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K J Netter

Publications and source records attributed to K J Netter.

At least 37 records · Page 2Linked to original sources
PubMed1991

Carbonyl reduction of metyrapone in human liver.

Carbonyl reduction was investigated in cytosolic and microsomal fractions of human liver using the ketone metyrapone as a substrate. The cytosolic enzyme has a stronger preference for NADPH over NADH than the microsomal enzyme: the former shows only 14% of the NADPH-supported activity while the latter exhibits 36% activity with NADH. Barbitone and quercitrin, the classic inhibitors of carbonyl reductases, do not affect metyrapone reduction in either fraction. Dicumarol and indomethacin, the specific inhibitors of NAD(P)H: quinone-oxidoreductase and dihydrodiol dehydrogenase, respectively, only slightly decreased metyrapol formation. In contrast, 5 alpha-dihydrotestosterone, the active form of the androgen steroid testosterone, inhibited metyrapone reduction very strongly in the microsomal fractions and is postulated to be the physiological substrate of the enzyme. This resembles the situation in mouse liver [E. Maser and K. J. Netter, Biochem Pharmacol 38: 3049-3054, 1989] where microsomal metyrapone reductase was inhibited by steroids and the purified enzyme was demonstrated to mediate androsterone oxidation. Immunoblot analysis revealed antigenic cross-reaction of antibodies against the 34 kDa metyrapone reductase from mouse liver microsomes with the homologous protein in human liver microsomes pointing to structural homologies between the respective enzymes of the two species. These results--together with previous findings, which have shown that there exist functional as well as structural relationships between microsomal mouse liver metyrapone reductase and 3 alpha-hydroxysteroid dehydrogenase from Pseudomonas testosteroni [E. Maser, U. Oppermann and K. J. Netter, Eur J Pharmacol 183:1366, 1990]--suggest that metyrapone reduction in human liver microsomes might be catalysed by a microsomal hydroxysteroid dehydrogenase.

Alcohol Oxidoreductases↗
PubMed1991

Heterogeneity of carbonyl reduction in subcellular fractions and different organs in rodents.

The pattern and distribution of carbonyl reduction in liver, kidney and adrenal gland subcellular fractions of NMRI mice, Wistar rats and Hartley guinea pigs were examined using the ketone compound metyrapone (2-methyl-1,2-di(3-pyridyl)1-propanone) commonly used as a diagnostic cytochrome P450 inhibitor. A direct HPLC method for alcohol metabolite determination instead of the indirect spectrophotometric recording of pyridine nucleotide oxidation at 340 nm was applied. All the tissues examined in these species rapidly reduced the employed compound but at the subcellular level no general distribution scheme of specific activity was found, although in all fractions metyrapol formation could be attributed to aldo-keto reductases. Cytosolic and microsomal metyrapone reducing enzymes are distinguished by their inhibitor sensitivity to phenobarbitone and quercitrin and thus can be characterized as aldehyde and ketone reductases according to the inhibitor subclassification of the aldo-keto reductase family. Moreover, the enzymes also differ with respect to their immunological cross-reactivity to anti-microsomal mouse liver metyrapone reductase antibodies. Immunological homologies were found between metyrapone reductases of liver microsomes from all species and kidney and adrenal gland microsomes from guinea pig. However, the protein of all the cytosolic fractions as well as that of kidney and adrenal gland microsomes from mouse and rat did not cross-react with the antibodies, indicating the absence of common antigenic determinants. From catalytic properties and functional data it is concluded that hydroxysteroid dehydrogenases present in the suspected subcellular fractions form a structurally and functionally related enzyme family which may have been conserved during evolution.

Adrenal Glands↗
PubMed1991

Reductive metabolism of metyrapone by a quercitrin-sensitive ketone reductase in mouse liver cytosol.

Mouse liver cytosol catalyses the reduction of metyrapone to the corresponding alcohol metabolite metyrapol. The enzyme involved was characterized as a NADPH-dependent carbonyl reductase which is strongly inhibited by the plant flavonoid quercitrin but which shows no sensitivity to phenobarbital. Thus, by inhibitor subdivision of carbonyl reductases the metyrapone reductase in mouse liver cytosol has to be classified as a ketone reductase rather than an aldehyde reductase, as it was shown previously for the analogous enzyme in mouse liver microsomes based on the same pattern of inhibitor classification. Moreover, immunological comparison of the metyrapone reductases from the two subcellular fractions reveal no common antigenic determinants indicating the structural difference between these enzymes. In conclusion, metyrapone undergoes reductive biotransformation mediated by two clearly distinct carbonyl reductases located in different subcellular compartments of mouse liver cells. Considering the convenient and sensitive HPLC-method for direct metyrapol determination, metyrapone may serve as a useful tool in the investigation of these enzymes, although their physiological roles remain to be determined.

Alcohol Oxidoreductases↗
PubMed1991

The occurrence of carbonyl reduction in continuous cell lines emphasizes the essentiality of this metabolic pathway.

Using the ketone compound metyrapone (MPON) as a substrate for carbonyl reduction it has been verified for the first time that various permanent cell lines in culture express carbonyl reducing activity. This is even true for the dedifferentiated and fibroblastoid cell line V79, emphasizing the essentiality of this metabolic pathway. MPON reducing enzyme activities are located in the endoplasmic reticulum as well as in the cytoplasm of the cells. Compared to MPON-reductase in rat liver microsomes, no immunological homology to microsomal C2REV7 rat liver hepatoma cell MPON-reductase could be detected, indicating differences in antigenic determinants between the enzymes of the solid organ and respective cells in continuous culture.

Animals↗
PubMed1991

Isolation, partial purification, and characterization of the cytochrome P-450-dependent monooxygenase system from the midgut of the earthworm Lumbricus terrestris.

1. Cytochrome P-450 was purified from microsomes of the midgut of the earthworm Lumbricus terrestris up to a maximal specific content of 5.5 nmol P-450/mg protein. 2. At least 3 different cytochromes P-450 with apparent molecular weights of 48,000, 51,000 and 53,000 were identified by SDS-PAGE. 3. Western blot analysis with various polyclonal antibodies did not show structural epitopes common to the cytochromes P-450 of rodents or yeast and L. terrestris. 4. The microsomes contained about 43 pmol P-450/mg protein corresponding to 0.51 nmol P-450/g midgut and 64 pmol P-450/g body weight, respectively, and converted benzyloxyresorufin into resorufin with a Vmax of 2.12 pmol resorufin/min.mg protein and a Km of 770 nM benzyloxyresorufin at 25 degrees C, pH 8.0. 5. The microsomes exhibited a NADPH-cytochrome P-450 reductase activity of 9.4 nmol cytochrome c/min.mg protein. 6. The apparent molecular weight of the threefold-purified reductase was 63,000.

Animals↗
PubMed1990

7-Alkoxyquinolines: new fluorescent substrates for cytochrome P450 monooxygenases.

A series of 7-alkoxyquinolines was synthesized and tested as substrates with hepatic microsomes prepared from male Wistar rats. Microsomal O-dealkylation rates and kinetic constants were determined for the 7-alkoxyquinolines with microsomes from control, 3-methylcholanthrene (MC)-pretreated, and phenobarbitone (PB)-pretreated rats. Structure-activity relationship studies indicated that the 7-benzyloxyquinoline was the most rapidly metabolized substrate for control microsomes and those from PB-pretreated rats, whereas the 7-ethoxy- and 7-propoxyquinolines were O-dealkylated more rapidly by microsomes of MC-pretreated animals. Differences in activities occurred in Vmax and apparent Km values; however, there does not appear to be a correlation between these two values for the different quinoline substrates. Apparent Km and Vmax values for the 7-alkoxyquinolines were: control microsomes, Km = 71-773 microM, Vmax = 0.37-8.4 nmol 7-quinolinol/min/mg protein; MC microsomes, Km = 0.5-14 microM, Vmax = 0.29-2.7 nmol 7-quinolinol/min/mg protein; PB microsomes, Km = 2.8-46 microM, Vmax = 0.9-12 nmol 7-quinolinol/min/mg protein. All of the quinoline substrates gave Type I binding spectra with control and MC microsomes. With PB microsomes, Type I. Reverse Type I, and a mixture of the two types of binding spectra were observed. Comparisons of the structure-activity relationships, levels of induction, and kinetic constants were made with 7-alkoxycoumarin and 7-alkoxyphenoxazone analogs. In addition, three new coumarin substrates (7-pentoxy-, 7-hexoxy-, and 7-benzyloxycoumarin) are described.

Animals↗
PubMed1990

Alteration of cytochrome P-450 by prolonged administration of imipramine and/or lithium to rats.

The aim of this study was to investigate imipramine-induced alterations of cytochrome P-450 and to determine whether prolonged concomitant administration of imipramine and lithium results in a pharmacokinetic interaction. Male Wistar rats received imipramine (10 mg/kg i.p.) at 12 h intervals or lithium chloride (100 mg/kg in drinking water) or they were treated with the combination of these drugs for 2 weeks. The long term treatment with imipramine produced a very complex alteration of cytochrome P-450: imipramine increased the level of the cytochrome, but it decreased the rate of its own aromatic hydroxylation in position 2. The rate of N-demethylation in the side chain was not changed. Consequently, in the case of both hydroxylation and demethylation, calculated molecular activities were decreased to 48% and 70% respectively. This differential change in activities corresponded well to the observed decrease of absorption in difference spectra (type I) produced in microsomes by imipramine. Carbamazepine-induced type I difference spectra were also decreased by imipramine pretreatment, but to a lesser extent. In contrast, hexobarbital type I binding was increased by imipramine treatment while type II difference spectra produced by metyrapone were not affected. The preliminary SDS-PAGE analysis of cytochrome P-450 isoenzymes of control and imipramine treated rats showed that the investigated antidepressant markedly intensified a protein band at 50.11 kD while bands at 51.28 kD, 56.20 kD and 56.88 kD were less intensive. These results indicate that the alteration of cytochrome P-450 by imipramine treatment is not only of quantitative but also of qualitative character. Lithium alone given to rats affected neither the concentration of cytochrome P-450 in microsomal protein nor the rate of imipramine metabolism in vitro. Lithium given jointly with imipramine reduced imipramine-induced elevation of cytochrome P-450.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

[Effect of untreated and non-irritating purified coffee and carbonic acid hydroxytryptamides on the gastric mucosa in the rat].

Commercial coffees frequently are processed to reduce the gastric irritation perceived by many coffee consumers. The influence of coffee infusions on gastric mucosa of rats was studied by observing the frequency of distention ulcers. Two kinds of coffee were used: untreated and treated coffee with decreased carbonic acid hydroxytryptamide-content. Infusions of untreated coffee given ad libitum induced severe gastric lesions after a period of at least 13 weeks, while treated coffee did not. In treated coffee the amount of carbonic acid hydroxytryptamides (CHT) is considerably decreased by the so-called CHT-procedure. Further, the ulcerogenic effect of CHT was studied by feeding experiments in rats with the same method resulting in severe lesions of the rat stomach after 3 and 8 weeks, respectively, of feeding. The possible causal effect of the coffee constituent CHT on the ulcerogenic effect of untreated roasted coffee is discussed.

Animals↗
PubMed1989

Purification and properties of a metyrapone-reducing enzyme from mouse liver microsomes--this ketone is reduced by an aldehyde reductase.

A ketone reducing enzyme was purified to homogeneity from female mouse liver microsomes, using the diagnostic cytochrome P-450 inhibitor metyrapone as a substrate. In contrast to the usually employed indirect spectrophotometric recording of pyridine nucleotide oxidation at 340 nm, a HPLC method was applied for direct alcohol metabolite determination. Purification of the carbonyl reductase resulted in a 360-fold increase in specific activity together with a single band in the 34 kD region after SDS-polyacrylamide gel electrophoresis. Phenobarbital, indomethacin, dicoumarol and 5 alpha-dihydrotestosterone inhibited the enzyme, whereas quercitrin did not affect the enzyme activity. Thus, by inhibitor classification of carbonyl reductases the ketone metyrapone is reduced by an aldehyde reductase, rather than by a ketone reductase. Dihydrotestosterone, the strongest inhibitor, is supposed to be the physiological substrate for the purified enzyme. It was demonstrated that during the steps of purification both NADPH and NADH can supply the required reducing equivalents, although the activity with NADH is weaker. The highest activity was obtained using an NADPH-regenerating system. Ethanol and the nonionic detergent Emulgen 913 led to an increased specific activity, indicating that the enzyme is bound to the membranes of the endoplasmic reticulum in a latent state. From these results it is concluded that the microsomal metyrapone-reducing enzyme belongs to the family of carbonyl reductases, but differs from the common patterns of their classification with regard to cofactor requirement and inhibitor susceptibility.

Alcohol Dehydrogenase↗
PubMed1989

Indium selectively increases the cytochrome P-450 dependent O-dealkylation of coumarin derivatives in male mice.

Indium pretreatment of rats and mice has been reported to decrease the concentration of cytochrome P-450, thereby reducing the activity of some cytochrome P-450 dependent enzymatic reactions. The present study reveals that pretreatment of C57Bl/6JHan mice of both sexes with one s.c. dose of 120 mg of In2(SO4)3.5 H2O per kg of body weight decreases the concentration of cytochrome P-450 to about 65% of control levels. Neither cytochrome b5 nor NADPH-cytochrome P-450 reductase is affected. Hepatic microsomal ethoxyresorufin O-deethylase activity declines to about 75% of control values. In contrast, with coumarin substrates, a sex dependence in the direction of change is observed: in female mice indium decreases the activity to about 75%, whereas in males it enhances the activity to 140%. Moreover, with 7-(methoxy-14C)coumarin as substrate, indium-pretreated male mice exhale about 180% and females about 65% of 14CO2 compared to the corresponding controls. A close correlation between the in vivo and in vitro effects of indium on the metabolism of the coumarin derivatives is suggested. After isolation and purification of cytochrome P-450, SDS-PAGE indicates in indium-pretreated male mice an intensification of a 48.5 kDa protein band which is decreased in females. Immunological studies using antibodies raised against control female cytochrome P-450 show cross reactivity among all microsomes used in these experiments. High percentages of inhibition occur in microsomes with high molecular activity towards coumarin derivatives. The in vitro kinetics of antibody-inhibited O-deethylation of 7-ethoxycoumarin seems to obey a non- or partial-competitive type of inhibition. Indium pretreatment of mice produces sex-dependent effects on the metabolism of coumarin derivatives.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1989

Brief maternal deprivation of rats reduces hepatic mixed function oxidase activities.

Deprivation of pups from mother and sibs for 3 min daily from day 5 to day 41 of life reduced activities of 4 hepatic mixed function oxidases (MFO) expressed per mg protein in male rats compared to unhandled control rats. These decreases, though generally small, 22.4% and under, reached statistical significance for the substrates aminopyrine, benzphetamine and ethoxycoumarin. This handling procedure did not consistently affect the inductive response to phenobarbital. Previously ignored as a source of variability in response to xenobiotics, "handling" appears from these results to merit further investigation as such a factor in uninduced rats. Differences among rats in "handling" could contribute to large day-to-day variations in their metabolism of xenobiotics.

Aminopyrine↗
PubMed1989

Interactions of heterocyclic Maillard products with the hepatic microsomal monooxygenase system.

1. Interactions of methyl-substituted pyrazines, and other constituents of Maillard products generated during heat treatment of food, with hepatic microsomal mixed-function oxygenases were studied in vitro. 2. Spectral interactions of N-containing heteroaromatic compounds with the cytochrome P-450 system are type I or type II depending on the state of induction, and are relatively weak. Inhibition of 7-ethoxycoumarin O-deethylation by these compounds is ten times lower than that of metyrapone, agreeing with the weak spectral interaction. Inhibition is competitive for 2,3-dimethylquinoxaline, and complex for 2,5-dimethylpyrazine and 2,3,5,6-tetramethylpyrazine. 3. Spectral and inhibitory interactions indicate biotransformation. This was studied with 2,3,5,6-tetramethylpyrazine; the metabolite formed was identified as 2-hydroxymethyl-3,5,6-trimethylpyrazine. Metabolism to the N-oxide did not occur.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1988

Metabolic interaction between imipramine and carbamazepine in vivo and in vitro in rats.

The pharmacokinetic consequences of the combination of carbamazepine with imipramine in male Wistar rats have been investigated. It was found that a 2-week treatment with the combination resulted in the increase of the concentrations of the parent compounds and a simultaneous decrease in their metabolites in blood plasma i.e. carbamazepine inhibited imipramine demethylation in the side chain while imipramine inhibited carbamazepine 10,11-epoxidation. The velocity of imipramine 2-hydroxylation and 10,11-epoxy-carbamazepine hydration did not seem to be changed by the combination. On the basis of studies in vitro it is concluded that the observed metabolic interaction between carbamazepine and imipramine is due to the competition of the drugs for the active centre of cytochrome P 450 and to a certain qualitative alteration of the enzyme by imipramine as can be deducted from the decrease of carbamazepine binding to the cytochrome.

Animals↗
PubMed1987

A qualitative study on vanadate effects in the tetrahydrofolate-dependent formate transfer in vitro and in vivo in mice.

Parenteral administration of sodium vanadate (NaVO3, 22 mg/kg b.w., i.p.) to mice depresses the oxidation rate of [14C]formate to [14C]CO2, as determined by radioactive breath analysis. The heavy metal-induced inhibition is relatively fast in onset, fairly intense (up to 80% inhibition), of short duration (about 2 h) and strongly correlated to the presence of vanadate (in the pentavalent state) in plasma. The [14C]CO2 exhalation rate from [14C]bicarbonate is less affected by vanadate in vivo, thereby suggesting a specific interference of vanadium in the intermediate step of formate oxidation to HCO3-. In vitro in mouse liver cytosolic fractions vanadate inhibits the enzymatic transfer of formate to tetrahydrofolic acid. The inhibition is accomplished by a vanadate-dependent oxidative degradation of tetrahydrofolate. In contrast, the concentrations of N5-methyltetrahydrofolate, dihydrofolate and folate remain unchanged upon in vitro-exposure to vanadate. The in vitro studies thus might explain the observed inhibition of formate oxidation to carbon dioxide in vivo by a vanadate-evoked depletion of its biological carrier tetrahydrofolic acid. Whether the interference in tetrahydrofolate metabolism also occurs under in vivo conditions, remains to be elucidated.

Animals↗