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.

Metagenomic analysis of nitrate-reducing bacteria in the oral cavity: implications for nitric oxide homeostasis.

The microbiota of the human lower intestinal tract helps maintain healthy host physiology, for example through nutrient acquisition and bile acid recycling, but specific positive contributions of the oral microbiota to host health are not well established. Nitric oxide (NO) homeostasis is crucial to mammalian physiology. The recently described entero-salivary nitrate-nitrite-nitric oxide pathway has been shown to provide bioactive NO from dietary nitrate sources. Interestingly, this pathway is dependent upon oral nitrate-reducing bacteria, since humans lack this enzyme activity. This pathway appears to represent a newly recognized symbiosis between oral nitrate-reducing bacteria and their human hosts in which the bacteria provide nitrite and nitric oxide from nitrate reduction. Here we measure the nitrate-reducing capacity of tongue-scraping samples from six healthy human volunteers, and analyze metagenomes of the bacterial communities to identify bacteria contributing to nitrate reduction. We identified 14 candidate species, seven of which were not previously believed to contribute to nitrate reduction. We cultivated isolates of four candidate species in single- and mixed-species biofilms, revealing that they have substantial nitrate- and nitrite-reduction capabilities. Colonization by specific oral bacteria may thus contribute to host NO homeostasis by providing nitrite and nitric oxide. Conversely, the lack of specific nitrate-reducing communities may disrupt the nitrate-nitrite-nitric oxide pathway and lead to a state of NO insufficiency. These findings may also provide mechanistic evidence for the oral systemic link. Our results provide a possible new therapeutic target and paradigm for NO restoration in humans by specific oral bacteria.

Dietary nitrite improves insulin signaling through GLUT4 translocation.

Diabetes mellitus type 2 is a syndrome of disordered metabolism with inappropriate hyperglycemia owing to a reduction in the biological effectiveness of insulin. Type 2 diabetes is associated with an impaired nitric oxide (NO) pathway that probably serves as the key link between metabolic disorders and cardiovascular disease. Insulin-mediated translocation of GLUT4 involves the PI3K/Akt kinase signal cascade that results in activation of endothelial NO synthase (eNOS). eNOS is dysfunctional during diabetes. We hypothesize that loss of eNOS-derived NO terminates the signaling cascade and therefore cannot activate GLUT4 translocation and that dietary nitrite may repair this pathway. In this study, we administered 50mg/L sodium nitrite to db/db diabetic mice for 4 weeks. After 4 weeks treatment, the db/db mice experienced less weight gain, improved fasting glucose levels, and reduced insulin levels. Cell culture experiments using CHO-HIRc-myc-GLUT4eGFP cell lines stably expressing insulin receptor and myc-GLUT4eGFP protein, as well as L6 skeletal muscle cells stably expressing rat GLUT4 with a Myc epitope (L6-GLUT4myc), showed that NO, nitrite, and GSNO stimulate GLUT4 translocation independent of insulin, which is inhibited by NEM. Collectively our data suggest that nitrite improves insulin signaling through restoration of NO-dependent nitrosation of GLUT4 signaling translocation. These data suggest that NO-mediated nitrosation of GLUT4 by nitrite or other nitrosating agents is necessary and sufficient for GLUT4 translocation in target tissue. Description of this pathway may justify a high-nitrate/nitrite diet along with the glycemic index to provide a safe and nutritional regimen for the management and treatment of diabetes.

Acute effects of hemodialysis on nitrite and nitrate: potential cardiovascular implications in dialysis patients.

Cardiovascular mortality in dialysis patients remains a serious problem. It is 10 to 20 times higher than in the general population. No molecular mechanism has been proven to explain this increased mortality, although nitric oxide (NO) has been implicated. The objective of our study was to determine the extent of the removal of the NO congeners nitrite and nitrate from plasma and saliva by hemodialysis, as this might disrupt physiological NO bioactivity and help explain the health disparity in dialysis patients. Blood and saliva were collected at baseline from patients on dialysis and blood was collected as it exited the dialysis unit. Blood and saliva were again collected after 4-5h of dialysis. In the 27 patients on dialysis, baseline plasma nitrite and nitrate by HPLC were 0.21±0.03 and 67.25±14.68 µM, respectively. Blood immediately upon exit from the dialysis unit had 57% less nitrite (0.09±0.03 µM; P=0.0008) and 84% less nitrate (11.04 µM; P=0.0003). After 4-5h of dialysis, new steady-state plasma levels of nitrite and nitrate were significantly lower than baseline, 0.09±0.01 µM (P=0.0002) and 16.72±2.27 µM (P=0.001), respectively. Dialysis also resulted in a significant reduction in salivary nitrite (232.58±75.65 to 25.77±10.88 µM; P=0.01) and nitrate (500.36±154.89 to 95.08±24.64 µM; P=0.01). Chronic and persistent depletion of plasma and salivary nitrite and nitrate probably reduces NO bioavailability and may explain in part the increased cardiovascular mortality in the dialysis patient.

Analytical techniques for assaying nitric oxide bioactivity.

Nitric oxide (NO) is a diatomic free radical that is extremely short lived in biological systems (less than 1 second in circulating blood). NO may be considered one of the most important signaling molecules produced in our body, regulating essential functions including but not limited to regulation of blood pressure, immune response and neural communication. Therefore its accurate detection and quantification in biological matrices is critical to understanding the role of NO in health and disease. With such a short physiological half life of NO, alternative strategies for the detection of reaction products of NO biochemistry have been developed. The quantification of relevant NO metabolites in multiple biological compartments provides valuable information with regards to in vivo NO production, bioavailability and metabolism. Simply sampling a single compartment such as blood or plasma may not always provide an accurate assessment of whole body NO status, particularly in tissues. The ability to compare blood with select tissues in experimental animals will help bridge the gap between basic science and clinical medicine as far as diagnostic and prognostic utility of NO biomarkers in health and disease. Therefore, extrapolation of plasma or blood NO status to specific tissues of interest is no longer a valid approach. As a result, methods continue to be developed and validated which allow the detection and quantification of NO and NO-related products/metabolites in multiple compartments of experimental animals in vivo. The established paradigm of NO biochemistry from production by NO synthases to activation of soluble guanylyl cyclase (sGC) to eventual oxidation to nitrite (NO(2)(-)) and nitrate (NO(3)(-)) may only represent part of NO's effects in vivo. The interaction of NO and NO-derived metabolites with protein thiols, secondary amines, and metals to form S-nitrosothiols (RSNOs), N-nitrosamines (RNNOs), and nitrosyl-heme respectively represent cGMP-independent effects of NO and are likely just as important physiologically as activation of sGC by NO. A true understanding of NO in physiology is derived from in vivo experiments sampling multiple compartments simultaneously. Nitric oxide (NO) methodology is a complex and often confusing science and the focus of many debates and discussion concerning NO biochemistry. The elucidation of new mechanisms and signaling pathways involving NO hinges on our ability to specifically, selectively and sensitively detect and quantify NO and all relevant NO products and metabolites in complex biological matrices. Here, we present a method for the rapid and sensitive analysis of nitrite and nitrate by HPLC as well as detection of free NO in biological samples using in vitro ozone based chemiluminescence with chemical derivitazation to determine molecular source of NO as well as ex vivo with organ bath myography.

Natural product nitric oxide chemistry: new activity of old medicines.

The use of complementary and alternative medicine (CAM) as a therapy and preventative care measure for cardiovascular diseases (CVD) may prove to be beneficial when used in conjunction with or in place of conventional medicine. However, the lack of understanding of a mechanism of action of many CAMs limits their use and acceptance in western medicine. We have recently recognized and characterized specific nitric oxide (NO) activity of select alternative and herbal medicines that may account for many of their reported health benefits. The ability of certain CAM to restore NO homeostasis both through enhancing endothelial production of NO and by providing a system for reducing nitrate and nitrite to NO as a compensatory pathway for repleting NO bioavailability may prove to be a safe and cost-effective strategy for combating CVD. We will review the current state of science behind NO activity of herbal medicines and their effects on CVD.

Concentration- and stage-specific effects of nitrite on colon cancer cell lines.

Colorectal cancer (CRC) is the second leading cause of cancer-related death in the United States. Nitrite in cured meats is thought to contribute to increased incidence of colon cancer. We sought to determine the effect of nitrite on human colon cancer cell lines at different stages. Our results indicate nitrite has no effect on proliferation of stage 1 SW116 colon cancer cells, while nitrite inhibits proliferation of stage 2 SW480 at 10 nM-100 µM and inhibits stage 3 HCT15 proliferation at 100 nM-1 µM, but promotes a significant increase in proliferation on stage 4 COLO205 cells at 100 µM. Furthermore, nitrite inhibited invasion into Matrigel® of stage 3 SW480 colon cancer cells in a concentration-dependent manner. However, it significantly promotes the invasion of stage 4 cells at 100 µM. Our FACS data demonstrated that nitrite decreased cell cycle progression in SW480 and HCT15 with arrested G2/M transition and delayed G1 phase entry in a concentration-dependent manner. However, 100 µM nitrite promoted cell cycle progression in COLO205 cells with increased S-phase entry. Taken together, our data indicate nitrite inhibits cancer cell progression at low concentrations and early stage but promotes cancer cell progression at higher concentrations in cells representing stage 4 colon carcinomas.

Nitric oxide and geriatrics: Implications in diagnostics and treatment of the elderly.

The nation's aging population is growing rapidly. By 2030, the number of adults age 65 and older will nearly double to 70 million. Americans are living longer and older adults can now live for many years with multiple chronic illnesses but with a substantial cost to health care. Twenty percent of the Medicare population has at least five chronic conditions i.e., hypertension, diabetes, arthritis, etc. Studies in experimental models and even humans reveal that constitutive production of nitric oxide (NO) is reduced with aging and this circumstance may be relevant to a number of diseases that plague the aging population. NO is a multifunctional signaling molecule, intricately involved with maintaining a host of physiological processes including, but not limited to, host defense, neuronal communication and the regulation of vascular tone. NO is one of the most important signaling molecules in our body, and loss of NO function is one of the earliest indicators or markers of disease. Clinical studies provide evidence that insufficient NO production is associated with all major cardiovascular risk factors, such as hyperlipidemia, diabetes, hypertension, smoking and severity of atherosclerosis, and also has a profound predictive value for disease progression including cardiovascular and Alzheimers disease. Thirty plus years after its discovery and over 13 years since a Nobel Prize was awarded for its discovery, there have been no hallmark therapeutic breakthroughs or even NO based diagnostics. We will review the current state of the science surrounding NO in the etiology of a number of different diseases in the geriatric patient. From these observations, it can be concluded that enzymatic production of NO declines steadily with increasing age in healthy human subjects. Implementing strategies to diagnose and treat NO insufficiency may provide enormous benefit to the geriatric patient.