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.     

The emerging roles of nitric oxide (NO) in plant mitochondria

In recent years nitric oxide (NO) has been recognized as an important signal molecule in plants. Both, reductive and oxidative pathways and different subcellular compartments appear involved in NO production. The reductive pathway uses nitrite as substrate, which is exclusively generated by cytosolic nitrate reductase (NR) and can be converted to NO by the same enzyme. The mitochondrial electron transport chain is another site for nitrite to NO reduction, operating specifically when the normal electron acceptor, O₂, is low or absent. Under these conditions, the mitochondrial NO production contributes to hypoxic survival by maintaining a minimal ATP formation. In contrast, excessive NO production and concomitant nitrosative stress may be prevented by the operation of NO-scavenging mechanisms in mitochondria and cytosol. During pathogen attacks, mitochondrial NO serves as a nitrosylating agent promoting cell death; whereas in symbiotic interactions as in root nodules, the turnover of mitochondrial NO helps in improving the energy status similarly as under hypoxia/anoxia. The contribution of NO turnover during pathogen defense, symbiosis and hypoxic stress is discussed in detail.     

AMPA receptors undergo channel arrest in the anoxic turtle cortex

Without oxygen, all mammals suffer neuronal injury and excitotoxic cell death mediated by overactivation of the glutamatergic N-methyl-D-aspartate receptor (NMDAR). The western painted turtle can survive anoxia for months, and downregulation of NMDAR activity is thought to be neuroprotective during anoxia. NMDAR activity is related to the activity of another glutamate receptor, the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR). AMPAR blockade is neuroprotective against anoxic insult in mammals, but the role of AMPARs in the turtle's anoxia tolerance has not been investigated. To determine whether AMPAR activity changes during hypoxia or anoxia in the turtle cortex, whole cell AMPAR currents, AMPAR-mediated excitatory postsynaptic potentials (EPSPs), and excitatory postsynaptic currents (EPSCs) were measured. The effect of AMPAR blockade on normoxic and anoxic NMDAR currents was also examined. During 60 min of normoxia, evoked peak AMPAR currents and the frequencies and amplitudes of EPSPs and EPSCs did not change. During anoxic perfusion, evoked AMPAR peak currents decreased 59.2 +/- 5.5 and 60.2 +/- 3.5% at 20 and 40 min, respectively. EPSP frequency (EPSP(f)) and amplitude decreased 28.7 +/- 6.4% and 13.2 +/- 1.7%, respectively, and EPSC(f) and amplitude decreased 50.7 +/- 5.1% and 51.3 +/- 4.7%, respectively. In contrast, hypoxic (Po(2) = 5%) AMPAR peak currents were potentiated 56.6 +/- 20.5 and 54.6 +/- 15.8% at 20 and 40 min, respectively. All changes were reversed by reoxygenation. AMPAR currents and EPSPs were abolished by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). In neurons pretreated with CNQX, anoxic NMDAR currents were reversibly depressed by 49.8 +/- 7.9%. These data suggest that AMPARs may undergo channel arrest in the anoxic turtle cortex.     

Expression of N-methyl-d-aspartate receptor and its effect on nitric oxide production of rat alveolar macrophages

We early show that glutamate (Glu) mediate hyperoxia-induced newborn rat lung injury through N-methyl-d-aspartate receptor (NMDAR). In this study, we search for evidence of NMDAR expression on newborn rat alveolar macrophages (AMs) and the difference between newborn and adult rat AMs, and the possible effect on nitric oxide (NO) production of AMs by exogenous NMDA. The protein of NMDAR was showed by immunocytochemistry, and the mRNA was examined by RT-PCR and real-time PCR. The results show that: (i) both newborn and adult rat AMs express NMDAR1 and the four NMDAR2 subtypes and newborn rat AMs are higher expression. (ii) NMDA administration increase NO production, inducible nitric oxide synthase (iNOS) activity and iNOS mRNA expression of AMs. (iii) NMDAR activation elevates NO secretion of AMs, which suggests that AM may be one of the key cellular origin of the elevated NO secretion in hyperoxia-induced lung injury.     

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.     

Endothelial nitric oxide production is tightly coupled to the citrulline–NO cycle

Nitric oxide (NO) is an important vasorelaxant produced along with L-citrulline from L-arginine in a reaction catalyzed by endothelial nitric oxide synthase (eNOS). Previous studies suggested that the recycling of L-citrulline to L-arginine is essential for NO production in endothelial cells. However, there is no direct evidence demonstrating the degree to which the recycling of L-citrulline to L-arginine is coupled to NO production. We hypothesized that the amount of NO formed would be significantly higher than the amount of L-citrulline formed due to the efficiency of L-citrulline recycling via the citrulline-NO cycle. To test this hypothesis, endothelial cells were incubated with [14C]-L-arginine and stimulated by various agents to produce NO. The extent of NO and [14C]-L-citrulline formation were simultaneously determined. NO production exceeded apparent L-citrulline formation of the order of 8 to 1, under both basal and stimulated conditions. As further support, alpha-methyl-DL-aspartate, an inhibitor of argininosuccinate synthase (AS), a component of the citrulline-NO cycle, inhibited NO production in a dose-dependent manner. The results of this study provide evidence for the essential and efficient coupling of L-citrulline recycling, via the citrulline-NO cycle, to endothelial NO production.     

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.     

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.     

Nitric oxide and protein nitration in the cystic fibrosis airway

Cystic fibrosis (CF), characterized by chronic airway infection and inflammation, ultimately leads to respiratory failure. Exhaled nitric oxide (NO), elevated in most inflammatory airway diseases, is decreased in CF, suggesting either decreased production or accelerated metabolism of NO. The present studies performed on two groups of CF patients provide further support for a disordered NO airway metabolism in CF respiratory tract disease. Despite confirmation of subnormal NOS2 in the CF airway epithelium, alternative isoforms NOS1 and NOS3 were present, and inflammatory cells in the CF airway expressed abundant NOS2. Increased immunohistochemical staining for nitrotyrosine was demonstrated in lung tissues from patients with CF as compared to control. To our knowledge, this is the first report localizing nitrotyrosine in diseased CF lung tissue. While the relative NOS2 deficiency in CF respiratory tract epithelium may contribute to the lower expired NO levels, these results suggest that increased metabolism of NO is also present in advanced CF lung disease. The significance of altered NO metabolism and protein nitration in CF remains to be fully elucidated.     

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.     

Effect of anoxia and pharmacological anoxia on whole-cell NMDA receptor currents in cortical neurons from the western painted turtle

The mammalian brain undergoes rapid cell death during anoxia that is characterized by uncontrolled Ca(2+) entry via N-methyl-D-aspartate receptors (NMDARs). In contrast, the western painted turtle is extremely anoxia tolerant and maintains close-to-normal [Ca(2+)](i) during periods of anoxia lasting from days to months. A plausible mechanism of anoxic survival in turtle neurons is the regulation of NMDARs to prevent excitotoxic Ca(2+) injury. However, studies using metabolic inhibitors such as cyanide (NaCN) as a convenient method to induce anoxia may not represent a true anoxic stress. This study was undertaken to determine whether turtle cortical neuron whole-cell NMDAR currents respond similarly to true anoxia with N(2) and to NaCN-induced anoxia. Whole-cell NMDAR currents were measured during a control N(2)-induced anoxic transition and a control NaCN-induced transition. During anoxia with N(2) normalized, NMDAR currents decreased to 35.3%+/-10.8% of control values. Two different NMDAR current responses were observed during NaCN-induced anoxia: one resulted in a 172%+/-51% increase in NMDAR currents, and the other was a decrease to 48%+/-14% of control. When responses were correlated to the two major neuronal subtypes under study, we found that stellate neurons responded to NaCN treatment with a decrease in NMDAR current, while pyramidal neurons exhibited both increases and decreases. Our results show that whole-cell NMDAR currents respond differently to NaCN-induced anoxia than to the more physiologically relevant anoxia with N(2).     

Immune cell-induced synthesis of NO and reactive oxygen species in lymphoma cells causes their death by apoptosis

Induction of apoptosis in a lymphoma cell line using immune cell-conditioned medium, etoposide or an nitric oxide (NO) donor, resulted in the production of reactive oxygen species (ROS). Agents that inhibited NO production or scavenged ROS or species formed by reaction of NO with ROS, protected the cells from apoptosis. These data support the suggestion that immune rejection of an immunogenic derivative of this lymphoma in vivo involves the induced synthesis of both NO and ROS by the tumour cells.     

Inducible nitric oxide synthase aggresome formation is mediated by nitric oxide

Nitric oxide (NO) generated by inducible NO synthase (iNOS) contributes critically to inflammatory injury and host defense. While previously thought as a soluble protein, iNOS was recently reported to form aggresomes inside cells. But what causes iNOS aggresome formation is unknown. Here we provide evidence demonstrating that iNOS aggresome formation is mediated by its own product NO. Exposure to inflammatory stimuli (lipopolysaccharide and interferon-γ) induced robust iNOS expression in mouse macrophages. While initially existing as a soluble protein, iNOS progressively formed protein aggregates as a function of time. Aggregated iNOS was inactive. Treating the cells with the NOS inhibitor N-nitro-l-arginine methyl ester (L-NAME) blocked NO production from iNOS without affecting iNOS expression. However, iNOS aggregation in cells was prevented by L-NAME. The preventing effect of NO blockade on iNOS aggresome formation was directly observed in GFP-iNOS-transfected cells by fluorescence imaging. Moreover, iNOS aggresome formation could be recaptured by adding exogenous NO to L-NAME-treated cells. These studies demonstrate that iNOS aggresome formation is caused by NO. The finding that NO induces iNOS aggregation and inactivation suggests aggresome formation as a feedback inhibition mechanism in iNOS regulation.     

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.     

Glucagon-like peptide-1 relaxes gastric antrum through nitric oxide in mice

Glucagon-like-peptide-1 (GLP-1) is a proglucagon-derived peptide expressed in the intestinal enteroendocrine-L cells and released after meal ingestion. GLP-1 reduces postprandial glycemia not only by its hormonal effects, but also by its inhibitory effects on gastrointestinal motility. Recently, we showed that GLP-1 acts in the enteric nervous system of mouse intestine. Therefore our working hypothesis was that GLP-1 may have also a direct influence on the gastric mechanical activity since the major part of experimental studies about its involvement in the regulation of gastric motility have been conducted in in vivo conditions. The purposes of this study were (i) to examine exogenous GLP-1 effects on mouse gastric mechanical activity using isolated whole stomach; (ii) to clarify the regional activity of GLP-1 using circular muscular strips from gastric fundus or antrum; (iii) to analyze the mechanism of action underlying the observed effects; (iv) to verify regional differences of GLP-1 receptors (GLP-1R) expression by RT-PCR. In the whole stomach GLP-1 caused concentration-dependent relaxation significantly anatagonized by exendin (9-39), an antagonist of GLP-1R and abolished by tetrodotoxin (TTX) or N_ω-nitro-l-arginine methyl ester (l-NAME), inhibitor of nitric oxide (NO) synthase. GLP-1 was without any effect in fundic strips, but it induced concentration-dependent relaxation in carbachol-precontracted antral strips. The effect was abolished by TTX or l-NAME. RT-PCR analysis revealed a higher expression of GLP-1R mRNA in antrum than in fundus. These results suggest that exogenous GLP-1 is able to reduce mouse gastric motility by acting peripherally in the antral region, through neural NO release.     

Analytical validation of a microplate reader-based method for the therapeutic drug monitoring of L-asparaginase in human serum

The enzyme L-asparaginase (ASNASE), which hydrolyzes L-asparagine (L-Asn) to ammonia and L-aspartic acid (L-Asp), is commonly used for remission induction in acute lymphoblastic leukemia. To correlate ASNASE activity with L-Asn reduction in human serum, sensitive methods for the determination of ASNASE activity are required. Using L-aspartic beta-hydroxamate (AHA) as substrate we developed a sensitive plate reader-based method for the quantification of ASNASE derived from Escherichia coli and Erwinia chrysanthemi and of pegylated E. coli ASNASE in human serum. ASNASE hydrolyzed AHA to L-Asp and hydroxylamine, which was determined at 710 nm after condensation with 8-hydroxyquinoline and oxidation to indooxine. Measuring the indooxine formation allowed the detection of 2 x 10(-5)U ASNASE in 20 microl serum. Linearity was observed within 2.5-75 and 75-1,250 U/L with coefficients of correlation of r(2)>0.99. The coefficients of variation for intra- and interday variability for the three different ASNASE enzymes were 1.98 to 8.77 and 1.73 to 11.0%. The overall recovery was 101+/-9.92%. The coefficient of correlation for dilution linearity was determined as r(2)=0.986 for dilutions up to 1:20. This method combined with sensitive methods for the quantification of L-Asn will allow bioequivalence studies and individualized therapeutic drug monitoring of different ASNASE preparations.     

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.     

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.     

Leptin improves membrane fluidity of erythrocytes in humans via a nitric oxide-dependent mechanism--an electron paramagnetic resonance investigation

Abnormalities in physical properties of the cell membranes may underlie the defects that are strongly linked to hypertension, stroke, and other cardiovascular diseases. Recently, there has been an indication that leptin, the product of the human obesity gene, actively participates not only in the metabolic regulations but also in the control of cardiovascular functions. In the present study, to assess the role of leptin in the regulation of membrane properties, the effects of leptin on membrane fluidity of erythrocytes in humans are examined. The membrane fluidity of erythrocytes in healthy volunteers by means of an electron paramagnetic resonance (EPR) and spin-labeling method is determined. In an in vitro study, leptin decreased the order parameter (S) for 5-nitroxide stearate (5-NS) and the peak height ratio (ho/h-1) for 16-NS obtained from EPR spectra of erythrocyte membranes in a dose-dependent manner in healthy volunteers. The finding indicated that leptin increased the membrane fluidity and improved the microviscosity of erythrocytes. The effect of leptin on the membrane fluidity was significantly potentiated by the nitric oxide (NO) donors, L-arginine and S-nitroso-N-acetylpenicillamine (SNAP), and a cyclic guanosine monophosphate (cGMP) analog, 8-bromo-cGMP. In contrast, the change evoked by leptin was significantly attenuated in the presence of the NO synthase inhibitors, N(G)-nitro-L-arginine-methyl-ester (L-NAME) and asymmetric dimethyl-L-arginine (ADMA). The results of the present study showed that leptin increased the membrane fluidity and improved the rigidity of cell membranes to some extent via an NO- and cGMP-dependent mechanism. Furthermore, the data also suggest that leptin might have a crucial role in the regulation of rheological behavior of erythrocytes and microcirculation in humans.     

NAD+-specific D-arabinose dehydrogenase and its contribution to erythroascorbic acid production in Saccharomyces cerevisiae

Erythroascorbic acid (eAsA) is a five-carbon analog of ascorbic acid, and it is synthesized from D-arabinose by D-arabinose dehydrogenase (ARA) and D-arabinono-gamma-lactone oxidase. We found an NAD+-specific ARA activity which is operative under submillimolar level of d-arabinose in the extracts of Saccharomyces cerevisiae. The hypothetical protein encoded by YMR041c showed a significant homology to a l-galactose dehydrogenase which plays in plant ascorbic acid biosynthesis, and we named it as Ara2p. Recombinant Ara2p showed NAD+-specific ARA activity with Km=0.78 mM to d-arabinose, which is 200-fold lower than that for the conventional NADP+-specific ARA, Ara1p. Gene disruptant of ARA2 lost entire NAD+-specific ARA activity and the conspicuous increase in intracellular eAsA by exogenous d-arabinose feeding, while the double knockout mutant of ARA1 and ARA2 still retained measurable amount of eAsA. It demonstrates that Ara2p, not Ara1p, mainly contributes to the production of eAsA from d-arabinose in S. cerevisiae.