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J L Boucher

Publications and source records attributed to J L Boucher.

At least 37 records · Page 2Linked to original sources
PubMed1998

Microsomal cytochrome P450 dependent oxidation of N-hydroxyguanidines, amidoximes, and ketoximes: mechanism of the oxidative cleavage of their C=N(OH) bond with formation of nitrogen oxides.

Oxidation by rat liver microsomes of 13 compounds involving a C=N(OH) function (including N-hydroxyguanidines, amidoximes, ketoximes, and aldoximes) was found to occur with the release of nitrogen oxides such as NO, NO2-, and NO3-. The greatest activities were observed with liver microsomes from dexamethasone-treated rats (up to 8 nmol of NO2- nmol of P450(-)1 min-1). A detailed study of the microsomal oxidation of some of these compounds was performed. Oxidation of N-(4-chlorophenyl)-N'-hydroxy-guanidine led to the formation of the corresponding urea and cyanamide in addition to NO, NO2-, and NO3-. Formation of all these products was dependent on NADPH, O2, and cytochromes P450. Oxidation of two arylamidoximes was found to occur with formation of the corresponding amides and nitriles in addition to nitrogen oxides. Oxidation of 4-(chlorophenyl)methyl ketone oxime gave the corresponding ketone and nitroalkane as well as NO, NO2-, and NO3-. These reactions were also dependent on cytochromes P450 and required NADPH and O2. Mechanistic experiments showed that microsomal oxidations of amidoximes to the corresponding nitriles and of ketoximes to the corresponding nitroalkanes are not inhibited by superoxide dismutase (SOD) and are performed by a cytochrome P450 active species, presumably the high-valent P450-iron-oxo complex. On the contrary, microsomal oxidation of N-hydroxyguanidines to the corresponding cyanamides was greatly inhibited by SOD and appeared to be mainly due to O2*- derived from the oxidase function of cytochromes P450. Similarly, microsomal oxidations of N-hydroxyguanidines and amidoximes to the corresponding ureas and amides were also found to be mainly performed by O2*-, as shown by the great inhibitory effect of SOD (70-100%) and the ability of the xanthine-xanthine oxidase system to give similar oxidation products. However, it is noteworthy that other species, such as the P450 Fe(II)-O2 complex, are also involved, to a minor extent, in the SOD-insensitive microsomal oxidative cleavages of compounds containing a C=N(OH) bond. Our results suggest a general mechanism for such oxidative cleavages of C=N(OH) bonds with formation of nitrogen oxides by cytochromes P450 and NO-synthases, with the involvement of O2*- and its Fe(III) complex [(FeIII-O2-) or (FeII-O2)] as main active species.

Amides↗
PubMed1998

Synthesis of boronic acid analogs of L-arginine as alternate substrates or inhibitors of nitric oxide synthase.

The asymmetric synthesis of an unprotected alpha-amino boronic acid analog of L-arginine 1a (boroarg-OH. 2 HCl) and its N alpha-acetyl derivative 1b (Ac-boroarg-OH. HCl) is described. These compounds were evaluated as substrates and inhibitors of recombinant nitric oxide synthases (NOS). Boroarg-OH 1a selectively inhibited inducible NOS (IC50 = 50 microM) compared to the neuronal isoform (IC50 = 300 microM).

Arginine↗
PubMed1998

Detection of a nitric oxide synthase possibly involved in the regulation of the Rhodococcus sp R312 nitrile hydratase.

Crude homogenates from Rhodococcus sp 312 catalyze the conversion of L-arginine into L-citrulline and NO2-, the usual oxidation product of NO under aerobic conditions. They also catalyze the conversion of N omega-hydroxy-L-arginine (NOHA) into L-citrulline and NO2- with similar rates (10-15 and 100-150 nmol of product.min-1.(mg of protein)-1 respectively for the crude homogenate and for a fraction obtained from ammonium sulfate precipitation). L-citrulline formation is strongly inhibited by classical inhibitors of mammalian nitric oxide synthases (NOSs) such as N omega-methyl-L-arginine (NMA) and thio-L-citrulline (TC). Finally, the lack of inhibitory effects of EGTA, a classical inhibitor of constitutive mammalian NOSs, and the specific immunodetection of a 100 kD protein from Rhodococcus cytosol by an antibody raised against human inducible NOS, is in favor of the presence of a NOS similar to inducible mammalian NOSs in Rhodococcus sp 312. This NOS should be responsible for the NO-dependent inactivation of Rhodococcus Nitrile Hydratase (NHase) in the absence of light; it could regulate the activity of the latter enzyme.

Bacterial Proteins↗
PubMed1998

Substrate specificity of NO synthases: detailed comparison of L-arginine, homo-L-arginine, their N omega-hydroxy derivatives, and N omega-hydroxynor-L-arginine.

A detailed comparison of the oxidation of five compounds closely related to L-arginine (Arg) by purified recombinant neuronal and macrophage NO synthases (NOS I and NOS II) was performed. Homo-L-arginine (homo-Arg) is oxidized by both NOSs in the presence of NADPH with major formation of NO and homo-L-citrulline, with a molar ratio of close to 1, and minor formation of N omega-hydroxyhomo-L-arginine (homo-NOHA). Oxidation of homo-NOHA by the two NOSs also leads to NO and homocitrulline in a 1:1 molar ratio. On the contrary, N omega-hydroxynor-L-arginine (nor-NOHA) is a very poor substrate of NOS I and II, which fails to produce significant amounts of nitrite. The catalytic efficiency of both NOSs markedly decreases in the order Arg > NOHA > homo-Arg > homo-NOHA, as shown by the 20- and 10-fold decrease of kcat/Km observed for NOS I and NOS II, respectively, when comparing Arg to homo-NOHA. The greater loss of catalytic efficiency for homo-Arg, when compared to that for Arg, appears to occur at the first step (N-hydroxylation) of the reaction. In that regard, it is noteworthy that the Vm values for NOHA and homo-NOHA oxidation are very similar (about 1 and 2 micromol of NO min-1 mg of protein-1 for NOS I and II, respectively). In fact, lengthening of the Arg chain by one CH2 leads not only to markedly decreased kcat/Km but also to clear disturbances in NOS functioning. This is shown by a greater accumulation of the N omega-hydroxyguanidine intermediate (homo-NOHA:homocitrulline ratio between 0.2 and 0.4) and an increased consumption of NADPH for NO formation (between 2.0 and 2.6 mol of NADPH consumed for the formation of 1 mol of NO in the case of homo-Arg, instead of 1.5 mol in the case of Arg). Most of the above results could be interpreted by comparing the possible positionings of the various substrates relative to the two NOS active oxygen species which are believed to be responsible for the two steps of the reaction.

Animals↗
PubMed1998

Formation of nitric oxide synthase-iron(II) nitrosoalkane complexes: severe restriction of access to the iron(II) site in the presence of tetrahydrobiopterin.

Nitric oxide synthases (NOS) are heme proteins, closely related to cytochromes P450, that catalyze oxidation of l-arginine (l-Arg) to nitric oxide (NO) and citrulline. To get further insight into their active site, we have studied the ability of recombinant mouse inducible NOS (iNOS) and rat brain neuronal NOS (nNOS), and of their oxygenase domains (iNOSoxy and nNOSoxy), to form Fe(II)-nitrosoalkane complexes. In the absence of BH4, iNOSoxy, nNOSoxy, and full-length iNOS readily form complexes characterized by Soret peaks around 448 nm, after reaction with various nitroalkanes and sodium dithionite. These complexes displayed physicochemical characteristics very similar to those of previously reported microsomal cytochrome P450-Fe(II)-nitrosoalkane complexes: (i) a Soret peak around 450 nm, (ii) a clear stability in the presence of CO, and (iii) a fast destruction upon oxidation of the iron by ferricyanide. Thus, in the absence of l-Arg and BH4, NOSs Fe(II) appear to be largely opened to even large R-NO ligands with R = cyclohexyl or p-Cl-C6H4-CH2CH(CH3) for instance, in a manner similar to microsomal P450s Fe(II). As expected, the presence of l-Arg inhibits the formation of NOSs Fe(II)-RNO complexes. More surprisingly, the presence of BH4 also strongly inhibits the formation of the NOSs Fe(II) complexes even with the smallest nitrosoalkane ligand, CH3NO (IC50 values of 0.5 and 4 microM for nNOSoxy and iNOSoxy, respectively). Accordingly, recombinant full-length nNOS containing BH4 and l-Arg is completely unable to form Fe(II)-nitrosoalkane complexes, even with CH3NO. These results suggest that, in the absence of l-Arg and BH4, the distal pocket of NOSs Fe(II) is largely opened even to bulky ligands, in a manner similar to that of microsomal cytochromes P450. On the contrary, the distal heme pocket of iNOS and nNOS seems to be closed after binding of l-Arg and BH4, particularly in the Fe(II) state. This results in a highly restricted access for Fe(II) ligands, except very small ones such as CO, NO, and O2. Such effects of BH4 in controlling the size of the distal heme pocket of NOS Fe(II) correspond to a new role of biopterins in biological systems.

Alkanes↗
PubMed1997

Strong inhibition of neuronal nitric oxide synthase by the calmodulin antagonist and anti-estrogen drug tamoxifen.

The anti-estrogen drug tamoxifen (TMX) was found to act as a strong inhibitor of purified neuronal nitric oxide synthase (nNOS) (IC50 = 2 +/- 0.5 microM), whereas it was inactive toward inducible macrophage NOS (IC50 > 100 microM). TMX affected the activation of NOS by calmodulin, as it not only inhibited L-arginine oxidation to nitric oxide and L-citrulline but also NADPH oxidation and calmodulin-dependent cytochrome c reduction catalyzed by nNOS. These results suggest that TMX could exert some of its biological effects by interfering with constitutive NOS-dependent formation of nitric oxide and/or superoxide ion in various tissues.

Animals↗
PubMed1997

Bromocriptine is a strong inhibitor of brain nitric oxide synthase: possible consequences for the origin of its therapeutic effects.

The ergot alkaloid bromocriptine (BKT) was found to act as a strong inhibitor of purified neuronal nitric oxide synthase (NOS) (IC50 = 10 +/- 2 microM) whereas it was poorly active towards inducible macrophage NOS (IC50 > 100 microM). BKT affects the activation of NOS by calmodulin, as it not only inhibits L-arginine oxidation to NO and L-citrulline but also NADPH oxidation and calmodulin-dependent cytochrome c reduction catalyzed by neuronal NOS. These results suggest that BKT could exert some of its therapeutic effects by interfering with the NOS-dependent formation of nitric oxide and/or superoxide ion in various tissues.

Animals↗
PubMed1997

Inhibition of arginase in rat and rabbit alveolar macrophages by N omega-hydroxy-D,L-indospicine, effects on L-arginine utilization by nitric oxide synthase.

1. Alveolar macrophages (AM phi) exhibit arginase activity and may, in addition, express an inducible form of nitric oxide (NO) synthase (iNOS). Both pathways may compete for the substrate. L-arginine. The present study tested whether two recently described potent inhibitors of liver arginase (N omega-hydroxy-D,L-indospicine and 4-hydroxyamidino-D,L-phenylalanine) might also inhibit arginase in AM phi and whether inhibition of arginase might affect L-arginine utilization by iNOS. 2. AM phi obtained by broncho-alveolar lavage of rat and rabbit isolated lungs were disseminated (2.5 or 3 x 10(6) cells per well) and allowed to adhere for 2 h. Thereafter, they were either used to study [3H]-L-arginine uptake (37 kBq, 0.1 microM, 2 min) or cultured for 20 h in the absence or presence of bacterial lipopolysaccharide (LPS). Cultured AM phi were incubated for 1 h with [3H]-L-arginine (37 kBq, 0.1 microM) and the accumulation of [3H]-L-citrulline (NOS activity) and [3H]-L-ornithine (arginase activity) was determined. 3. During 1 h incubation of rabbit AM phi with [3H]-L-arginine, no [3H]-L-citrulline, but significant amounts of [3H]-L-ornithine (150 d.p.m x 1000) were formed. N omega-hydroxy-D,L-indospicine and 4-hydroxyamidino-D,L-phenylalanine, present during incubation, concentration-dependently reduced [3H]-L-ornithine formation (IC50: 2 and 45 microM, respectively). 4. N omega-hydroxy-D,L-indospicine (up to 100 microM) had no effect on [3H]-L-arginine uptake into rabbit AM phi, whereas 4-hydroxyamidino-D,L-phenylalanine caused a concentration-dependent inhibition (IC50: 300 microM). 5. Rat AM phi, cultured in the absence of LPS, formed significant amounts of [3H]-L-citrulline and [3H]-L-ornithine (133 and 212 d.p.m x 1000, respectively) when incubated for 1 h with [3H]-L-arginine. When AM phi had been cultured in the presence of 0.1 or 1 microgram ml-1 LPS, the formation of [3H]-L-citrulline was enhanced by 37 +/- 8.3 and 99 +/- 12% and that of [3H]-L-ornithine reduced by 21 +/- 8.7 and 70 +/- 2.5%, respectively. 6. In rat AM phi, cultured in the absence or presence of LPS, N omega-hydroxy-D,L-indospicine (10 and 30 microM) greatly reduced formation of [3H]-L-ornithine (by 80-95%) and this was accompanied by increased formation of [3H]-L-citrulline. However, only 20-30% of the [3H]-L-arginine not metabolized to [3H]-L-ornithine after inhibition of arginase was metabolized to [3H]-L-citrulline, when the AM phi had been cultured in the absence of LPS (i.e. low level of iNOS). On the other hand, when the AM phi had been cultured in the presence of LPS (i.e. high level of iNOS), all the [3H]-L-arginine not metabolized by the inhibited arginase was metabolized to [3H]-L-citrulline. 7. In conclusion, N omega-hydroxy-D,L-indospicine is a potent and specific inhibitor of arginase in AM phi. In cells in which, in addition to arginase, iNOS is expressed, inhibition of arginase can cause a shift of L-arginine metabolism to the NOS pathway. However, the extent of this shift appears to depend in a complex manner on the level of iNOS.

Animals↗
PubMed1996

White adipose tissue nitric oxide synthase: a potential source for NO production.

Nitric oxide synthase (NOS) activity was detected in soluble and membranous fractions of adipose tissue homogenates of control rats. After LPS-treatment, this activity was (i) markedly increased (about 10-fold) in both fractions, (ii) unaltered after dexamethasone pretreatment, (iii) partly calcium-calmodulin sensitive, and (iv) almost entirely accounted by the NOS activity found in isolated adipocytes. In adipose tissue homogenates from control rats, Western blot analysis demonstrated the presence of the endothelial (eNOS) isoform in the membranous fraction of control rats and of the inducible (iNOS) isoform in the soluble and membranous fractions. After LPS treatment, the amount of immunoreactive iNOS protein was dramatically increased, suggesting that adipose tissue is an important site of NO production during the endotoxic shock.

Adipose Tissue↗
PubMed1996

In vitro hydroxylation and epoxidation of some isomeric lauric acid analogs by rat liver microsomes. Identification of metabolites and effects of clofibrate or phenobarbital pretreatment.

The primary metabolites of a series of unsaturated lauric acid analogs (8-, 9-, 10-, and 11-dodecenoic acids) used as radiolabeled substrates for rat liver microsomes were quantitated by TLC and reverse phase-HPLC analysis, and identified by chemical derivation and GC/MS. Isomeric epoxidodecanoic acids and omega- and (omega-1)-monohydroxydodecenoic acids were essentially the only products formed from the incubations of the unsaturated fatty acids. Rat liver microsomes predominantly oxidized the terminal carbons of all substrates, leading to omega- and (omega-1)-hydroxylated metabolites, with the exception of 11-dodecenoic acid, which was efficiently converted to the epoxide. The E and Z isomers of dodecenoic acids were metabolized with the same efficiency and gave rise to the same pattern of hydroxylated vs. epoxidized products. The hydroxylation/epoxidation ratio was directly related to the position, but not to the geometry of the double bond in the aliphatic chain. Clofibrate pretreatment of the animals resulted in a strong induction of omega-oxidation, with a decrease in the ability to catalyze epoxidation of internal olefins, whereas phenobarbital pretreatment only stimulated (omega-1)-hydroxylation without any effect on epoxidation. In contrast to higher plants in which carbon 9 is the major target, rat liver cytochromes P450 selectively carried out hydroxylation (or epoxidation) at carbons 12 and 11 of lauric acid, as well as its unsaturated isomeric analogs.

Animals↗
PubMed1995

On the mechanism of nitric oxide formation upon oxidative cleavage of C = N(OH) bonds by NO-synthases and cytochromes P450.

Microsomal liver cytochromes P450 catalyze the oxidative cleavage of the C = NOH bond of many ketoximes, amidoximes and guanidoximes, and NO synthases catalyze the oxidation of N omega-hydroxy-L-arginine to citrulline and NO. All these oxidations appear to be performed either by the FE(II) O2 complex of these hemoproteins or by O2.- which is formed by its decomposition. This leads to a unifying view of the mechanisms of P450- and NOS-dependent oxidative cleavage of C = NOH bonds, the relative contribution of Fe(II) O2.- being very different in NO-synthase and cytochromes P450.

Amino Acid Sequence↗
PubMed1995

Nitric oxide formation during the cytochrome P-450-dependent reductive metabolism of 18-nitro-oxyandrostenedione.

18-Nitro-oxyandrostenedione (18-ONO2A), a potential mechanism-based inhibitor of the last steps of aldosterone biosynthesis, is well recognized by different cytochrome P-450s, which are able to metabolize it reductively into nitric oxide (NO) and 18-hydroxyandrostenedione. Rat liver microsomal P-450s are able to carry out this reaction with increased efficiency under anaerobic conditions. P-450 3A isozymes induced upon treatment of rats with dexamethasone or troleandomycin were best able to bind and metabolize 18-ONO2A. This reaction was shown to occur in the presence of dioxygen as well, suggesting that it may be of physiological relevance. The formation of NO was detected as a transient P-450-Fe(II)NO complex by UV-visible and EPR spectroscopy. In addition, steroidogenic tissues containing cytochrome P-450s such as bovine adrenal mitochondria or human placental microsomes also were capable of binding and metabolizing 18-ONO2A as judged by the formation of an Fe(II)NO complex. This recognition of a steroid nitrate, a potential antialdosterone and its subsequent metabolism under reductive conditions to generate NO both in hepatic and steroidogenic tissues, can be of pharmacological interest, because NO has been demonstrated to modulate steroidogenesis in addition to other processes such as vascular relaxation, neurotransmission or cytostasis. A nitrate derivative of a steroid could perhaps act as a vectorized NO precursor in which the steroid moiety is targeted specifically to steroid receptors or steroidogenic tissues, thus leading to localized NO liberation.

Adrenal Glands↗
PubMed1994

N omega-hydroxyl-L-arginine, an intermediate in the L-arginine to nitric oxide pathway, is a strong inhibitor of liver and macrophage arginase.

N omega-Hydroxy-L-arginine (L-NOHA) is a potent inhibitor of the hydrolysis of L-arginine (L-Arg) to L-ornithine (L-Orn) catalyzed by purified bovine liver arginase (BLA). It appears as one of the most powerful arginase inhibitors reported so far (Ki = 150 microM). The other products of NO synthase are either without effect (NO2-, NO3-) or much weaker inhibitors (L-citrulline (L-Cit) and NO) of BLA. Products derived from a possible hydrolysis of L-Arg (L-Orn and urea) or of L-NOHA (L-Cit, hydroxyurea and hydroxylamine) are also inactive toward BLA at concentrations up to 2 mM. The configuration of L-NOHA is important as D-NOHA is much less active, and its free -COOH and alpha-NH2 functions are required for recognition of BLA. L-NOHA is also a potent inhibitor of the arginase activity of rat liver homogenates and of murine macrophages (IC50 of 150 and 450 microM, respectively). These remarkable properties of L-NOHA could play a role in the modulation of the biosynthesis of the biological mediator NO by increasing local L-Arg concentrations.

Animals↗
PubMed1994

Dehydration of alkyl- and arylaldoximes as a new cytochrome P450-catalyzed reaction: mechanism and stereochemical characteristics.

The Z isomers of benzaldoxime and 4-(hexyloxy)benzaldoxime were dehydrated into the corresponding nitriles in the presence of rat liver microsomes and NADPH or dithionite. Their E isomers remained unchanged under identical conditions. Alkylaldoximes, like phenylacetaldoxime and heptanaldoxime, are also dehydrated under these conditions, the alkylaldoximes being more rapidly transformed than the arylaldoximes. A genetically well-defined P450 expressed in yeast, P450 3A4, the major P450 isozyme in human liver, was also found to be catalytically active for dehydration of (Z)-benzaldoxime. All these reactions were found to be catalyzed by P450 Fe(II) as they required the use of intact microsomes in the presence of NADPH or dithionite and were strongly inhibited by O2 and CO as well as by classical P450 inhibitors. A P450 complex characterized by a Soret peak at 442 nm was detected during these reactions; its disappearance was found to be concomitant with the consumption of the aldoxime and the formation of the corresponding nitrile. (E)-benzaldoximes and all the studied ketoximes failed to give such complexes with P450 Fe(II). On the basis of these results, a possible mechanism for this new P450 reaction is proposed. It involves a P450 Fe(II)<--N(OH)=CHR complex as a key intermediate and a charge transfer from P450 Fe(II) to the aldoxime C=N bond which results in a cleavage of the aldoxime N-O bond.

Animals↗
PubMed1993

Particular ability of liver P450s3A to catalyze the oxidation of N omega-hydroxyarginine to citrulline and nitrogen oxides and occurrence in no synthases of a sequence very similar to the heme-binding sequence in P450s.

Liver microsomes from rats pretreated with various inducers of P450 isoforms exhibit very different abilities to catalyze the oxidation of N omega-hydroxy-L-arginine (NOHA) by NADPH and O2 with formation of citrulline and nitrogen oxides. Treatment of rats with dexamethasone, a classical inducer of P450 3A, leads to a spectacular 7-fold increase of the activity found for untreated rats, while induction by phenobarbital causes a much lower increase of this activity and induction by 3-methylcholanthrene or clofibrate decreases it. Specific inhibitors of P450s3A as troleandomycin and dihydroergotamine strongly inhibit NOHA oxidation whereas metyrapone, an inhibitor of other P450 subfamilies, was without effect. These data show the particular ability of P450s of the 3A subfamily to catalyze the second step of the oxidation of L-arginine by NO synthases (NOS). This analogy between NOSs and P450s3A is further substantiated by a protein sequence comparison which shows that a 9-amino acid segment present in all NOSs exhibits a strong similarity with the sequence mainly responsible for heme binding in P450s3A which is well conserved in all P450s. This segment contains all the structural factors which are thought to be crucial for heme binding in P450s.

Amino Acid Oxidoreductases↗
PubMed1993

EPR characterization of molecular targets for NO in mammalian cells and organelles.

Nitric oxide is synthesized in mammalian cells from L-arginine or from pharmaceutical drugs. It forms paramagnetic complexes with some metalloproteins, inhibiting key enzymes in DNA synthesis, mitochondrial respiration, iron metabolism, etc. This article reviews how electron paramagnetic resonance spectroscopy helps to detect unambiguously such specific molecular targets for NO in mammalian whole cells and organelles. EPR has also been used for the detection of spin adducts of free NO by spin-trapping methods.

Animals↗
PubMed1993

Lack of effect of phenobarbital on ex vivo leukocyte oxidative metabolism in healthy volunteers.

Oxidative metabolism in activated human polymorphonuclears catabolizes leukotriene B4 by a cytochrome P450 omega-hydroxylase and procainamide by a myeloperoxidase. The percentage of leukotriene B4 metabolized by activated human polymorphonuclears and the apparent enzymatic parameters of procainamide metabolism were studied ex vivo in six healthy volunteers before and after phenobarbital intake (100 mg/day) for 10 days and in six healthy control volunteers. No differences were found between groups for the difference in percentage of leukotriene B4 metabolized between day 11 and day 1. The apparent enzymatic parameters, Km and Vm, of procainamide oxidation did not differ significantly between the groups both on day 1 and day 11. These results do not show any evidence of inducibility of leukotriene B4 and procainamide metabolism in human polymorphonuclears. However, a positive correlation between 6 beta OH-cortisol excretion and percentage of leukotriene B4 metabolized was observed on day 11. This study suggests that human polymorphonuclears cytochrome P450 leukotriene B4 omega-hydroxylase and procainamide metabolism is not a useful method to study cytochrome P450 induction in clinical pharmacology.

17-Hydroxycorticosteroids↗