PL11 Sulfide and mitochondrial bioenergetics

Abstracts / Nitric Oxide 31 (2013) S11–S65

PL08 Homocysteine to Hydrogen Sulfide: in vitro, ex vivo and in vivo Suresh C. Tyagi Department of Physiology and Biophysics, University of Louisville, Kentucky, USA Hydrogen sulfide (H2S) is identified as a regulator of various physiological events, including the hypertension. Endogenously, H2S is produced as a metabolite of homocysteine (Hcy) by cystathionine bsynthase (CBS), cystathionine c-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST). Although Hcy is recognized as vascular risk factor at an elevated level [hyperhomocysteinemia (HHcy)] and contributes to vascular injury leading to hypertension, the exact mechanism was unclear. To determine whether conversion of Hcy to H2S mitigates hypertension. Ex vivo renal artery culture with CBS, CSE, and 3MST triple gene therapy generated more H2S in the presence of Hcy, and these arteries were more responsive to endothelial-dependent vasodilation compared with nontransfected arteries treated with high Hcy. Cross section of triple gene-delivered renal arteries immunostaining suggested increased expression of CD31 and VEGF and diminished expression of the antiangiogenic factor endostatin. In vitro endothelial cell culture demonstrated increased mitophagy during high levels of Hcy and was mitigated by triple gene delivery. Upregulated matrix metalloproteinases-13 and downregulated tissue inhibitor of metalloproteinase-1 in HHcy were normalized by overexpression of triple genes. Together, these results suggest that H2S plays a key role in hypertension during HHcy. http://dx.doi.org/10.1016/j.niox.2013.06.018

PL09 Sulfhydration of P66Shc is critical to the anti-oxidative effect of hydrogen sulfide Jin-Song Bain Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, 117597 Singapore, Singapore It has been well demonstrated that hydrogen sulfide (H2S) produces anti-oxidant effect. However, the underlying molecular mechanisms are still unclear. P66Shc protein is an upstream activator of mitochondrial redox signaling. Phosphorylation of this protein may switch on mitochondrial ROS production. We found in the present study that H2O2 induced mitochondrial reactive oxygen species (ROS) production, p66Shc protein phosphorylation and cellular senescence in SH-SY5Y neuroblastoma and HEK293 cells. These effects were largely attenuated by pretreatment with NaHS, an H2S donor. We further examined the effect of H2S on activities of protein kinase C-bII (PKCbII) and PP2A, both of which are critical to regulate p66Shc activity. Although we found NaHS treatment failed to affect the activities of PKCbII and PP2A, co-immunoprecipitation assay and immunofluoresence staining revealed that H2S treatment impaired the binding of PKCbII to p66Shc. Western blotting analysis showed that NaHS induced S-sulfhydration of p66Shc protein. These data suggest that the anti-oxidant effect of H2S may result from the disruption of the interaction between PKCbII and p66Shc due to S-sulfhydration of p66Shc. This was further confirmed with a mutation at Cysteine 59, which is located in the N-terminal CH2 domain of p66Shc. We found that C59S abolished the inhibitory effect of H2S on H2O2-induced ROS production and p66Shc phosphorylation. Over-expression of cystathionine b-synthase produced similar effects as those observed with exogenous application of NaHS. Taken

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together, this study revealed a unique mechanism for the regulatory effect of H2S on mitochondrial ROS generation and may provide a better understanding on homeostatic regulation of oxidative stress. http://dx.doi.org/10.1016/j.niox.2013.06.019

PL10 Hydrogen sulfide, glucose, and methylglyoxal formation Lingyun (Lily) Wu Department of Health Sciences, Lakehead University, Thunder Bay Regional Research Institute, Thunder Bay, Canada Hydrogen sulfide, an important gasotransmitter, has been implicated in the pathogenesis of diabetes. Increased methylglyoxal (MG) level is linked to the development of type 2 diabetes and hypertension. As a member of the reactive carbonyl species, MG is formed mainly through the nonenzymatic conversion of triosephosphates, such as dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GA3P). The triosephosphate pool, in turn, is regulated by cellular levels of glucose and fructose. The present study investigated whether MG level was altered in mice with cystathionine rlyase knockout (CSE-KO) and its underlying mechanisms. We measured plasma and renal MG levels in both CSE-KO and wild type (WT) mice at different age groups (6–22 weeks). We also evaluated the role of fructose-1,6-bisphosphatase (FBPase) and related signaling pathway in the regulation of MG formation. We observed a significant decrease in plasma glucose levels along with a significant increase in plasma MG levels in all three age groups (6–8, 14–16, and 20–22 week-old) of the CSE-KO mice. Renal MG, DHAP and GA3P were increased, whereas renal FBPase activity and its mRNA levels were decreased in CSE-KO mice. Decreased FBPase activity was accompanied by lower levels of its product, fructose-6-phosphate, and higher levels of its substrate, fructose-1,6-bisphosphate, in renal extracts from CSE-KO mice. These data indicate an important role of hydrogen sulfide in the regulation of glucose metabolism and MG generation. http://dx.doi.org/10.1016/j.niox.2013.06.020

PL11 Sulfide and mitochondrial bioenergetics Frédéric Bouillaud a, Céline Ransy a, Mireille Andriamihaja b, François Blachier b a INSERM U1016, CNRS UMR8104, Université Paris Descartes, Paris, France b INRA-AgroParisTech., Paris, France Sulfide shows the same toxicity as cyanide. This toxic effect results from the inhibition of the complex IV of the mitochondrial respiratory chain (cytochrome oxidase) that reduces oxygen into water. With the isolated enzyme the toxic effect is exerted in the high nanomolar range. However, significant inhibition of cellular/mitochondrial respiration needs lM concentrations (5–20 lM). In contrast at lower concentrations (nM) sulfide is an hydrogen donor used as a substrate by mitochondria in a majority of tissues/cells. The enzyme involved is a sulfide quinone reductase (SQR) associated with a dioxygenase and a sulfur transferase. Therefore according to the concentration sulfide has two opposite effects on respiration: it increases oxygen consumption and drives ATP production at low (nM) concentration whereas at high (lM) concentration sulfide inhibits respiration and has adverse effects on cellular bioenergetics.

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Abstracts / Nitric Oxide 31 (2013) S11–S65

The present estimation of the endogenous sulfide release rates, the occurence of SQR in a large majority of the tissues/cells explored so far and its high affinity for sulfide make the SQR and mitochondrial respiration the major pathway maintaining intracellular sulfide in an acceptable non toxic low range of concentrations. Therefore mitochondrial respiration is both the sulfide sink and the target of sulfide toxicity. This paves the way for positive feedback effects making the orientation towards one or another of the opposite consequences of a given sulfide exposure highly sensitive to various factors. SQR is thus likely to interfere with any endogenous sulfide signaling and definitely needs to be taken into account when pharmacological intervention involves sulfide donors. The lumen of the colon hosts a bacterial communauty releasing sulfide and a value of 60 lM is proposed for the concentration of free sulfide in the human colon. Accordingly, the epithelial cells (colonocytes) are exposed to toxic sulfide concentrations. These colonocytes show a high SQR activity and cellular bioenergetics adaptations to increase their tolerance to sulfide. The mechanisms involved in mitochondrial sulfide bioenergetics as well as their consequences with regard to the signaling role of sulfide and to the use of sulfide donors will be explained. http://dx.doi.org/10.1016/j.niox.2013.06.021

wound healing, while reduced H2S production (mice deficient in CSE) delayed it. H2S -mediated and angiogenesis in vivo was suppressed by pharmacological inhibition (L-NAME) or genetic ablation of eNOS (eNOS / mice). Conclusions: H2S is a pro-angiogenic endogenous hormone. Administration of H2S promotes wound healing. NO and H2S are mutually required for the control of angiogenesis and wound healing. H2S supplementation may be beneficial to facilitate post-burn wound healing. Potential modes of delivery may include topical or systemic administration. However, the angiogenic effect of H2S is not expected to be optimal in conditions where endogenous NO production is impaired (i.e. conditions associated with endothelial dysfunction syndrome). In such conditions, simultaneous supplementation of both gasotransmitters may be possible. In addition to wound healing, the pro-angiogenic effects of H2S may be utilized to improve revascularization following ischemic conditions, whereas inhibition of H2S production (and associated angiogenic responses) may be of therapeutic interest in conditions associated with pathological increases in angiogenesis (e.g. in the context of tumor angiogenesis or in diabetic retinopathy). Acknowledgements This work was supported by a Grant (#8661) of the Shriners Hospitals of North America to C.S. http://dx.doi.org/10.1016/j.niox.2013.06.022

PL12 Molecular mechanisms and therapeutic implications of the pro-angiogenic effect of hydrogen sulfide Csaba Szabo Department of Anesthesiology, University of Texas Medical Branch and Shriners Hospital for Children, Galveston, TX, USA Introduction: Angiogenesis is a key process in a variety of biological processes ranging from wound healing to tumor biology. Hydrogen sulfide (H2S) and nitric oxide (NO) are recognized as essential, interacting, endogenous gaseous signaling molecules. Here we review the existing data demonstrating the effect of exogenous and endogenous H2S on angiogenesis and overview the molecular pathways involved. We are also presenting a range of therapeutic context where these mechanisms can be exploited in the future. Methods: Murine bEnd.3 endothelial cells were used for in vitro studies; burn-induced wound healing in rats and mice was used for in vivo studies. Results: Exposure of endothelial cells to a H2S donor increased intracellular cGMP in a NO-dependent manner, activated protein kinase G (PKG) and induced the phosphorylation of its downstream substrate, VASP. Inhibition of eNOS or PKG abolished the H2S-stimulated angiogenic response. Thus, there is a convergence of the vascular actions of H2S and NO to the cGMP/PKG pathway. Silencing of the H2S-producing enzyme cystathionine-gamma-lyase (CSE) abolished NO-stimulated cGMP accumulation and angiogenesis, indicating the requirement of H2S in the vascular actions of NO. VEGF, a pro-angiogenic and vasorelaxant hormone stimulated the production of both NO and H2S: VEGF-induced angiogenic and vasorelaxant responses were attenuated either by the inhibition of NO or by the silencing of CSE. A key site of the cooperative interaction between NO and H2S involves the cGMP/PKG pathway: H2S exerted a potent inhibitory effect on phosphodiesterase 5 activity in vitro leading to cGMP elevation and PKG activation. Another site of the interaction between H2S and NO is Akt: H2S increased the phosphorylation (activation) of Akt, as well as the phosphorylation of eNOS at Ser1177. In vivo H2S administration promoted post-burn

PL13 Saying NO to H2S Jan Miljkovic a, Mirjam Eberhardt b,c, Martin Herrmann d, Karl Messlinger b, Peter Reeh b, Ivana Ivanovic-Burmazovic a, Milos R. Filipovic a a Department of Chemistry and Pharmacy, Fridrich-Alexander University, Erlangen, Germany b Institute of Physiology and Pathophysiology, Fridrich-Alexander University, Erlangen, Germany c Medical Clinic 3 – Rheumatology and Immunology, Fridrich-Alexander University, Erlangen, Germany d Hannover Medical School, Hannover, Germany Hydrogen sulfide (H2S) emerged recently as an important signaling molecule involved in regulation of blood pressure and neuronal activity. Additionally, H2S and its donors possess huge pharmacological potential to prevent ischemia–reperfusion injuries and, as it has been speculated, to induce suspended animation-like state in small animals. However, the real (bio) chemical events behind all these observed phenomena remained elusive. In this talk, physiological effects of H2S in context of its interaction with another physiological gassotransmitter, nitric oxide (NO), and its metabolites (peroxynitrite, S-nitrosothiols, nitrite) will be addressed, providing the evidence for the existence of a strong cross-talk of these molecules. Applying state-of-the art analytical techniques, as the products of these reactions, we identify thionitric (HSNO2) and thionitrous acid (HSNO) as well as nitroxyl (HNO), and show on cellular level that they too serve as important signaling molecules. Finally on the animal level we prove our chemistry and show that H2S-induced vasodilatory effects are directly NO dependent, identifying therefore completely new signaling cascade for systemic neurovascular control. http://dx.doi.org/10.1016/j.niox.2013.06.023