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.     

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.     

l-Lactate generates hydrogen peroxide in purified rat liver mitochondria due to the putative l-lactate oxidase localized in the intermembrane space

In order to ascertain whether and how mitochondria can produce hydrogen peroxide (H₂O₂) as a result of l-lactate addition, we monitored H₂O₂ generation in rat liver mitochondria and in submitochondrial fractions free of peroxisomal and cytosolic contamination. We found that H₂O₂ is produced independently on the respiratory chain with 1:1 stoichiometry with pyruvate, due to a putative flavine-dependent l-lactate oxidase restricted to the intermembrane space. The l-lactate oxidase reaction shows a hyperbolic dependence on l-lactate concentration and is inhibited by NAD⁺ in a competitive manner, being the enzyme different from the l-lactate dehydrogenase isoenzymes as shown by their pH profiles.     

The regulatory effect of hydrogen sulfide on hypoxic pulmonary hypertension in rats

Hypoxic pulmonary hypertension (HPH) is an important pathophysiological process. The mechanism of HPH is still not fully understood. Recent studies showed that hydrogen sulfide (H(2)S) could relax vascular smooth muscles and inhibit the proliferation of cultured vascular smooth muscle cells. Our study showed that both the gene expression of cystathionine gamma-lyase (CSE), one of the H(2)S generating enzymes, and the activity of CSE were suppressed in lung tissues during HPH. And the plasma level of H(2)S was decreased during HPH. Exogenous supply of H(2)S could increase the plasma level of H(2)S, enhance CSE activity, and up-regulate CSE gene expression in lung tissue. At the same time, exogenous supply of H(2)S could oppose the elevation of pulmonary arterial pressure and lessen the pulmonary vascular structure remodeling during HPH. The results showed that endogenous H(2)S system was involved and exogenous H(2)S could exert beneficial effect on the pathogenesis of HPH.     

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.     

The regulatory effect of endogenous hydrogen sulfide on pulmonary vascular structure and gasotransmitters in rats with high pulmonary blood flow

The study aimed to explore the regulatory effect of endogenous hydrogen sulfide (H(2)S), a novel gasotransmitter, on pulmonary vascular structure and gasotransmitters in rats with high pulmonary blood flow. Thirty-two Sprague-Dawley rats were randomly divided into a sham group, shunt group, sham+PPG (propargylglycine, an inhibitor of cystathionine-gamma-lyase) group and shunt+PPG group. Rats in the shunt and shunt+PPG groups underwent abdominal aorta-inferior vena cava shunting. Rats in the shunt+PPG and sham+PPG groups were intraperitoneally injected with PPG. After 4 weeks of shunting, mean pulmonary artery pressure (MPAP) and pulmonary vascular structural remodeling (PVSR) were evaluated. H(2)S, nitric oxide (NO) and carbon monoxide (CO) contents were measured in lung tissues. Meanwhile, nitric oxide synthase (eNOS), heme oxygenase (HO-1) and proliferative cell nuclear antigen (PCNA) protein expressions and ERK activation were evaluated. After 4 weeks of shunting, rats showed PVSR with increased lung tissue H(2)S and NO content but decreased CO content. After the PPG treatment, MPAP further increased and PVSR was aggravated. Meanwhile, PCNA expression and ERK activation were augmented with decreased lung tissue CO and HO-1 protein production but increased lung tissue NO production and eNOS expression. H(2)S exerted a protective effect on PVSR, and the inhibition of the NO/NOS pathway and the augmentation of the CO/HO pathway might be involved in the mechanisms by which H(2)S regulates PVSR in rats with high pulmonary flow.     

内源性硫化氢在大鼠早期高肺血流性肺动脉高压中的动态变化

目的:探讨大鼠早期高肺血流性肺动脉高压形成中内源性硫化氢体系的动态变化规律。方法:雄性SD大鼠80只,体重140~160 g,随机分为分流组(40只)和对照组(40只)。分流组大鼠经下腔静脉-腹主动脉穿刺术建立高肺血流动物模型。分别在术后1 d、3 d、1周、4周及8周各实验时间点测量肺动脉收缩压(SPAP)、肺组织匀浆中硫化氢(H2S)含量及CSE mRNA相对含量。结果:SPAP于分流后1周开始升高,8周明显升高;肺组织H2S及CSE mRNA含量于分流后3 d及4周显著升高;SPAP与肺组织H2S、CSEm RNA含量于分流后1周、4周及8周呈明显负相关。结论:大鼠高肺血流性动物模型可导致肺动脉高压,早期伴随内源性肺组织H2S及CSEmRNA含量的变化,提示内源性H2S体系可能与高肺血流性肺动脉高压形成有关,并在其中发挥保护性的调节作用。     

Chitosan induces different L-arginine metabolic pathways in resting and inflammatory macrophages

Chitosan is a linear polymer of N-acetyl-D-glucosamine and deacetylated glucosamine widely used as a wound-healing accelerator in clinical and veterinary medicine. Chitosan enhances the functions of inflammatory cells such as macrophages (Mphi), inducing the production of cytokines as well as the expression of activation markers, Fc receptors and mannose receptor. In this work we studied the effects of chitosan on the arginine metabolic pathways of both resident and inflammatory (proteose-peptone elicited) rat Mphi. Our results show that low molecular weight (LMW) chitosan activated moderately both the inducible nitric oxide synthase (iNOS) and arginase pathways in resident Mphi. In inflammatory Mphi treated with chitosan instead, the arginase activity was strongly enhanced. Supernatants of chitosan-stimulated Mphi enhanced the proliferation of the rat cell line C6. These findings suggest that the healing activity of chitosan could rely on the enhanced arginase activity observed in a wound-associated inflammatory milieu.     

Interaction of L-arginine with dihexadecylphosphate unilamellar liposomes: the effect of the lipid phase organization

The interaction of L-arginine with unilamellar liposomes of dihexadecylphosphate sodium salt (DHP-Na) has been investigated using calorimetric, light scattering, fluorescence spectroscopy and zeta-potential techniques. Heating from room temperature, the bilayer exhibits a phase transition from a subgel (L(c)) to the gel (L(beta')) phase as well as a pre-transition (L(beta')-P(beta')), which is followed by the main lipid phase transition (P(beta')-L(alpha)). Direct studies of the interaction of L-arginine with the DHP-Na bilayers via isothermal titration calorimetry at 27 degrees C depict significant differences between samples in the L(c) and the L(beta') phases reflecting the effect of molecular organization of the lipids upon the interaction. While L-arginine has only a small impact upon the L(c) to L(beta') phase transition, it affects more significantly the transition temperature as well as the shape of the DSC peaks of the main lipid phase transition. Based on fluorescence and zeta-potential studies, the permeability of L-arginine through the liposomal membrane is higher within the temperature range of the main lipid phase transition. Encapsulated l-arginine obstructs the formation of the subgel phase.     

Glucose-induced production of hydrogen sulfide may protect the pancreatic beta-cells from apoptotic cell death by high glucose

We examined the expression of the major H₂S-producing enzymes, cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE). CBS was ubiquitously distributed in the mouse pancreas, but CSE was found only in the exocrine. Freshly isolated islets expressed CBS, while CSE was faint. However, high glucose increased the CSE expression in the beta-cells. l-Cysteine or NaHS suppressed islet cell apoptosis with high glucose, and increased glutathione content in MIN6 beta-cells. Pretreatment with l-cysteine improved the secretory responsiveness following stimulation with glucose. The CSE inhibitor dl-propargylglycine antagonized these l-cysteine effects. We suggest H₂ S may function as an ‘intrinsic brake’ which protects beta-cells from glucotoxicity.     

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.     

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.     

Brain hydrogen sulfide is severely decreased in Alzheimer's disease

Although hydrogen sulfide (H2S) is generally thought of in terms of a poisonous gas, it is endogenously produced in the brain from cysteine by cystathionine beta-synthase (CBS). H2S functions as a neuromodulator as well as a smooth muscle relaxant. Here we show that the levels of H2S are severely decreased in the brains of Alzheimer's disease (AD) patients compared with the brains of the age matched normal individuals. In addition to H2S production CBS also catalyzes another metabolic pathway in which cystathionine is produced from the substrate homocysteine. Previous findings, which showed that S-adenosyl-l-methionine (SAM), a CBS activator, is much reduced in AD brain and that homocysteine accumulates in the serum of AD patients, were confirmed. These observations suggest that CBS activity is reduced in AD brains and the decrease in H2S may be involved in some aspects of the cognitive decline in AD.     

The influence of selected O-alkyl derivatives of cyclodextrins on the enzymatic decomposition of l-tryptophan by l-tryptophan indole-lyase

A series of O-alkyl derivatives of cyclodextrin: heksakis[2,3,6-tri-O-(2′-methoxyethyl)]-α-cyclodextrin; heksakis(2,3-di-O-methyl)-α-cyclodextrin; heptakis(2,3-di-O-methyl)-β-cyclodextrin; heksakis[2,3-di-O-methyl-6-O-(2′-methoxyethyl)]-α-cyclodextrin; heptakis[2,3-di-O-methyl-6-O-(2′-methoxyethyl)]-β-cyclodextrin; heksakis[2,3-di-O-(2′-methoxyethyl)]-α-cyclodextrin and heptakis[2,3-di-O-(2′-methoxyethyl)]-β-cyclodextrin have been synthesized. Purity and composition of the obtained substances were examined. The cyclodextrin derivatives listed above as well as (2-hydroxypropyl)-α-cyclodextrin and (2-hydroxypropyl)-β-cyclodextrin, the two commercially available ones, have been investigated as the additives in the course of enzymatic decomposition of l-tryptophan by l-tryptophan indole-lyase. It has been found that each of cyclodextrin derivatives causes the inhibition of enzymatic process, both competitive and non-competitive. The competitive inhibition is connected with the formation of inclusion complexes between cyclodextrins and l-tryptophan, related to the geometry of these complexes. The mechanism of the non-competitive inhibition is not so evident; it could be related to the formation of the cyclodextrin complexes on the surface of the enzyme, leading to the change in the flexibility of the enzyme molecule.     

Cardiovascular and pulmonary effects of NOS inhibition in endotoxemic conscious rats subjected to swimming training

Sepsis is characterized by systemic hypotension, hyporeactiveness to vasoconstrictors, impaired tissue perfusion, and multiple organ failure. During exercise training (ET), dynamic cardiovascular adjustments take place to maintain proper blood pressure and adjust blood supply to different vascular beds. The aim of this study was to investigate whether ET protects against the cardiovascular abnormalities induced by LPS, a model of experimental endotoxemia, and to evaluate the role of nitric oxide (NO) in pulmonary edema. Wistar rats were subjected to swimming training (up to 1 h/day, 5 days/week for 4 weeks) after which their femoral artery and vein were catheterized. LPS (5 mg/kg, i.v.), injected in control (C) and trained animals (ET), promoted 3 distinct phases in mean arterial pressure (MAP) and heart rate (HR). After ET the alterations in MAP were attenuated. The ET animals showed a lower pulmonary edema index (PEI) after LPS (C=0.65+/-0.01; ET=0.60+/-0.02), which was attenuated after treatment with aminoguanidine in both groups (C=0.53+/-0.02; ET=0.53+/-0.02, p<0.05). After l-NAME, PEI was enhanced numerically in the C and was statistically higher in the ET group (C=0.73+/-0.05; ET=1.30+/-0.3, p<0.05). 7-nitroindazole did not promote any alteration in either group. The adaptations promoted by ET seem to be beneficial, counteracting the cardiovascular abnormalities and pulmonary edema seen in septicemia induced by LPS. The results suggest that iNOS aggravates and cNOS protects against this pulmonary edema.     

Production of L-xylose from L-xylulose using Escherichia coli L-fucose isomerase

l-Xylulose was used as a raw material for the production of l-xylose with a recombinantly produced Escherichia colil-fucose isomerase as the catalyst. The enzyme had a very alkaline pH optimum (over 10.5) and displayed Michaelis-Menten kinetics for l-xylulose with a K_m of 41 mM and a V_max of 0.23 μmol/(mg min). The half-lives determined for the enzyme at 35 °C and at 45 °C were 6 h 50 min and 1 h 31 min, respectively. The reaction equilibrium between l-xylulose and l-xylose was 15:85 at 35 °C and thus favored the formation of l-xylose. Contrary to the l-rhamnose isomerase catalyzed reaction described previously [14]l-lyxose was not detected in the reaction mixture with l-fucose isomerase. Although xylitol acted as an inhibitor of the reaction, even at a high ratio of xylitol to l-xylulose the inhibition did not reach 50%.     

Oxidation of L-serine and L-threonine by bis(hydrogen periodato)argentate(III) complex anion: a mechanistic study

Oxidation of l-serine and l-threonine by a silver(III) complex anion, [Ag(HIO(6))(2)](5-), has been studied in aqueous alkaline medium. The oxidation products of the amino acids have been identified as ammonia, glyoxylic acid and aldehyde (formaldehyde for serine and acetaldehyde for threonine). Kinetics of the oxidation reactions has been followed by the conventional spectrophotometry in the temperature range of 20.0-35.0 degrees C and the reactions display an overall second-order behavior: first-order with respect to both Ag(III) and the amino acids. Analysis of influences of [OH(-)] and [periodate] on the second-order rate constants k' reveals an empirical rate expression: k(')=(k(a)+k(b)[OH(-)])K(1)/([H(2)IO(6)(3-)](e)+K(1)), where [H(2)IO(6)(3-)](e) is equilibrium concentration of periodate, and where k(a)=6.1+/-0.5M(-1)s(-1), k(b)=264+/-6M(-2)s(-1), and K(1)=(6.5+/-1.3)x10(-4)M for serine and k(a)=12.6+/-1.7M(-1)s(-1), k(b)=(5.5+/-0.2)x10(2)M(-2)s(-1), and K(1)=(6.2+/-1.5)x10(-4)M for threonine at 25.0 degrees C and ionic strength of 0.30M. Activation parameters associated with k(a) and k(b) have also been derived. A reaction mechanism is proposed to involve two pre-equilibria, leading to formation of an Ag(III)-periodato-amino acid ternary complex. The ternary complex undergoes a two-electron transfer from the coordinated amino acid to the metal center via two parallel pathways: one pathway is spontaneous and the other is assisted by a hydroxide ion. Potential applications of the Ag(III) complex as a reagent for modifications of peptides and proteins are implicated.     

Solubilization of active green fluorescent protein from insoluble particles by guanidine and arginine

Expression of green fluorescent protein (GFP) in Escherichia coli (E. coli) resulted in only small amount of soluble and fluorescent GFP protein and hence most of the protein in insoluble particles. The expressed GFP in insoluble particles, however, was fluorescent, indicating that it is at least in part folded with an intact chromophore. The GFP in insoluble particles could not be solubilized by an aqueous (denaturant-free) buffer. Solubilization of active GFP from insoluble particles was then attempted with guanidine hydrochloride (GdnHCl), a strong protein-denaturant, or L-arginine, an aggregation suppressor. Solubilization from insoluble particles by 6M GdnHCl led to complete denaturation of the GFP existing in insoluble particles, while GdnHCl solution at lower concentration could solubilize fluorescent GFP. Solubilization of fluorescently active GFP from insoluble particles was also achieved by L-arginine. It is noteworthy that L-arginine was stronger in solubilizing insoluble GFP than GdnHCl below 2M. These results demonstrate that some proteins expressed in E. coli may form insoluble particles containing native conformation and L-arginine may be used to recover the proteins in the native form from such insoluble particles.     

Dictyostelium phenylalanine hydroxylase is activated by its substrate phenylalanine

We have studied the regulatory function of Dictyostelium discoideum Ax2 phenylalanine hydroxylase (dicPAH) via characterization of domain structures. Including the full-length protein, partial proteins truncated in regulatory, tetramerization, or both, were prepared from Escherichia coli as his-tag proteins and examined for oligomeric status and catalytic parameters for phenylalanine. The proteins were also expressed extrachromosomally in the dicPAH knockout strain to examine their in vivo compatibility. The results suggest that phenylalanine activates dicPAH, which is functional in vivo as a tetramer, although cooperativity was not observed. In addition, the results of kinetic study suggest that the regulatory domain of dicPAH may play a role different from that of the domain in mammalian PAH.

Structured summary of protein interactions

dicPAH and dicPAHbind by molecular sieving (View Interaction: 1, 2, 3, 4)     

Intracellular l-arginine concentration does not determine NO production in endothelial cells: Implications on the “l-arginine paradox”

We examined the relative contributory roles of extracellular vs. intracellular l-arginine (ARG) toward cellular activation of endothelial nitric oxide synthase (eNOS) in human endothelial cells. EA.hy926 human endothelial cells were incubated with different concentrations of ¹⁵N₄-ARG, ARG, or l-arginine ethyl ester (ARG-EE) for 2 h. To modulate ARG transport, siRNA for ARG transporter (CAT-1) vs. sham siRNA were transfected into cells. ARG transport activity was assessed by cellular fluxes of ARG, ¹⁵N₄-ARG, dimethylarginines, and l-citrulline by an LC–MS/MS assay. eNOS activity was determined by nitrite/nitrate accumulation, either via a fluorometric assay or by¹⁵N-nitrite or estimated ¹⁵N₃-citrulline concentrations when ¹⁵N₄-ARG was used to challenge the cells. We found that ARG-EE incubation increased cellular ARG concentration but no increase in nitrite/nitrate was observed, while ARG incubation increased both cellular ARG concentration and nitrite accumulation. Cellular nitrite/nitrate production did not correlate with cellular total ARG concentration. Reduced ¹⁵N₄-ARG cellular uptake in CAT-1 siRNA transfected cells vs. control was accompanied by reduced eNOS activity, as determined by ¹⁵N-nitrite, total nitrite and ¹⁵N₃-citrulline formation. Our data suggest that extracellular ARG, not intracellular ARG, is the major determinant of NO production in endothelial cells. It is likely that once transported inside the cell, ARG can no longer gain access to the membrane-bound eNOS. These observations indicate that the “l-arginine paradox” should not consider intracellular ARG concentration as a reference point.