a strategy to increase cell viability and 1 longevity . Decrease on nutrient levels without promoting mal2 nutrition was showed to increase eNOS expression . Thus, the aim of this study is to investigate how a decrease on nutrient levels in a metastatic tumor colon cell line SW620 regulates NO production and how this event controls cell proliferation and viability. We confirmed previous observations that SW620 cells were positive for iNOS expression and negative for eNOS and nNOS expression. SW620 cells cultured under nutrients restriction characterized by the absence or the presence of low concentrations (1%) of fetal bovine serum (FBS), exhibit increasing levels of intracellular NO. In addition, these cells showed high viability although lower proliferation rates when compared to cells growing in 10% FBS. To examine the participation of iNOS in this survival response displayed by SW620 cells submitted to nutrients restriction, we silenced the expression of iNOS through permanent transfection of cells with shRNA. Contrarily to our observations in wild type cells, iNOS silenced cells showed no differences on the intracellular levels of NO and on cell viability when cultured in the absence or presence of FBS (10%). Altogether, our results suggest that NO and iNOS may play an important role in the viability of SW620 colon tumor cells submitted to nutrients restriction. 1 Mech Ageing Dev. 131:494-502; 2010.

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Science. 310:314-317; 2005. Financial Support: This work was supported by the Brazilian institutions: CNPq, FAPESP, and CAPES.

doi:10.1016/j.freeradbiomed.2012.10.507

466 Potential Role for 8-Nitro-CGMP and Protein SGuanylation in Nitrite-Dependent Signaling 1

Benjamin Y. Owusu , Ryan Stapley1, Takaaki Akaike2, and Rakesh Patel1 1 2 University of Alabama at Birmingham, Kumamoto University, Japan Recent studies have shown that 8-nitro-cGMP can act as a novel second messenger in nitric oxide (NO)-dependent signaling paradigms. Unlike cGMP, 8-nitro-cGMP is resistant to PDE5dependent degradation and has the potential to elicit an NOinduced signal transduction via a new chemical modification of proteins involving ‘S-guanylation’ of critical cysteine residues. An increasing body of literature has underscored the potential for nitrite to elicit both acute and chronic NO-dependent effects especially at lower oxygen tensions; however the mechanisms remain unclear. In this study we tested if nitrite could promote NO-signaling via nitration of cGMP and subsequent Sguanylation. A one-time intraperitoneal injection of nitrite into rats led to a time-dependent increase in protein S-guanylation (detected by immunostaining) in the heart that increased within 30min, reaching maximal levels at 12h. Using isolated rat thoracic aorta as a model to assess NO-dependent signaling, nitrite stimulated vasodilation at 21% O2 (oxygenated) in a dosedependent manner. This response was significantly improved at 1% O2 (deoxygenated) conditions. Interestingly, pre-treatment of vascular strips with the PDE5 inhibitor, Zaprinast (5μM) improved nitrite-dependent vasodilation by ∼2.5 fold. However, Zaprinast had no effect on nitrite-dependent vasodilation at 1% O2, indicating a hypoxia-dependent shift from a PDE5 sensitive to insensitive mechanism for nitrite-dependent vasodilation. Immunostaining for 8-nitro-cGMP in vessels exposed to nitrite showed the highest intensity of staining at low oxygen tension (1%). Collectively, these data provide evidence for a novel role for 8-nitro-cGMP and S-guanylation in nitrite-signaling paradigms.

doi:10.1016/j.freeradbiomed.2012.10.508

467 Effect of Blood Nitrite and Nitrate Levels on Platelet Function in Mice 1

Ji-Won Park , Barbora Piknova1, and Alan N. Schechter1 1 Molecular Medicine Branch, NIDDK/NIH It has been known that nitric oxide (NO) plays an important role in the regulation of thrombosis and hemostasis by inhibiting platelet function. The new mechanism for NO generation by which nitrate (NO3Ǧ) and nitrite (NO2Ǧ) can be reduced to NO via non-enzymatic as well as enzymatic pathways has obtained increasing attention with respect to the potential beneficial effect in cardiovascular diseases. We have previously shown that nitrite anions at 0.1ȝM inhibit aggregation and activation of human platelet preparations in the presence of red blood cells and this effect was enhanced by deoxygenation. In the present study, we hypothesized that the major NO metabolites, nitrite and nitrate derived from the diet, could also alter platelet function upon their conversion to NO. To manipulate the levels of nitrite and nitrate in mouse blood, we

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