Steady-state nitric oxide concentrations during denitrification

Three species of denitrifying bacteria, Paracoccus denitrificans, Pseudomonas stutzeri strain JM300, and Achromobacter cycloclastes, were allowed to reduce nitrate or nitrite in anaerobic, closed vials while the equilibration of gases between aqueous and gas phases was facilitated by vigorous stirring. The gas phase was sampled and analyzed for NO with use of a chemiluminescence detector calibrated against bottled NO standards or against NO produced by the nitrite-iodide reaction. [NOaq] was inferred from [NOg] and the solubility of NO. NO was detected only during denitrification in amounts that, once established, did not change with time, were independent of the initial concentration of nitrate or nitrite, and were largely independent of cell concentration, at least when nitrate was the oxidant. The usual level of NO was promptly re-established following the addition of exogenous NO or following the loss of NO by sparging. The aforementioned properties are expected for a steady-state intermediate in denitrification. Steady-state [NOaq] ranged between 1 and 65 nM depending on species and conditions. Similar results were also obtained in a related experiment in which P. stutzeri strain ZoBell respired nitrite under growth conditions. The very low steady-state [NOaq] observed during denitrification imply that the maximum activity of nitric oxide reductase in vivo, if it could be realized, would be large relative to that for nitrite reductase. This circumstance allows NO to be an intermediate without reaching toxic steady-state levels.     

Nitric oxide and nitrous oxide production and cycling during dissimilatory nitrite reduction by Pseudomonas perfectomarina

The denitrifier Pseudomonas perfectomarina reduced nitrite under conditions of kinetic competition between cells and gas sparging for extracellular dissolved nitric and nitrous oxides, NOaq and N2Oaq, in a chemically defined marine medium. Time courses of nitrite reduction and NOg and N2Og alpha removal were integrated to give NOg and N2Og yields. At high sparging rates, the NOg yield was greater than 50% of nitrite-N reduced, and the yield of NOg + N2Og was approximately 75%. Hence interrupted denitrification yields NOaq and N2Oaq as major products. The yields varied with sparging rates in agreement with a quantitative model of denitrification (Betlach, M. P., and Tiedje, J.M. (1981) Appl. Environ. Microbiol. 42, 1074-1084) that applies simplified Michaelis-Menten kinetics to NO2-----NOaq----N2Oaq----N2. The fit gave an estimate of the maximum scavengeable NOaq yield of 73 +/- 8% of nitrite-N. Thus a minor path independent of NOaq is also required. The fit of the model to data at lower sparging rates, where normal denitrification products predominate, implies that the extracellular NOaq pool yield is independent of gas sparging rate. Thus in P. perfectomarina NOaq and N2Oaq are intermediates, or facilely equilibrate with true intermediates, during complete denitrification. The recovery of most nitrite-N as NO and/or N2O under perturbed conditions is not an artifact of irreversible product removal, but an attribute of denitrification in this species, and most probably it is characteristic of denitrification in other species as well.     

Structure, biochemistry and biology of hepoxilins: an update

Hepoxilins are biologically relevant epoxy-hydroxy eicosanoids synthesized through the 12S-lipoxygenase (12S-LOX) pathway of the arachidonic acid (AA) metabolism. The pathway is bifurcated at the level of 12S-hydroperoxy-eicosatetraenoic acid (12S-HpETE), which can either be reduced to 12S-hydro-eicosatetraenoic acid (12S-HETE) or converted to hepoxilins. The present review gives an update on the biochemistry, biology and clinical aspects of hepoxilin-based drug development. The isolation, cloning and characterization of a rat leukocyte-type 12S-LOX from rat insulinoma RINm5F cells revealed a 12S-LOX possessing an intrinsic 8S/R-hydroxy-11,12-epoxyeicosa-5Z,9E,14Z-trienoic acid (HXA(3)) synthase activity. Site-directed mutagenesis studies on rat 12S-LOX showed that the HXA(3) synthase activity was impaired when the positional specificity of AA was altered. Interestingly, amino acid Leu353, and not conventional sequence determinants Met419 and Ile418, was found to be a crucial sequence determinant for AA oxygenation. The regulation of HXA(3) formation is dependent on the cellular overall peroxide tone. Cellular glutathione peroxidases (cGPxs) compete with HXA(3) synthase for 12S-HpETE as substrate either to reduce to 12S-HETE or to convert to HXA(3), respectively. Therefore, RINm5F cells, which are devoid of GPxs, are capable of converting AA or 12S-HpETE to HXA(3) under basal conditions, whereas cells overexpressing cGPx are unable to do so. HXA(3) exhibits a myriad of biological effects, most of which are associated with the stimulation of intracellular calcium or the transport of calcium across the membrane. The activation of HXA(3)-G-protein-coupled receptors explains many of the extracellular effects of HXA(3), including AA- and diacylglycerol (DAG) release in human neutrophils, insulin secretion in rat pancreatic beta-cells or islets, and synaptic actions in the brain. The availability of stable analogs of HXA(3), termed 10-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoic acid derivatives (PBTs), recently made several animal studies possible and explored the role of HXA(3) as a therapeutic in treatment of diseases. Thus, PBT-3 induced apoptosis in K562 tumour cells and inhibited growth of K562 CML solid tumours in nude mice. HXA(3) inhibited bleomycin-evoked lung fibrosis and inflammation in mice and the raised insulin level in the circulation of rats. At low glucose concentrations (0-3 mm), HXA(3) also stimulated insulin secretion in RINm5F cells through the activation of IRE1alpha, an endoplasmic reticulum-resident kinase. The latter regulates the protein folding for insulin biosynthesis. In conclusion, HXA(3)-mediated signaling may be involved in normal physiological functions, and hepoxilin-based drugs may serve as therapeutics in diseases such as type II diabetes and idiopathic lung fibrosis.     

Hepoxilin A(3) protects β-cells from apoptosis in contrast to its precursor, 12-hydroperoxyeicosatetraenoic acid

Pancreatic β-cells have a deficit of scavenging enzymes such as catalase (Cat) and glutathione peroxidase (GPx) and therefore are susceptible to oxidative stress and apoptosis. Our previous work showed that, in the absence of cytosolic GPx in insulinoma RINm5F cells, an intrinsic activity of 12 lipoxygenase (12(S)-LOX) converts 12S-hydroperoxyeicosatetraenoic acid (12(S)-HpETE) to the bioactive epoxide hepoxilin A(3) (HXA(3)). The aim of the present study was to investigate the effect of HXA(3) on apoptosis as compared to its precursor 12(S)-HpETE and shed light upon the underlying pathways. In contrast to 12(S)-HpETE, which induced apoptosis via the extrinsic pathway, we found HXA(3) not only to prevent it but also to promote cell proliferation. In particular, HXA(3) suppressed the pro-apoptotic BAX and upregulated the anti-apoptotic Bcl-2. Moreover, HXA(3) induced the anti-apoptotic 12(S)-LOX by recruiting heat shock protein 90 (HSP90), another anti-apoptotic protein. Finally, a co-chaperone protein of HSP90, protein phosphatase 5 (PP5), was upregulated by HXA(3), which counteracted oxidative stress-induced apoptosis by dephosphorylating and thus inactivating apoptosis signal-regulating kinase 1 (ASK1). Taken together, these findings suggest that HXA(3) protects insulinoma cells from oxidative stress and, via multiple signaling pathways, prevents them from undergoing apoptosis.     

Hepoxilin A3 (HXA3) synthase deficiency is causative of a novel ichthyosis form

Non-bullous congenital ichthyosis erythroderma (NCIE) and lamellar ichthyosis (LI) are characterized by mutations in 12R-lipoxygenase (12R-LOX) and/or epidermal lipoxygenase 3 (eLOX3) enzymes. The eLOX3 lacks oxygenase activity, but is capable of forming hepoxilin-type products from arachidonic acid-derived hydroperoxide from 12R-LOX, termed 12R-hydroperoxyeicosa-5,8,10,14-tetraenoic acid (12R-HpETE). Mutations in either of two enzymes lead to NCIE or LI. Moreover, 12R-LOX-deficient mice exhibit severe phenotypic water barrier dysfunctions. Here, we demonstrate that 12R-HpETE can also be transformed to 8R-HXA(3) by hepoxilin A(3) (HXA(3)) synthase (12-lipoxygenase), which exhibits oxygenase activity. We also presented a novel form of ichthyosis in a patient, termed hepoxilin A(3) synthase-linked ichthyosis (HXALI), whose scales expressed high levels of 12R-LOX, but were deficient of HXA(3) synthase.     

Anandamide metabolism by Tetrahymena pyriformis in vitro. Characterization and identification of a 66 kDa fatty acid amidohydrolase

Fatty acid amidohydrolase, a membrane-bound enzyme found in a variety of mammalian cells, is responsible for the catabolism of neuromodulatory fatty acid amides, including anandamide. In an earlier study we reported that Tetrahymena pyriformis was able to secrete a FAAH-like activity in starvation medium (Karava V., Fasia L., Siafaka-Kapadai A., FEBS Lett. 508 (2001) 327-331). In this study the endocannabinoid anandamide, was found to be metabolized by T. pyriformis homogenate by the action of a FAAH-like enzyme, in a time- and concentration-dependent manner. The main metabolic products of [3H]anandamide hydrolysis were [3H]arachidonic acid and ethanolamine. Amidohydrolase activity was maximal at pH 9-10, it was inhibited by phenylmethylsulfonyl fluoride and arachidonyltrifluoromethyl ketone and was Ca2+ and Mg(2+)-independent. Kinetic experiments demonstrated that the enzyme had an apparent K(m) of 2.5 microM and V(max) of 20.6 nmol/min mg. Subcellular fractionation of T. pyriformis homogenate showed that the activity was present in every subcellular fraction with highest specific activity in the microsomal as well as in non-microsomal membrane fraction. Immunoblot analysis of selected subcellular fractions, using an anti-FAAH polyclonal antibody, revealed the presence of an immunoreactive protein with a molecular mass approximately 66 kDa similar to the molecular mass of the mammalian enzyme. In conclusion, this study demonstrates that a FAAH similar to the mammalian enzyme is present in a unicellular eukaryote, indicating the importance of FAAH activity throughout evolution. It also supports the notion that Tetrahymena species may be a suitable model for metabolic studies on endocannabinoids, as well as for the study of drugs targeted towards FAAH.     

Biological role of hepoxilins: upregulation of phospholipid hydroperoxide glutathione peroxidase as a cellular response to oxidative stress?

The 12S-lipoxygenase (12S-LOX) pathway of arachidonic acid (AA) metabolism is bifurcated at 12(S)-hydroperoxy-5Z,8Z,10E (12S-HpETE) in the reduction route to form 12S-hydroxy-eicosatetraenoic acid (12S-HETE) and in 8(S/R)-hydroxy-11(S),12S-trans-epoxyeicosa-5Z,9E,14Z-trienoic acid (HXA3) synthase pathway, previously known as isomerization route, to form hepoxilins. Earlier we showed that the HXA3 formation is restricted to cellular systems devoid of hydroperoxide reducing enzymes, e.g. GPxs, thus causing a persistent oxidative stress situation. Here, we show that HXA3 at as low as 100 nM concentration upregulates phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA and protein expressions, whereas other metabolites of AA metabolism 12S-HpETE and 12S-HETE failed to stimulate the PHGPx. Moreover, the decrease in 12S-HpETE below a threshold value of the hydroperoxide tone causes both suppression of the overall 12S-LOX activity and a shift from HXA3 formation towards 12S-HETE formation. We therefore propose that under persistent oxidative stress the formation of HXA3 and the HXA3-induced upregulation of PHGPx constitute a compensatory defense response to protect the vitality and functionality of the cell.     

High affinity binding sites on plasma membrane obtained from the lymphoblastoid cultured 1301 cell line for highly radioactive serum thymic factor

The interaction of the synthetic serum thymic factor (FTS, facteur thymique sérique) with a plasma membrane preparation of human T lymphocytes from the lymphoblastoid T cell line 1301 was studied using ³H-labelled FTS (specific activity 120 Ci/mmol). The binding is temperature dependent and function of the concentration of both ³H-labelled FTS and membrane proteins. At 37°C, using 1 nM of ³H-labelled FTS a steady state is observed within 80 min. The binding is reversible, specific and saturable. Scatchard analysis reveals the existence of at least two binding sites with respective K_d of the order of 0.516±0.2 nM and 110±27.8 nM with concentrations of 0.186±0.045 pmol and 2.026±0.367 pmol per mg of membrane protein.