Uncovering Novel Protein Partners of Inducible Nitric Oxide Synthase in Human Testis.

Peroxidative damage to human spermatozoa has been shown to be the primary cause of male infertility. The possible role of nitric oxide (NO) in affecting sperm motility, capacitation, and acrosome reaction has been reported, too. The overproduction of NO by the enzyme inducible nitric oxide synthase (iNOS) could be responsible as it has been implicated in the pathogenesis of many diseases. There have been many studies on regulating iNOS function in various tissues, especially by protein-protein interaction; however, no study has looked for iNOS-interacting proteins in the human testis. Here, we have reported the identification of two proteins that interact with iNOS. We initially undertook a popular yeast two-hybrid assay to screen a human testis cDNA library in yeast using an iNOS-peptide fragment (amino acids 181-335) as bait. We verified our data using the mammalian chemiluminescent co-IP method; first, employing the same peptide and, then, a full-length protein co-expressed in HEK293 cells in addition to the candidate protein. In both cases, these two protein partners of iNOS were revealed: (a) sperm acrosome-associated 7 protein and (b) retinoblastoma tumor-suppressor binding protein.

H2S Prodrug, SG-1002, Protects against Myocardial Oxidative Damage and Hypertrophy In Vitro via Induction of Cystathionine ß-Synthase and Antioxidant Proteins.

Endogenously produced hydrogen sulfide (H2S) is critical for cardiovascular homeostasis. Therapeutic strategies aimed at increasing H2S levels have proven cardioprotective in models of acute myocardial infarction (MI) and heart failure (HF). The present study was undertaken to investigate the effects of a novel H2S prodrug, SG-1002, on stress induced hypertrophic signaling in murine HL-1 cardiac muscle cells. Treatment of HL-1 cells with SG-1002 under serum starvation without or with H2O2 increased the levels of H2S, H2S producing enzyme, and cystathionine ß-synthase (CBS), as well as antioxidant protein levels, such as super oxide dismutase1 (SOD1) and catalase, and additionally decreased oxidative stress. SG-1002 also decreased the expression of hypertrophic/HF protein markers such as atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), galectin-3, TIMP1, collagen type III, and TGF-ß1 in stressed HL-1 cells. Treatment with SG-1002 caused a significant induction of cell viability and a marked reduction of cellular cytotoxicity in HL-1 cells under serum starvation incubated without or with H2O2. Experimental results of this study suggest that SG-1002 attenuates myocardial cellular oxidative damage and/or hypertrophic signaling via increasing H2S levels or H2S producing enzymes, CBS, and antioxidant proteins.

Zofenopril Protects Against Myocardial Ischemia-Reperfusion Injury by Increasing Nitric Oxide and Hydrogen Sulfide Bioavailability.

BACKGROUND: Zofenopril, a sulfhydrylated angiotensin-converting enzyme inhibitor (ACEI), reduces mortality and morbidity in infarcted patients to a greater extent than do other ACEIs. Zofenopril is a unique ACEI that has been shown to increase hydrogen sulfide (H2S) bioavailability and nitric oxide (NO) levels via bradykinin-dependent signaling. Both H2S and NO exert cytoprotective and antioxidant effects. We examined zofenopril effects on H2S and NO bioavailability and cardiac damage in murine and swine models of myocardial ischemia/reperfusion (I/R) injury. METHODS AND RESULTS: Zofenopril (10 mg/kg PO) was administered for 1, 8, and 24 hours to establish optimal dosing in mice. Myocardial and plasma H2S and NO levels were measured along with the levels of H2S and NO enzymes (cystathionine ß-synthase, cystathionine γ-lyase, 3-mercaptopyruvate sulfur transferase, and endothelial nitric oxide synthase). Mice received 8 hours of zofenopril or vehicle pretreatment followed by 45 minutes of ischemia and 24 hours of reperfusion. Pigs received placebo or zofenopril (30 mg/daily orally) 7 days before 75 minutes of ischemia and 48 hours of reperfusion. Zofenopril significantly augmented both plasma and myocardial H2S and NO levels in mice and plasma H2S (sulfane sulfur) in pigs. Cystathionine ß-synthase, cystathionine γ-lyase, 3-mercaptopyruvate sulfur transferase, and total endothelial nitric oxide synthase levels were unaltered, while phospho-endothelial nitric oxide synthase(1177) was significantly increased in mice. Pretreatment with zofenopril significantly reduced myocardial infarct size and cardiac troponin I levels after I/R injury in both mice and swine. Zofenopril also significantly preserved ischemic zone endocardial blood flow at reperfusion in pigs after I/R. CONCLUSIONS: Zofenopril-mediated cardioprotection during I/R is associated with an increase in H2S and NO signaling.

Nebivolol Acts as a S-Nitrosoglutathione Reductase Inhibitor: A New Mechanism of Action.

BACKGROUND AND PURPOSE: Published data on nebivolol reveal selective ß1 adrenergic selectively along with novel nitric oxide (NO)-dependent vasodilatory properties. However, the exact molecular mechanism is unknown. Protein S-nitrosylation constitutes a large part of the ubiquitous influence of NO on cellular signal transduction and is involved in a number of human diseases. More recently, protein denitrosylation has been shown to play a major role in controlling cellular S-nitrosylation (SNO). Several enzymes have been reported to catalyze the reduction of SNOs and are viewed as candidate denitrosylases. One of the first described is known as S-nitrosoglutathione reductase (GSNOR). Importantly, GSNOR has been shown to play a role in regulating SNO signaling downstream of the ß-adrenergic receptor and is therefore operative in cellular signal transduction. Pharmacological inhibition or genetic deletion of GSNOR leads to enhanced vasodilation and characteristic of known effects of nebivolol. Structurally, nebivolol is similar to known inhibitors of GSNOR. Therefore, we hypothesize that some of the known effects of nebivolol may occur through this mechanism. EXPERIMENTAL APPROACH: Using cell culture systems, tissue organ bath, and intact animal models, we report that nebivolol treatment leads to a dose-dependent accumulation of nitrosothiols in cells, and this is associated with an enhanced vasodilation by S-nitrosoglutathione. KEY RESULTS: These data suggest a new mechanism of action of nebivolol that may explain in part the reported NO activity. CONCLUSIONS AND IMPLICATIONS: Because exogenous mediators of protein SNO or denitrosylation can substantially affect the development or progression of disease, this may call for new utility of nebivolol.

Hydrogen Sulfide Levels and Nuclear Factor-Erythroid 2-Related Factor 2 (NRF2) Activity Are Attenuated in the Setting of Critical Limb Ischemia (CLI).

BACKGROUND: Cystathionine γ-lyase, cystathionine ß-synthase, and 3-mercaptopyruvate sulfurtransferase are endogenous enzymatic sources of hydrogen sulfide (H2S). Functions of H2S are mediated by several targets including ion channels and signaling proteins. Nuclear factor-erythroid 2-related factor 2 is responsible for the expression of antioxidant response element-regulated genes and is known to be upregulated by H2S. We examined the levels of H2S, H2S-producing enzymes, and nuclear factor-erythroid 2-related factor 2 activation status in skeletal muscle obtained from critical limb ischemia (CLI) patients. METHODS AND RESULTS: Gastrocnemius tissues were attained postamputation from human CLI and healthy control patients. We found mRNA and protein levels of cystathionine γ-lyase, cystathionine ß-synthase, and 3-mercaptopyruvate sulfurtransferase were significantly decreased in skeletal muscle of CLI patients as compared to control. H2S and sulfane sulfur levels were significantly decreased in skeletal muscle of CLI patients. We also observed significant reductions in nuclear factor-erythroid 2-related factor 2 activation as well as antioxidant proteins, such as Cu, Zn-superoxide dismutase, catalase, and glutathione peroxidase in skeletal muscle of CLI patients. Biomarkers of oxidative stress, such as malondialdehyde and protein carbonyl formation, were significantly increased in skeletal muscle of CLI patients as compared to healthy controls. CONCLUSIONS: The data demonstrate that H2S bioavailability and nuclear factor-erythroid 2-related factor 2 activation are both attenuated in CLI tissues concomitant with significantly increased oxidative stress. Reductions in the activity of H2S-producing enzymes may contribute to the pathogenesis of CLI.

Therapeutic potential of sustained-release sodium nitrite for critical limb ischemia in the setting of metabolic syndrome.

Nitrite is a storage reservoir of nitric oxide that is readily reduced to nitric oxide under pathological conditions. Previous studies have demonstrated that nitrite levels are significantly reduced in cardiovascular disease states, including peripheral vascular disease. We investigated the cytoprotective and proangiogenic actions of a novel, sustained-release formulation of nitrite (SR-nitrite) in a clinically relevant in vivo swine model of critical limb ischemia (CLI) involving central obesity and metabolic syndrome. CLI was induced in obese Ossabaw swine (n = 18) by unilateral external iliac artery deployment of a full cross-sectional vessel occlusion device positioned within an endovascular expanded polytetrafluoroethylene-lined nitinol stent-graft. At post-CLI day 14, pigs were randomized to placebo (n = 9) or SR-nitrite (80 mg, n = 9) twice daily by mouth for 21 days. SR-nitrite therapy increased nitrite, nitrate, and S-nitrosothiol in plasma and ischemic skeletal muscle. Oxidative stress was reduced in ischemic limb tissue of SR-nitrite- compared with placebo-treated pigs. Ischemic limb tissue levels of proangiogenic growth factors were increased following SR-nitrite therapy compared with placebo. Despite the increases in cytoprotective and angiogenic signals with SR-nitrite therapy, new arterial vessel formation and enhancement of blood flow to the ischemic limb were not different from placebo. Our data clearly demonstrate cytoprotective and proangiogenic signaling in ischemic tissues following SR-nitrite therapy in a very severe model of CLI. Further studies evaluating longer-duration nitrite therapy and/or additional nitrite dosing strategies are warranted to more fully evaluate the therapeutic potential of nitrite therapy in peripheral vascular disease.

Hydrogen sulfide cytoprotective signaling is endothelial nitric oxide synthase-nitric oxide dependent.

Previous studies have demonstrated that hydrogen sulfide (H2S) protects against multiple cardiovascular disease states in a similar manner as nitric oxide (NO). H2S therapy also has been shown to augment NO bioavailability and signaling. The purpose of this study was to investigate the impact of H2S deficiency on endothelial NO synthase (eNOS) function, NO production, and ischemia/reperfusion (I/R) injury. We found that mice lacking the H2S-producing enzyme cystathionine γ-lyase (CSE) exhibit elevated oxidative stress, dysfunctional eNOS, diminished NO levels, and exacerbated myocardial and hepatic I/R injury. In CSE KO mice, acute H2S therapy restored eNOS function and NO bioavailability and attenuated I/R injury. In addition, we found that H2S therapy fails to protect against I/R in eNOS phosphomutant mice (S1179A). Our results suggest that H2S-mediated cytoprotective signaling in the setting of I/R injury is dependent in large part on eNOS activation and NO generation.

Regulation of nuclear factor κB (NF-κB) in the nucleus of cardiomyocytes by G protein-coupled receptor kinase 5 (GRK5).

G protein-coupled receptor kinase 5 GRK5 plays a key role in regulating cardiac signaling and its expression is increased in heart failure. GRK5 activity in the nucleus of myocytes has been shown to be detrimental in the setting of pressure-overload hypertrophy. The ubiquitous nuclear transcription factor κB (NF-κB) is involved in the regulation of numerous genes in various tissues, and activation of NF-κB has been shown to be associated with heart disease. Herein, we investigated whether GRK5 can specifically regulate the NF-κB signaling pathway in myocytes. We found that overexpression of GRK5 increased the levels of NF-κB -p50 and p65 in vitro and in vivo, whereas loss of GRK5 resulted in lower cardiac NF-κB levels. Furthermore, increased GRK5 expression induced the phosphorylation status of p65, increased the activity of a NF-κB reporter, and increased NF-κB DNA binding activity in cultured neonatal rat ventricular myocytes. Importantly, siRNA against GRK5 presented with the opposite results in neonatal rat ventricular myocytes as p65 and p50 were decreased, and there was a loss of NF-κB DNA binding activity. The influence of GRK5 on NF-κB expression and activity was dependent on its nuclear localization as overexpression of a mutant GRK5 that cannot enter the nucleus was devoid of NF-κB activation or DNA binding. Our study demonstrates that a novel pathological consequence of GRK5 up-regulation in the injured and failing heart is the induction of NF-κB expression and activity.

Involvement of nuclear factor κB (NF-κB) signaling pathway in regulation of cardiac G protein-coupled receptor kinase 5 (GRK5) expression.

G protein-coupled receptor kinase 5 (GRK5) plays a key role in cardiac signaling regulation, and its expression is increased in heart failure. Recently, increased expression of GRK5 in the myocardium of mice has been shown to be detrimental in the setting of pressure-overload hypertrophy. The ubiquitous nuclear transcription factor κB (NF-κB) is involved in the regulation of numerous genes in various tissues, and activation of NF-κB has been shown to be associated with heart disease. Here, we investigated the role of NF-κB signaling in the regulation of the GRK5 gene and expression of this kinase in cardiomyocytes. First, in analyzing the 5'-flanking DNA of GRK5, the presence of a potential NF-κB binding site was observed in the promoter region. Phorbol myristate acetate, a known stimulator of NF-κB, increased the levels of GRK5 in myocytes whereas treatment of cells with N-acetyl cysteine, a known inhibitor of NF-κB, or with SC 514, an inhibitor of IκB kinase 2 decreased GRK5. Utilizing EMSA or ChIP assays, we found that both p50 and p65 NF-κB could interact with the promoter of GRK5 following myocytes NF-κB activation. Importantly, short interfering RNA (siRNA)-mediated loss of p65 in myocytes decreased the stimulated increased levels of GRK5 mRNA and protein. Finally, adenovirus-mediated overexpression of a dominant-negative IκBα in myocytes inhibited the levels of GRK5. Taken together, our study demonstrates that NF-κB plays a critical role in the regulation of GRK5 transcription in myocytes and that this may translate to the significant expression changes seen in heart disease.

Glucocorticoid inhibition of SP-A gene expression in lung type II cells is mediated via the TTF-1-binding element.

Induction of surfactant protein-A (SP-A) gene expression in fetal lung type II cells by cAMP and IL-1 is mediated by increased binding of thyroid transcription factor-1 (TTF-1) and NF-B proteins p50 and p65 to the TTF-1-binding element (TBE) at -183 bp. In type II cell transfections, dexamethasone (Dex) markedly inhibits cAMP-induced expression of rabbit SP-A:human growth hormone (hGH) fusion genes containing as little as 300 bp of the SP-A 5'-flanking sequence. Dex inhibition is blocked by RU-486, suggesting a role of the glucocorticoid receptor (GR). The present study was undertaken to define the mechanisms for GR inhibition of SP-A expression. Cotransfection of primary cultures of type II cells with a GR expression vector abrogated cAMP induction of SP-A promoter activity while, at the same time, causing a 60-fold induction of cotransfected mouse mammary tumor virus (MMTV) promoter. In lung cells transfected with a fusion gene containing three TBEs fused to the basal SP-A promoter, Dex prevented the stimulatory effect of IL-1 on TTF-1 induction of SP-A promoter activity, suggesting that the GR inhibits SP-A promoter activity through the TBE. In gel shift assays using nuclear extracts from human fetal type II cells cultured in the absence or presence of cAMP, Dex markedly reduced binding of nuclear proteins to the TBE and blocked the stimulatory effect of cAMP on TBE-binding activity. Our finding that Dex increased expression of the NF-kappaB inhibitory partner IkappaB-alpha suggests that the decrease in TBE-binding activity may be caused, in part, by GR inhibition of NF-kappaB interaction with this site.