Immunofluorescence of NADPH-cytochrome c (P-450) reductase in rat and minipig tissues injected with phenobarbital.

The enzyme NADPH-cytochrome c (P-450) reductase was identified by indirect immunofluorescence in hepatocytes, bronchioles, and proximal tubules of liver, lung, and kidney, respectively, of rats and minipigs that had been injected with phenobarbital or saline. The distribution of this component of the cytochrome P-450-mediated microsomal system may be relevant to sites of drug toxicity and carcinogenesis.

[Nitric oxide synthase, typical flavohemoproteins and their complicated enzymology]. / Syntázy oxidu dusnatého, typické hemoflavoproteiny a jejich komplikovaná enzymologie.

Nitric oxide is a diatomic gaseous molecule with unpaired electron in the molecule. Physical properties such as solubility, diffusibility and half-life decide the chemical reactivity of nitric oxide. Nitric oxide is the unstable free radical in vessels, immune system and central nervous system. The reactivity of nitric oxide under physiological and pathological conditions depends upon its concentration and site of production. Nitric oxide is thought to play a role in many pathological situations: septic shock, cardiovascular diseases, arthritis, diabetes, multiple sclerosis, asthma, and hypertension. Nitric oxide synthase is a self-sufficient flavohemoprotein capable of producing nitric oxide from L-arginine by two successive monooxygenation steps. Although the N-terminal heme domain functionally resembles cytochromes P450, no structural similarities exist between cytochrome P450 and nitric oxide synthases heme domains. The C-terminal domain of nitric oxide synthases containing flavin adenine dinucleotide and flavin mononucleotide as cofactors exhibits a high degree of sequence similarity with NADPH-cytocrome P450 reductase. The reductase domains serve as an intermediary for the transfer of electrons from NADPH for the catalytic reaction. The connecting domain between the oxygenase and the reductase domains of nitric oxide synthase isoforms binds calmodulin in the presence of calcium. The binding of calmodulin to all nitric oxide synthase isoforms is obligatory for the production of nitric oxide. At the same time, the presence of one or more phosphorylation sites in nitric oxide synthase puts them among the kinase-mediated signaling pathways. This also means that nitric oxide synthases are regulated indirectly by the events that regulate kinases. This field of research of nitric oxide synthase regulation has become one of the most actively pursued and much has been learned from basic biochemical mechanisms to physiological processes and to medical applications, but many more questions still remain to be answered.

Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin and phenobarbital on the occurrence and distribution of four cytochrome P-450 isozymes in rabbit kidney, lung, and liver.

The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and of phenobarbital (PB) on the distribution and occurrence of four cytochrome P-450 isozymes, Forms 2, 3, 4, and 6, in the kidney, lung, and liver of adult male rabbits was investigated using immunofluorescence. In the kidney, Forms 2 and 3 were localized in the proximal tubules of both untreated and PB-treated animals, while antibodies to Forms 4 and 6 showed weak to negative staining. In TCDD-pretreated animals, Forms 4 and 6 appeared in the renal endothelium, in addition to staining the proximal tubular epithelium intensely. Form 2 was the only isoenzyme of those studied found to be present in the lungs of normal and PB-pretreated rabbits; it was also present in lungs of TCDD-pretreated rabbits. Form 3 was not detected in any of the rabbit lungs examined. Forms 4 and 6, while not apparent in the lungs of normal or PB-treated animals, were found in the lungs of TCDD-treated animals and also appeared in the endothelium of the pulmonary arteries and veins. All forms tested were present in control liver. The staining for Form 2 was intense in the livers of PB-pretreated animals, as was the staining for Forms 4 and 6 in the livers of TCDD-pretreated animals. Our results indicate that, while PB altered the intensity of staining for Form 2 in the liver and kidney, TCDD altered both the staining intensity and distribution of the isozymes in kidney, lung, and liver, producing, for example, a localization of Forms 4 and 6 in the endothelium of both the kidney and lung which was not seen in either untreated or PB-pretreated rabbits.

Assay of isoforms of Escherichia coli-expressed nitric oxide synthase.

The techniques described herein have added to our repertoire of experimental approaches for the characterization of the NOSs. These procedures have reinforced our conviction that the NOSs are structurally suited to perform unique functions in their cellular milieux and that these differences have physiological consequences.

Electron spin resonance spin-trapping detection of superoxide generated by neuronal nitric oxide synthase.

NOS is a ubiquitous enzyme that has an oxygenase and reductase activity. NOS reduces electron acceptors, at the reductase domain, by a one-electron mechanism that is not inhibited by SOD. One example of this activity is the direct reduction of ferricytochrome c by nNOS. Redox cycling electron acceptors (EA in Scheme 1), such as lucigenin and NBT, are reduced by NOS to generate an intermediate radical (EAred). This radical can then be reoxidized to the parent compound by oxygen, and in the process generate superoxide. Consequently, both NBT and lucigenin will enhance NADPH-dependent superoxide generation in the presence of flavoprotein reductases such as NOS. The artificial generation of superoxide from lucigenin and NBT is a major pitfall in the use of these compounds as superoxide probes. We conclude that the use of ESR spin-trapping techniques, although not free of problems, is a viable technique for the detection and quantification of superoxide in systems containing nNOS.

N(omega)-Nitroarginine-containing dipeptide amides. Potent and highly selective inhibitors of neuronal nitric oxide synthase.

Selective inhibition of the isoforms of nitric oxide synthase (NOS) could be therapeutically useful in the treatment of certain disease states arising from the overproduction of nitric oxide (NO). Recently, we reported the dipeptide methyl ester, D-Phe-D-Arg(NO)()2-OMe (19), as a modest inhibitor of nNOS (K(i) = 2 microM), but with selectivity over iNOS as high as 1800-fold (Silverman, R. B.; Huang, H.; Marletta, M. A.; Martasek, P. J. Med. Chem. 1997, 40, 2813-2817). Here a library of 152 dipeptide amides containing nitroarginine and amino acids other than Phe are synthesized and screened for activity. Excellent inhibitory potency and selectivity for nNOS over eNOS and iNOS is achieved with the dipeptide amides containing a basic amine side chain (20-24), which indicates a possible electrostatic (or hydrogen bonding) interaction at the enzyme active site. The most potent nNOS inhibitor among these compounds is L-Arg(NO)()2-L-Dbu-NH(2) (23) (K(i) = 130 nM), which also exhibits the highest selectivity over eNOS (>1500-fold) with a 192-fold selectivity over iNOS. These compounds do not exhibit time-dependent inhibition. The order and the chirality of the amino acids in the dipeptide amides have profound influences on the inhibitory potency as well as on the isoform selectivity. These dipeptide amide inhibitors open the door to the design of potent and highly selective inhibitors of nNOS.

Mechanism-based inhibitors of prostaglandin omega-hydroxylase: (R)- and (S)-12-hydroxy-16-heptadecynoic acid and 2,2-dimethyl-12-hydroxy-16-heptadecynoic acid.

12-Hydroxy-16-heptadecynoic acid has been shown to selectively inactivate cytochrome P450 4A4, a pulmonary cytochrome P450 enzyme that catalyzes the omega-hydroxylation of prostaglandins [Muerhoff, A. S.; Williams, D. E.; Reich, N. O.; CaJacob, C. A.; Ortiz de Montellano, P. R.; Masters, B. S. S. J. Biol. Chem. 1989, 264, 749-756]. Potent, specific inhibitors of this enzyme are required to explore its physiological role. In a continuing effort to develop such agents, the two enantiomers of 12-hydroxy-16-heptadecynoic acid have been stereospecifically synthesized, their absolute stereochemistry confirmed, and the dependence of enzyme inactivation on absolute stereochemistry determined using cytochrome P450 4A4 purified from the lungs of pregnant rabbits. The 12S enantiomer is roughly twice as active (KI = 1.8 microM, t1/2 = 0.7 min) as the 12R enantiomer (KI = 3.6 microM, t1/2 = 0.8 min), but the chirality of the hydroxyl group is not a major determinant of the specificity for the prostaglandin omega-hydroxylase. The flexibility of the acyclic skeleton of the inhibitor may account for the relatively low enantiomeric discrimination. 2,2-Dimethyl-12-hydroxy-16-heptadecynoic acid, an analogue that cannot undergo beta-oxidation, has also been synthesized as a potential in vivo inhibitor of the enzyme and has been shown to inactivate the purified enzyme with KI = 4.9 microM and t1/2 = 1.0 min. These acetylenic agents, particularly the dimethyl analog, are promising in vivo inhibitors of cytochrome P450 4A4.

Sex differences in cytochrome P-450 isozyme composition and activity in kidney microsomes of mature rainbow trout.

Kidney microsomes from sexually mature male, as opposed to female, rainbow trout displayed an approximately 20-fold higher cytochrome P-450 specific content, NADPH-cytochrome c reductase activity, and rates of hydroxylation of lauric acid, testosterone, progesterone and aflatoxin B1. Little or no sex difference in metabolism was observed with benzo[a]pyrene or benzphetamine as substrates. A similar pattern was observed in hepatic microsomes from these fish, but the difference was much less striking (approximately 2-fold higher activity in males). Juvenile trout (both sexes) possessed activities intermediate between mature males and females. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of kidney and liver microsomes of juvenile and sexually mature male and female trout suggested that the striking sex difference in kidney could be due to the high amount of trout P-450 isozyme LM2 in sexually mature males. Immunoquantitation of LM2, performed by Western Blotting and immunostaining with rabbit anti-trout LM2-IgG, confirmed that mature male kidney contained much higher levels of P-450 LM2 than juvenile or female kidney, or even of liver microsomes of all three groups. The amount of P-450 LM2 in mature female kidney microsomes was barely detectable. The high amount of LM2 in male trout kidney is consistent with the high activity of these microsomes towards lauric acid and aflatoxin B1, which have been shown previously to be preferentially metabolized by trout P-450 LM2. It is suggested that rainbow trout may serve as an alternative to the rat as an animal model for the study of sex-dependent differences in cytochromes P-450.

Regulation of aromatase activity of rat granulosa cells: induction of synthesis of NADPH-cytochrome P-450 reductase by FSH and dibutyryl cyclic AMP.

Aromatase is an enzyme complex that is composed of a specific form of cytochrome P-450 and a flavoprotein, NADPH-cytochrome P-450 reductase. Aromatase activity of granulosa cells is increased markedly by follicle-stimulating hormone (FSH) and by analogs of cyclic AMP. It was the objective of the present study to investigate the effects of FSH and dibutyryl cyclic AMP (Bt2cAMP) on the synthesis of NADPH-cytochrome P-450 reductase in rat granulosa cells maintained in vitro. Granulosa cells were obtained from the ovaries of diethylstilbestrol (DES)-treated immature rats and were incubated in the presence of DES (10(-7) M), DES + FSH (250 ng/ml), or DES + Bt2cAMP (1 mM) for up to 72 h. After 72 h of incubation, aromatase activity of cells incubated with DES alone was 5 pmoles estrogen formed 2 h-1 mg-1 protein and was increased greater than 60-fold in cells incubated with FSH or Bt2cAMP. NADPH-cytochrome P-450 reductase was immunoisolated from [35S]methionine-labeled lysates of granulosa cells incubated for 72 h in the absence or presence of stimulatory factors. The rate of synthesis of reductase was found to be increased about 3-fold in cells incubated with DES + FSH or DES + Bt2cAMP as compared to cells incubated with DES alone. By immunoblot analysis we found that the cellular content of reductase was increased about 2-fold by FSH and Bt2cAMP treatment. Reductase specific activity was 10 nmoles min-1 mg-1 protein in membrane fractions of DES-treated cells and was increased 1.6-fold by FSH treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Mapping the active site polarity in structures of endothelial nitric oxide synthase heme domain complexed with isothioureas.

Analyzing the active site topology and plasticity of nitric oxide synthase (NOS) and understanding enzyme-drug interactions are crucial for the development of potent, isoform-selective NOS inhibitors. A small hydrophobic pocket in the active site is identified in the bovine eNOS heme domain structures complexed with potent isothiourea inhibitors: seleno analogue of S-ethyl-isothiourea, S-isopropyl-isothiourea, and 2-aminothiazoline, respectively. These structures reveal the importance of nonpolar van der Waals contacts in addition to the well-known hydrogen bonding interactions between inhibitor and enzyme. The scaffold of a potent NOS inhibitor should be capable of donating hydrogen bonds to as well as making nonpolar contacts with amino acids in the NOS active site.