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Tetrahydrobiopterin and Cardiovascular Disease
Abstract
Tetrahydrobiopterin (BH4) is an essential cofactor for the aromatic amino acid hydroxylases, which are essential in the formation of neurotransmitters, and for nitric oxide synthase. It is presently used clinically to treat some forms of phenylketonuria (PKU) that can be ameliorated by BH4 supplementation. Recent evidence supports potential cardiovascular benefits from BH4 replacement for the treatment of hypertension, ischemia-reperfusion injury, and cardiac hypertrophy with chamber remodeling. Such disorders exhibit BH4 depletion because of its oxidation and/or reduced synthesis, which can result in functional uncoupling of nitric oxide synthase (NOS). Uncoupled NOS generates more oxygen free radicals and less nitric oxide, shifting the nitroso-redox balance and having adverse consequences on the cardiovascular system. While previously difficult to use as a treatment because of chemical instability and cost, newer methods to synthesize stable BH4 suggest its novel potential as a therapeutic agent. This review discusses the biochemistry, physiology, and evolving therapeutic potential of BH4 for cardiovascular disease.
... NOS is a dimer of two identical monomers tethered by tetrahedral coordination of a zinc ion at two CysXXXXCys motifs (each motif is contributed by a monomer). Through the motif, NOS binds the substrate L-arginine and cofactor tetrahydrobiopterin (BH 4 ) which facilitates dimerization, substrate binding, and enzymatic function [34,40]. The constitutive NOS-1 and -3, but not inducible NOS-2, are bound by calmodulin in response to increased calcium levels, which induces conformational changes to activate the enzymatic function [34,41]. ...
... At lower NO concentrations, the activities of NO manifest through its direct interaction with the biological target, which are likely to lead to anti-oxidant effects. At higher NO concentrations, conversely, the activities of NO are indirectly mediated by RNOS derived from the reaction of NO with [40]. (Left) In the normal coupled state, two NOS monomers are tethered by BH4, which also increases the binding affinity of the substrate L-arginine. ...
... Then, instead of producing NO, the heme of the oxygenase domain now produces superoxide (O2• − ) (Figure 1, right), which further lowers the BH4 level by oxidizing it to dihydrobiopterin (BH2) [43,44]. In fact, deficiency of BH4 is a major cause of NO deficiency in chronic disorders, such as diabetes [45], obesity [46], cardiovascular disease [40,44], as well as cancer [47]. NO deficiency could lead to tissue fibrosis and stiffening [48], which also increases the cancer risks of patients [49]. ...
Nitric oxide (NO) is a highly reactive molecule, generated through metabolism of L-arginine by NO synthase (NOS). Abnormal NO levels in mammalian cells are associated with multiple human diseases, including cancer. Recent studies have uncovered that the NO signaling is compartmentalized, owing to the localization of NOS and the nature of biochemical reactions of NO, including S-nitrosylation. S-nitrosylation is a selective covalent post-translational modification adding a nitrosyl group to the reactive thiol group of a cysteine to form S-nitrosothiol (SNO), which is a key mechanism in transferring NO-mediated signals. While S-nitrosylation occurs only at select cysteine thiols, such a spatial constraint is partially resolved by transnitrosylation, where the nitrosyl moiety is transferred between two interacting proteins to successively transfer the NO signal to a distant location. As NOS is present in various subcellular locales, a stress could trigger concerted S-nitrosylation and transnitrosylation of a large number of proteins involved in divergent signaling cascades. S-nitrosylation is an emerging paradigm of redox signaling by which cells confer protection against oxidative stress.
... Superoxide formed by the NAD(P)H oxidases appears to play a more dominant role in impaired EDD noted in arterial vessels in a variety of pathologic states. An interesting and important feature of eNOS is its ability to produce superoxide under certain conditions, such as L-arginine or tetrahydrobiopterin (BH 4 ) deficiency, dephosphorylation of threonine 495, and formation of eNOS monomers (Forstermann, 2006;Moens & Kass, 2006;Munzel, Daiber, Ullrich & Mulsch, 2005). Functional eNOS converts L-arginine and O 2 to NO and L-citrulline, an enzymatic process that consumes NADPH and requires Ca2+/calmodulin, flavin adenine dinucleotide, flavin monocleotide, and BH 4 . ...
... Applying a force to integrins has been shown to activate Nox by an Ang II-dependent mechanism (Browe & Baumgaarten, 2004, 2006. Of high significance to our central hypothesis (Figure 2) is the growing body of evidence supporting the notion that the products of the Nox's and eNOS (superoxide and NO, respectively) rapidly interact to form ONOO-, which can oxidize the essential NOS co-factor, BH 4 , to biologically inactive products (Forstermann, 2006;Moens & Kass, 2006). As a consequence, oxygen reduction uncouples from NO synthesis, thereby transforming functional, NOproducing eNOS into a dysfunctional superoxide-producing enzyme. ...
... As a consequence, oxygen reduction uncouples from NO synthesis, thereby transforming functional, NOproducing eNOS into a dysfunctional superoxide-producing enzyme. Indeed, Nox-driven eNOS uncoupling by oxidation of BH 4 has been implicated in the vascular dysfunction associated with hypertension, hypercholesterolemia, and diabetes, which can be attenuated by administration of exogenous BH 4 (Elrod, Duranski, Langston, Greer, Tao, Dugas, et al 2006;Forstermann, 2006;Moens & Kass, 2006;Munzel Daiber, Ullrich & Mulsch, 2005;Stokes, Dugas, Tang, Garg, Guidry, Bryan, 2009). Moreover, myocardial infarct size, cardiac superoxide generation, and coronary artery endothelial dysfunction are attenuated by provision of exogenous BH 4 , findings which support the concept that eNOS uncoupling contributes to I/R injury in the heart (Dumitrescu, Biondi, Xia, Cardounel, Druhan, Ambrosio et al, 2007;Forstermann, 2006;Moens & Kass, 2006). ...
Ischemia/reperfusion (I/R) induces leukocyte/endothelial cell adhesive interactions (LECA) in postcapillary venules and impaired endothelium-dependent, NO-mediated dilatory responses (EDD) in upstream arterioles. A large body of evidence has implicated reactive oxygen species, adherent leukocytes, and proteases in postischemic EDD dysfunction in conduit arteries. However, arterioles represent the major site for the regulation of vascular resistance but have received less attention with regard to the mechanisms underlying their reduced responsiveness to EDD stimuli in I/R. Even though leukocytes do not roll along, adhere to, or emigrate across arteriolar endothelium in postischemic intestine, recent work indicates that I/R-induced venular LECA is causally linked to EDD in arterioles. An emerging body of evidence suggests that I/R-induced EDD in arterioles occurs by a mechanism that is triggered by LECA in postcapillary venules and involves the formation of signals in the interstitium elicited by the proteolytic activity of emigrated leukocytes. This activity releases matricryptins from or exposes matricryptic sites in the extracellular matrix that interact with the integrin αvβ3 to induce mast cell chymase-dependent formation of angiotensin II (Ang II). Subsequent activation of NAD(P)H oxidase by Ang II leads to the formation of oxidants which inactivate NO and leads to eNOS uncoupling, resulting in arteriolar EDD dysfunction. This work establishes new links between LECA in postcapillary venules, signals generated in the interstitium by emigrated leukocytes, mast cell degranulation, and impaired EDD in upstream arterioles. These fundamentally important findings have enormous implications for our understanding of blood flow dysregulation in conditions characterized by I/R.
... This enzyme belongs to an enzymes group that catalyze the production of NO and citrulline from oxygen and L-arginine as substrates. Uncoupled NOS generates more ROS and less NO, modifying the nitroso-redox balance and causing adverse consequences in the cardiovascular system, while playing a key role in ischemia/reperfusion injury, cardiac hypertrophy, and cardiac remodeling [81]. Conversely, increased NO bioavailability may be considered as one of the universal mechanisms for cardiovascular protection against cardiac impairment. ...
... It has been suggested that nNOS-derived NO may inhibit Ca 2+ influx through L-type Ca 2+ channels and stimulate Ca 2+ re-uptake in the sarcoplasmic reticulum by promoting phospholamban (PLN) phosphorylation. The nNOSderived NO may also modulate the inotropic response to β-adrenergic stimulation and inhibit XOR activity, thereby limiting myocardial oxidative stress and, indirectly, increasing NO availability within the myocardium [81]. Finally, NO derived from iNOS isoform is considered to have detrimental effects on the myocardium. ...
Heart disease requires a surgical approach sometimes. Cardiac-surgery patients develop heart failure associated with ischemia induced during extracorporeal circulation. This complication could be decreased with anesthetic drugs. The cardioprotective effects of halogenated agents are based on pre- and postconditioning (sevoflurane, desflurane, or isoflurane) compared to intravenous hypnotics (propofol). We tried to put light on the shadows walking through the line of the halogenated anesthetic drugs’ effects in several enzymatic routes and oxidative stress, waiting for the final results of the ACDHUVV-16 clinical trial regarding the genetic modulation of this kind of drugs.
... Folate and biopterin coenzymes have structural and functional similarities, with metabolic interactions occurring between their pathways. BH4 cellular bioavailability is indeed derived from de novo synthesis starting from guanidine triphosphate (GTP) and guided by guanidine triphosphate cyclohydrolase (GTPCH), a magnesium, zinc and NADPH-dependent enzyme, or alternatively from the salvage pathway that regenerates BH4 from the BH2 oxidized form through the enzymatic activity of dihydrofolate reductase, involved in the folate metabolism [63,64]. Then, folate enhances regeneration of BH4 from the inactive form BH2, participating in the maintenance of adequate levels of BH4 for TH activity and dopamine synthesis. ...
Dolutegravir (DTG) is one of the most prescribed antiretroviral drugs for treating people with HIV infection, including women of child-bearing potential or pregnant. Nonetheless, neuropsychiatric symptoms are frequently reported. Early reports suggested that, probably in relation to folic acid (FA) shortage, DTG may induce neural tube defects in infants born to women taking the drug during pregnancy. Subsequent reports did not definitively confirm these findings. Recent studies in animal models have highlighted the association between DTG exposure in utero and congenital anomalies, and an increased risk of neurologic abnormalities in children exposed during in utero life has been reported. Underlying mechanisms for DTG-related neurologic symptoms and congenital anomalies are not fully understood. We aimed to deepen our knowledge on the neurodevelopmental effects of DTG exposure and further explore the protective role of FA by the use of zebrafish embryos. We treated embryos at 4 and up to 144 h post fertilization (hpf) with a subtherapeutic DTG concentration (1 μM) and observed the disruption of the anterior–posterior axis and several morphological malformations in the developing brain that were both prevented by pre-exposure (2 hpf) and rescued by post-exposure (10 hpf) with FA. By whole-mount in situ hybridization with riboprobes for genes that are crucial during the early phases of neurodevelopment (ntl, pax2a, ngn1, neurod1) and by in vivo visualization of the transgenic Tg(ngn1:EGFP) zebrafish line, we found that DTG induced severe neurodevelopmental defects over time in most regions of the nervous system (notochord, midbrain–hindbrain boundary, eye, forebrain, midbrain, hindbrain, spinal cord) that were mostly but not completely rescued by FA supplementation. Of note, we observed the disruption of ngn1 expression in the dopaminergic regions of the developing forebrain, spinal cord neurons and spinal motor neuron projections, with the depletion of the tyrosine hydroxylase (TH)+ dopaminergic neurons of the dorsal diencephalon and the strong reduction in larvae locomotion. Our study further supports previous evidence that DTG can interfere with FA pathways in the developing brain but also provides new insights regarding the mechanisms involved in the increased risk of DTG-associated fetal neurodevelopmental defects and adverse neurologic outcomes in in utero exposed children, suggesting the impairment of dopaminergic pathways.
... Supplementation with BH4 has been shown to have beneficial effects in improving conditions like hypertension and cardiac dysfunctions. By supporting NOS3 function, BH4 promotes the production of NO, thereby improving cardiovascular health (190). ...
Cancer survivors undergone treatment face an increased risk of developing atherosclerotic cardiovascular disease (CVD), yet the underlying mechanisms remain elusive. Recent studies have revealed that chemotherapy can drive senescent cancer cells to acquire a proliferative phenotype known as senescence-associated stemness (SAS). These SAS cells exhibit enhanced growth and resistance to cancer treatment, thereby contributing to disease progression. Endothelial cell (EC) senescence has been implicated in atherosclerosis and cancer, including among cancer survivors. Treatment modalities for cancer can induce EC senescence, leading to the development of SAS phenotype and subsequent atherosclerosis in cancer survivors. Consequently, targeting senescent ECs displaying the SAS phenotype hold promise as a therapeutic approach for managing atherosclerotic CVD in this population. This review aims to provide a mechanistic understanding of SAS induction in ECs and its contribution to atherosclerosis among cancer survivors. We delve into the mechanisms underlying EC senescence in response to disturbed flow and ionizing radiation, which play pivotal role in atherosclerosis and cancer. Key pathways, including p90RSK/TERF2IP, TGFβR1/SMAD, and BH4 signaling are explored as potential targets for cancer treatment. By comprehending the similarities and distinctions between different types of senescence and the associated pathways, we can pave the way for targeted interventions aim at enhancing the cardiovascular health of this vulnerable population. The insights gained from this review may facilitate the development of novel therapeutic strategies for managing atherosclerotic CVD in cancer survivors.
... NADPH oxidase is regarded as the most important provider of ROS in vascular walls and endothelial cells and has an indispensable role in the pathogenesis of endothelial dysfunction and vascular remodeling. There is another crucial source of eNOS, xanthine oxidase and mitochondrial uncoupling, that helps to explain the increase in ROS production in different vascular diseases (133,134). Previous evidence demonstrated that ROS regulates the arrangement of various proteins and the role of signal pathways in cells and that this redox biology is precisely and spatially regulated to influence the individual healthy conditions. Moreover, disordered physiological ROS production can cause a variety of diseases, including hypertension. ...
Hypertension is regarded as the most prominent risk factor for cardiovascular diseases, which have become a primary cause of death, and recent research has demonstrated that chronic inflammation is involved in the pathogenesis of hypertension. Both innate and adaptive immunity are now known to promote the elevation of blood pressure by triggering vascular inflammation and microvascular remodeling. For example, as an important part of innate immune system, classically activated macrophages (M1), neutrophils, and dendritic cells contribute to hypertension by secreting inflammatory cy3tokines. In particular, interferon-gamma (IFN-γ) and interleukin-17 (IL-17) produced by activated T lymphocytes contribute to hypertension by inducing oxidative stress injury and endothelial dysfunction. However, the regulatory T cells and alternatively activated macrophages (M2) may have a protective role in hypertension. Although inflammation is related to hypertension, the exact mechanisms are complex and unclear. The present review aims to reveal the roles of inflammation, immunity, and oxidative stress in the initiation and evolution of hypertension. We envisage that the review will strengthen public understanding of the pathophysiological mechanisms of hypertension and may provide new insights and potential therapeutic strategies for hypertension.
... Similarly, docked confirmation of the ligands metformin and Sirt1 revealed that the ligands made significant bonded and non-bonded interactions with the residues of the target enzyme Sirt1, resulting in higher docking scores -5.34.Sirt1, metformin, and SER682, PRO688, and hydrophobic interactions with PHE698,ASP707,PRO271,VAL285 and GLY385. In terms of Sirt1 transcripts, ADMA concentration, and p21, p53 expression in endothelial cells, metformin had a higher docking score than the most active molecule, indicating that the antagonistic effect of the most active drug could be due to mechanisms other than PRMT1 inhibition 36 . We performed HPLC measurement of intracellular NAD+ and ADMA levels to further confirm our findings on senescence. ...
Metformin is intended to function as an agonist of SIRT1, a nicotinamide adenine dinucleotide (NAD +)-dependent deacetylase that mediates a number of beneficial metabolic responses. We investigated the effect of metformin in DAHP (GTPCH1 inhibitor) treated EAhy926 endothelial cells on cellular senescence. Cellular senescence was evaluated through senescence associated parameters viz., namely Beta galactosidase assay, p21 and p53 mRNA expression, nicotinamide (NAD+ content), asymmetric dimethylarginine content (ADMA) content, protein arginine methylation (PRMT1) and Sirt 1 protein expression. We also performed an in silico investigation of the possible interactions between metformin and SIRT1 that focuses on molecular docking which revealed that metformin binds with Sirt1 and that the binding affinity of metformin with Sirt1 is prominent through docking score. Oxidative stress (OS) indices such as intracellular biopterin concentrations (tetrahydrobiopterin-BH4 and dihydrobiopterin-BH2) were also determined. Metformin treatment exhibited distinct anti senescence effect in endothelial cells by downregulating the senescence markers such as beta galactosidase activity, p21 and p53 gene expression and PRMT1 protein expression while upregulating NAD+ content and Sirt1 content compared to the respective controls. We postulate that metformin restores early onset of cellular senescence potentially through oxidative stress mediated cellular events in endothelial cells, one of a kind report.
... The de novo biosynthesis pathway takes place with three major enzymes, and the same is true in vivo and in vitro. These enzymes are respectively GCHI, PTPS, and SPR (Moens & Kass, 2006;Celik, 2018). ...
Phenylketonuria (PKU) is a disease caused by phenylalanine hydroxylase enzyme deficiency in newborn infants and is the most important cause of treatable mental retardation. One of the causes of the disease comes from the defects of the PTPS in the metabolic pathway of phenilalanine metabolisms. Treatment of the disease is not feasible, and life-time tetrahydrobiopterin loading is performed in chronic patients. Today, tetrahydrobiopterin is chemically synthesized. Biological production is a different point of view due to the long duration of chemical synthesis, costs, and exposure to chemical pollutants. For this reason, the gch-I gene from the thermophilic A. flavithermus DSM 2641T was identified by PCR method. We cloned the gchI gene that was 603 bp and its open reading frame has 200 amino acids. The gene was cloned into pET28a(+) expression vector with 6xHis tags and transform in E. coli BL21(DE3)pLys host cells to express with 1 mM IPTG induction. After purification with Ni-NTA resin, we determined that GCH-I is 24 kDa, its optimum pH is 8.0 and temperature is 65C.Under optimal conditions, GCH-I exhibited enzymatic activity with Km- and Vmax- values of 243 ± 23,25 μM and 100,93 ± 3,5 nM/min/mg protein, respectively.
... ROS induces the oxidation of BH4 to BH2, which reduces the level of BH4 in endothelial cells (Najjar et al. 2013). The subsequent decrease in the BH4-to-BH2 ratio leads to the inhibition of eNOS while uncoupling arginine as its substrate, thereby allowing it to contact environmental oxygen and increased production of superoxide ions (Najjar et al. 2013;Moens and Kass 2006). As mentioned above, O 2− in turn combines with NO to form ONOO--, which promotes the oxidative conversion of BH4 to BH2, which further reduces the activity of eNOS, forming a positive feedback loop (Chen et al. 2010). ...
The blood–brain barrier (BBB) is an important barrier that maintains homeostasis within the central nervous system. Brain microvascular endothelial cells are arranged to form vessel walls and express tight junctional complexes that limit the paracellular pathways of the BBB and therefore play a crucial role in ensuring brain function. These vessel walls tightly regulate the movement of ions, molecules, and cells between the blood and the brain, which protect the neural tissue from toxins and pathogens. Primary damage caused by BBB dysfunction can disrupt the expression of tight junctions, transport proteins and leukocyte adhesion molecules, leading to brain edema, disturbances in ion homeostasis, altered signaling and immune infiltration, which can lead to neuronal cell death. Various neurological diseases are known to cause BBB dysfunction, but the mechanism that causes this disorder is not clear. Recently, ferroptosis has been found to play an important role in BBB dysfunction. Ferroptosis is a new form of regulatory cell death, which is caused by the excessive accumulation of lipid peroxides and iron-dependent reactive oxygen species. This review summarizes the role of ferroptosis in BBB dysfunction and the latest progress of ferroptosis mechanism, and further discusses the influence of various factors of ferroptosis on the severity and prognosis of BBB dysfunction, which may provide better therapeutic targets for BBB dysfunction.
Graphical Abstract
... BH 4 can be regenerated from its oxidized form BH 2 via the salvage pathway by dihydrofolate reductase (DHFR) [37]. Cardiovascular diseases are associated with oxidative stress, oxidation of BH 4 to BH 2 , and eNOS uncoupling, implicating that the salvage pathway seems to have particular importance in their pathogenesis [115]. Indeed, DHFR has been suggested to play a critical role in regulating the BH 4 /BH 2 ratio and eNOS coupling and activity in endothelial cells [116,117]. ...
Cardiovascular diseases (CVDs) are the leading cause of death worldwide. The initial stage of CVDs is characterized by endothelial dysfunction, defined as the limited bioavailability of nitric oxide (NO). Thus, any factors that interfere with the synthesis or metabolism of NO in endothelial cells are involved in CVD pathogenesis. It is well established that hypoxia is both the triggering factor as well as the accompanying factor in cardiovascular disease, and diminished tissue oxygen levels have been reported to influence endothelial NO bioavailability. In endothelial cells, NO is produced by endothelial nitric oxide synthase (eNOS) from L-Arg, with tetrahydrobiopterin (BH4) as an essential cofactor. Here, we discuss the mechanisms by which hypoxia affects NO bioavailability, including regulation of eNOS expression and activity. What is particularly important is the fact that hypoxia contributes to the depletion of cofactor BH4 and deficiency of substrate L-Arg, and thus elicits eNOS uncoupling—a state in which the enzyme produces superoxide instead of NO. eNOS uncoupling and the resulting oxidative stress is the major driver of endothelial dysfunction and atherogenesis. Moreover, hypoxia induces impairment in mitochondrial respiration and endothelial cell activation; thus, oxidative stress and inflammation, along with the hypoxic response, contribute to the development of endothelial dysfunction.
... During lactation, NO is even secreted into the breast milk as an essential component for neonatal immunity and growth [68]. On the other hand, NO production is downmodulated in many types of chronic conditions, such as aging [69], diabetes [70], obesity [71], cardiovascular disease [72,73] and cancer [23,74]. In fact, these chronic conditions frequently succumb to the formation of tissues/organs fibrosis, which is directly linked to increased cancer risk [75][76][77]. ...
Simple Summary
Fibrosis, which is often caused by chronic diseases and environmental substances, is closely associated with cancer. Thus, the development of a robust method allowing for deep studies of the linkage between fibrosis and cancer is essential. Here, we tested whether our novel three-dimensional (3D) co-culture of breast epithelia and fibroblasts would be a suitable model for that purpose. We compared the phenotypic effects of L-NAME, an inhibitor of nitric oxide (NO) production, on 3D mono- and co-cultures. We previously reported that prolonged NO depletion with L-NAME caused fibrosis and tumorigenesis in mouse mammary glands. Such in vivo effects of L-NAME were well recapitulated in 3D co-cultures, but not in 3D mono-cultures of epithelia and fibroblasts. These results support not only the essential roles of the presence of the stroma in cancer development, but also the utility of this co-culture in studying the causal relationship between fibrosis and cancer.
Abstract
Excessive myofibroblast activation, which leads to dysregulated collagen deposition and the stiffening of the extracellular matrix (ECM), plays pivotal roles in cancer initiation and progression. Cumulative evidence attests to the cancer-causing effects of a number of fibrogenic factors found in the environment, diseases and drugs. While identifying such factors largely depends on epidemiological studies, it would be of great importance to develop a robust in vitro method to demonstrate the causal relationship between fibrosis and cancer. Here, we tested whether our recently developed organotypic three-dimensional (3D) co-culture would be suitable for that purpose. This co-culture system utilizes the discontinuous ECM to separately culture mammary epithelia and fibroblasts in the discrete matrices to model the complexity of the mammary gland. We observed that pharmaceutical deprivation of nitric oxide (NO) in 3D co-cultures induced myofibroblast differentiation of the stroma as well as the occurrence of epithelial–mesenchymal transition (EMT) of the parenchyma. Such in vitro response to NO deprivation was unique to co-cultures and closely mimicked the phenotype of NO-depleted mammary glands exhibiting stromal desmoplasia and precancerous lesions undergoing EMT. These results suggest that this novel 3D co-culture system could be utilized in the deep mechanistic studies of the linkage between fibrosis and cancer.
... 47 When oxidative stress occurs, eNOS is inactivated by oxidation of the essential cofactor tetrahydrobiopterin and is thus rendered incapable of producing NO. 48 Inducible NOS is activated by the depolarization of the endothelial cell membrane induced by the loss of shear stress of pulmonary vasculature during ischemia. 49 As shown in several models, eNOS and iNOS activity exerts an antiinflammatory effect and reduces leukocyte recruitment. ...
Case reports from as early as the 1970s have shown that intravenous injection of even a small dose of volatile anesthetics result in fatal lung injury. Direct contact between volatile anesthetics and pulmonary vasculature triggers chemical damage in the vessel walls. A wide variety of factors are involved in lung ischemia-reperfusion injury (LIRI), such as pulmonary endothelial cells, alveolar epithelial cells, alveolar macrophages, neutrophils, mast cells, platelets, proinflammatory cytokines, and surfactant. With a constellation of factors involved, the assessment of the protective effect of volatile anesthetics in LIRI is difficult. Multiple animal studies have reported that with regards to LIRI, sevoflurane demonstrates an anti-inflammatory effect in immunocompetent cells and an anti-apoptotic effect on lung tissue. Scattered studies have dismissed a protective effect of desflurane against LIRI. While a single-center randomized controlled trial (RCT) found that volatile anesthetics including desflurane demonstrated a lung-protective effect in thoracic surgery, a multicenter RCT did not demonstrate a lung-protective effect of desflurane. LIRI is common in lung transplantation. One study, although limited due to its small sample size, found that the use of volatile anesthetics in organ procurement surgery involving “death by neurologic criteria” donors did not improve lung graft survival. Future studies on the protective effect of volatile anesthetics against LIRI must examine not only the mechanism of the protective effect but also differences in the effects of different types of volatile anesthetics, their optimal dosage, and the appropriateness of their use in the event of marked alveolar capillary barrier damage.
... The functional disturbance of the enzyme results in the production of superoxide anion (O 2 − ·) rather than NO, a phenomenon named eNOS uncoupling [37,38]. The ability of eNOS to generate NO can be disabled by the deficiency of tetrahydrobiopterin (BH4), an essential enzyme co-factor, which transforms eNOS into an oxidantproducing enzyme of O 2 − · [39,40]. ROS may induce oxidative changes of BH4 to dihydrobiopterin (BH2), a BH4 competing compound ineffective as eNOS co-factor. ...
Cardiovascular mortality is a major cause of death among in type 2 diabetes (T2DM). Endothelial dysfunction (ED) is a well-known important risk factor for the development of diabetes cardiovascular complications. Therefore, the prevention of diabetic macroangiopathies by preserving endothelial function represents a major therapeutic concern for all National Health Systems. Several complex mechanisms support ED in diabetic patients, frequently cross-talking each other: uncoupling of eNOS with impaired endothelium-dependent vascular response, increased ROS production, mitochondrial dysfunction, activation of polyol pathway, generation of advanced glycation end-products (AGEs), activation of protein kinase C (PKC), endothelial inflammation, endothelial apoptosis and senescence, and dysregulation of microRNAs (miRNAs). Metformin is a milestone in T2DM treatment. To date, according to most recent EASD/ADA guidelines, it still represents the first-choice drug in these patients. Intriguingly, several extraglycemic effects of metformin have been recently observed, among which large preclinical and clinical evidence support metformin’s efficacy against ED in T2DM. Metformin seems effective thanks to its favorable action on all the aforementioned pathophysiological ED mechanisms. AMPK pharmacological activation plays a key role, with metformin inhibiting inflammation and improving ED. Therefore, aim of this review is to assess metformin’s beneficial effects on endothelial dysfunction in T2DM, which could preempt development of atherosclerosis.
... Furthermore, BH 4 levels are determined by a balance in the rate of synthesis, oxidation and recycling of BH 2 to BH 4 . Production of BH 4 is regulated by guanosine-5'-triphosphatecyclohydrolase I (GCH I) [91]. AMPK also affects this aspect of eNOS regulation by suppressing the 26S proteasome-dependent GTPCH I degradation in vitro, reversing diabetes-induced endothelial dysfunction [90]. ...
Cardiovascular diseases represent the leading cause of global deaths and life years spent with a severe disability. Endothelial dysfunction and vascular oxidative stress are early precursors of atherosclerotic processes in the vascular wall, all of which are hallmarks in the development of cardiovascular diseases and predictors of future cardiovascular events. There is growing evidence that inflammatory processes represent a major trigger for endothelial dysfunction, vascular oxidative stress and atherosclerosis and clinical data identified inflammation as a cardiovascular risk factor on its own. AMP-activated protein kinase (AMPK) is a central enzyme of cellular energy balance and metabolism that has been shown to confer cardio-protection and antioxidant defense which thereby contributes to vascular health. Interestingly, AMPK is also redox-regulated itself. We have previously shown that AMPK largely contributes to a healthy endothelium, confers potent antioxidant effects and prevents arterial hypertension. Recently, we provided deep mechanistic insights into the role of AMPK in cardiovascular protection and redox homeostasis by studies on arterial hypertension in endothelial and myelomonocytic cell-specific AMPK knockout (Cadh5CrexAMPKfl/fl and LysMCrexAMPKfl/fl) mice. Using these cell-specific knockout mice, we revealed the potent anti-inflammatory properties of AMPK representing the molecular basis of the antihypertensive effects of AMPK. Here, we discuss our own findings in the context of literature data with respect to the anti-inflammatory and antioxidant effects of AMPK in the specific setting of arterial hypertension as well as cardiovascular diseases in general.
... Uncoupled NOS generates more ROS and less NO, shifting the nitrosoredox balance and leading to adverse consequences on the cardiovascular system. Thus, reduced BH 4 and uncoupled NOS play an important role in I/R injury, cardiac hypertrophy, and remodeling [46]. Conversely, an increased NO bioavailability can be considered a universal mechanism for cardioprotection against these types of damage. ...
Reactive oxygen species (ROS) are highly reactive chemical species containing oxygen, controlled by both enzymatic and nonenzymatic antioxidant defense systems. In the heart, ROS play an important role in cell homeostasis, by modulating cell proliferation, differentiation, and excitation-contraction coupling. Oxidative stress occurs when ROS production exceeds the buffering capacity of the antioxidant defense systems, leading to cellular and molecular abnormalities, ultimately resulting in cardiac dysfunction. In this review, we will discuss the physiological sources of ROS in the heart, the mechanisms of oxidative stress-related myocardial injury, and the implications of experimental studies and clinical trials with antioxidant therapies in cardiovascular diseases.
... There is little information on regulatory mechanisms of GTPCH and DHFR gene expression or activity. BH4 and high concentrations of BH 2 inhibit GTPCH-1 and subsequently de novo synthesis of BH 4 , while insulin and mediators such as interferon gamma (IFN-γ), TNF-α, and interleukin-1 beta (IL-1β) can upregulate its activity and expression [77][78][79][80]. Expression of DHFR can be downregulated by angiotensin II [81]. ...
Atherosclerosis and its clinical complications constitute the major healthcare problems of the world population. Due to the central role of endothelium throughout the atherosclerotic disease process, endothelial dysfunction is regarded as a common mechanism for various cardiovascular (CV) disorders. It is well established that patients with rheumatic autoimmune diseases are characterized by significantly increased prevalence of cardiovascular morbidity and mortality compared with the general population. The current European guidelines on cardiovascular disease (CVD) prevention in clinical practice recommend to use a 1,5-factor multiplier for CV risk in rheumatoid arthritis as well as in other autoimmune inflammatory diseases. However, mechanisms of accelerated atherosclerosis in these diseases, especially in the absence of traditional risk factors, still remain unclear. Oxidative stress plays the major role in the endothelial dysfunction and recently is strongly attributed to endothelial NO synthase dysfunction (eNOS uncoupling). Converted to a superoxide-producing enzyme, uncoupled eNOS not only leads to reduction of the nitric oxide (NO) generation but also potentiates the preexisting oxidative stress, which contributes significantly to atherogenesis. However, to date, there are no systemic analyses on the role of eNOS uncoupling in the excess CV mortality linked with autoimmune rheumatic diseases. The current review paper addresses this issue.
... We tested whether supplementing sepiapterin, an endogenously-produced precursor of the NOS cofactor BH 4 (14), could redirect arginine metabolism from the pathway synthesizing polyamines to that of synthesizing NO in mammary tumors. BH 4 plays essential roles in the formation of the functional dimer, substrate binding and enzymatic functions of NOS [34][35][36]. ...
Immunotherapy is a first-line treatment for many tumor types. However, most breast tumors are immuno-suppressive and only modestly respond to immunotherapy. We hypothesized that correcting arginine metabolism might improve the immunogenicity of breast tumors. We tested whether supplementing sepiapterin, the precursor of tetrahydrobiopterin (BH4)—the nitric oxide synthase (NOS) cofactor—redirects arginine metabolism from the pathway synthesizing polyamines to that of synthesizing nitric oxide (NO) and make breast tumors more immunogenic. We showed that sepiapterin elevated NO but lowered polyamine levels in tumor cells, as well as in tumor-associated macrophages (TAMs). This not only suppressed tumor cell proliferation, but also induced the conversion of TAMs from the immuno-suppressive M2-type to immuno-stimulatory M1-type. Furthermore, sepiapterin abrogated the expression of a checkpoint ligand, PD-L1, in tumors in a STAT3-dependent manner. This is the first study which reveals that supplementing sepiapterin normalizes arginine metabolism, improves the immunogenicity and inhibits the growth of breast tumor cells.
... ONOO is a powerful oxidant that uncouples eNOS through oxidation of tetra hydro biopterine (BH4). Thus, a vicious circle of superoxide anion generation is created [105]. Early-life NO-ROS imbalance is capable of programming adult hypertension and kidney disease [106,107]. ...
Cardiovascular diseases are being included in the study of developmental origins of health and disease (DOHaD) and essential systemic hypertension has also been added to this field. Epigenetic modifications are one of the main mechanisms leading to early programming of disease. Different environmental factors occurring during critical windows in the early stages of life may leave epigenetic cues, which may be involved in the programming of hypertension when individuals reach adulthood. Such environmental factors include pre-term birth, low weight at birth, altered programming of different organs such as the blood vessels and the kidney, and living in disadvantageous conditions in the programming of hypertension. Mechanisms behind these factors that impact on the programming include undernutrition, oxidative stress, inflammation, emotional stress, and changes in the microbiota. These factors and their underlying causes acting at the vascular level will be discussed in this paper. We also explore the establishment of epigenetic cues that may lead to hypertension at the vascular level such as DNA methylation, histone modifications (methylation and acetylation), and the role of microRNAs in the endothelial cells and blood vessel smooth muscle which participate in hypertension. Since epigenetic changes are reversible, the knowledge of this type of markers could be useful in the field of prevention, diagnosis or epigenetic drugs as a therapeutic approach to hypertension.
... Increasing the availability of BH4 by modulating GcH-1 has been recognized as a novel strategy for protecting the heart during post-infarction remodeling, dilated myopathic remodeling and cardiac hypertrophy (23). certain studies have demonstrated that the treatment of exogenous BH4 or BH4 precursor SP may promote NO production and NOS-dependent coronary flow recovery following ischemia (6,24). ...
Anesthetic preconditioning (APC) may decrease the myocardium injury nearly 50% following ischemia/reperfusion (I/R) by enhancing recovery of cardiac function, reducing myocardial enzyme release and lowering infarct size when utilized as pretreatment or posttreatment agents. I/R increases nitric oxide (NO) production through endothelial NO synthase (NOS3) and heat shock protein 90 (HSP90). The present study aimed to observe the role of BH4 availability and the association of HSP90 with NOS3 in APC‑mediated cardioprotection against I/R injury. Isolated rat hearts were subjected to no‑flow ischemia for 30 min and reperfusion for 120 min. Sevoflurane (3.5%) was administered for 15 min followed by a 15 min washout prior to ischemia. 2,4-Diamino-6-hydroxypyrimidine (DAHP) or sepiapterin (SP) was administered for 40 min until the onset of ischemia. The results revealed that compared with pre‑ischemic basal levels, BH4 levels decreased and BH2 levels increased following I/R. BH4 levels were significantly increased and BH2 levels were significantly decreased in the APC + I/R hearts compared with the I/R group hearts. The BH4:BH2 ratio in the APC‑treated hearts was also increased compared with that in the I/R group hearts. SP increased the recovery of contractile function and the production of NO, and decreased the production of superoxide anion (O2·‑) in I/R heart, but did not elicit these effects in APC‑treated hearts. DAHP treatment inhibited the APC‑mediated recovery of contractile function, increased O2·‑ levels and decreased NO production, but had no effect in I/R hearts. The cardioprotection of APC was demonstrated to be modulated by the BH4 precursor SP, which increased BH4 levels, or DAHP, which inhibited GTP cyclohydrolase I. Both APC and SP treatments increased the combination of HSP90 and NOS3, which improved the NOS3 activity and function. The results suggested that BH4, which servesas a cofactor for NOS, mediated the resistance of APC to I/R injury by promoting the binding of HSP90 and NOS3.
... Significant downregulation of sodiumrelated NO production has been confirmed in several other investigations [25,26]. This contributes to deterioration of endothelial function and increases ROS production and vasoconstriction [27,28]. Reactive oxygen species themselves are also directly related to the development of hypercoagulability [10,11]. ...
Background:
Both hyponatremia and portal vein thrombosis (PVT) reflect the severity of liver dysfunction and are independently associated with increased morbidity in cirrhotic patients. In this study, we analyzed effects of hyponatremia on PVT development.
Methods:
Data on adult liver transplants (LTs) in the Model for End-Stage Liver Disease era through September 2016 were obtained. Receiver operating curves and multivariable logistic regression models were constructed to evaluate the association between serum sodium level and PVT. Based on the receiver operating curves, hyponatremia was defined as a sodium level below 125 mEq/L.
Results:
Of the 49,155 recipients included, 16% had hyponatremia (n = 7828) and 9% had PVT (n = 4414) at transplant. Subjects with hyponatremia had lower rates of PVT at the time of LT (4.4% vs 10.1%, P < .001), incidence of nonalcoholic steatohepatitis (10.8% vs 16.5%, P < .001), diabetes (19.7% vs 24.3%, P < .001), and need for dialysis (8.8% vs 16.0%, P < .001) as well as higher rates of chronic hepatitis C and B (37.6% vs 29.1%, P < .001 and 2.9% vs 1.7%, P < .001). Multivariable regression analysis confirmed that hyponatremia was independently associated with a decreased likelihood of PVT (odds ratio [OR], 0.44, P < .001). African American patients had a lower incidence of PVT (OR, 0.70; P < .001). Variables associated with a higher incidence of PVT were: nonalcoholic steatohepatitis (OR, 1.15; P = .005), moderate-to-severe ascites (OR, 1.10; P = .008), and Hispanic ethnicity (OR, 1.2; P < .001).
Conclusion:
Hyponatremia is associated with a lower rate of PVT independent of severity of liver disease and other thrombotic risk factors. This protective effect should be taken into consideration during the perioperative management of hyponatremia in patients undergoing LT.
... 2,[16][17][18] In fact, it has been proven experimentally and clinically that the female sex hormones have a protective role on the kidneys through a lesser stimulation of reninangiotensin-aldosterone system, an increase of the rate of oxyde nitrogen and metalloproteases. [19][20][21][22][23][24][25][26][27] In our series, the sex ratio was 1.6; a result that is consistent with most published series. ...
The increased incidence of the acute kidney injury (AKI) in the elderly is becoming a disturbing reality in our days, mainly with the aging of the general population, and the predisposition of old persons to chronic diseases, drug toxicity, and infections. The aim of this study was to investigate the epidemiological, clinical, and biological features and to assess variant etiologies and outcomes of AKI in the elderly. Data were collected from the medical records of patients older than 65 years age having AKI admitted in the Internal Medicine Department of the Military Hospital of Tunis from January 2006 to June 2014. One hundred and seventeen cases were included in the study. The median age was 74.2 years. Male:female ratio was 1.6. Hypertension and diabetes were the most frequently found comorbidities seen in 69.2% and 48.7% of patients, respectively. The percentage of patients having acute-on-chronic renal failure was 13.7%. The AKI was manifested by nausea or vomiting in 33.3% of cases. One patient had hematemesis. The other symptoms were dyspnea in 14.5% of cases, uremic encephalopathy in 6.8% of cases, and oligoanuria in 16.2% of cases. The AKI was discovered fortuitously in 31.6% of cases and was diagnosed early within the 48 h after admission in 94% of cases and after this delay, in 6% of cases. It was ranked Acute Kidney Injury Network 1, 2, or 3, respectively, in 29.9%, 24.8%, and 45.3% of cases. Organic etiologies were observed more frequently (53.8%) followed by functional etiologies (37.6%) and then by obstructive ones (8.5%). Hemodialysis was performed for 9.4% of the patients. Of all the patients, 70.1% had favorable outcome, 49.6% of patients recovered totally. There was aggravation of the AKI in 29.9% of cases. Death occurred in eight cases (6.8%). The epidemiological, clinical, biological, and etiological profile of AKI in the elderly emphasizes the effect of aging of the human being on determining the pathology.
... The mechanism underpinning depleted BH 4 levels under such conditions involves ROS-induced oxidation of BH 4 to dihydrobiopterin (BH 2 ), subsequently reducing levels of the former molecule in the endothelium of the BBB (Najjar et al. 2013). The subsequent decrease in the BH 4 to BH 2 ratio results in the inhibition of eNOS while simultaneously uncoupling arginine as its substrate thereby enabling engagement with environmental oxygen and increased production of superoxide ions (Bouloumie et al. 1999;Moens and Kass 2006;Najjar et al. 2013). The resultant combination of superoxide ions with NO results in further increases in levels of ONOO − , thereby inducing increased oxidation of BH 4 to BH 2 , further decreasing the activity of eNOS in an escalating positive feedback loop (Chen et al. 2010;Szabó et al. 2007). ...
A model of the development and progression of chronic fatigue syndrome (myalgic encephalomyelitis), the aetiology of which is currently unknown, is put forward, starting with a consideration of the post-infection role of damage-associated molecular patterns and the development of chronic inflammatory, oxidative and nitrosative stress in genetically predisposed individuals. The consequences are detailed, including the role of increased intestinal permeability and the translocation of commensal antigens into the circulation, and the development of dysautonomia, neuroinflammation, and neurocognitive and neuroimaging abnormalities. Increasing levels of such stress and the switch to immune and metabolic downregulation are detailed next in relation to the advent of hypernitrosylation, impaired mitochondrial performance, immune suppression, cellular hibernation, endotoxin tolerance and sirtuin 1 activation. The role of chronic stress and the development of endotoxin tolerance via indoleamine 2,3-dioxygenase upregulation and the characteristics of neutrophils, monocytes, macrophages and T cells, including regulatory T cells, in endotoxin tolerance are detailed next. Finally, it is shown how the immune and metabolic abnormalities of chronic fatigue syndrome can be explained by endotoxin tolerance, thus completing the model.
... The mechanism underpinning reductions of BH 4 levels involves ROS-induced oxidation of BH 4 to dihydrobiopterin (BH 2 ), thereby decreasing levels of this molecule in the endothelium (Najjar et al., 2013). The subsequent decrease in the BH 4 to BH 2 ratio inhibits the activity of eNOS while uncoupling arginine as its substrate and thus allowing engagement with molecular oxygen and increased production of O 2 (Bouloumie et al., 1999;Moens and Kass, 2006;Najjar et al., 2013). As mentioned above, O 2 − , in turn, combines with NO to form ONOO − , thereby further increasing the oxidative conversion of BH 4 to BH 2 , which further lowers eNOS activity in a positive feedback loop Szabó et al., 2007). ...
Background:
The blood-brain barrier acts as a highly regulated interface; its dysfunction may exacerbate, and perhaps initiate, neurological and neuropsychiatric disorders.
Methods:
In this narrative review, focussing on redox, inflammatory and mitochondrial pathways and their effects on the blood-brain barrier, a model is proposed detailing mechanisms which might explain how increases in blood-brain barrier permeability occur and can be maintained with increasing inflammatory and oxidative and nitrosative stress being the initial drivers.
Results:
Peripheral inflammation, which is causatively implicated in the pathogenesis of major psychiatric disorders, is associated with elevated peripheral pro-inflammatory cytokines, which in turn cause increased blood-brain barrier permeability. Reactive oxygen species, such as superoxide radicals and hydrogen peroxide, and reactive nitrogen species, such as nitric oxide and peroxynitrite, play essential roles in normal brain capillary endothelial cell functioning; however, chronically elevated oxidative and nitrosative stress can lead to mitochondrial dysfunction and damage to the blood-brain barrier. Activated microglia, redox control of which is mediated by nitric oxide synthases and nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, secrete neurotoxic molecules such as reactive oxygen species, nitric oxide, prostaglandin, cyclooxygenase-2, quinolinic acid, several chemokines (including monocyte chemoattractant protein-1 [MCP-1], C-X-C motif chemokine ligand 1 [CXCL-1] and macrophage inflammatory protein 1α [MIP-1α]) and the pro-inflammatory cytokines interleukin-6, tumour necrosis factor-α and interleukin-1β, which can exert a detrimental effect on blood-brain barrier integrity and function. Similarly, reactive astrocytes produce neurotoxic molecules such as prostaglandin E2 and pro-inflammatory cytokines, which can cause a 'leaky brain'.
Conclusion:
Chronic inflammatory and oxidative and nitrosative stress is associated with the development of a 'leaky gut'. The following evidence-based approaches, which address the leaky gut and blood-brain barrier dysfunction, are suggested as potential therapeutic interventions for neurological and neuropsychiatric disorders: melatonin, statins, probiotics containing Bifidobacteria and Lactobacilli, N-acetylcysteine, and prebiotics containing fructo-oligosaccharides and galacto-oligosaccharides.
... NO formation is dependent on the presence of tetrahydrobiopterin (BH 4 ) (34,52), which is required to maintain optimal function of NO synthase (NOS) for the production of NO. In conditions where BH 4 bioavailability is limited, such as HF secondary to higher levels of oxidative stress (46), NOS becomes structurally unstable and produces superoxide rather than NO (13). ...
Heart failure (HF) patients are susceptible to heat strain during exercise, secondary to blunted skin blood flow (SkBF) responses which may be explained by impaired nitric oxide (NO)-dependent vasodilation. Folic acid improves vascular endothelial function and SkBF through NO-dependent mechanisms in healthy older individuals and cardiovascular disease patients. We examined the effect of folic acid supplementation (5 mg/d for 6wk) on vascular function (brachial artery flow-mediated dilation [FMD]), and SkBF responses (cutaneous vascular conductance [CVC]) during 60-min of exercise at a fixed metabolic heat production (300 ẆHprod) in a 30{degree sign}C environment in ten HF (New York Heart Association Class I-II) patients and ten healthy controls (CON). Serum folic acid concentration increased in HF (pre-intervention: 1.4{plus minus}0.2; post-intervention: 8.9{plus minus}6.7 ng/ml, p=0.01) and CON (pre-intervention: 1.3{plus minus}0.6; post-intervention: 5.2{plus minus}4.9 ng/ml, p=0.03). FMD improved by 2.1 {plus minus} 1.3% in HF (p<0.01), but no change was observed in CON post-intervention (p=0.20). During exercise, the external workload performed on the cycle ergometer to attain the fixed level of Hprod for exercise was similar between groups (HF: 60 {plus minus} 13; CON: 65 {plus minus} 20 Ẇext, p = 0.52). Increases in CVC during exercise were similar in HF (pre: 0.89{plus minus}0.43; post: 0.83{plus minus}0.45au/mmHg, p=0.80) and CON (pre: 2.01{plus minus}0.79; post: 2.03{plus minus}0.72au/mmHg, p=0.73), although the values were consistently lower in HF for both pre- and post-intervention measurement intervals (p<0.05). These findings demonstrate that folic acid improves vascular endothelial function in patients with HF, but does not enhance SkBF during exercise at a fixed metabolic heat production in a warm environment.
... This was followed by washout and then exposure ( dilator responses to acetylcholine or DETA NONOate. Additional adipose arterioles were exposed to co-treatment of medin 5 mM, antioxidant polyethylene glycol superoxide dismutase (PEG-SOD, 300 U/ml, Sigma-Aldritch, St. Louis, MO), tetrahydrobiopterin (BH4, 100 mM, an essential cofactor for endothelial nitric oxide synthase [eNOS] function and maintenance of eNOS coupling, 12,13 Schircks Laboratories, Jona, Switzerland) or FPS-ZM1 (100 mM, a high-affinity RAGE-specific inhibitor, 14 Calbiochem, San Diego, CA). The maximum medin dose selected in this study (5 mM) is at par with physiologic/pathologic levels determined from our aortic tissue assays. ...
Aims:
Medin is a common amyloidogenic protein in humans that accumulates in arteries with advanced age and has been implicated in vascular degeneration. Medin's effect on endothelial function remains unknown. The aims are to assess medin's effects on human arteriole endothelial function and identify potential mechanisms underlying medin-induced vascular injury.
Methods and results:
Ex vivo human adipose and leptomeningeal arterioles were exposed (1 h) to medin (0.1, 1, or 5 µM) without or with FPS-ZM1 [100 µM, receptor for advanced glycation endproducts (RAGE)-specific inhibitor] and endothelium-dependent function (acetylcholine dilator response) and endothelium-independent function (dilator response to nitric oxide donor diethylenetriamine NONOate) were compared with baseline control. Human umbilical vein endothelial cells were exposed to medin without or with FPS-ZM1 and oxidative and nitrative stress, cell viability, and pro-inflammatory signaling measures were obtained. Medin caused impaired endothelial function (vs. baseline response: -45.2 ± 5.1 and -35.8 ± 7.9% in adipose and leptomeningeal arterioles, respectively, each P < 0.05). Dilator response to NONOate was not significantly changed. Medin decreased arteriole and endothelial cell nitric oxide production, increased superoxide production, reduced endothelial cell viability, proliferation, and migration. Medin increased gene and protein expression of interleukin-6 and interleukin-8 via activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB). Medin-induced endothelial dysfunction and oxidative stress were reversed by antioxidant polyethylene glycol superoxide dismutase and by RAGE inhibitor FPS-ZM1.
Conclusions:
Medin causes human microvascular endothelial dysfunction through oxidative and nitrative stress and promotes pro-inflammatory signaling in endothelial cells. These effects appear to be mediated via RAGE. The findings represent a potential novel mechanism of vascular injury.
... ROS are originated from various supplies in distinctive cellular compartments. NADPH oxidase considered as the leading source for ROS in the blood vessels wall and endothelial cells and is mainly essential in pathology processes of endothelial dysfunction and hypertension, also another important sources uncoupling of the endothelial NO synthase (eNOS), xanthine oxidase and mitochondria contribute to the increased ROS production observed in different vascular maladies [34,35], including hypertension [36]. A large numbers of literatures published over the past two decades suppose that ROS adjust the action of a massive arrangement of proteins within the cells and signaling pathways, and this redox biology is precisely and spatially adjusted to formulate distinctive cellular influences in both disease and health states. ...
Hypertension is considered as the most common risk factor for cardiovascular diseases, also is regarded as a leading cause of the mortality and morbidity worldwide. The mechanisms underlying the pathological process of hypertension are not completely explained. However, there is growing evidence that increased oxidative stress plays an important role in the pathophysiology of hypertension. Several preclinical studies and clinical trials have indicated that antioxidant therapy is important for management of hypertension, using antioxidants compounds such as alpha tocopherol (Vit E) and ascorbic acid (Vit C), polyphenols with others and some antihypertensive drugs that are now in clinical use (e.g., ACEIs, ARBs, novel B-blockers, dihydropyridine CCBs) which have antioxidative pleiotropic effects. The purpose of this review is to highlight the importance of antioxidant therapy for management of oxidative stress induced hypertension. Furthermore, reviewing the current knowledge in the oxidative stress and its significance in hypertension.
... Adequate eNOS coupling requires the interaction of eNOS with multiple co-factors. Tetrahydrobiopterin (BH4) is an essential cofactor for all members of the nitric oxide synthase (NOS) family (7)(8). BH4 deficiency uncouples eNOS, which leads to superoxide formation instead of NO formation, when NOS is activated. ...
Background:
Bronchopulmonary dysplasia (BPD) is a major morbidity in premature infants, and impaired angiogenesis is considered a major contributor to BPD. Early caffeine treatment decreases the incidence of BPD; the mechanism remains incompletely understood.
Methods:
Sprague-Dawley rat pups exposed to normoxia or hyperoxia since birth were treated daily with either 20 mg/kg caffeine or normal saline by intraperitoneal injection from day 2 of life. Lungs were obtained for studies at day 10 and 21.
Results:
Hyperoxia impaired somatic growth and lung growth in the rat pups. The impaired lung growth during hyperoxia was associated with decreased levels of cyclic AMP (cAMP) and tetrahydrobiopterin (BH4) in the lungs. Early caffeine treatment increased cAMP levels in the lungs of hyperoxia-exposed pups. Caffeine also increased the levels of phosphorylated endothelial nitric oxide synthase (eNOS) at serine(1177), total and serine(51) phosphorylated GTP-cyclohydrolase-1 (GCH1), and BH4 levels, with improved alveolar structure and angiogenesis in hyperoxia-exposed lungs. Reduced GCH1 levels in hyperoxia were due, in part, to increased degradation by the ubiquitin-proteasome system.
Conclusion:
Our data support the notion that early caffeine treatment can protect immature lungs from hyperoxia-induced damage by improving eNOS activity through increased BH4 bioavailability.Pediatric Research accepted article preview online, 11 April 2017. doi:10.1038/pr.2017.89.
... Substantial evidence also suggests that limited vascular BH4 bioavailability causes uncoupling of nitric oxide production from arginine oxidation, resulting in the generation of superoxide radicals and contributing to the pathogenesis of endothelial dysfunction [35]. Among others, BH4 and its derivatives have been implicated in various diseases, including phenylketonuria [36], neurological disorders [37], cardiovascular disease [38,39] and diabetes [40], which have been reviewed elsewhere [23]. Enzymatic deficiencies in the BH4 biosynthesis and regeneration pathways can also give rise to additional pteridine derivatives. ...
Pteridines and their derivatives function as intermediates in the metabolism of several vitamins and cofactors, and their relevance to disease has inspired new efforts to study their roles as disease biomarkers. Recent analytical advances, such as the emergence of sensitive mass spectrometry techniques, new workflows for measuring pteridine derivatives in their native oxidation states and increased multiplexing capacities for the simultaneous determination of many pteridine derivatives, have enabled researchers to explore the roles of urinary pteridines as disease biomarkers at much lower levels with greater accuracy than with previous technologies or methods. As a result, urinary pteridines are being increasingly studied as putative cancer biomarkers with promising results being reported from exploratory studies. In addition, the role of urinary neopterin as a universal biomarker for immune system activation is being investigated in new diseases where it is anticipated to become a useful supplementary marker in clinical diagnostic settings. In summary, this review provides an overview of recent developments in the clinical study of urinary pteridines as disease biomarkers, covers the most promising aspects of advanced analytical techniques being developed for the determination of urinary pteridines and discusses the major challenges associated with implementing pteridine biomarkers in clinical laboratory settings.
... Conversely, H 2 O 2 reduced SOD activity (Fig. 2D), which was abolished by pretreatment of cells with XMJ. is determined by BH4 21 . Intracellular BH4 levels are dictated by a balance of de novo synthesis, BH4 oxidation to BH2 22 . De novo biosynthesis of BH4 is regulated by GTP cyclohydrolase 1 (GTPCH1), a homodecameric protein consisting of 25 kDa subunits in mammalian cells 23 . ...
Endothelial dysfunction, which is caused by endothelial nitric oxide synthase (eNOS) uncoupling, is an initial step in atherosclerosis. This study was designed to explore whether Chinese medicine xin-mai-jia (XMJ) recouples eNOS to exert anti-atherosclerotic effects. Pretreatment of XMJ (25, 50, 100 μg/ml) for 30 minutes concentration-dependently activated eNOS, improved cell viabilities, increased NO generations, and reduced ROS productions in human umbilical vein endothelial cells incubated with H2O2 for 2 hours, accompanied with restoration of BH4. Importantly, these protective effects produced by XMJ were abolished by eNOS inhibitor L-NAME or specific eNOS siRNA in H2O2-treated cells. In ex vivo experiments, exposure of isolated aortic rings from rats to H2O2 for 6 hours dramatically impaired acetylcholine-induced vasorelaxation, reduced NO levels and increased ROS productions, which were ablated by XMJ in concentration-dependent manner. In vivo analysis indicated that administration of XMJ (0.6, 2.0, 6.0 g/kg/d) for 12 weeks remarkably recoupled eNOS and reduced the size of carotid atherosclerotic plaque in rats feeding with high fat diet plus balloon injury. In conclusion, XMJ recouples eNOS to prevent the growth of atherosclerosis in rats. Clinically, XMJ is potentially considered as a medicine to treat patients with atherosclerosis.
... Recent evidence also indicated that immune activation is associated with an increased Phe/Tyr ratio in cardiovascular disease patients (Mangge et al., 2013;Murr et al., 2014). Other studies have also related a decrease in BH4 levels to hypertension development and cardiovascular disease (Du, Guan, Alp, Channon, & Chen, 2008;Moens & Kass, 2006). Moreover, BH4 is a regulator of endothelial nitric oxide synthase (eNOS), thus a decrease in BH4 will reduce bioavailability of endothelial-derived NO, implicated in cardiovascular disease (Bendall, Douglas, McNeill, Channon, & Crabtree, 2014 Guanosine-triphosphate-cyclohydrolase-1 Figure 1. ...
Defensive coping (DefS), oxidative stress, inflammation, and related amino acids (phenylalanine [Phe] and tyrosine [Tyr]) have been implicated in cardiovascular disease. This study assessed whether inflammation, oxidative stress, changes in essential amino acids, and altered coping strategies are correlated with subclinical vascular changes in African (n = 82) and Caucasian (n = 100) men from the Sympathetic Activity and Ambulatory Blood Pressure in Africans (SABPA) study. The Coping Strategy Indicator questionnaire identified DefS participants. Ambulatory blood pressure (BP) was monitored for 24 h, whereas carotid intima media thickness (CIMT) and cross-sectional wall area (CSWA) were determined ultrasonically. Essential amino acids were analyzed with a liquid chromatography tandem mass spectrometry method. Oxidative-inflammatory markers were measured by spectrophotometry. African men had poorer health than Caucasian men, including higher alcohol abuse, elevated BP, abdominal obesity, physical inactivity, and elevated inflammation. Phe (p < .001) and Phe/Tyr ratio (p = .006) as well as CIMT (p = .032) were higher in African men. DefS African men had higher levels of Phe (p = .002) and Phe/Tyr (p = .009) compared to DefS Caucasian men; these differences were not observed in non-DefS men. Systolic BP and inflammation (C-reactive protein) were positively associated with left (L-) CSWA, while Phe/Tyr was negatively associated with L-CSWA in DefS African men. African males presented with elevated Phe and Phe/Tyr ratio, catecholamine precursors, worsening during DefS—possibly driven by inflammation and BP contributing to structural vascular abnormalities.
... Oxidative stress imposed on endothelial cells causes depletion of BH 4 and eNOS uncoupling. The effect of eNOS uncoupling has been investigated in a wide variety of in vitro models, animal models of CVD, and human subjects with cardiovascular risk factors [10,69,70]. Inhibition of oxidative stress to endothelial cells and subsequent occurrence of NOS uncoupling provide an ameliorative effect on endothelial function. ...
Oxidative stress has been implicated in pathophysiology of aging and age-associated disease. Antioxidative medicine has become a practice for prevention of atherosclerosis. However, limited success in preventing cardiovascular disease (CVD) in individuals with atherosclerosis using general antioxidants has prompted us to develop a novel antioxidative strategy to prevent atherosclerosis. Reducing visceral adipose tissue by calorie restriction (CR) and regular endurance exercise represents a causative therapy for ameliorating oxidative stress. Some of the recently emerging drugs used for the treatment of CVD may be assigned as site-specific antioxidants. CR and exercise mimetic agents are the choice for individuals who are difficult to continue CR and exercise. Better understanding of molecular and cellular biology of redox signaling will pave the way for more effective antioxidative medicine for prevention of CVD and prolongation of healthy life span.
Ferroptosis is a new form of regulated cell death caused by iron‐dependent accumulation of lethal polyunsaturated phospholipids peroxidation. It has received considerable attention owing to its putative involvement in a wide range of pathophysiological processes such as organ injury, cardiac ischemia/reperfusion, degenerative disease and its prevalence in plants, invertebrates, yeasts, bacteria, and archaea. To counter ferroptosis, living organisms have evolved a myriad of intrinsic efficient defense systems, such as cyst(e)ine‐glutathione‐glutathione peroxidase 4 system (cyst(e)ine‐GPX4 system), guanosine triphosphate cyclohydrolase 1/tetrahydrobiopterin (BH 4 ) system (GCH1/BH 4 system), ferroptosis suppressor protein 1/coenzyme Q 10 system (FSP1/CoQ 10 system), and so forth. Among these, GPX4 serves as the only enzymatic protection system through the reduction of lipid hydroperoxides, while other defense systems ultimately rely on small compounds to scavenge lipid radicals and prevent ferroptotic cell death. In this article, we systematically summarize the chemical biology of lipid radical trapping process by endogenous chemicals, such as coenzyme Q 10 (CoQ 10 ), BH 4 , hydropersulfides, vitamin K, vitamin E, 7‐dehydrocholesterol, with the aim of guiding the discovery of novel ferroptosis inhibitors.
Chronic kidney disease (CKD) is a major public health concern, underscoring a need to identify pathogenic mechanisms and potential therapeutic targets. Reactive oxygen species (ROS) are derivatives of oxygen molecules that are generated during aerobic metabolism and are involved in a variety of cellular functions that are governed by redox conditions. Low levels of ROS are required for diverse processes, including intracellular signal transduction, metabolism, immune and hypoxic responses, and transcriptional regulation. However, excess ROS can be pathological, and contribute to the development and progression of chronic diseases. Despite evidence linking elevated levels of ROS to CKD development and progression, the use of low-molecular-weight antioxidants to remove ROS has not been successful in preventing or slowing disease progression. More recent advances have enabled evaluation of the molecular interactions between specific ROS and their targets in redox signalling pathways. Such studies may pave the way for the development of sophisticated treatments that allow the selective control of specific ROS-mediated signalling pathways.
Nitric oxide (NO) is a diffusible free radical and universal messenger that is produced from L-arginine by three different isoforms of nitric oxide synthases (NOS), neuronal (nNOS), inducible (iNOS) and endothelial NOS (eNOS). NO plays an important role in the regulation of variety of physiological functions including myocardial contractility, vascular tone, blood pressure, cell growth, proliferation and platelet aggregation. Most of the effects of NO are mediated through the activation of soluble guanylate cyclase–cGMP system, however, cGMP-independent pathways have also been shown to be responsible in mediating its effects. The levels of NO are regulated by several factors and cofactors required for the activation of NOS, however, reduced bioavailability of these factors results in the decreased levels of NO and thereby endothelial dysfunction leading to the pathogenesis of cardiovascular diseases including hypertension, diabetes, atherosclerosis etc. This review will focus on the role of NO in physiology and pathophysiology of cardiovascular system including vascular remodeling, hypertension and the underlying molecular mechanisms contributing to these functions.
Sickle cell disease (SCD) is associated with repeated bouts of vascular insufficiency leading to organ dysfunction. Deficits in revascularization following vascular injury are evident in SCD patients and animal models. We aimed to elucidate whether enhancing nitric oxide bioavailability in SCD mice improves outcomes in a model of vascular insufficiency. Townes AA (wild type) and SS (sickle cell) mice were treated with either L-Arginine (5% in drinking water), L-NAME (N(ω)-nitro-L-arginine methyl ester; 1 g/L in drinking water) or NO-generating hydrogel (PA-YK-NO), then subjected to hindlimb ischemia via femoral artery ligation and excision. Perfusion recovery was monitored over 28 days via LASER Doppler perfusion imaging. Consistent with previous findings, perfusion was impaired in SS mice (63 ± 4% of non-ischemic limb perfusion in AA vs 33 ± 3% in SS; day 28; P < 0.001; n = 5-7) and associated with increased necrosis. L-Arginine treatment had no significant effect on perfusion recovery or necrosis (n = 5-7). PA-YK-NO treatment led to worsened perfusion recovery (19 ± 3 vs. 32 ± 3 in vehicle-treated mice; day 7; P < 0.05; n = 4-5), increased necrosis score (P < 0.05, n = 4-5) and a 46% increase in hindlimb peroxynitrite (P = 0.055, n = 4-5). Interestingly, L-NAME worsened outcomes in SS mice with decreased in vivo lectin staining following ischemia (7 ± 2% area in untreated vs 4 ± 2% in treated mice, P < 0.05, n = 5). Our findings demonstrate that L-arginine and direct NO delivery both fail to improve postischemic neovascularization in SCD. Addition of NO to the inflammatory, oxidative environment in SCD may result in further oxidative stress and limit recovery.
Aims:
Tetrahydrobiopterin (BH4) is a critical determinant of the biological function of endothelial nitric oxide synthase. The present study was to investigate the role of valvular endothelial cell (VEC)-derived BH4 in aortic valve calcification.
Methods and results:
Plasma and aortic valve BH4 concentrations and the BH4:BH2 ratio were significantly lower in calcific aortic valve disease patients than in controls. There was a significant decrease of the two key enzymes of BH4 biosynthesis, guanosine 5'-triphosphate cyclohydrolase I (GCH1) and dihydrofolate reductase (DHFR), in calcified aortic valves compared with the normal ones. Endothelial cell-specific deficiency of Gch1 in Apoe-/- (Apoe-/-Gch1fl/flTie2Cre) mice showed a marked increase in transvalvular peak jet velocity, calcium deposition, runt-related transcription factor 2 (Runx2), dihydroethidium (DHE), and 3-nitrotyrosine (3-NT) levels in aortic valve leaflets compared with Apoe-/-Gch1fl/fl mice after a 24-week western diet (WD) challenge. Oxidized LDL (ox-LDL) induced osteoblastic differentiation of valvular interstitial cells (VICs) co-cultured with either si-GCH1- or si-DHFR-transfected VECs, while the effects could be abolished by BH4 supplementation. Deficiency of BH4 in VECs caused peroxynitrite formation increase and 3-NT protein increase under ox-LDL stimulation in VICs. SIN-1, the peroxynitrite generator, significantly up-regulated alkaline phosphatase (ALP) and Runx2 expression in VICs via tyrosine nitration of dynamin-related protein 1 (DRP1) at Y628. Finally, folic acid (FA) significantly attenuated aortic valve calcification in WD-fed Apoe-/- mice through increasing DHFR and salvaging BH4 biosynthesis.
Conclusion:
The reduction in endothelial-dependent BH4 levels promoted peroxynitrite formation, which subsequently resulted in DRP1 tyrosine nitration and osteoblastic differentiation of VICs, thereby leading to aortic valve calcification. Supplementation of FA in diet attenuated hypercholesterolaemia-induced aortic valve calcification by salvaging BH4 bioavailability.
Key question:
Tetrahydrobiopterin (BH4) insufficiency-dependent endothelial nitric oxide synthase dysfunction mediates endothelial dysfunction and leads to vascular diseases. The role of BH4 in aortic valve calcification has not been explored.
Key finding:
Tetrahydrobiopterin and guanosine 5'-triphosphate cyclohydrolase I are reduced in calcified aortic valves. Endothelial cell-specific deficiency of Gch1 promotes murine aortic valve calcification. Tetrahydrobiopterin reduction in valvular endothelial cells promotes valvular interstitial cells (VICs) osteogenesis via ONOO- formation. Folic acid suppresses murine aortic valve calcification by salvaging BH4 bioavailability.
Take-home message:
Tetrahydrobiopterin is closely associated with the development of aortic valve calcification. Valvular endothelial cells dysfunction leads to ONOO- formation, resulting in osteoblastic differentiation of VICs. Salvaging BH4 bioavailability may be a potential therapeutic strategy for aortic valve calcification.
Paraquat (PQ) is a toxic, organic herbicide for which there is no specific antidote. Although banned in some countries, it is still used as an irreplaceable weed killer in others. The lack of understanding of the precise mechanism of its toxicity has hindered the development of treatments for PQ exposure. While toxicity is thought to be related to PQ-induced oxidative stress, antioxidants are limited in their ability to ameliorate the untoward biological responses to this agent. Summarized in this review are data on the absorption, distribution, metabolism, excretion, and toxicity (ADME/T) of PQ, focusing on the essential roles of individual transporters and enzymes in these processes. Based on these findings, strategies are proposed to design and test specific and effective antidotes for the clinical management of PQ poisoning.
Nitric oxide (NO.) has been widely studied as an active agent of many physiological and pathological processes. Currently, NO. divides attention with its sibling molecule, nitroxyl (HNO), mainly due to their differences in physiological responses broadening their applications. In order for NO. and HNO to have their multiple biological effects, they must reach quite specific concentrations in the body. This key issue makes it essential to develop strategies for delivering these molecules in a controlled and selective manner. The wide range of activities of these compounds along with smart strategies in the development of NO./HNO donors have made them a hot spot. There are some NO. donor strategies in clinical use and also others in clinical trial, while HNO donors are further behind, illustrating the opportunities to come. Along these lines, we reviewed some current exciting NO. and HNO donor species, including organic‐ and inorganic‐based compounds, as well as nanomaterial platforms and NO. donor devices. This update may provide an overview of the systems currently available and how far we have come to meet multiple pharmacological needs.
S-nitrosylation is a selective and reversible post-translational modification of protein thiols by nitric oxide (NO), which is a bioactive signaling molecule, to exert a variety of effects. These effects include the modulation of protein conformation, activity, stability, and protein-protein interactions. S-nitrosylation plays a central role in propagating NO signals within a cell, tissue, and tissue microenvironment, as the nitrosyl moiety can rapidly be transferred from one protein to another upon contact. This modification has also been reported to confer either tumor-suppressing or tumor-promoting effects and is portrayed as a process involved in every stage of cancer progression. In particular, S-nitrosylation has recently been found as an essential regulator of the tumor microenvironment (TME), the environment around a tumor governing the disease pathogenesis. This review aims to outline the effects of S-nitrosylation on different resident cells in the TME and the diverse outcomes in a context-dependent manner. Furthermore, we will discuss the therapeutic potentials of modulating S-nitrosylation levels in tumors.
This review provides an updated overview of the emerging roles of neopterin derivatives in atherosclerosis. Neopterin, a metabolite of guanosine triphosphate, is produced by interferon-γ-activated macrophages and is expressed at high levels in atheromatous plaques within the human carotid and coronary arteries as well as in the aorta. Plasma concentrations of neopterin are higher in patients with carotid, cerebral, and coronary artery diseases as well as aortic aneurysm. The concentration of neopterin is positively correlated with the severity of coronary artery disease. However, a prospective cohort study showed that neopterin contributes to protection against plaque formation in carotid arteries in patients with atherosclerosis. Moreover, using both in vitro and in vivo experiments, a recent study has shown the atheroprotective effects of neopterin. Neopterin suppresses the expression of monocyte chemotactic protein-1, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 in endothelial cells, and thereby suppresses the adhesion of monocytes to endothelial cells. It also suppresses the inflammatory phenotype of monocyte-derived macrophages. In addition, neopterin suppresses oxidized low-density lipoprotein-induced foam cell formation in macrophages and the migration and proliferation of vascular smooth muscle cells. Neopterin injection into apolipoprotein E-deficient (Apoe-/-) mice suppresses the development of atherosclerotic lesions. A neopterin derivative tetrahydroneopterin (BH4), also known as a cofactor for nitric oxide (NO) synthases, suppresses atherosclerosis and vascular injury-induced neointimal hyperplasia in Apoe-/- mice. BH4 administration improves endothelial dysfunction in patients with coronary artery disease. These findings suggest that neopterin production may increase to counteract the progression of atherosclerosis, as neopterin contributes to atheroprotection. Otherwise, the increased neopterin levels in atherosclerosis may reflect a compensatory mechanism associated with inducible NO synthase upregulation in macrophages to supply BH4 for high output NO production caused by decreased endothelial NO synthase in atherosclerosis. Therefore, neopterin derivatives are a novel therapeutic target for atherosclerosis and related diseases.
Based on work-done in the rostral ventrolateral medulla (RVLM), this review presents four lessons learnt from studying the differential impacts of oxidative stress and nitrosative stress on sympathetic vasomotor tone and their clinical and therapeutic implications. The first lesson is that an increase in sympathetic vasomotor tone because of augmented oxidative stress in the RVLM is responsible for the generation of neurogenic hypertension. On the other hand, a shift from oxidative stress to nitrosative stress in the RVLM underpins the succession of increase to decrease in sympathetic vasomotor tone during the progression towards brain stem death. The second lesson is that, by having different cellular sources, regulatory mechanisms on synthesis and degradation, kinetics of chemical reactions, and downstream signaling pathways, reactive oxygen species and reactive nitrogen species should not be regarded as a singular moiety. The third lesson is that well-defined differential roles of oxidative stress and nitrosative stress with distinct regulatory mechanisms in the RVLM during neurogenic hypertension and brain stem death clearly denote that they are not interchangeable phenomena with unified cellular actions. Special attention must be paid to their beneficial or detrimental roles under a specific disease or a particular time-window of that disease. The fourth lesson is that, to be successful, future antioxidant therapies against neurogenic hypertension must take into consideration the much more complicated picture than that presented in this review on the generation, maintenance, regulation or modulation of the sympathetic vasomotor tone. The identification that the progression towards brain stem death entails a shift from oxidative stress to nitrosative stress in the RVLM may open a new vista for therapeutic intervention to slow down this transition.
Phytotherapy has the potential to give patients long term benefits with less or no side effects. This is the second volume of the series. This volume brings 11 chapters that cover updates on general phytotherapy, traditional Chinese medicine as well as information on anti-diabetic and antihypertensive herbs (including Senna spp., Curcumin, Carum carvi, Premna serratifolia, Eugenia jambolana and more). The monographs presented within this volume give several details necessary for pharmacopoeial data for quality assurance of pharmaceutical products derived from these specific plant sources: botanical features, distribution, identity tests, purity requirements, chemical assays, active or major chemical constituents, clinical applications, pharmacology, contraindications, warnings, precautions, potential adverse reactions, and posology. Hence academic and professional pharmacologists or clinicians will find comprehensive information on a variety of therapeutic agents along with guidelines for applying them in practical phytotherapy of diabetes and hypertension.
Aims
Exogenous tetrahydrobiopterin (BH4), an indispensable cofactor of endothelial nitric oxide synthase (eNOS), supplementation has been proved to be of advantage to improve cardiovascular function. Nevertheless, due to its highly redox-sensitive and easy to be oxidized, there is an urgent need to develop an appropriate BH4 formulation for clinical therapy. Gum Arabic (GA) has been considered as an alternative biopolymer for the stabilization and coating of drugs. The effects of GA on protecting BH4 from being oxidized were investigated in a rat model of myocardial ischemia-reperfusion (I/R).
Main methods
Rats were subjected to 60-min of in vivo left coronary artery occlusion and varying periods of reperfusion with or without pre-ischemic GA-coated BH4 supplementation (10 mg/kg, oral). Myocardial infarction, fibrotic area and left ventricle ejection fraction were correlated with cardiac BH4 content, eNOS protein, NOS enzyme activity, and ROS/NO generation.
Key findings
Pretreatment of rats with GA-coated 6R-BH4, 24 h before myocardial ischemia, resulted in smaller myocardial infarction, improved left ventricular function and inhibited fibrosis, correlated with maintained high levels of cardiac BH4 content, preserved eNOS activation and dimerization, and decreased ROS generation. However in uncoated group, 6R-BH4 treatment did not reduce acute and chronic myocardial I/R injury compared with control I/R rats, which was closely related with the marked loss of myocardial BH4 levels during I/R.
Significance
These findings provide evidence that in vivo pre-ischemic oral GA-coated BH4 administration preserves eNOS function secondary to maintaining cardiac BH4 content, and confers cardioprotection after I/R.
The present study examined whether exendin‑4 (Ex4) can improve the endothelial dysfunction of apolipoprotein E knockout (APOE‑KO) mice fed a high‑cholesterol diet and the potential mechanism by which it acts. Genetically wild‑type (WT) C57BL/6 mice and APOE‑KO mice of C57BL/6 background, were each randomly assigned to receive either Ex4 treatment (Ex4‑treated, for 8 weeks) or not (control). The 4 groups were fed the same high‑cholesterol diet for 8 weeks. The following were measured at the end of the eighth week: Endothelium‑dependent vasodilation of the arteries; plasma nitric oxide (NO) and metabolic index; levels of endothelial NO synthase (eNOS); phosphorylated eNOS (p‑eNOS; Ser‑1,177); guanosine triphosphate cyclohydrolase‑1 (GCH1); and tetrahydrobiopterin (THB). Ex4 treatment was associated with higher p‑eNOS levels in the WT group and in the APOE‑KO group, and higher vascular expression of GCH1 and higher arterial THB content, compared with baseline values. The results of the present study suggested that Ex4 may exert cardioprotective effects by reversing high‑cholesterol diet‑induced endothelial dysfunction in APOE‑KO mice. The protective mechanism is probably associated with the promotion of the expression levels of GCH1 protein and THB that maintain the normal function of eNOS.
Background: Folic acid supplementation can increase the
concentration of serum folate and decrease the concentration
of plasma total homocysteine (p-tHcy), which is
considered a risk factor for cardiovascular diseases. The aim
of this study was to investigate whether genetic polymorphisms
involved in folate metabolism and endothelial
nitric oxide synthase (eNOS) affect the concentration of serum folate,
plasma total homocysteine and serum nitrate in healthy subjects after folic
acid supplementation.
Method: It was a randomized, double blind, cross over study. Half of the
participants were given folic acid 800 µg/day and the other half of the
participants received placebo for 2 weeks.
Results: The polymorphisms in folate gene and eNOS gene had a
significant increase in the concentration of serum folate and decrease
in p-tHcy after folic acid supplementation. However, these gene polymorphisms
did not affect the concentration of serum nitrate.
Conclusion: Polymorphisms in folate gene and eNOS gene affect the
concentration of serum folate and p-tHcy but do not have any effect on the
concentration of serum nitrate in healthy individuals after folic acid
supplementation.
It has been 60 yr since the discovery of reactive oxygen species (ROS) in biology and the beginning of the scientific community's attempt to understand the impact of the unpaired electron of ROS molecules in biological pathways, which was eventually noted to be toxic. Several studies have shown that the presence of ROS is essential in triggering or acting as a secondary factor for numerous pathologies, including metabolic and genetic diseases; however, it was demonstrated that chronic treatment with antioxidants failed to show efficacy and positive effects in the prevention of diseases or health complications that result from oxidative stress. On the contrary, such treatment has been shown to sometimes even worsen the disease. Because of the permanent presence of ROS in organisms, elaborate mechanisms to adapt with these reactive molecules and to use them without necessarily blocking or preventing their actions have been studied. There is now a large body of evidence that shows that living organisms have conformed to the presence of ROS and, in retrospect, have adapted to the bioactive molecules that are generated by ROS on proteins, lipids, and DNA. In addition, ROS have undergone a shift from being molecules that invoked oxidative damage in regulating signaling pathways that impinged on normal physiological and redox responses. Working in this direction, this review unlocks a new conception about the involvement of cellular oxidants in the maintenance of redox homeostasis in redox regulation of normal physiological functions, and an explanation for its essential role in numerous patho-physiological states is noted.-Roy, J., Galano, J.-M., Durand, T., Le Guennec, J.-Y., Lee, J. C.-Y. Physiological role of reactive oxygen species as promoters of natural defenses.
Atherosclerosis is a progressive and multifactorial disease which occurs under the influence of various risk factors including endothelial dysfunction (ED), oxidative stress, and low-density lipoprotein (LDL) oxidation. In contract to the initial hypotheses on the usefulness of vitamin E supplementation for cardiovascular disease prevention, large outcome trials showed consumption of vitamin E has no obvious effect on cardiovascular disease and, in some cases, it may even increase the rate of mortality. This seemingly unexpected finding may be due to the opposite effects of vitamin E compounds. Vitamin E is a group of compounds which have different and even opposing effects, yet in most of the studies, the exact consumed component of vitamin E is not determined. It appears that the combined consumption of gamma-tocopherol, vitamin C, D, and tetrahydrobiopterin (BH4) may be extremely effective in both preventing atherogenesis and suppressing plaque development. In this regard, one of main issues is effect of vitamins E and D deficiency on microRNAs network in atherosclerosis. Various studies have indicated that miRNAs have key roles in atherosclerosis pathogenesis. The deficiency of vitamins E and D could provide a deregulation for miRNAs network and these events could lead to progression of atherosclerosis. Here, we highlighted a variety of mechanisms involve in the progression of atherosclerosis and effects of vitamins D and E on these mechanisms. Moreover, we summarized miRNAs involve in atherosclerosis and their regulation by vitamins E and D deficiency.
Upon heat exposure, the thermoregulatory system evokes reflex increases in sweating and skin blood flow responses to facilitate heat dissipation and maintain heat balance to prevent the continuing rise in core temperature. These heat dissipating responses are mediated primarily by autonomic and cardiovascular adjustments; which if attenuated, may compromise thermoregulatory control. In patients with heart failure (HF), the neurohumoral and cardiovascular dysfunction that underpins this condition may potentially impair thermoregulatory responses and, consequently, place these patients at a greater risk of heat-related illness. The aim of this review is to describe thermoregulatory mechanisms and the factors that may increase the risk of heat-related illness in patients with HF. An understanding of the mechanisms responsible for impaired thermoregulatory control in HF patients is of particular importance, given the current and projected increase in frequency and intensity of heat waves, as well as the promotion of regular exercise as a therapeutic modality. Furthermore, novel therapeutic strategies that may improve thermoregulatory control in HF, and the clinical relevance of this work in this population will be discussed.
In addition to its well-known metabolic effects, insulin has been demonstrated to exert direct effects on the vasculature that may play a role in enhancing its own delivery to target tissues and promoting glucose disposal. Defects in these vascular effects of insulin may be responsible for the close association between insulin resistance and hypertension. Compelling evidence has now revealed that insulin produces vasodilator effects that are dependent on endothelium-derived nitric oxide (NO). However, the exact mechanism underlying insulin-induced vasodilatation has yet to be discovered. Due to the importance of tetrahydrobiopterin (BH 4) (an absolute cofactor requirement for NO synthase activation) in NO synthesis, we examined the possibility that insulin may increase endothelial NO synthesis by modulating the availability of BH 4 in the present study. In isolated tissue bath preparations, we studied the effects of insulin (150 nM) on femoral arterial reactivity to norepinephrine (NE; 10 -9-10 -4 M) in Sprague Dawley rats in the presence and absence of 2×10 -3 M 2,4-diamino-6-hydroxypyrimidine (DAHP), a specific inhibitor of BH 4 production. Insulin caused a significant reduction in contractile responses of femoral arteries to NE (% maximum contraction 10 -6 M NE]: without insulin 60.7 ±4.2; with insulin 17.7 ±4.3*, *p < 0.05), and the effect of insulin was inhibited by the presence of DAHP (% maximum contraction 10 -6 M [NE]: with insulin + DAHP 41.7 ±7.4, vs with insulin, p<O.05). Our data indicate that inhibition of BH4 synthesis results in a marked attenuation in the vasodepressor effect of insulin. One possibility is that insulin may regulate NO production by increasing cofactor (BH 4) availability for activation of NO synthase.
The nitric-oxide synthase (NOS; EC 1.14.13.39) reaction is formulated as a partially tetrahydrobiopterin (H4Bip)-dependent 5-electron oxidation of a terminal guanidino nitrogen of L-arginine (Arg) associated with stoichiometric consumption of dioxygen (O2) and 1.5 mol of NADPH to form L-citrulline (Cit) and nitric oxide (\cdot NO). Analysis of NOS activity has relied largely on indirect methods such as quantification of nitrite/nitrate or the coproduct Cit; we therefore sought to directly quantify\cdot NO formation from purified NOS. However, by two independent methods, NOS did not yield detectable \cdot NO unless superoxide dismutase (SOD; EC 1.15.1.1) was present. In the presence of H4Bip, internal \cdot NO standards were only partially recovered and the dismutation of superoxide (O2{\cdot}), which otherwise scavenges \cdot NO to yield ONOO-, was a plausible mechanism of action of SOD. Under these conditions, a reaction between NADPH and ONOO- resulted in considerable overestimation of enzymatic NADPH consumption. SOD lowered the NADPH:Cit stoichiometry to 0.8-1.1, suggesting either that additional reducing equivalents besides NADPH are required to explain Arg oxidation to \cdot NO or that \cdot NO was not primarily formed. The latter was supported by an additional set of experiments in the absence of H4Bip. Here, recovery of internal \cdot NO standards was unaffected. Thus, a second activity of SOD, the conversion of nitroxyl (NO-) to \cdot NO, was a more likely mechanism of action of SOD. Detection of NOS-derived nitrous oxide (N2O) and hydroxylamine (NH2OH), which cannot arise from \cdot NO decomposition, was consistent with formation of an \cdot NO precursor molecule such as NO-. When, in the presence of SOD, glutathione was added, S-nitrosoglutathione was detected. Our results indicate that \cdot NO is not the primary reaction product of NOS-catalyzed Arg turnover and an alternative reaction mechanism and stoichiometry have to be taken into account.
Constitutive nitric oxide synthase (cNOS) with insufficient cofactor (6R)-5,6,7,8-tetrahydrobiopterin (H4B) may generate damaging superoxide (O2-). This study was designed to determine whether cNOS-dependent generation of O2- occurs in spontaneously hypertensive rats (SHR) before the onset of hypertension. Aortas from 4-wk-old SHR and Wistar-Kyoto rats were used. cNOS was stimulated by calcium ionophore A23187. In situ measurements of nitric oxide and hydrogen peroxide by electrochemical sensors and O2- production by chemiluminescence method were performed. Isometric tension was continuously recorded. H4B by high performance liquid chromatography and [3H]citrulline assay were determined in homogenized tissue. The A23187-stimulated production of O2- and its superoxide dismutase product hydrogen peroxide were significantly higher, whereas nitric oxide release was reduced in SHR aortas, with opposite results in the presence of exogenous H4B. Furthermore, NG-monomethyl-L-arginine inhibited the generation of cNOS-dependent O2- by approximately 70%. Natural H4B levels were similar in both strains; however, equivalent cNOS activity required additional H4B in SHR. The endothelium-dependent relaxations to A23187 were significantly inhibited by catalase, and enhanced by superoxide dismutase, only in SHR; however, these enzymes had no effect in the presence of H4B. Thus, dysfunctional cNOS may be a source of O2- in prehypertensive SHR and contribute to the development of hypertension and its vascular complications.
The oxygenase domain (amino acids 1-498) of inducible nitric oxide synthase (iNOSox) is a hemeprotein that binds L-arginine (L-Arg) and tetrahydrobiopterin (H4B). During NO synthesis, the heme iron must bind and activate O2, but it also binds self-generated No to form an inactive complex. To better understand how L-Arg and H4B affect heme iron function in iNOSox, we utilized stopped-flow spectroscopy to study heme reactivity with CO and NO and the properties of the resulting CO and NO complexes. CO and NO binding to ferrous and ferric (NO only) iNOSox and subsequent complex stability was studied under four conditions: in the absence of L-Arg and H4B and in the presence of either or both molecules. Ferric iNOSox without L-Arg or H4B was dimeric and contained low-spin heme iron, while in H4B- or L-Arg-saturated iNOSox, the heme iron was partially or almost completely high-spin, respectively. In the presence of L-Arg or H4B, the rate of CO binding to ferrous iNOSox was slowed considerably, indicating that these molecules restrict CO access to the heme iron. In contrast, rates of NO binding were minimally affected. Under all conditions, the off rates for CO and NO were very high as compared to other hemeproteins. The six-coordinate FeII-CO and -NO complexes that initially formed were unstable and converted either slowly (CO) or quickly (NO) to their respective 5-coordinate complexes. However, this transition was largely prevented by either L-Arg or H4B and was reversed upon air oxidation of the complex in the presence of these molecules. Thus, H4B and L-Arg both promote a conformational change in the distal heme pocket of iNOSox that can greatly reduce ligand access to the heme iron. The ability of H4B and L-Arg to prevent formation of a five-coordinate heme Fe-NO complex, along with the high off rates observed for NO, help explain why iNOS can remain active despite forming a complex with NO during its normal catalysis.
Nitric oxide (NO) synthesis is induced in vascular smooth muscle cells by lipopolysaccharide (LPS) where it appears to mediate a variety of vascular dysfunctions. In some cell types tetrahydrobiopterin (BH4) synthesis has also been found to be induced by cytokines. Because BH4 is a cofactor for NO synthase, we investigated whether BH4 synthesis is required for LPS-induced NO production in rat aortic smooth muscle cells (RASMC). The total biopterin content (BH4 and more oxidized states) of untreated RASMC was below our limit of detection. However, treatment with LPS caused a significant rise in biopterin levels and an induction of NO synthesis; both effects of LPS were markedly potentiated by interferon-gamma. 2,4-Diamino-6-hydroxypyrimidine (DAHP), a selective inhibitor of GTP cyclohydrolase I, the rate-limiting enzyme for de novo BH4 synthesis, completely abolished the elevated biopterin levels induced by LPS. DAHP also caused a concentration-dependent inhibition of LPS-induced NO synthesis. Inhibition of NO synthesis by DAHP was reversed by sepiapterin, an agent which circumvents the inhibition of biopterin synthesis by DAHP by serving as a substrate for BH4 synthesis via the pterin salvage pathway. The reversal by sepiapterin was overcome by methotrexate, an inhibitor of the pterin salvage pathway. Sepiapterin, and to a lesser extent BH4, dose-dependently enhanced LPS-induced NO synthesis, indicating that BH4 concentration limits the rate of NO production by LPS-activated RASMC. Sepiapterin also caused LPS-induced NO synthesis to appear with an abbreviated lag period phase, suggesting that BH4 availability also limits the onset of NO synthesis. In contrast to the stimulation of LPS-induced NO synthesis, observed when sepiapterin was given alone, sepiapterin became a potent inhibitor of NO synthesis in the presence of methotrexate. This is attributable to a direct inhibitory action of sepiapterin on GTP cyclohydrolase I, an activity which is only revealed after blocking the metabolism of sepiapterin to BH4. Further studies with sepiapterin, methotrexate, and N-acetylserotonin (an inhibitor of the BH4 synthetic enzyme, sepiapterin reductase) indicated that the BH4 is synthesized in RASMC predominantly from GTP; however, a lesser amount may derive from pterin salvage. We demonstrate that BH4 synthesis is an absolute requirement for induction of NO synthesis by LPS in vascular smooth muscle. Our findings also suggest that pterin synthesis inhibitors may be useful for the therapy of endotoxin- and cytokine-induced shock.
Nitric oxide (NO) is synthesized within the immune, vascular, and nervous systems, where it acts as a wide-ranging mediator of mammalian physiology. The NO synthases (EC 1.14.13.39) isolated from neurons or endothelium are calmodulin dependent. Calmodulin binds reversibly to neuronal NO synthase in response to elevated Ca2+, triggering its NO production by an unknown mechanism. Here we show that calmodulin binding allows NADPH-derived electrons to pass onto the heme group of neuronal NO synthase. Calmodulin-triggered electron transfer to heme was independent of substrate binding, caused rapid enzymatic oxidation of NADPH in the presence of O2, and was required for NO synthesis. An NO synthase isolated from cytokine-induced macrophages that contains tightly bound calmodulin catalyzed spontaneous electron transfer to its heme, consistent with bound calmodulin also enabling electron transfer within this isoform. Together, these results provide a basis for how calmodulin may regulate NO synthesis. The ability of calmodulin to trigger electron transfer within an enzyme is unexpected and represents an additional function for calcium-binding proteins in biology.
We have examined cytokine regulation of nitric oxide synthase (NOS) in human umbilical vein endothelial cells (HUVEC). 24-h treatment with IFN-gamma (200 U/ml) plus TNF (200 U/ml) or IL-1 beta (5 U/ml) increased NOS activity in HUVEC lysates, measured as conversion of [14C]L-arginine to [14C]L-citrulline. Essentially, all NOS activity in these cells was calcium dependent and membrane associated. Histamine-induced nitric oxide release, measured by chemiluminescence, was greater in cytokine-treated cells than in control cells. Paradoxically, steady-state mRNA levels of endothelial NOS fell by 94 +/- 2.0% after cytokine treatment. Supplementation of HUVEC lysates with exogenous tetrahydrobiopterin (3 microM) greatly increased total NOS activity, and under these assay conditions, cytokine treatment decreased maximal NOS activity. IFN-gamma plus TNF or IL-1 beta increased endogenous tetrahydrobiopterin levels and GTP cyclohydrolase I activity, the rate-limiting enzyme of tetrahydrobiopterin synthesis. Intracellular tetrahydrobiopterin levels were higher in freshly isolated HUVEC than in cultured cells, but were still limiting. We conclude that inflammatory cytokines increase NOS activity in cultured human endothelial cells by increasing tetrahydrobiopterin levels in the face of falling total enzyme; similar regulation appears possible in vivo.
Nitric oxide (NO) synthases contain FAD, FMN, heme, and (6R)-5,6,7,8-tetrahydro-L-biopterin as prosthetic groups. We have characterized the pteridine-binding site of purified brain NO synthase, using 3H-labeled (6R)-5,6,7,8-tetrahydro-L-biopterin as radioligand. Association of [3H]tetrahydrobiopterin followed second-order kinetics (kon = 1.3 x 10(6) M-1 min-1), the dissociation reaction was reversible and first-order (koff = 3.2 x 10(-1) min-1), yielding a kinetic KD of 0.25 microM. Binding of the radioligand was competitively antagonized by several pteridine derivatives with the following order of potency (KI): 7,8-dihydro-L-biopterin (2.2 microM), (6S)-5,6,7,8-tetrahydro-L-biopterin (19 microM), (6R,S)-6-methyl-5,6,7,8-tetrahydropterin (240 microM), and 6,7-dimethyl-5,6,7,8-tetrahydropterin (> 1 mM). The affinity of NO synthase for tetrahydrobiopterin was increased 6-fold in the presence of 0.1 mM L-arginine (KD = 37 nM), and, conversely, tetrahydrobiopterin enhanced the affinity of the enzyme for 3H-labeled NG-nitro-L-arginine about 2-fold. 7-Nitroindazole, which presumably binds to the heme group of NO synthase, competitively inhibited binding of [3H]tetrahydrobiopterin and [3H]NG-nitro-L-arginine with similar Ki values (0.1 microM). Functional as well as binding studies revealed that 7-nitroindazole was competitive with both L-arginine and tetrahydrobiopterin. Our data indicate that brain NO synthase exhibits a highly specific binding site for (6R)-5,6,7,8-tetrahydro-L-biopterin, which allosterically interacts with the substrate domain and may be located proximal to the prosthetic heme group of NO synthase.
The nitric-oxide synthase (NOS; EC 1.14.13.39) reaction is formulated as a partially tetrahydrobiopterin (H4Bip)-dependent 5-electron oxidation of a terminal guanidino nitrogen of L-arginine (Arg) associated with stoichiometric consumption of dioxygen (O2) and 1.5 mol of NADPH to form L-citrulline (Cit) and nitric oxide (.NO). Analysis of NOS activity has relied largely on indirect methods such as quantification of nitrite/nitrate or the coproduct Cit; we therefore sought to directly quantify .NO formation from purified NOS. However, by two independent methods, NOS did not yield detectable .NO unless superoxide dismutase (SOD; EC 1.15.1.1) was present. In the presence of H4Bip, internal .NO standards were only partially recovered and the dismutation of superoxide (O2-.), which otherwise scavenges. .NO to yield ONOO-, was a plausible mechanism of action of SOD. Under these conditions, a reaction between NADPH and ONOO- resulted in considerable overestimation of enzymatic NADPH consumption. SOD lowered the NADPH:Cit stoichiometry to 0.8-1.1, suggesting either that additional reducing equivalents besides NADPH are required to explain Arg oxidation to .NO or that .NO was not primarily formed. The latter was supported by an additional set of experiments in the absence of H4Bip. Here, recovery of internal .NO standards was unaffected. Thus, a second activity of SOD, the conversion of nitroxyl (NO-) to .NO, was a more likely mechanism of action of SOD. Detection of NOS-derived nitrous oxide (N2O) and hydroxylamine (NH2OH), which cannot arise from .NO decomposition, was consistent with formation of an .NO precursor molecule such as NO-. When, in the presence of SOD, glutathione was added, S-nitrosoglutathione was detected. Our results indicate that .NO is not the primary reaction product of NOS-catalyzed Arg turnover and an alternative reaction mechanism and stoichiometry have to be taken into account.
In hypercholesterolemia, impaired nitric oxide activity has been associated with increased nitric oxide degradation by oxygen radicals. Deficiency of tetrahydrobiopterin, an essential cofactor of nitric oxide synthase, causes both impaired nitric oxide activity and increased oxygen radical formation. In this study we tested whether tetrahydrobiopterin deficiency contributes to the decreased nitric oxide activity observed in hypercholesterolemic patients. Therefore, L-mono-methyl-arginine to inhibit basal nitric oxide activity, serotonin to stimulate nitric oxide activity, and nitroprusside as endothelium-independent vasodilator were infused in the brachial artery of 13 patients with familial hypercholesterolemia and 13 matched controls. The infusions were repeated during coinfusion of L-arginine (200 microg/kg/min), tetrahydrobiopterin (500 microg/min), or the combination of both compounds. Forearm vasomotion was assessed using forearm venous occlusion plethysmography and expressed as ratio of blood flow between measurement and control arm (M/C ratio). Tetrahydrobiopterin infusion alone did not alter M/C ratio. Both the attenuated L-mono-methyl-arginine-induced vasoconstriction as well as the impaired serotonin-induced vasodilation were restored in patients during tetrahydrobiopterin infusion. Tetrahydrobiopterin had no effect in controls. In conclusion, this study demonstrates restoration of endothelial dysfunction by tetrahydrobiopterin suppletion in hypercholesterolemic patients.
Research on the biological roles of nitric oxide has revealed that it functions as an important signal and effector molecule in a variety of physiologic and pathologic settings. In animals, nitric oxide is synthesized enzymatically from L-arginine through the actions of the nitric oxide synthases (NOSs). The three known NOS isoforms are all dimeric, bi-domain enzymes that contain iron protoporphyrin IX, flavin adenine dinucleotide, flavin mononucleotide, and tetrahydrobiopterin as bound prosthetic groups. This chapter summarizes information regarding the structure-function aspects of the NOSs, which includes composition of the domains, the protein residues and regions involved in prosthetic group binding, catalytic properties of the domains, the relationship between dimeric structure and prosthetic group binding and function, and factors that control assembly of NOS in cells. A general model for NOS structure and assembly is presented.
Nitric oxide synthases (NOS) are hemeproteins that catalyze oxidation of l-arginine to nitric oxide (NO) and citrulline. The NOS heme iron is expected to participate in oxygen activation during catalysis,
but its interactions with O2 are not characterized. We utilized the heme-containing oxygenase domain of neuronal NOS (nNOSoxy) and stopped-flow methods
to study formation and autooxidative decomposition of the nNOSoxy oxygenated complex at 10 °C. Mixing ferrous nNOSoxy with
air-saturated buffer generated a transient species with absorption maxima at 427 and ∼560 nm. This species decayed within
1 s to form ferric nNOSoxy. Its formation was first order with respect to O2, monophasic, and gave rate constants fork
on = 9 × 105
m
−1 s−1 andk
off = 108 s−1 for anl-arginine- and tetrahydrobiopterin (H4B)-saturated nNOSoxy. Omission of l-arginine and/or H4B did not greatly effect O2 binding and dissociation rates. Decomposition of the oxygenated intermediate was independent of O2 concentration and was either biphasic or monophasic depending on sample conditions. l-Arginine stabilized the oxygenated intermediate (decay rate = 0.14 s−1), while H4B accelerated its decay by a factor of 70 irrespective of l-arginine. The spectral and kinetic properties of the intermediate identify it as the FeIIO2 complex of nNOSoxy. Destabilization of a metallo-oxy species by H4B is unprecedented and may be important regarding the role of this cofactor in NO synthesis.
We investigated how pH affects rat brain neuronal nitric oxide synthase (nNOS) with regard to spin-state equilibrium and the thiolate ligand bond of the haem group, catalytic activity, and monomerleft and right arrow dimer equilibrium. At neutral pH, nNOS containing 1 equiv. of (6R)-5,6,7,8-tetrahydro-l-biopterin (BH4) per dimer was mostly high-spin (lambdamax at 398 nm), whereas the BH4-free enzyme consisted of a mixture of the high-spin and two low-spin forms (lambdamax at 418 nm, and at 376 and 456 nm respectively). With BH4-free nNOS, an appreciable high-spin fraction was only observed between pH 7 and 8; at pH 6 and 9, the 418 and 376/456 nm low-spin forms predominated respectively. With nNOS containing 1 equiv. of BH4 per dimer, similar observations were made, but these involved only half of the enzyme; the other half, presumably the BH4-containing subunits, remained high-spin. Since the spin state in the BH4-free subunit appeared little affected by the state of the other subunit, we conclude that, in dimeric nNOS, the two haem groups function independently. Low pH destabilized thiolate binding and the interaction between NOS subunits, as indicated by CO-binding studies and gel electrophoresis respectively. Formation of l-citrulline was optimal between pH 7.0 and 7.5; the decrease in NOS activity at lower pH proved to be due to uncoupling of NADPH oxidation, resulting in increased formation of H2O2. At high pH strict coupling of l-arginine and NADPH oxidation was maintained, even in the absence of exogenous BH4. The possible pathophysiological implications of the uncoupling at low pH are discussed.
Inducible nitric-oxide synthase (iNOS) is a hemeprotein that requires tetrahydrobiopterin (H4B) for activity. The influence of H4B on iNOS structure-function is complex, and its exact role in nitric oxide (NO) synthesis is unknown. Crystal structures of the mouse iNOS oxygenase domain (iNOSox) revealed a unique H4B-binding site with a high degree of aromatic character located in the dimer interface and near the heme. Four conserved residues (Arg-375, Trp-455, Trp-457, and Phe-470) engage in hydrogen bonding or aromatic stacking interactions with the H4B ring. We utilized point mutagenesis to investigate how each residue modulates H4B function. All mutants contained heme ligated to Cys-194 indicating no deleterious effect on general protein structure. Ala mutants were monomers except for W457A and did not form a homodimer with excess H4B and Arg. However, they did form heterodimers when paired with a full-length iNOS subunit, and these were either fully or partially active regarding NO synthesis, indicating that preserving residue identities or aromatic character is not essential for H4B binding or activity. Aromatic substitution at Trp-455 or Trp-457 generated monomers that could dimerize with H4B and Arg. These mutants bound Arg and H4B with near normal affinity, but Arg could not displace heme-bound imidazole, and they had NO synthesis activities lower than wild-type in both homodimeric and heterodimeric settings. Aromatic substitution at Phe-470 had no significant effects. Together, our work shows how hydrogen bonding and aromatic stacking interactions of Arg-375, Trp-457, Trp-455, and Phe-470 influence iNOSox dimeric structure, heme environment, and NO synthesis and thus help modulate the multiple effects of H4B.
The biosynthesis of nitric oxide (NO) is catalyzed by homodimeric NO synthases (NOS). For unknown reasons, all NOS co-purify with substoichiometric amounts of (6R)-5,6,7,8-tetrahydrobiopterin (H(4)Bip) and require additional H(4)Bip for maximal activity. We examined the effects of H(4)Bip and pterin-derived inhibitors (anti-pterins) on purified neuronal NOS-I quaternary structure and H(4)Bip content. During L-arginine turnover, NOS-I dimers time dependently dissociated into inactive monomers, paralleled by a loss of enzyme-associated pterin. Dimer dissociation was inhibited when saturating levels of H(4)Bip were added during catalysis. Similar results were obtained with pterin-free NOS-I expressed in Escherichia coli. This stabilizing effect of H(4)Bip was mimicked by the anti-pterin 2-amino-4,6-dioxo-3,4,5,6,8,8a,9, 10-octahydro-oxazolo[1,2f]-pteridine (PHS-32), which also displaced NOS-associated H(4)Bip in a competitive manner. Surprisingly, H(4)Bip not only dissociated from NOS during catalysis, but was only partially recovered in the solute (50.0 +/- 16.5% of control at 20 min). NOS-associated H(4)Bip appeared to react with a NOS catalysis product to a derivative distinct from dihydrobiopterin or biopterin. Under identical conditions, reagent H(4)Bip was chemically stable and fully recovered (95.5 +/- 3.4% of control). A similar loss of both reagent and enzyme-bound H(4)Bip and dimer content was observed by NO generated from spermine NONOate. In conclusion, we propose a role for H(4)Bip as a dimer-stabilizing factor of neuronal NOS during catalysis, possibly by interfering with enzyme destabilizing products.
Objective— Endothelial nitric oxide synthase (eNOS) activity is supported by tetrahydrobiopterin (BH4), which appears to be important for generating protective NO but decreases uncoupling formation of superoxide. We investigated the effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, or statins, in terms of BH4 metabolism in human umbilical vein endothelial cells (HUVECs).
Methods and Results— We measured the mRNA levels of GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme in the first step of de novo BH4 synthesis, by real-time polymerase chain reaction. The mRNA of GTPCH, as well as of eNOS, was upregulated in HUVECs treated with cerivastatin. This increase was time and dose dependent. Fluvastatin was also observed to enhance GTPCH and eNOS mRNA levels. In parallel with this observation, cerivastatin increased intracellular BH4. Incubating HUVECs with tumor necrosis factor (TNF-α) was observed to increase GTPCH mRNA while decreasing eNOS mRNA. In the presence of cerivastatin, the TNF-α–mediated increase in GTPCH mRNA was enhanced, and the TNF-α–mediated decrease in eNOS mRNA was attenuated. Cerivastatin increased the stability of eNOS mRNA. However, it did not alter the stability of GTPCH mRNA but increased GTPCH gene transcription, as shown by nuclear run-on assays. Preteatment of HUVECs with the selective GTPCH inhibitor, 2,4-diamino-6-hydroxypyrimidine, caused a decrease in intracellular BH4 and decreased citrulline formation after stimulation with ionomycin. Furthermore, the potentiating effect of cerivastatin was decreased by limiting the cellular availability of BH4.
Conclusions— Our data demonstrate that statins elevate GTPCH mRNA, thereby increasing BH4 levels in vascular endothelial cells. In addition to augmenting eNOS expression, statins potentiate GTPCH gene expression and BH4 synthesis, thereby increasing NO production and preventing relative shortages of BH4.
Objective: To examine the effect of tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide synthase, on coronary artery endothelial function in hypercholesterolaemic patients. Design: Quantitative coronary angiography and Doppler flowmetry were used to examine the effects of intracoronary infusion of BH4 on vascular response to acetylcholine (ACh). Setting: Tertiary cardiology centre. Patients: 18 patients with angiographically normal coronary arteries, of whom nine had hypercholesterolaemia and nine had noromocholesterolaemia. Interventions: ACh (3 and 30 μg/min) was infused for two minutes into the left coronary ostium. ACh was then simultaneously infused with BH4 (1 mg/min) before and after infusion of L-NG-monomethyl-L-arginine (L-NMMA) (40 μmol/min for five minutes). Main outcome measures: Diameter of the epicardial coronary arteries and coronary blood flow. Results: In hypercholesterolaemic patients, BH4 attenuated the ACh induced decrease in coronary diameter (p < 0.05) and restored the ACh induced increase in coronary blood flow (p < 0.05). In normocholesterolaemic patients, BH4 did not affect the ACh induced changes in coronary diameter or coronary blood flow. In both groups, L-NMMA decreased the baseline coronary diameter (p < 0.05) and baseline coronary blood flow (p < 0.05). In hypercholesterolaemic patients, L-NMMA inhibited both the BH4 mediated attenuation of the ACh induced decrease in coronary diameter (p < 0.05) and the BH4 mediated enhancement of the ACh induced increase in coronary blood flow (p < 0.01). Conclusions: Intracoronary infusion of BH4 restores coronary endothelial function by improving the bioavailability of endothelium derived nitric oxide in hypercholesterolaemic patients.
The nitric oxide synthase cofactor tetrahydrobiopterin (BH4) is involved in the regulation of endothelium-dependent vascular functions mediated by nitric oxide. Vascular endothelial cells synthesize and secrete large amounts of BH4 on cytokine activation. There is scant knowledge about molecular mechanisms of cytokine-triggered BH4 production in endothelial cells.
Pteridine production, mRNA expression of GTP cyclohydrolase (GTPCH) and 6-pyruvoyltetrahydropterin synthase (PTPS) (both key enzymes of BH4 biosynthesis), and PTPS activity were studied in human umbilical vein endothelial cells (HUVECs) exposed to inflammatory cytokines. BH4 levels were </=140-fold enhanced on treatment of HUVECs with combined interferon-gamma/tumor necrosis factor-alpha/interleukin-1 (IFN/TNF/IL-1). Specific PTPS activity was approximately 3-fold higher in cytokine-treated HUVECs than in untreated cells. Reverse-transcription/limiting-dilution polymerase chain reaction analysis showed that in response to IFN/TNF/IL-1, mRNA abundance of GTPCH and PTPS was increased approximately 64-fold and 10-fold, respectively.
The present study demonstrates for the first time the cytokine-dependent regulation of PTPS, the second enzyme in BH4 synthesis. Although GTPCH is believed to be the rate-limiting step, control of endothelial PTPS expression by cytokines may play an important role in regulating BH4-dependent nitric oxide production in the vascular system.
OBJECTIVES
We investigated whether abnormal pteridine metabolism is related to coronary endothelial dysfunction in insulin-resistant subjects.BACKGROUND
Depletion of tetrahydrobiopterin (BH4) and elevation of the 7,8-dihydrobiopterin (BH2) (activating and inactivating cofactors of nitric oxide synthase [NOS], respectively) contribute to impairment of NO-dependent vasodilation through reduction of NOS activity as well as increased superoxide anion generation in insulin-resistant rats.METHODS
Thirty-six consecutive nondiabetic, normotensive and nonobese subjects with angiographically normal coronary vessels were studied. Traditional coronary risk factors, plasma pteridine levels, activities of erythrocyte dihydropteridine reductase (DHPR), the recycling enzyme that converts BH2 to BH4 and lipid peroxide (LPO) levels were measured and coronary endothelial function was assessed with graded infusions of acetylcholine (ACh).RESULTSWhen we divided patients into tertiles based on insulin sensitivity, we observed stepwise decreases in the maximal ACh-induced vasodilation and plasma BH4/7,8-BH2 ratio, and increases in coronary LPO production as insulin sensitivity decreased. The ACh-induced vasodilation was positively correlated with insulin sensitivity, BH4/7,8-BH2 ratio and DHPR activity. Furthermore, BH4/7,8-BH2 was inversely correlated with DHPR activity and insulin sensitivity. In multiple stepwise regression analysis, BH4/BH2 was independently related to ACh-induced vasodilation and accounted for 39% of the variance. However, no significant correlation existed between other traditional risk factors and BH4/7,8-BH2.CONCLUSIONS
These results indicate that both abnormal pteridine metabolism and vascular oxidative stress are linked to coronary endothelial dysfunction in the insulin-resistant subjects.
Biopterins production during three different protocols for adoptive immunotherapy for human cancer was investigated. Adoptive immunotherapy treatment with interleukin-2 (IL-2) was carried out for 13 patients with malignant melanoma; eight with metastatic renal cell carcinoma; and three with metastatic colon cancer. The authors estimated total biopterins in plasma and lymphokine (IL-2)-activated killer cells (LAK) from these patients before and during various treatment phases to determine if increased biopterins production reflects leukocyte activation by IL-2 or antitumor activity. They noted an increased synthesis of total “biopterins,” i.e., biopterin; 7,8-dehydrobiopterin; and L-neopterin, in LAK cells and plasma which correlated with IL-2 exposure. Mean plasma biopterins were normal (1.2 ± 0.5 ng/ml) before therapy; in contrast, biopterins increased significantly to 3.4 ± 1.9 ng/ml and 3.9 ± 1.9 ng/ml during IL-2 and IL-2 + LAK treatment each, respectively. Similar biopterin elevations were noted irrespective of the different adoptive immunotherapy protocols used. Elevated biopterins decreased to normal levels (1.2 ± 0.7 ng/ml) when IL-2 treatment was omitted. Tumor regression with adoptive immunotherapy did not correlate with increased plasma biopterins. Increased biopterins production was also associated with increase in plasma catecholamine after IL-2 treatment during adoptive immunotherapy. Conceivably increased biopterins, induced by IL-2 activation of a leukocyte population, is a cell-mediated consequence not necessarily serving as a signal for the antitumor effect associated with adoptive immunotherapy.
Tetrahydrobiopterin and the folate coenzymes can reciprocally interact in ways that would be useful to the metabolic pathways subserved by both of these coenzymes. Thus, through one of the reactions catalyzed by methylene tetrahydrofolate reductase, 5-CH3-H4-folate can regenerate BH4 from q-BH2 and q-BH2 can provide an escape from the "methyl trap."
Biopterins production during three different protocols for adoptive immunotherapy for human cancer was investigated. Adoptive immunotherapy treatment with interleukin-2 (IL-2) was carried out for 13 patients with malignant melanoma; eight with metastatic renal cell carcinoma; and three with metastatic colon cancer. The authors estimated total biopterins in plasma and lymphokine (IL-2)-activated killer cells (LAK) from these patients before and during various treatment phases to determine if increased biopterins production reflects leukocyte activation by IL-2 or antitumor activity. They noted an increased synthesis of total "biopterins," i.e., biopterin; 7,8-dehydrobiopterin; and L-neopterin in LAK cells and plasma which correlated with IL-2 exposure. Mean plasma biopterins were normal (1.2 +/- 0.5 ng/ml) before therapy; in contrast, biopterins increased significantly to 3.4 +/- 1.9 ng/ml and 3.9 +/- 1.9 ng/ml during IL-2 and IL-2 + LAK treatment each, respectively. Similar biopterin elevations were noted irrespective of the different adoptive immunotherapy protocols used. Elevated biopterins decreased to normal levels (1.2 +/- 0.7 ng/ml) when IL-2 treatment was omitted. Tumor regression with adoptive immunotherapy did not correlate with increased plasma biopterins. Increased biopterins production was also associated with increase in plasma catecholamine after IL-2 treatment during adoptive immunotherapy. Conceivably increased biopterins, induced by IL-2 activation of a leukocyte population, is a cell-mediated consequence not necessarily serving as a signal for the antitumor effect associated with adoptive immunotherapy.
A hyperphenylalaninemic mouse mutant, hph-1, has been identified in the progeny of mice treated with the mutagen ethylnitrosourea. Phenylalanine hydroxylase activity levels in mutant liver lysates are reduced relative to normal, but correction for the amount of enzyme protein present demonstrates that the specific activity of this enzyme is normal in mutant mice. Quinonoid-dihydropteridine reductase activity is also normal. GTP-cyclohydrolase activity levels are essentially absent early in life and greatly diminished later in life. This finding has significant implications for the study of catecholamine neurotransmitter synthesis because GTP-cyclohydrolase catalyzes an important step in the de novo synthesis of tetrahydrobiopterin, an enzyme cofactor required for the synthesis of 3,4-dihydroxyphenylalanine (DOPA) and serotonin.
Soluble tryptophan hydroxylating enzymes have been obtained from both the beef pineal gland and rat brainstem. The enzymes have a pH optimum of 7.5 and appear to be typical aromatic ring hydroxylases. Although aniron requirement of the enzymes can be demonstrated by iron chelators, exogenous iron is not absolutely required for activity. Tetrahydrobiopterin was more effective than 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8 tetrahydropteridine as a cofactor for the enzymes. The beef pineal enzyme hydroxylates phenylalanine at approximately the same rate as it does tryptophan, whereas the brain enzyme showed no detectable activity toward phenylalanine. Finally, catechol compounds are effective inhibitors of the enzymes, presumably because of their ability to chelate iron.
Mammalian cells and tissues were found to have two pathways for the biosynthesis of tetrahydrobiopterin (BH4): (i) the conversion of GTP to BH4 by a methotrexate-insensitive de novo pathway, and (ii) the conversion of sepiapterin to BH4 by a pterin salvage pathway dependent on dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) activity. In a Chinese hamster ovary cell mutant lacking dihydrofolate reductase (DUKX-B11), endogenous formation of BH4 proceeds normally but, unlike the parent cells, these cells or extracts of them do not convert sepiapterin or 7,8-dihydrobiopterin to BH4. KB cells, which do not contain detectable levels of GTP cyclohydrolase or BH4 but do contain dihydrofolate reductase, readily convert sepiapterin to BH4 and this conversion is completely prevented by methotrexate. In supernatant fractions of bovine adrenal medulla, the conversion of sepiapterin to BH4 is completely inhibited by methotrexate. Similarly, this conversion in rat brain in vivo is methotrexate-sensitive. Sepiapterin and 7,8-dihydrobiopterin apparently do not enter the de novo pathway of BH4 biosynthesis and may be derived from labile intermediates which have not yet been characterized.
Since there is no nutritional requirement for the biopterin cofactor, we attempted to create a drug-induced deficiency in rats in order to study the role of tetrahydrobiopterin in regulating the biosynthesis of dopamine and serotonin. The hypothesis that dihydrofolate reductase (EC 1.5.1.3) mediates the final step in the de novo synthesis of tetrahydrobiopterin was tested by treating rats with methotrexate along with leucovorin as a protective agent; there was no reduction in total biopterin or in the fraction present as tetrahydrobiopterin in adrenal medulla, adrenal cortex, pituitary, brain, or pineal glands. Similar results were obtained with metoprine, a lipid-soluble inhibitor of dihydrofolate reductase which readily enters the central nervous system. Treatment with loading doses of phenylalanine along with methotrexate reduced the level of tetrahydrobiopterin in liver. Neuroblastoma N115 cells growing in medium supplemented with thymidine and hypoxanthine continued to form normal amounts of tetrahydrobiopterin in the presence of concentrations of methotrexate which completely inhibited dihydrofolate reductase; higher concentrations of methotrexate increased the tetrahydrobiopterin content of the cells 2-fold and the total biopterin in the medium 3-fold. Although attempts to create a drug-induced deficiency were unsuccessful, the evidence indicates that the de novo synthesis of tetrahydrobiopterin proceeds by a pathway independent of dihydrofolate reductase and that folate antagonists, such as methotrexate are unlikely to impair the hydroxylation of tyrosine and tryptophan, which is dependent upon the availability of the biopterin cofactor.
Guanosine triphosphate cyclohydrolase, the enzyme that is apparently rate-limiting in biopterin biosynthesis, is increased in adrenal cortex and medulla of rats treated with insulin or reserpine. Denervation and hypophysectomy block the increase in medullary and cortical enzyme activity, respectively, whereas cycloheximide presents the increase in both tissues. These results provide evidence for induction and regulation of guanosine triphosphate cyclohydrolase.
(6R)-5,6,7,8-Tetrahydro-L-biopterin(H4biopterin) is well known as a cofactor of enzymes that hydroxylate aromatic amino acids. More recent work has revealed an essential role of H4biopterin in the biosynthesis of nitric oxide (NO), an intercellular messenger molecule synthesized from L-arginine by different NO synthase isozymes in many species and tissues. While the function of H4biopterin in aromatic amino acid hydroxylation is well established, the role of this pteridine in NO synthesis is, as yet, elusive. Current experimental evidence hints at a dual mode of action of H4biopterin, involving both an allosteric effect on the NO synthase protein and participation as a reactant in L-arginine oxidation. As discussed in detail in the present article, the latter effect of this pteridine may be related to the protection of NO synthase from feedback inhibition by NO.
The murine macrophage cell line RAW 264 constitutively synthesizes tetrahydrobiopterin (BH4), the cofactor required for the hydroxylation of the aromatic amino acids and for the production of nitric oxide. Stimulation of the cells with interferon-gamma and lipopolysaccharide induced the production of nitric oxide and increased BH4 levels further. When the cells were stimulated in the presence of 2,4-diamino-6-hydroxypyrimidine (DAHP), an inhibitor of BH4 biosynthesis, biopterin levels decreased by 90% within 6 hr, whereas nitrite production was essentially unaffected. Pretreatment of the cells for 12 hr with DAHP decreased intracellular BH4 concentrations by > 95% yet inhibited the cytokine-stimulated production of nitric oxide by only 50%. However, pretreatment with DAHP plus N-acetylserotonin, an inhibitor of sepiapterin reductase, the terminal enzyme of the BH4 biosynthetic pathway, decreased biopterin levels by > 99% and inhibited nitric oxide synthesis by 90%. This inhibition could be reversed by loading the cells with dihydrobiopterin, a precursor of BH4 via the dihydrofolate reductase salvage pathway. In addition, these studies revealed that N-acetylserotonin has a direct inhibitory effect on nitric oxide synthesis, acting in a BH4-independent manner. The results presented here support previous suggestions, based on experiments with isolated enzymes, that BH4 is absolutely required for cytokine-stimulated nitric oxide production in macrophages and they suggest that only a small fraction of the total intracellular BH4 pool in macrophages is utilized in the production of fully active nitric oxide synthase.
Biosynthesis of tetrahydrobiopterin starts from guanosine triphosphate by the action of guanosine triphosphate cyclohydrolase I, which yields the first intermediate, 7,8-dihydroneopterin triphosphate. This compound is then converted by subsequent enzymes, 6-pyruvoyl tetrahydropterin synthase and sepiapterin reductase, to tetrahydrobiopterin, the biologically active metabolite. Cytokines such as gamma-interferon or tumor necrosis factor-alpha strongly stimulate the activity of guanosine triphosphate cyclohydrolase I in murine and human cells, yielding a potentiation of intracellular tetrahydrobiopterin concentrations. In human cells, particularly in human monocytes and macrophages, the low activity of 6-pyruvoyl tetrahydropterin synthase leads to the additional accumulation of neopterin derivatives, which leak from the cells after dephosphorylation and are found increased in body fluids of humans with diseases challenging cell-mediated immunity. A functional role for the stimulation of tetrahydrobiopterin biosynthesis by cytokines is the formation of a limiting cofactor required for the enzymatic conversion of L-arginine to citrulline and nitric oxide.
Smoking impairs the endothelium-dependent relaxation of arteries and veins, with the maximum relaxation in response to the calcium ionophore A23187 of saphenous vein rings being reduced from 53 +/- 4% in nonsmokers to 27 +/- 5% in smokers. We have investigated whether this endothelial dysfunction was attributable to altered activity or concentration of nitric oxide synthase (NOS). The concentration of NOS in saphenous vein endothelium, determined by Western blotting and immunohistochemistry, was not different in nonsmokers and smokers. Nitrite production from vein strips stimulated with A23187 was higher in nonsmokers (median 23.6 nmol.cm-2.h-1) than smokers (median 3.3 nmol.cm-2.h-1), P=.001, this difference being abolished when vein strips were preincubated in the presence of NG-monomethyl-L-arginine. Organ chamber studies to monitor the endothelium-dependent relaxation of vein rings in response to A23187 showed that preincubation of rings from smokers with either L-arginine (3mmol/L) or superoxide dismutase (250 U/mL) did not improve the maximum relaxation. In contrast, preincubation of vein rings from smokers with 20 micromol/L tetrahydrobiopterin increased the maximum relaxation from 27 +/- 5% to 51 +/- 6%, P=.01. Preincubation of vein from smokers with tetrahydrobiopterin also significantly increased nitrite and cGMP production in response to stimulation with A23187. The impaired endothelium-dependent relaxation of saphenous vein rings from smokers appears to be caused by a reduction in the activity of endothelial NOS that is attributable to an inadequate supply of the coenzyme tetrahydrobiopterin.
Tetrahydrobiopterin is a cofactor for nitric oxide synthase. In low concentrations of this cofactor, nitric oxide synthase is known to produce less nitric oxide and, correspondingly, enhanced quantities of the oxidant species, hydrogen peroxide. In this study, we tested the hypothesis that an exogenous tetrahydrobiopterin derivative might improve endothelial nitric oxide synthase activity in diabetic endothelium. Diabetes was induced in Sprague-Dawley rats with intravenous injections of streptozotocin. After 8 weeks, endothelium-dependent relaxation was assessed in aortic rings by using acetylcholine, whereas endothelium-independent relaxation was assessed by using nitroglycerin. Acetylcholine-induced relaxation was impaired in diabetic rings, whereas nitroglycerin-induced relaxation was unimpaired. Exposure of rings for 30 min with 100 microM of the pteridine derivative, 6-methyl-5,6,7,8-tetrahydropterin (in the presence of diethylenetriaminepentaacetic acid to inhibit oxidation), followed by washing and equilibration in control media, augmented relaxation induced by acetylcholine in diabetic rings but had no effect on relaxation in control rings. Pteridine exposure did not alter relaxation or sensitivity to nitroglycerin in control rings either with or without endothelium. In diabetic rings, pteridine exposure augmented maximal relaxation to nitroglycerin in rings with or without endothelium while increasing the sensitivity only in rings with endothelium but not in rings without endothelium. In contrast, there was no effect of pteridine exposure on relaxation or sensitivity to nitroglycerin in diabetic rings (with or without endothelium) that are pretreated with L-nitroarginine. In summary, tetrahydrobiopterin availability can play a key role in the regulation of nitric oxide production by diabetic endothelium.
The substrate binding site in nitric oxide synthase (NOS) can accommodate the physiological substrates, L-arginine and N(omega)-hydroxy L-arginine as well as many substrate analogues and inhibitors. Resonance Raman spectra of carbon monoxide-bound NOS were measured to determine how these substrates and analogues interact with heme, the prosthetic group which activates oxygen for the catalytic generation of NO and citrulline from arginine in the enzyme. Two distinct conformations of the Fe-C-O moiety were detected in the resonance Raman spectra, although in the optical absorption spectra the two species are indistinguishable. In one, termed the beta-form, the Fe-CO stretching frequency and the C-O stretching frequency, located at approximately 487 and approximately 1949 cm(-1), respectively, demonstrate that the Fe-C-O group adopts a linear conformation perpendicular to the heme plane ("open" structure). In the other, termed the alpha-form, frequencies of approximately 502 and approximately 1929 cm(-1), respectively, indicate that the binding properties of bound CO are significantly affected by its immediate environment thereby leading to a "closed" structure. In the presence of L-arginine or N(omega)-OH-L-arginine all of the molecules exhibit the closed structure, indicating that the substrates exert a strong polar (and/or steric) effect on the heme-bound ligand. In the absence of any substrate or inhibitor only half of the heme population adopts the open structure whereas the rest of the heme content retains the closed conformation. It is proposed that in this population with the closed structure tetrahydrobiopterin, a cofactor of NO synthase, may reside in close proximity to the heme-bound ligand and interact with it in a similar manner as do substrates. The inverse correlation between the Fe-CO and C-O stretching modes suggests that in NOS the bonding of the cysteine to the heme iron may be weaker, as found in chloroperoxidase, than in cytochrome P-450 enzymes. This work continually proves resonance Raman spectroscopy as a powerful probe for the interactions between substrate/inhibitor and the heme active site of proteins.
The characteristics of tetrahydrobiopterin (H4biopterin) binding to pteridine-free recombinant macrophage inducible nitric oxide synthase expressed in Escherichia coli were investigated with a special focus given to effects caused by 2,4-diamino-5,6,7, 8-tetrahydro-6-(l-erythro-1,2-dihydroxypropyl)pteridine (4-amino-H4biopterin), a novel pterin-based inhibitor of nitric oxide synthase. The 4-amino compound completely inhibited enzyme stimulation by 10 microM H4biopterin with a half-maximally active concentration of 7.2 +/- 0.39 microM, whereas H2biopterin and sepiapterin were much less potent. Binding studies using [3H]H4biopterin at 4 degrees C revealed biphasic association of the radioligand according to two first-order reactions with apparent rate constants of 2.2 and 0.05 min-1, each accounting for approximately 50% of total binding. Dissociation of [3H]H4biopterin occurred with rate constants of 0.005 and 0.0028 min-1 in the absence and presence of l-arginine, respectively. Specific binding of 10 nM [3H]H4biopterin was antagonized by unlabeled H4biopterin and its 4-amino analog with half-maximal effects at 84 +/- 6 and 34 +/- 3.2 nM, respectively. Binding of H4biopterin and 4-amino-H4biopterin was accompanied by a partial low spin to high spin conversion of the heme that was completed by l-arginine. Similarly, the active cofactor and the inhibitory 4-amino derivative both induced significant formation of stable protein dimers that survived during SDS electrophoresis, suggesting that the allosteric effects caused by H4biopterin do not explain sufficiently the essential role of the pteridine cofactor in NO biosynthesis.
We have cloned the human liver inducible isoform of nitric oxide synthase (NOS) into an Escherichia coli expression vector and have expressed and purified the enzyme. The protein has been expressed with and without a polyhistidine tail. In both cases, expression of functional protein requires coexpression with calmodulin and inclusion of tetrahydrobiopterin (H4B) in the purification buffers. Unlike the constitutive isoforms of NOS, this isoform is unstable in the absence of L-arginine (L-Arg) and H4B toward loss of the heme group and the formation of a low-spin species spectroscopically distinct from that of the cofactor-bound protein. The enzyme purified in the presence of both L-Arg and H4B is highly active, with a Vmax of approximately 800 nmol NO min(-1) mg(-1) and a Km for L-Arg of 22 microM. The cytochrome c reductase activity is 38,000 nmol x min(-1) mg(-1). Similar values are obtained for the enzyme with and without the polyhistidine tail. Ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid does not inhibit the activity of the protein, nor is the activity of the enzyme increased by the addition of exogenous calmodulin and/or Ca2+. These findings contrast with an earlier report, based on experiments with extracts of COS-1 cells expressing the recombinant enzyme, that the enzyme responds to changes in the Ca2+ concentration. The human hepatic isoform is similar in its properties to the inducible NOS isoform purified from macrophages.
Tetrahydrobiopterin is an essential cofactor required for activity of nitric oxide synthases. Existing evidence suggests that, during activation of constitutive and inducible isoforms of nitric oxide synthase, tetrahydrobiopterin is needed for allosteric and redox activation of enzymatic activity. However, precise mechanisms underlying the role of tetrahydrobiopterin in regulation of nitric oxide formation is not fully understood. In cerebral and peripheral arteries, increased availability of tetrahydrobiopterin can augment production of nitric oxide. In contrast, in arteries depleted of tetrahydrobiopterin, production of nitric oxide is impaired. Proinflammatory cytokines enhance mRNA expression of the rate-limiting enzyme of tetrahydrobiopterin biosynthesis, GTP cyclohydrolase I and stimulate production of tetrahydrobiopterin. The ability of vascular tissues to synthesize tetrahydrobiopterin plays an important role in regulation of nitric oxide synthase under physiological conditions as well as during inflammation and sepsis. More recent studies concerning expression and function of recombinant nitric oxide synthases suggest that availability of tetrahydrobiopterin is important for production of nitric oxide in genetically engineered blood vessels. In this review, mechanisms regulating availability of intracellular tetrahydrobiopterin and its role in control of vascular tone under physiological and pathological conditions will be discussed.
Nitric oxide (NO) produced by the endothelial isoform of nitric oxide synthase (NOS III) is a key determinant of the anti-atherosclerotic properties of the endothelium. Recent in vivo studies suggest that NOS III may also be a source of superoxide production, which would limit its role as a NO-producing enzyme. In the current study we examined both the NO and the superoxide generating potential of recombinant NOS III obtained from a baculovirus/Sf9 expression system. Using lucigenin chemiluminesence we could indeed demonstrate (superoxide dismutase inhibitable) superoxide production by NOS III. This superoxide production was not affected by administration of L-arginine, but could be inhibited dose-dependently by the co-factor tetrahydrobiopterin (BH4). BH4 also dose dependently decreased superoxide generation by hypoxanthine/xantine oxidase suggesting a direct antioxidant effect. Superoxide generation by NOS III could be completely inhibited by diphenyleneiodonium (DPI), an inhibitor of the flavin moiety of the enzyme, indicating that this group is a main source of superoxide production by the enzyme. Using measurement of [3H-L-arginine] conversion to [3H-L-citrulline], it appeared that BH4 directly increased the production of NO by NOS III. In addition, we observed that BH4 stablized the NOS III in its dimeric form, suggesting that an effect on allosteric conformation could be involved in this effect on NO production. NOS III thus appears to be a superoxide generating enzyme probably through its flavin moiety, as well as a BH4-dependent NO producing enzyme.
The key role of tetrahydrobiopterin (BH4) in the synthesis of nitric oxide by human umbilical vein endothelial cells (HUVEC) has been demonstrated. We characterized the induction of BH4 synthesis in a cell line (ECV) derived from HUVEC and primary HUVEC. A significant induction of guanosine triphosphate cyclohydrolase I (GTPCH) mRNA was observed in response to TNF, IL-1beta, and IFNgamma in ECV and HUVEC. The induction of GTPCH mRNA was abolished by actinomycin D. The cytokines led to an increased accumulation of BH4 in ECV. This effect was prevented by 2,4-diamino-6-hydroxypyrimidine, a selective inhibitor of GTPCH, as well as by actinomycin D and by cycloheximide. Results provide evidence for an increase in GTPCH activity and in BH4 levels in response to immunostimulants in human endothelial cells.
In vascular endothelial cells, tetrahydrobiopterin serves as an essential cofactor required for enzymatic activity of nitric oxide synthase. GTP cyclohydrolase I is the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin. Previous studies have demonstrated that proinflammatory cytokines stimulate production of tetrahydrobiopterin in endothelial cells. Long-term regulation of GTP cyclohydrolase I gene expression in endothelium has not been studied. The present study was designed to determine whether the cytokines tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (INF-gamma), and interleukin-1beta (IL-1beta) stimulate tetrahydrobiopterin synthesis by increasing expression of GTP cyclohydrolase I mRNA in endothelial cells. The relative reverse transcription polymerase chain reaction was used to quantify expression of GTP cyclohydrolase I mRNA in cultured human umbilical vein endothelial cells. Nuclear run-on assay was performed to determine the transcription rate of GTP cyclohydrolase I gene. GTP cyclohydrolase I enzymatic activity and production of tetrahydrobiopterin were measured in cell extracts. After incubation with TNF-alpha (2 microg/mL), INF-gamma (200 U/mL), and IL-1beta (5 U/mL) for 24 hours, significantly increased expression of GTP cyclohydrolase I mRNA was detected. Cytokines increased the transcription rate of GTP cyclohydrolase I 3.6-fold. This increase was associated with increased GTP cyclohydrolase I enzymatic activity and elevation of intracellular levels of tetrahydrobiopterin. An RNA synthesis inhibitor, actinomycin D (2 microg/mL), inhibited cytokine-induced expression of GTP cyclohydrolase I gene. A protein synthesis inhibitor, cycloheximide (0.5 microg/mL), did not affect expression of GTP cyclohydrolase I mRNA but blocked the increase in enzyme activity, as well as production of tetrahydrobiopterin. Incubation of endothelial cells for 24 hours in the presence of 8-bromoadenosine 3':5'-cyclic monophosphate (10[-3] mol/L) did not affect expression of GTP cyclohydrolase I mRNA. These results demonstrate that in vascular endothelial cells, cytokines increase production of tetrahydrobiopterin by stimulating expression of GTP cyclohydrolase I gene. This effect is apparently due to increased transcription rather than stabilization of mRNA. Regulation of GTP cyclohydrolase I gene expression by cytokines may play an important role in control of endothelial nitric oxide synthesis.
Insulin has been shown to elicit vasodilation through increases in nitric oxide (NO) production. To examine whether insulin may modulate the availability of tetrahydrobiopterin (BH4) (an absolute cofactor requirement for NO synthase activation), we studied the effects of insulin (150 nmol/L) on femoral arterial reactivity (to norepinephrine [NE]) in the presence and absence of 2,4-diamino-6-hydroxypyrimidine (DAHP), a specific inhibitor of BH4 production. Our data indicate that inhibition of BH4 synthesis results in an attenuation in the vasodepressor effect of insulin. One possibility is that insulin may regulate NO production by increasing cofactor (BH4) availability for activation of NO synthase.
Murine macrophage nitric oxide synthase (NOS) was expressed in E. coli and purified in the presence (holoNOS) or absence (H4B-free NOS) of (6R)-tetrahydro-L-biopterin (H4B). Isolation of active enzyme required the coexpression of calmodulin. Recombinant holoNOS displayed similar spectral characteristics and activity as the enzyme isolated from murine macrophages. H4B-free NOS exhibited a Soret band at approximately 420 nm and, by analytical gel filtration, consisted of a mixture of monomers and dimers. H4B-free NOS catalyzed the oxidation of NG-hydroxy-L-arginine (NHA) with either hydrogen peroxide (H2O2) or NADPH and O2 as substrates. No product formation from arginine was observed under either condition. The amino acid products of NHA oxidation in both the H2O2 and NADPH/O2 reactions were determined to be citrulline and Ndelta-cyanoornithine (CN-orn). Nitrite and nitrate were also formed. Chemiluminescent analysis did not detect the formation of nitric oxide (*NO) in the NADPH/O2 reaction. The initial inorganic product of the NADPH/O2 reaction is proposed to be the nitroxyl anion (NO-) based on the formation of a ferrous nitrosyl complex using the heme domain of soluble guanylate cyclase as a trap, and the formation of a ferrous nitrosyl complex of H4B-free NOS during turnover of NHA and NADPH. NO- is unstable and, under the conditions of the reaction, is oxidized to nitrite and nitrate. At 25 degreesC, the H2O2-supported reaction had a specific activity of 120 +/- 14 nmol min-1 mg-1 and the NADPH-supported reaction had a specific activity of 31 +/- 6 nmol min-1 mg-1 with a KM,app for NHA of 129 +/- 9 microM. HoloNOS catalyzed the H2O2-supported reaction with a specific activity of 815 +/- 30 nmol min-1 mg-1 and the NADPH-dependent reaction to produce *NO and citrulline at 171 +/- 20 nmol min-1 mg-1 with a KM, app for NHA in the NADPH reaction of 36.9 +/- 0.3 microM.
Subsaturating levels of tetrahydrobiopterin (BH(4)), an essential cofactor for nitric oxide synthase (NOS), can lead to endothelial dysfunction as a result of decreased production of nitric oxide. Furthermore, insufficient BH(4) can also result in NOS-uncoupled production of reactive oxygen intermediates, such as superoxide anion and hydrogen peroxide. Nitric oxide and superoxide react rapidly to form peroxynitrite, which may be the reactive species responsible for many of the toxic effects of nitric oxide. Here we show that BH(4) is a primary target for peroxynitrite-catalyzed oxidation because at pH 7.4, physiologically relevant concentrations of BH(4) are oxidized rapidly by low concentrations of peroxynitrite. Peroxynitrite oxidizes BH(4) to quinonoid 5,6-dihydrobiopterin and a large proportion of the quinonoid isomer readily loses its side chain to form 7,8-dihydropterin which is not a cofactor for nitric oxide synthase. Thus, abnormally low levels of BH(4) can promote a cycle of its own destruction mediated by nitric oxide synthase-dependent formation of peroxynitrite. This mechanism might contribute to vascular endothelial dysfunction induced by oxidative stress.