(±)-Praeruptorin A enantiomers exert distinct relaxant effects on isolated rat aorta rings dependent on endothelium and nitric oxide synthesis

Praeruptorin A is a coumarin compound naturally occurring in the roots of Peucedanum praeruptorum Dunn., a commonly used traditional Chinese medicine for the treatment of certain respiratory diseases and hypertension. Although previous studies indicated the relaxant effects of (±)-praeruptorin A on tracheal and arterial preparations, little is known about the functional characteristics of the enantiomers. In the present study, the two enantiomers were successfully isolated and identified by using a preparative Daicel Chiralpak AD-H column, and their relaxant effects on aorta rings were observed and compared. (+)-Praeruptorin A showed more potent relaxation than (?)-praeruptorin A against KCl- and phenylephrine-induced contraction of rat isolated aortic rings with intact endothelium. Removal of the endothelium remarkably reduced the relaxant effect of (+)-praeruptorin A but not that of (?)-praeruptorin A. Pretreatment of aortic rings with N^ω-nitro-l-arginine methyl ester (l-NAME, an inhibitor of nitric oxide synthase) or methylene blue (MB, a soluble guanylyl cyclase inhibitor) resulted in similar changes of the relaxant effects of the two enantiomers to endothelium removal. Molecular docking studies also demonstrated that (+)-praeruptorin A was in more agreement to nitric oxide synthase pharmacophores than (?)-praeruptorin A. On the other hand, the two enantiomers of praeruptorin A could slightly attenuate the contraction of rat aortic rings induced by internal Ca²⁺ release from sarcoplasmic reticulum (SR). These findings indicated that (+)-praeruptorin A and (?)-praeruptorin A exerted distinct relaxant effects on isolated rat aorta rings, which might be mainly attributed to nitric oxide synthesis catalyzed by endothelial nitric oxide synthase.     

Killing of Bacillus spores is mediated by nitric oxide and nitric oxide synthase during glycoconjugate–enhanced phagocytosis

Nitric oxide (NO) is a signaling and defense molecule of major importance. NO endows macrophages with bactericidal, cytostatic as well as cytotoxic activity against various pathogens. Bacillus spores can produce serious diseases, which might be attenuated if macrophages were able to kill the spores on contact. Present research was carried out to study whether glycoconjugates stimulated NO and nitric oxide synthase (NOS2) production during phagocytosis killing of Bacillus spores. Murine macrophages exposed to glycoconjugate-treated spores induced NOS2 and NO production that was correlated with high viability of macrophages and killing rate of bacterial spores. Increased levels of inducible NOS2 and NO production by macrophages in presence of glycoconjugates suggested that the latter provide an activation signal directed to macrophages. Glycoconjugates were shown to exert a protective influence, sparing macrophages from spore-induced cell death. In presence of glycoconjugates, macrophages efficiently kill the organisms. Without glycoconjugate activation, murine macrophages were ineffective at killing Bacillus spores. These results suggest that glycoconjugates promote killing of Bacillus spores by blocking spore-induced macrophage cell death, while increasing their activation level and NO and NOS2 production. Glycoconjugates suggest novel antimicrobial approaches to prevention and treatment of infection caused by bacterial spores.     

α-Glucosidase inhibitory and nitric oxide production inhibitory activities of alkaloids isolated from a twig extract of Polyalthia cinnamomea

The first phytochemical investigation of Polyalthia cinnamomea led to the isolation and identification of two new oxoprotoberberine alkaloids, (−)-(13aS)-polyalthiacinnamines A and B, together with eleven known compounds. The structures of the new compounds were elucidated by extensive spectroscopic methods. The absolute configuration of miliusacunine E and consanguine B was established by X-ray diffraction analysis using Cu Kα radiation and ECD spectra, whereas the absolute configurations of polyalthiacinnamines A and B were established by comparison of their ECD spectra and specific rotations with those of miliusacunine E and consanguine B. Compounds 1–4, 6, and 8 exhibited α-glucosidase inhibitory activities (IC₅₀ values ranging from 11.3 to 57.9 µM) better than a positive control (acarbose, IC₅₀ 83.5 μM). Compound 2 also exhibited NO production inhibitory activity with an IC₅₀ value of 24.4 μM (indomethacin, a positive control, IC₅₀ = 32.2 μM).     

Anti-inflammatory effect of soyasaponins through suppressing nitric oxide production in LPS-stimulated RAW 264.7 cells by attenuation of NF-κB-mediated nitric oxide synthase expression

The anti-inflammatory properties of soyasaponins (especially soyasaponins with different chemical structures) have scarcely been investigated. We investigated the inhibitory effects of five structural types of soyasaponins (soyasaponin A¹, A², I and soyasapogenol A, B) on the induction of nitric oxide (NO) and inducible NO synthase (iNOS) in murine RAW 264.7 cells activated with lipopolysaccharide (LPS). Soyasaponin A¹, A² and I (25-200 μg/mL) dose-dependently inhibited the production of NO and tumor necrosis factor α (TNF-α) in LPS-activated macrophages, whereas soyasapogenol A and B did not. Furthermore, soyasaponin A¹, A² and I suppressed the iNOS enzyme activity and down-regulated the iNOS mRNA expression both in a dose-dependent manner. The reporter gene assay revealed that soyasaponin A¹, A² and I decreased LPS-induced nuclear factor kappa B (NF-κB) activity. Soyasaponin A¹, A² and I exhibit anti-inflammatory properties by suppressing NO production in LPS-stimulated RAW 264.7 cells through attenuation of NF-κB-mediated iNOS expression. It is proposed that the sugar chains present in the structures of soyasaponins are important for their anti-inflammatory activities. These results have important implication for using selected soyasaponins towards the development of effective chemopreventive and anti-inflammatory agents.     

Why is the reduction of NO in cytochrome c dependent nitric oxide reductase (cNOR) not electrogenic?

The membrane-bound enzyme cNOR (cytochrome c dependent nitric oxide reductase) catalyzes the reduction of NO in a non-electrogenic process. This is in contrast to the reduction of O₂ in cytochrome c oxidase (CcO), the other member of the heme-copper oxidase family, which stores energy by the generation of a membrane gradient. This difference between the two enzymes has not been understood, but it has been speculated to be of kinetic origin, since per electron the NO reduction is more exergonic than the O₂ reduction, and the energy should thus be enough for an electrogenic process. However, it has not been clear how and why electrogenicity, which mainly affects the thermodynamics, would slow down the very exergonic NO reduction. Quantum chemical calculations are used to construct a free energy profile for the catalytic reduction of NO in the active site of cNOR. The energy profile shows that the reduction of the NO molecules by the enzyme and the formation of N₂O are very exergonic steps, making the rereduction of the enzyme endergonic and rate-limiting for the entire catalytic cycle. Therefore the NO reduction cannot be electrogenic, i.e. cannot take electrons and protons from the opposite sides of the membrane, since it would increase the endergonicity of the rereduction when the gradient is present, thereby increasing the rate-limiting barrier, and the reaction would become too slow. It also means that proton pumping coupled to electron transfer is not possible in cNOR. In CcO the corresponding rereduction of the enzyme is very exergonic.     

Activation of macrophage nuclear factor-κB and induction of inducible nitric oxide synthase by LPS

Background  

Chronic lung disease (CLD) of prematurity is a major problem of neonatal care. Bacterial infection and inflammatory response have been thought to play an important role in the development of CLD and steroids have been given, with some benefit, to neonates with this disease. In the present study, we assessed the ability of lipopolysaccharide (LPS) to stimulate rat alveolar macrophages to produce nitric oxide (NO), express inducible nitric oxide synthase (iNOS) and activate nuclear factor-κB (NF-κB) in vitro. In addition, we investigated the impact of dexamethasone and budesonide on these processes.     

Gastroprotective mechanisms of Citrus lemon (Rutaceae) essential oil and its majority compounds limonene and β-pinene: involvement of heat-shock protein-70, vasoactive intestinal peptide, glutathione, sulfhydryl compounds, nitric oxide and prostaglandin E₂

Citrus lemon (CL) belongs to Rutaceae family and is popularly known in Brazil as limão siciliano. The phytochemical analysis of CL fruit bark essential oil showed two majority components, limonene (LIM) and β-pinene (PIN). This study aimed to evaluate the gastroprotective mechanism of action from CL, LIM and PIN in ethanol- and indomethacin-induced gastric ulcers and its in vitro anti-Helicobacter pylori activity. After ethanol-induced gastric ulcer, the ulcer area was measured and the stomachs were destined to histology (HE and PAS), immunohistochemistry for HSP-70 and VIP and glutathione (GSH) measurement. The involvement of nitric oxide (NO) and sulfhydryl (SH) compounds was determined. The ulcer area for indomethacin-induced gastric ulcers was measured. PGE₂ concentration was biochemically measured. The minimum inhibitory concentration (MIC) against H. pylori was determined in vitro. In ethanol model, CL and LIM demonstrated 100% of gastroprotection, while PIN did not exert effective gastroprotection (53.26%). In the indomethacin model, CL and LIM offered effective gastroprotection but PIN did not show gastroprotective effect. The gastric ulcer area of rats pretreated with NO-synthase inhibitor or SH-blocker was decreased in comparison to the control group. The MIC obtained for CL was 125 μg/mL, for LIM was 75 μg/mL and for PIN was 500 μg/mL. The gastroprotective effect of CL and LIM was involved with increasing in mucus secretion, HSP-70 and VIP, but not with GSH, NO or SH compounds. CL gastroprotective mechanism is involved with PGE₂. PIN did not present gastroprotective activity.     

Arbuscular mycorrhizal symbiosis modifies the effects of a nitric oxide donor (sodium nitroprusside;SNP) and a nitric oxide synthesis inhibitor (Nω-nitro-L-arginine methyl ester;L-NAME) on lettuce plants under well watered and drought conditions

Arbuscular mycorrhizal (AM) symbiosis is known to help the host plant to overcome environmental stresses as drought by a combination of multiple mechanisms including enhancing of root water uptake capacity. On the other hand, Nitric oxide (NO) is involved in regulating the response of plants to environmental stresses and colonization process of AM fungi. The objective of this research was to study how AM and non-AM lettuce plants responded to a NO donor (sodium nitroprusside; SNP) or to a NO synthesis inhibitor (Nω-nitro-L-arginine methyl ester hydrochloride; L-NAME) under well watered and drought conditions. Most remarkable results were that L-NAME increased the percentage of AM colonized roots under both water regimes and AM plants modified the shoot:root ratio by both chemicals under well watered conditions. Also, the deleterious effects of SNP treatment were partially prevented by AM symbiosis. Moreover, NO could be involved in the diminution of leaf water content under drought conditions, and SNP treatment seems to favor apoplastic water path inside roots. Therefore, different outcomes of relative water content, stomatal conductance and root hydraulic conductivity observed between AM and non-AM plants could be mediated by NO.     

Expression of the cystine-glutamate exchanger (xc −) in retinal ganglion cells and regulation by nitric oxide and oxidative stress

The cystine-glutamate exchanger, system x_c ⁻, mediates the Na⁺-independent exchange of cystine into cells, coupled to the efflux of intracellular glutamate. System x_c ⁻ plays a critical role in glutathione homeostasis. Early studies of brain suggested that system x_c ⁻ was present primarily in astrocytes but not neurons. More recent work indicates that certain brain neurons have an active system x_c ⁻. In the retina, system x_c ⁻ has been demonstrated in Müller and retinal pigment epithelial cells. We have recently suggested that two protein components of system x_c ⁻, xCT and 4F2hc, are present in ganglion cells of the intact retina. Here, we have used (1) molecular and immunohistochemical assays to determine whether system x_c ⁻ is present in primary ganglion cells isolated from neonatal mouse retinas and (2) functional assays to determine whether its activity is regulated by oxidative stress in a retinal ganglion cell line (RGC–5). Primary mouse ganglion cells and RGC–5 cells express xCT and 4F2hc. RGC–5 cells take up [³H]glutamate in the absence of Na⁺, and this uptake is blocked by known substrates of system x_c ⁻ (glutamate, cysteine, cystine, quisqualic acid). Treatment of RGC–5 cells with NO and reactive oxygen species donors leads to increased activity of system x_c ⁻ associated with an increase in the maximal velocity of the transporter with no significant change in the substrate affinity. This is the first report of system x_c ⁻ in primary retinal ganglion cells and RGC–5 cells. Oxidative stress upregulates this transport system in RGC–5 cells, and the process is associated with an increase in xCT mRNA and protein but no change in 4F2hc mRNA or protein. This work was supported by National Institutes of Health grants EY014560 and EY012830.     

Regulation of glycolysis and pentose–phosphate pathway by nitric oxide: Impact on neuronal survival

Besides its essential role at regulating neural functions through cyclic GMP, nitric oxide is emerging as an endogenous physiological modulator of energy conservation for the brain. Thus, nitric oxide inhibits cytochrome c oxidase activity in neurones and glia, resulting in down-regulation of mitochondrial energy production. The subsequent increase in AMP facilitates the activation of 5′-AMP-dependent protein kinase, which rapidly triggers the activation of 6-phosphofructo-1-kinase – the master regulator of the glycolytic pathway – and Glut1 and Glut3 — the main glucose transporters in the brain. In addition, nitric oxide activates glucose-6-phosphate dehydrogenase, the first and rate-limiting step of the pentose–phosphate pathway. Here, we review recent evidences suggesting that nitric oxide exerts a fine control of neuronal energy metabolism by tuning the balance of glucose-6-phosphate consumption between glycolysis and pentose–phosphate pathway. This may have important implications for our understanding of the mechanisms controlling neuronal survival during oxidative stress and bioenergetic crisis.     

Coordinated regulation of endothelial calcium signaling and shear stress-induced nitric oxide production by PKCβ and PKCη

BackgroundProtein Kinase C (PKC) is a promiscuous serine/threonine kinase regulating vasodilatory responses in vascular endothelial cells. Calcium-dependent PKCbeta (PKCβ) and calcium-independent PKCeta (PKCη) have both been implicated in the regulation and dysfunction of endothelial responses to shear stress and agonists.ObjectiveWe hypothesized that PKCβ and PKCη differentially modulate shear stress-induced nitric oxide (NO) production by regulating the transduced calcium signals and the resultant eNOS activation. As such, this study sought to characterize the contribution of PKCη and PKCβ in regulating calcium signaling and endothelial nitric oxide synthase (eNOS) activation after exposure of endothelial cells to ATP or shear stress.MethodsBovine aortic endothelial cells were stimulated in vitro under pharmacological inhibition of PKCβ with LY333531 or PKCη targeting with a pseudosubstrate inhibitor. The participation of PKC isozymes in calcium flux, eNOS phosphorylation and NO production was assessed following stimulation with ATP or shear stress.ResultsPKCη proved to be a robust regulator of agonist- and shear stress-induced eNOS activation, modulating calcium fluxes and tuning eNOS activity by multi-site phosphorylation. PKCβ showed modest influence in this pathway, promoting eNOS activation basally and in response to shear stress. Both PKC isozymes contributed to the constitutive and induced phosphorylation of eNOS. The observed PKC signaling architecture is intricate, recruiting Src to mediate a portion of PKCη's control on calcium entry and eNOS phosphorylation. Elucidation of the importance of PKCη in this pathway was tempered by evidence of a single stimulus producing concurrent phosphorylation at ser1179 and thr497 which are antagonistic to eNOS activity.ConclusionsWe have, for the first time, shown in a single species in vitro that shear stress- and ATP-stimulated NO production are differentially regulated by classical and novel PKCs. This study furthers our understanding of the PKC isozyme interplay that optimizes NO production. These considerations will inform the ongoing design of drugs for the treatment of PKC-sensitive cardiovascular pathologies.     

Porcine oviduct sperm binding glycoprotein and its deleterious effect on sperm: a mechanism for negative selection of sperm?

In their journey through the oviduct some subpopulations of sperm are preserved in a reservoir, while others are negatively selected. Sperm binding glycoprotein (SBG) is a pig oviductal epithelial cell glycoprotein that produces, under capacitating conditions, acrosome alteration, p97 tyrosine-phosphorylation and reduction of the motility of sperm. In this paper, we show that SBG is accessible at the extracellular surface of the oviductal epithelial cells, supporting a sperm interaction biological role in situ. We analyze the possible dependence of the tyrosine-phosphorylation of p97 on the PKA mechanism, finding that apparently it is not PKA dependent. Also, after SBG treatment the phosphorylated proteins locate mainly at the detached periacrosomal region and at the tail of sperm; the latter may be related to SBG's motility reduction effect. The study of the time course effect of SBG on sperm as detected by chlortetracycline (CTC) staining and of its binding to sperm by immunodetection in conjunction with CTC, shows results in agreement with the hypothesis that this glycoprotein is involved in the alteration of acrosomes in a specific sperm subpopulation. The results suggest that SBG may be part of a mechanism for negative selection of sperm.     

Effect of an inhibitor of nitric oxide production on Cu−Zn/SOD and its cofactors in diabetic rats

The aim of this study was to investigate the effect of an inhibitor of nitric oxide production, N(omega)-nitro-L-arginine methyl esther (L-NAME) on Cu-Zn/SOD (superoxide dismutase) enzyme activity and copper and zinc concentrations in diabetes-induced rats. The control group consisted of 12 male albino Sprague-Dawley rats, 10-12 wk of age and weighing 300 g. Twenty-six albino Sprague-Dawley rats, 10-12 wk of age and weighing 315 g, constituted the experimental group. The experimental group was divided into two groups. The first group (n=12) constituted streptozotocininduced (55 mg/kg, intraperitoneally) diabetic rats and the second group (n=14) was administered L-NAME (1 mg/kg/d) after streptozotocin induction. For determination of Cu-Zn/SOD activity, spectrophotometry was used. Zinc and copper concentrations were determined by atomic absorption spectrophotometry. Results showed that Cu-Zn/SOD activity was increased significantly in both experimental groups compared to controls, and the increase in the second group was higher than in the first group (p<0.01, p<0.01, p<0.05). Plazma zinc concentration was increased in the second group when compared with controls (p<0.05). Plasma copper was decreased significantly in the second group compared to controls and the first group (p<0.001, p<0.001). Red cell copper concentration was decreased significantly in the first group compared to controls (p<0.05). This study showed that L-NAME administration has ensured an additive effect on the antioxidant defense system, which was proved by the increase in Cu-Zn/SOD activity. This increase might have a protective effect against tissue damage in the acute period, with corresponding changes in zinc and copper concentrations.     

Are cyclooxygenase-2 and nitric oxide involved in the dyskinesia of Parkinson's disease induced by l-DOPA?

Inflammatory mechanisms are proposed to play a role in l-DOPA-induced dyskinesia. Cyclooxygenase-2 (COX2) contributes to inflammation pathways in the periphery and is constitutively expressed in the central nervous system. Considering that inhibition of nitric oxide (NO) formation attenuates l-DOPA-induced dyskinesia, this study aimed at investigating if a NO synthase (NOS) inhibitor would change COX2 brain expression in animals with l-DOPA-induced dyskinesia. To this aim, male Wistar rats received unilateral 6-hydroxydopamine microinjection into the medial forebrain bundle were treated daily with l-DOPA (21 days) combined with 7-nitroindazole or vehicle. All hemi-Parkinsonian rats receiving l-DOPA showed dyskinesia. They also presented increased neuronal COX2 immunoreactivity in the dopamine-depleted dorsal striatum that was directly correlated with dyskinesia severity. Striatal COX2 co-localized with choline-acetyltransferase, calbindin and DARPP-32 (dopamine-cAMP-regulated phosphoprotein-32), neuronal markers of GABAergic neurons. NOS inhibition prevented l-DOPA-induced dyskinesia and COX2 increased expression in the dorsal striatum. These results suggest that increased COX2 expression after l-DOPA long-term treatment in Parkinsonian-like rats could contribute to the development of dyskinesia.     

N(α)-Acetylation of yeast ribosomal proteins and its effect on protein synthesis

N(α)-Acetyltransferases (NATs) cause the N(α)-acetylation of the majority of eukaryotic proteins during their translation, although the functions of this modification have been largely unexplored. In yeast (Saccharomyces cerevisiae), four NATs have been identified: NatA, NatB, NatC, and NatD. In this study, the N(α)-acetylation status of ribosomal protein was analyzed using NAT mutants combined with two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry (MS). A total of 60 ribosomal proteins were identified, of which 17 were N(α)-acetylated by NatA, and two by NatB. The N(α)-acetylation of two of these, S17 and L23, by NatA was not previously observed. Furthermore, we tested the effect of ribosomal protein N(α)-acetylation on protein synthesis using the purified ribosomes from each NAT mutant. It was found that the protein synthesis activities of ribosomes from NatA and NatB mutants were decreased by 27% and 23%, respectively, as compared to that of the normal strain. Furthermore, we have shown that ribosomal protein N(α)-acetylation by NatA influences translational fidelity in the presence of paromomycin. These results suggest that ribosomal protein N(α)-acetylation is necessary to maintain the ribosome's protein synthesis function.     

Can phosphorylation of phosphatidate phosphohydrolase by a cyclic AMP-dependent mechanism regulate its activity and subcellular distribution and control hepatic glycerolipid synthesis?

Incubating the particle-free supernatant of rat liver with alkaline phosphatase decreased the activity of phosphatidate phosphohydrolase by 21-29%. When the particle-free supernatant was incubated with various combinations of Mg2+, ATP, cyclic AMP and cyclic AMP-dependent protein kinase this failed to alter significantly phosphatidate phosphohydrolase activity under the conditions employed. The incubation of hepatocytes in monolayer culture with 0.5 mM-8-(4-chlorophenylthio)adenosine 3',5'-monophosphate increased the total activity of phosphatidate phosphohydrolase as measured in vitro. This also decreased the proportion of the phosphohydrolase that was associated with the membrane fraction of the cells and increased that in the cytosolic fraction. Adding 1 mM-oleate to the hepatocytes promoted the translocation of phosphatidate phosphohydrolase from the cytosol to the membrane-associated compartment. Oleate overcame the effect of the cyclic AMP analogue in favouring the cytosolic distribution of the phosphohydrolase. These results are discussed in relation to the interaction of hormonal balance and substrate supply in controlling the synthesis of phosphatidylcholine and triacylglycerol in the liver in stress and in diabetes. It is proposed that the cytosolic phosphatidate phosphohydrolase activity represents a reservoir of potential activity that becomes expressed when the enzyme translocates to the membranes on which the synthesis of glycerolipids occurs.