Inhibition of intestinal peroxidase activity by nonsteroidal antiinflammatory drugs

The peroxidase activity of the mitochondrial fraction of rat intestine is inhibited in vitro by non-steroidal antiinflammatory drugs (NSAIDs), such as indomethacin (IMN) and acetylsalicylic acid (ASA), the former being more potent than the latter. The peroxidase was solubilised by cetab-NH₄Cl extraction and purified to apparent homogeneity by Sephadex G-150 gel filtration and affinity chromatography on Con-A Sepharose. The purified enzyme activity was 80% inhibited by 150 μM IMN and 50% by 2.67 mM ASA. IMN could also inhibit lactoperoxidase activity to the same extent but not the horseradish peroxidase activity. The inhibition of peroxidase-catalysed iodide oxidation by IMN and ASA was optimal at pH 5.5 and 4.5, respectively. Kinetic studies revealed that the inhibition by IMN was competitive with respect to iodide or guaiacol, while the inhibition by ASA was noncompetitive and reversible in nature. Studies of some structural analogues showed that indole-3-acetic acid was as effective as IMN, while salicylic acid was more potent than ASA. Spectral studies showed a small bathochromic shift of the Soret band of the enzyme by IMN, suggesting its possible interaction at or near the heme moiety. The competitive nature of IMN may be explained as due to its oxidation by the peroxidase to a product absorbing at 412 nm, the formation of which is inhibited by iodide. We suggest that IMN inhibits intestinal peroxidase activity by acting as a competitive substrate for the enzyme. As intestinal peroxidase is mainly contributed by the invading eosinophils, NSAIDs may affect the host defence mechanism by inhibiting the activity of the enzyme.     

Metallothionein induction by nonsteroidal antiinflammatory drugs

In the present study we report on the effects of commonly used nonsteroidal antiinflammatory drugs on metallothionein (MT) and MT-I mRNA levels. A single dose of chloroquine (100 mg/kg), diclofenac (100 mg/kg), indomethacin (10 mg/kg), or piroxicam (100 mg/kg) was administered ip to C57B1 mice. After 18 h, MT levels were determined with a Cd-saturation radioassay. MT-I mRNA levels were measured by Northern Blot analyses using a probe containing the mouse MT-I gene. All drugs tested caused an increase in the MT content of the liver but not of the kidneys and lung. The lowest and highest effects were observed with chloroquine (8 times the control value) and diclofenac (18 times), respectively. In accordance with the stimulation of MT synthesis, increased accumulation of hepatic MT-I mRNA could be demonstrated. These results indicate that elevated MT levels may contribute to the effectiveness of nonsteroidal antiinflammatory drugs in the treatment of rheumatoid arthritis (RA).     

Purification and properties of 3 alpha-hydroxysteroid dehydrogenase from rat brain cytosol. Inhibition by nonsteroidal anti-inflammatory drugs and progestins

The 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) of rat brain cytosol has been purified to apparent homogeneity. The purification procedure involves six successive steps, includes one affinity chromatography, and yields enzyme which displays a 1,550-fold enhancement in specific activity. The homogeneous enzyme has a Km of 8.0 microM for 5 alpha-dihydrotestosterone, a Vmax of 1.3 mumol of 3 alpha-androstanediol formed per h/mg of protein, and displays a preference for NADPH. It appears to be the major activity responsible for the reduction of 5 alpha-dihydrotestosterone in this tissue and may play a pivotal role in brain androgen metabolism. The homogeneous enzyme has several properties in common with the 3 alpha-hydroxysteroid dehydrogenase purified from rat liver cytosol (Penning, T. M., Mukharji, I., Barrows, S., and Talalay, P. (1984) Biochem. J. 222, 601-611). It is a monomer with a molecular weight of 31,000, it has a pI of 5.5, and it is potently inhibited by the nonsteroidal anti-inflammatory drugs (IC50 value for indomethacin = 2.0 microM). The potency of inhibition observed for the brain enzyme parallels that observed for cyclooxygenase: indomethacin greater than fenamates greater than l-methylpyrrole acetic acids greater than arylpropionic acids greater than salicylates greater than acetaminophen. Examination of a variety of steroidal contraceptives as modulators of the dehydrogenase indicates that ethinylestradiol is a very poor inhibitor (IC50 = 100 microM), while 6-medroxyprogesterone acetate (Provera) is an extremely potent inhibitor (IC50 = 0.2 microM). The possibility exists that brain androgen metabolism may be altered by the nonsteroidal anti-inflammatory drugs and synthetic progestins.     

Soybean lipoxygenase inhibition by nonsteroidal antiinflammatory drugs

Eighteen known nonsteroidal antiinflammatory drugs (NSAID) were tested for their action against soybean lipoxygenase (E.C.1.13.11.12) using linoleic acid as substrate. It was found that the best inhibitors of lipoxygenase were naproxen, BW 755C, indomethacin and isoxicam. Drugs with intermediate potency were meclofenamic acid, phenyl-butazone and benoxaprofen. Other drugs such as ibuprofen and zomepirac were only weakly active in the test.     

Inhibition of the human placental NAD- and NADP-linked 15-hydroxyprostaglandin dehydrogenases by nonsteroidal anti-inflammatory drugs

A number of nonsteroidal anti-inflammatory drugs are non-competitive or mixed inhibitors of human placental dehydrogenases. - and -sulindac sulfide and - and -sulindac inhibit the NAD-linked enzyme as well or better than they inhibit various cyclooxygenases . The remainder of the compounds tested are at least one order of magnitude less effective as inhibitors of the 15-hydroxyprostaglandin dehydrogenases than they are as inhibitors of cyclooxygenases. - and -sulindac sulfide are sufficiently strong inhibitors of the NAD-linked enzyme (K_is of 7.8 μM and 6.8 μM respectively) to raise the possibility that they might also inhibit this enzyme .     

Inhibition of the human placental NAD- and NADP-linked 15-hydroxyprostaglandin dehydrogenases by nonsteroidal anti-inflammatory drugs

A number of nonsteroidal anti-inflammatory drugs are non-competitive or mixed inhibitors of human placental NAD- and NADP-linked 15-hydroxyprostaglandin dehydrogenases. Cis- and trans-sulindac sulfide and cis- and trans-sulindac inhibit the NAD-linked enzyme as well or better than they inhibit various cyclooxygenases in vitro. The remainder of the compounds tested are at least one order of magnitude less effective as inhibitors of the 15-hydroxyprostaglandin dehydrogenases than they are as inhibitors of cyclooxygenases. Cis- and trans-sulindac sulfide are sufficiently strong inhibitors of the NAD-linked enzyme (Kis of 7.8 microM and 6.8 microM respectively) to raise the possibility that they might also inhibit this enzyme in vivo.     

Signal peptide peptidase: biochemical properties and modulation by nonsteroidal antiinflammatory drugs

Signal peptide peptidase (SPP) is an intramembrane aspartyl protease that cleaves remnant signal peptides after their release by signal peptidase. SPP contains active site motifs also found in presenilin, the catalytic component of the gamma-secretase complex of Alzheimer's disease. However, SPP has a membrane topology opposite that of presenilin, cleaves transmembrane substrates of opposite directionality, and does not require complexation with other proteins. Here we show that, upon isolation of membranes and solubilization with detergent, the biochemical characteristics of SPP are remarkably similar to gamma-secretase. The majority of the SPP-catalyzed cleavages occurred at a single site in a synthetic substrate based on the prolactin (Prl) signal sequence. However, as seen with cleavage of substrates by gamma-secretase, additional cuts at other minor sites are also observed. Like gamma-secretase, SPP is inhibited by helical peptidomimetics and apparently contains a substrate-binding site that is distinct from the active site. Surprisingly, certain nonsteroidal antiinflammatory drugs known to shift the site of proteolysis by gamma-secretase also alter the cleavage site of Prl by SPP. Together, these findings suggest that SPP and presenilin share certain biochemical properties, including a conserved drug-binding site for allosteric modulation of substrate proteolysis.     

Identification of two dihydrodiol dehydrogenases associated with 3(17)alpha-hydroxysteroid dehydrogenase activity in mouse kidney

Dihydrodiol dehydrogenase activity was detected in the cytosol of various mouse tissues, among which kidney exhibited high specific activity comparable to the value for liver. The enzyme activity in the kidney cytosol was resolved into one major and three minor peaks by Q-Sepharose chromatography: one minor form cross-reacted immunologically with hepatic 3 alpha-hydroxysteroid dehydrogenase and another with aldehyde reductase. The other minor form was partially purified and the major form was purified to homogeneity. These two forms, although different in their charges, were monomeric proteins with the same molecular weight of 39,000 and had similar catalytic properties. They oxidized cis-benzene dihydrodiol and alicyclic alcohols as well as trans-dihydrodiols of benzene and naphthalene in the presence of NADP+ or NAD+, and reduced several xenobiotic aldehydes and ketones with NAD(P)H as a cofactor. The enzymes also catalyzed the oxidation of 3 alpha-hydroxysteroids and epitestosterone, and the reduction of 3- and 17-ketosteroids, showing much lower Km values (10(-7)-10(-6) M) for the steroids than for the xenobiotic alcohols. The results of mixed substrate experiments, heat stability, and activity staining on polyacrylamide gel electrophoresis suggested that, in the two enzymes, both dihydrodiol dehydrogenase and 3(17)alpha-hydroxysteroid dehydrogenase activities reside on a single enzyme protein. Thus, dihydrodiol dehydrogenase existed in four forms in mouse kidney cytosol, and the two forms distinct from the hepatic enzymes may be identical to 3(17)alpha-hydroxysteroid dehydrogenases.     

Histochemical distribution of delta5-3beta- and 17beta-hydroxysteroid dehydrogenases in hamster trophoblast.

The histochemical distribution of delta5-3beta- and 17beta-hydroxysteroid dehydrogenases was demonstrated in hamster trophoblast between Days 8 and 15 of pregnancy. The delta5-3beta-hydroxysteroid dehydrogenase activity in the ectoplacental trophoblast of 8-day embryos was demonstrated by use of delta5-pregnenolone and dehydroepiandrosterone as substrates; between Days 11 and 15, activity was demonstrated in the trophoblastic giant cells of the placenta and in the intra-arterial trophoblast cells when delta5-pregnenolone was the substrate. Between Days 11 and 15, 17beta-hydroxysteroid activity was present in the spongiotrophoblast, labyrinth, placental giant cells and intra-arterial trophoblast cells, as shown by use of testosterone and oestradiol as substrates. Both enzymes were demonstrated in ectopic trophoblast cells, indicating that these activities are autonomous.     

Inhibition of neutrophil priming and tyrosyl phosphorylation by cepharanthine, a nonsteroidal antiinflammatory drug.

Receptor-mediated superoxide (O2.-)-generation and tyrosyl phosphorylation of neutrophil proteins, such as 58, 65, 84, 108 and 115 kDa, were enhanced by priming cells with granulocyte colony stimulating factor (G-CSF) [Akimura, K. et al. Arch. Biochem. Biophys. 298: 703-709, 1992]. To elucidate the possible involvement of tyrosyl phosphorylation of neutrophil proteins in the enhancing mechanism of O2.- generation, the effect of cepharanthine, a biscoclaurine alkaloid that inhibits phorbol 12-myristate 13-acetate (PMA)- and receptor-mediated O2.- generation, on the priming of human peripheral neutrophils (HPPMN) was studied. Both enhancement of formyl-methionyl-leucyl- phenylalanine (FMLP)-mediated O2.- generation and tyrosyl phosphorylation of some neutrophil proteins, i.e., 115, 108 and 84 kDa proteins, by HHPMN after treatment with G-CSF were strongly inhibited by cepharanthine in a concentration- and treatment-time-dependent manner. In contrast, inhibition of PMA-mediated O2.- generation by cepharanthine was weak and independent of treatment time. These results suggest that cepharanthine might inhibit the priming step of neutrophil activation concomitantly with its inhibition of the tyrosyl phosphorylation of some neutrophil proteins that might underlie the mechanism for priming of neutrophils with G-CSF.     

Inhibition of 3(17)alpha-hydroxysteroid dehydrogenase (AKR1C21) by aldose reductase inhibitors

Mouse 3(17)alpha-hydroxysteroid dehydrogenase (AKR1C21) is a member of the aldo-keto reductase superfamily that catalyses the oxido-reduction of steroid hormones such as estrogens, androgens and neurosteroids. Inhibitors of aldose reductase (AR), a member of the same superfamily, were evaluated against AKR1C21. Models of the enzyme-inhibitor complexes suggest that Tyr118 and Phe311 are important residues for inhibitor recognition and orientation in the active site of AKR1C21.     

Development of affinity labeling agents based on nonsteroidal anti-inflammatory drugs: labeling of the nonsteroidal anti-inflammatory drug binding site of 3 alpha-hydroxysteroid dehydrogenase.

Nonsteroidal anti-inflammatory drugs (NSAIDs) exert their effect by inhibiting the target enzyme cyclooxygenase (prostaglandin H2 synthase); however, little is known about the peptides comprising its NSAID binding site. Hydroxyprostaglandin dehydrogenases also bind NSAIDs, but their NSAID binding sites have not been well characterized. Using existing synthetic strategies, we have incorporated the bromoacetoxy affinity labeling moiety around the perimeter of two potent NSAIDs, indomethacin and mefenamate, a N-phenylanthranilate. The compounds synthesized were 1-(4-(bromoacetamido)benzyl)-5-methoxy-2-methylindole-3-acetic acid (1), 3-(2-(2-bromoacetoxy)ethyl)-1-(4-chlorobenzyl)-5-methoxy-2-methylindole (2), 4-(bromoacetamido)-N-(2,3-dimethylphenyl)anthranilic acid (3), N-(3-(bromoacetamido)phenyl)-anthranilic acid (4), and N-(4-(bromoacetamido)phenyl)anthranilic acid (5). To access whether these compounds have general utility in labeling NSAID binding sites, the compounds were evaluated as affinity labeling agents for 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) from rat liver cytosol. This enzyme displays 9-, 11-, and 15-hydroxyprostaglandin dehydrogenase activity, is inhibited potently by NSAIDs, and is homologous to bovine lung prostaglandin F synthase. Compounds 1-5 were shown to affinity label the NSAID binding site of 3 alpha-HSD. They inactivated 3 alpha-HSD through an E.I complex in a time- and concentration-dependent manner with t1/2 values ranging from seconds to hours. Ligands that compete for the active site of 3 alpha-HSD (NAD+ and indomethacin) afforded protection against inactivation, and the inactivators could demonstrate competitive kinetics against 3 alpha-hydroxysteroid substrates by forming an E.NAD+.I complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

An improved method for the determination of cytoplasmic 3 alpha- and 17 beta- and microsomal 3 alpha-hydroxysteroid dehydrogenase activities in rat liver.

This paper described a modified method for the radiometric determination of hydroxysteroid dehydrogenase activities in rat liver. The principle advantages of this method are the improved precision and a radical reduction in the time involved in performing the assay. The procedure comprises the following steps: incubation of 14C-labelled substrate with coenzyme and cell fraction under optimized conditions; termination of the reaction by addition of organic solvent containing a defined amount of 3H-labelled reaction product; removal of precipitated protein and coenzyme by centrifugation; paper chromatographic isolation of the product; direct quantitation of 14C activity in the product zone of the paper chromatogram. The assay systems have been applied to elucidate and quantitate sex and strain differences in the activities of the above enzymes in Chbb/THOM and Sprague-Dawley rats.     

Electrophoretic and immunochemical characterization of 3 alpha-hydroxysteroid/dihydrodiol dehydrogenases of rat tissues.

The properties of 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase from Sprague-Dawley rat liver cytosol have been re-examined in light of several reports which suggest that multiple forms of the enzyme may exist in this tissue. During enzyme purification, chromatography on DE-52 cellulose and chromatofocusing columns indicated the existence of only one form of the protein. Re-chromatography of the purified enzyme by either of these techniques failed to resolve the protein into additional forms. When the purified enzyme was subjected to SDS/polyacrylamide-gel electrophoresis a single band corresponding to Mr 34,000 was detected. Two-dimensional gels showed one predominant protein with a pI of 5.9. Using the homogeneous enzyme as antigen, high-titre polyclonal antibody was raised in rabbits. Western-blot analysis of cytosolic proteins prepared from male and female Sprague-Dawley rat liver indicated the presence of a single immunoreactive band with an Mr of 34,000 in both sexes. All of the 3 alpha-hydroxysteroid dehydrogenase activity present in rat liver cytosol could be immunotitrated with the antibody and the resulting titration curve was superimposable on the titration curve obtained with the purified enzyme. Western-blot analysis of cytosolic proteins prepared from livers of male Wistar and Fischer rats also revealed the presence of a single immunoreactive protein with an Mr of 34,000. These data indicate that, contrary to previous reports, only one form of the dehydrogenase may exist in liver cytosols prepared from a variety of rat strains. Although 3 alpha-hydroxysteroid dehydrogenase activity is known to be widely distributed in male Sprague-Dawley rat tissues, Western blots indicate that only the liver, lung, testis and small intestine contain immunoreactive protein with an Mr of 34,000. The levels of immunoreactive protein in these tissues follow the distribution of dihydrodiol dehydrogenase.