Nitric oxide improves membrane fluidity of erythrocytes in essential hypertension: An electron paramagnetic resonance investigation

It has been shown that rheological abnormality might be an etiological factor in hypertension. Recent studies have revealed that human erythrocytes possess a nitric oxide (NO) synthase and that this activation might be involved in the regulation of rheological properties of erythrocytes. The present study was undertaken to investigate the role of NO in the regulation of membrane functions of erythrocytes in patients with essential hypertension by means of an electron paramagnetic resonance (EPR) and spin-labeling method. The NO donor S-nitroso-N-acetylpenicillamine (SNAP) decreased the order parameter (S) for 5-nitroxide stearate (5-NS) and the peak height ratio (h(0)/h(-1)) for 16-NS obtained from EPR spectra of erythrocyte membranes in a dose-dependent manner. The finding indicated that the NO donor increased the membrane fluidity of erythrocytes. In addition, the effect of SNAP was significantly potentiated by 8-bromo-cyclic guanosine monophosphate. By contrast, the change of the fluidity induced by SNAP was reversed in the presence of L-N(G)-nitroarginine methyl ester and asymmetric dimethyl L-arginine. In patients with essential hypertension, the membrane fluidity of erythrocytes was significantly lower than in the normotensive subjects. The effect of SNAP was more pronounced in essential hypertension than in normotensive subjects. These results showed that NO increased the membrane fluidity and decreased the rigidity of cell membranes. Furthermore, the greater effect of NO on the fluidity in essential hypertension suggests that NO might actively participate in the regulation of rheological behavior of erythrocytes and have a crucial role in the improvement of microcirculation in hypertension.     

Comparison of wild type neuronal nitric oxide synthase and its Tyr588Phe mutant towards various l-arginine analogues

Crystal structures of nitric oxide synthases (NOS) isoforms have shown the presence of a strongly conserved heme active-site residue, Tyr588 (numbering for rat neuronal NOS, nNOS). Preliminary biochemical studies have highlighted its importance in the binding and oxidation to NO of natural substrates L-Arg and N^ω-hydroxy-l-arginine (NOHA) and suggested its involvement in mechanism. We have used UV-visible and EPR spectroscopy to investigate the effects of the Tyr588 to Phe mutation on the heme-distal environment, on the binding of a large series of guanidines and N-hydroxyguanidines that differ from L-Arg and NOHA by the nature of their alkyl- or aryl-side chain, and on the abilities of wild type (WT) and mutant to oxidize these analogues with formation of NO. Our EPR experiments show that the heme environment of the Tyr588Phe mutant differs from that of WT nNOS. However, the addition of L-Arg to this mutant results in EPR spectra similar to that of WT nNOS. Tyr588Phe mutant binds L-Arg and NOHA with much weaker affinities than WT nNOS but both proteins bind non α-amino acid guanidines and N-hydroxyguanidines with close affinities. WT nNOS and mutant do not form NO from the tested guanidines but oxidize several N-hydroxyguanidines with formation of NO in almost identical rates. Our results show that the Tyr588Phe mutation induces structural modifications of the H-bonds network in the heme-distal site that alter the reactivity of the heme. They support recent spectroscopic and mechanistic studies that involve two distinct heme-based active species in the two steps of NOS mechanism.     

Synergism between hydrogen sulfide (H(2)S) and nitric oxide (NO) in vasorelaxation induced by stonustoxin (SNTX), a lethal and hypotensive protein factor isolated from stonefish Synanceja horrida venom

Stonustoxin (SNTX) is a 148 kDa, dimeric, hypotensive and lethal protein factor isolated from the venom of the stonefish Synanceja horrida. SNTX (10-320 ng/ml) progressively causes relaxation of endothelium-intact, phenylephrine (PE)-precontracted rat thoracic aortic rings. The SNTX-induced vasorelaxation was inhibited by L-N(G)-nitro arginine methyl ester (L-NAME), suggesting that nitric oxide (NO) contributes to the SNTX-induced response. Interestingly, D, L-proparglyglycine (PAG) and beta-cyano-L-alanine (BCA), irreversible and competitive inhibitors of cystathionine-gamma-lyase (CSE) respectively, also inhibited SNTX-induced vasorelaxation, indicating that H(2)S may also play a part in the effect of SNTX. The combined use of L-NAME with PAG or BCA showed that H(2)S and NO act synergistically in effecting SNTX-induced vasorelaxation.     

Diazepam effects on carrageenan-induced inflammatory paw edema in rats: role of nitric oxide

High doses of diazepam (10.0-20.0 mg/kg) were shown to reduce the volume of acute inflammatory paw edema in rats as a response to carrageenan administration. This effect was attributed to an action of diazepam on the peripheral-type benzodiazepine receptor (PBR) present in the adrenal and/or immune/inflammatory cells. The present study was undertaken to analyze the involvement of nitric oxide (NO) on the effects of diazepam on carrageenan-induced paw edema in rats (CIPE) and to look for the presence of PBR and inducible/constitutive NO synthases (NOS) on slices taken from the inflamed paws of diazepam-treated rats. For that, an acute inhibition of NO biosynthesis was achieved using 50.0 mg/kg No mega-nitro-L-arginine (L-NAME), L-arginine (300.0 mg/kg), the true precursor of NO, and D-arginine (300.0 mg/kg), its false substrate, were also used. The following results were obtained: (1) diazepam (10.0 and 20.0 mg/kg) decreased CIPE values in a dose- and time-dependent way; (2) diazepam effects on CIPE were increased by L-NAME pretreatment; (3) treatment with L-arginine but not with D-arginine reverted at least in part the decrements of CIPE values observed after diazepam administration; (4) PBR were found in endothelial and inflammatory cells that migrated to the inflammatory site at the rat paw; (5) confocal microscopy showed the presence of both PBR and NOS in endothelial and inflammatory cells taken from inflamed paw tissues of rats treated with diazepam a finding not observed in tissues provided from rats treated with diazepam's control solution. These results suggest an important role for NO on the effects of diazepam on CIPE. Most probably, these effects reflect a direct action of diazepam on PBR present in the endothelium of the microvascular ambient and/or on immune/inflammatory cells. An action like that would lead, among other factors, to a decrease in NO, generated by NO synthase, and thus in the mechanisms responsible for CIPE.     

Low levels of mammalian TGF-beta1 are protective against malaria parasite infection, a paradox clarified in the mosquito host

Nitric oxide (NO), derived from catalysis of inducible NO synthase (iNOS), limits malaria parasite growth in mammals. Transforming growth factor (TGF)-beta1 suppresses iNOS in cells in vitro as well as in vivo in mice, but paradoxically severe malaria in humans is associated with low levels of TGF-beta1. We hypothesized that this paradox is a universal feature of infection and occurs in the mosquito Anopheles stephensi, an invertebrate host for Plasmodium that also regulates parasite development with inducible NO synthase (AsNOS). We show that exogenous human TGF-beta1 dose-dependently regulates mosquito AsNOS expression and that parasite killing by low dose TGF-beta1 depends on AsNOS catalysis. Furthermore, induction of AsNOS expression by TGF-beta1 is regulated by NO synthesis. These results suggest that TGF-beta1 plays similar roles during parasite infection in mammals and mosquitoes and that this role is linked to the effects of TGF-beta1 on inducible NO synthesis.     

Ascorbic acid synthesis due to L-gulono-1,4-lactone oxidase expression enhances NO production in endothelial cells

As a primary antioxidant, ascorbic acid (AA) provides beneficial effects for vascular health mitigating oxidative stress and endothelial dysfunction. However, the association of intracellular AA with NO production occurring inside the endothelial cells remains unclear. In the present study, we addressed this issue by increasing intracellular AA directly through de novo synthesis. To restore AA synthesis pathway, bovine aortic endothelial cells were transfected with the plasmid vector encoding L-gulono-1,4-lactone oxidase (GULO, EC 1.1.3.8), the missing enzyme converting L-gulono-1,4-lactone (GUL) to AA. Functional expression of GULO was verified by Western blotting and in vitro enzyme activity assay. GULO expression alone did not lead to AA synthesis but the supply of GUL resulted in a marked increase of intracellular AA. When the cells were stimulated with calcium ionophore, A23187, NO production was more active in the GULO-expressing cells supplied with GUL, in comparison with the cells without GULO expression or without GUL supply, indicating that intracellular AA regulated NO production. Enhancement of NO production by intracellular AA was further verified in aortic endothelial cells obtained from eNOS knockout mice that were cotransfected with eNOS and GULO constructs. GULO-dependent AA synthesis also elevated intracellular tetrahydrobiopterin content, implicating that this essential cofactor of endothelial nitric oxide synthase (eNOS) might mediate the AA effect. The present study strongly suggests that intracellular AA plays critical roles in vascular physiology through enhancing endothelial NO production.     

Mitochondrial metabolic states and membrane potential modulate mtNOS activity

The mitochondrial metabolic state regulates the rate of NO release from coupled mitochondria: NO release by heart, liver and kidney mitochondria was about 40-45% lower in state 3 (1.2, 0.7 and 0.4 nmol/min mg protein) than in state 4 (2.2, 1.3 and 0.7 nmol/min mg protein). The activity of mtNOS, responsible for NO release, appears driven by the membrane potential component and not by intramitochondrial pH of the proton motive force. The intramitochondrial concentrations of the NOS substrates, l-arginine (about 310 μM) and NADPH (1.04-1.78 mM) are 60-1000 times higher than their K_M values. Moreover, the changes in their concentrations in the state 4-state 3 transition are not enough to explain the changes in NO release. Nitric oxide release was exponentially dependent on membrane potential as reported for mitochondrial H₂O₂ production [S.S. Korshunov, V.P. Skulachev, A.A. Satarkov, High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria. FEBS Lett. 416 (1997) 15-18.]. Agents that decrease or abolish membrane potential minimize NO release while the addition of oligomycin that produces mitochondrial hyperpolarization generates the maximal NO release. The regulation of mtNOS activity, an apparently voltage-dependent enzyme, by membrane potential is marked at the physiological range of membrane potentials.     

A study of l-leucine, l-phenylalanine and l-alanine transport in the perfused rat mammary gland: possible involvement of LAT1 and LAT2

The transport of l-leucine, l-phenylalanine and l-alanine by the perfused lactating rat mammary gland has been examined using a rapid, paired-tracer dilution technique. The clearances of all three amino acids by the mammary gland consisted of a rising phase followed by a rapid fall-off, respectively, reflecting influx and efflux of the radiotracers. The peak clearance of l-leucine was inhibited by BCH (65%) and d-leucine (58%) but not by l-proline. The inhibition of l-leucine clearance by BCH and d-leucine was not additive. l-leucine inhibited the peak clearance of radiolabelled l-leucine by 78%. BCH also inhibited the peak clearance of l-phenylalanine (66%) and l-alanine (33%) by the perfused mammary gland. Lactating rat mammary tissue was found to express both LAT1 and LAT2 mRNA. The results suggest that system L is situated in the basolateral aspect of the lactating rat mammary epithelium and thus probably plays a central role in neutral amino acid uptake from blood. The finding that l-alanine uptake by the gland was inhibited by BCH suggests that LAT2 may make a significant contribution to neutral amino acid uptake by the mammary epithelium.     

Myocardial infarction increases reactivity to phenylephrine in isolated aortic rings of ovariectomized rats

Clinical studies demonstrated that the incidence of cardiovascular disease is low in premenopausal women, rises in postmenopausal women, and is reduced to premenopausal levels in postmenopausal women who receive estrogen therapy. The interaction between gender and myocardial infarction indicates that the survival advantage of women is modified by the occurrence of myocardial infarction. Therefore, the effect of myocardial infarction on mortality is greater in women than men. The aim of our study was to investigate the influence of the ovariectomy on the reactivity to phenylephrine in aortic rings of female rats post-myocardial infarction. Animals were divided in four groups: Control (Cont), Ovariectomized (Ovx), Infarcted (Inf) and Ovariectomized and Infarcted (Ovx-Inf). Aortic rings were studied 60 days after ovariectomy and infarction surgery. The infarct area was similar among groups. The maximal response to phenylephrine was increased in the Ovx-Inf group compared to all the other groups (Cont = 2.411+/-0.131 (N = 11); Ovx = 2.863+/-0.121(N = 15); Inf = 2.794+/-0.102 (N = 13); Ovx-Inf = 3.40+/-0.201* (N = 12) g; *P < 0.05). In the absence of endothelium and L-NAME perfusion, the maximal response to phenylephrine was similarly increased in all groups. Relaxation to acetylcholine was also similar. The indirect evaluation of NO bioavailability analyzed by the area under the curve demonstrated a reduction on NO on the Ovx-Inf group that could contributes to increased response to phenylephrine. In conclusion our results showed that ovariectomy associated to a myocardial infarction leads to an increment of aorta reactivity to phenylephrine associated to a reduction of basal NO bioavailability in spite of a normal endothelium-dependent relaxation induced by acetylcholine.     

Thermodynamic determination of the binding constants of angiotensin-converting enzyme inhibitors by a displacement method

Somatic angiotensin I-converting enzyme (s-ACE) plays a central role in blood pressure regulation and has been the target of most antihypertensive drugs. A displacement isothermal titration calorimetry method has been used to accurately determine the binding constant of three strong s-ACE inhibitors. Under the experimental conditions studied in this work, the relative potency of the inhibitors was determined to be enalaprilat>lisinopril>captopril. We analyze the thermodynamic behaviour of the binding process using the new structural information provided by the ACE structures, as well as the conformational changes that occur upon binding.     

Biophysical characterization of genistein–membrane interaction and its correlation with biological effect on cells — The case of EYPC liposomes and human erythrocyte membranes

With application of EPR and ¹H NMR techniques genistein interaction with liposomes formed with egg yolk lecithin and with erythrocyte membranes was assessed. The present study addressed the problem of genistein localization and its effects on lipid membrane fluidity and protein conformation. The range of microscopic techniques was employed to study genistein effects on HeLa cells and human erythrocytes. Moreover, DPPH bioassay, superoxide anion radical test and enzymatic measurements were performed in HeLa cells subjected to genistein. The gathered results from both EPR and NMR techniques indicated strong ordering effect of genistein on the motional freedom of lipids in the head group region and the adjacent hydrophobic zone in liposomal as well as in red blood cell membranes. EPR study of human ghost showed also the changes in the erythrocyte membrane protein conformation. The membrane effects of genistein were correlated with the changes in internal membranes arrangement of HeLa cells as it was noticed using transmission electron microscopic and fluorescent techniques. Scanning electron and light microscopy methods showed that one of the aftermaths of genistein incorporation into membranes was creation of echinocytic form of the red blood cells with reduced diameter. Genistein improved redox status of HeLa cells treated with H₂O₂ by lowering radicals' level.     

Reduction of Nomega-hydroxy-L-arginine to L-arginine by pig liver microsomes, mitochondria, and human liver microsomes

Nomega-Hydroxy-L-arginine, the intermediate in nitric oxide formation from L-arginine catalyzed by NO synthase, can be released into the extracellular space. It has been suggested that it can circulate and exert paracrine effects. Since it cannot only be used as substrate by NO synthases, but can also be oxidized by cytochrome P450 and other hemoproteins in a superoxide-dependent manner, it has been proposed that it can serve as NO donor. In the present study, the in vitro reduction of Nomega-hydroxy-L-arginine was examined. Pig and human liver microsomes as well as pig liver mitochondria were capable of reducing Nomega-hydroxy-L-arginine to L-arginine in an oxygen-insensitive enzymatic reaction. These results demonstrate that this metabolic pathway has to be considered when suggesting Nomega-hydroxy-L-arginine as NO-precursor. The reconstituted liver microsomal system of a pig liver CYP2D enzyme, the benzamidoxime reductase, was unable to replace microsomes to produce L-arginine from Nomega-hydroxy-L-arginine.     

Relationship between exercise intervention and NO pathway in patients with heart failure with preserved ejection fraction

Objective: Elevated levels of arginine derivatives in the NO pathway, such as asymmetric dimethylarginine (ADMA), are related to disease severity and reduced exercise capacity in heart failure (HF). We investigated the influence of exercise intervention on these parameters and on L-arginine (L-Arg) and L-homoarginine (L-hArg) in HF with preserved ejection fraction (HFpEF) patients.

Material and methods: Sixty-two patients (65?±?6 years) were included in this analysis and randomized to supervised endurance/resistance training (ET) or to usual care (UC). EDTA-plasma was analysed for NO metabolites.

Results: There were baseline associations for adjusted values of maximum workload with ADMA (r=??0.322, p?=?0.028) and L-Arg/ADMA ratio (r?=?0.331, p?=?0.015), and for the 6-min walk test (6MWT) with ADMA (r=??0.314, p?=?0.024) and L-Arg/ADMA ratio (r?=?0.346, p?=?0.015). No significant differences between UC and ET changes of NO parameters were observed at 3-month follow-up. Higher L-hArg levels were associated with a greater improvement in peak oxygen uptake (peak O₂) at follow-up: 3.4?±?2.8 vs. 1.1?±?2.9?mL/min/kg (p?=?0.005).

Conclusions: Exercise intervention did not influence NO parameters in HFpEF patients, but L-hArg was related to change in peak O₂.     

The effects on plasma L-arginine levels of combined oral L-citrulline and L-arginine supplementation in healthy males

We investigated the effects of combining 1 g of l-citrulline and 1 g of l-arginine as oral supplementation on plasma l-arginine levels in healthy males. Oral l-citrulline plus l-arginine supplementation more efficiently increased plasma l-arginine levels than 2 g of l-citrulline or l-arginine, suggesting that oral l-citrulline and l-arginine increase plasma l-arginine levels more effectively in humans when combined.     

Base catalysed isomerisation of aldoses of the arabino and lyxo series in the presence of aluminate

Base-catalysed isomerisation of aldoses of the arabino and lyxo series in aluminate solution has been investigated. L-Arabinose and D-galactose give L-erythro-2-pentulose (L-ribulose) and D-lyxo-2-hexulose (D-tagatose), respectively, in good yields, whereas lower reactivity is observed for 6-deoxy-D-galactose (D-fucose). From D-lyxose, D-mannose and 6-deoxy-L-mannose (L-rhamnose) are obtained mixtures of ketoses and C-2 epimeric aldoses. Small amounts of the 3-epimers of the ketoses were also formed. 6-Deoxy-L-arabino-2-hexulose (6-deoxy-L-fructose) and 6-deoxy-L-glucose (L-quinovose) were formed in low yields from 6-deoxy-L-mannose and isolated as their O-isopropylidene derivatives. Explanations of the differences in reactivity and course of the reaction have been suggested on the basis of steric effects.     

A specific enzymatic high-performance liquid chromatography method to determine N-methyl-D-aspartic acid in biological tissues

Recently we demonstrated that N-methyl-D-aspartic acid (NMDA) is present as an endogenous compound in the nervous tissues and endocrine glands of the rat where it plays a role in the regulation of the luteinizing hormone, growth hormone, and prolactin (FASEB J. 14 (2000) 699; Endocrinology 141 (2000) 3861). Based on the prediction that NMDA could have future importance in neuroendocrinology, we have devised an improved method for the specific and routine determination of NMDA in biological tissue. This method is based on the detection by HPLC of methylamine (CH(3)NH(2)) which comes from the oxidation of NMDA by D-aspartate oxidase, an enzyme which specifically oxidizes NMDA, yielding CH(3)NH(2) as one of the oxidative products of the reaction. The sensitivity of the method permits the accurate determination of NMDA in the supernatant of a tissue homogenate at levels of about 5-10 picomol/assay. However, for those tissues in which the concentration of NMDA is less than 1nmol/g, the sample must be further purified by treatment with o-phthaldialdehyde in order to separate the NMDA from the other amino acids and amino compounds and then concentrated and analyzed by HPLC. Using this method we have conducted a comparative study in order to measure the amount of NMDA in neuroendocrine and other tissues of various animal phyla from mollusks to mammals.     

Biochemical characterization of the HydE and HydG iron-only hydrogenase maturation enzymes from Thermatoga maritima

Fe-only hydrogenases contain a di-iron active site complex, in which the two Fe atoms have carbon monoxide and cyanide ligands and are linked together by a putative di(thiomethyl)amine molecule. We have cloned, purified and characterized the HydE and HydG proteins, thought to be involved in the biosynthesis of this peculiar metal site, from the thermophilic organism Thermotoga maritima. The HydE protein anaerobically reconstituted with iron and sulfide binds two [4Fe-4S] clusters, as characterized by UV and EPR spectroscopy. The HydG protein binds one [4Fe-4S] cluster, and probably an additional one. Both enzymes are able to reductively cleave S-adenosylmethionine (SAM) when reduced by dithionite, confirming that they are Radical-SAM enzymes.     

Sex differences in modulating blood brain barrier permeability by NO in pentylenetetrazol-induced epileptic seizures

Susceptibility to epilepsy as well as BBB dysfunction in some pathological conditions varies depending on sex difference. It has recently been shown that systemically given NO donor and antagonists modify the nature of seizures induced by PTZ (pentylenetetrazol) differently in male and female rats. This study investigates the role of NO on BBB permeability in PTZ seizures with sex differences using NO donor, sodium nitroprusside (SNP), and NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME). Nitrite+nitrate levels as indices of NO generation in the brain were also assessed. L-NAME prolonged seizure latency in male rats, seizure intensity and seizure duration were lessened. L-NAME depicted opposite effects in seizure nature in female rats. SNP prolonged seizure latency, while seizure intensity and duration were lessened only in female rats. L-NAME in male rats increased L-NAME use in female rats (not in male rats) which resulted in a more leaky BBB especially in midbrain, thalamus, hippocampus, corpus striatum and cerebellum whereas SNP use in male rats and not in female rats resulted in pronounced BBB opening in all brain regions studied than PTZ per se. L-NAME while decreasing nitrite+nitrate levels in male rat brains, acted in an opposite fashion in females. SNP use depicted an inverse picture with respect to L-NAME, with an opposite action in different sexes. This study reveals that NO effect on BBB in PTZ-induced seizures depends unequivocally on sex difference. The sex-dependent action of NO in seizures and in CNS pathologies warrants further investigation.     

Efficient synthesis of 2-deoxy-L-erythro-pentose (2-deoxy-L-ribose) from L-arabinose

An efficient and practical route for the large-scale synthesis of 2-deoxy-L-erythro-pentose (2-deoxy-L-ribose) starting from L-arabinose was developed using Barton-type free-radical deoxygenation reaction as a key step. The radical precursor, a phenoxythiocarbonyl ester, was prepared in situ, and the most efficient deoxygenation was achieved by slow addition of tributyltin hydride to the reaction mixture.