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PubMed1986

Some properties of lipoxygenase activities in cytosol and microsomal fractions of mouse epidermal homogenate.

Lipoxygenase activity in microsomal fraction of mouse epidermal homogenate was characterized in comparison with cytosol lipoxygenase activity. The major activity was identified as 12-lipoxygenase in microsomal fraction as well as in cytosol fraction by the analyses with high-performance liquid chromatography and gas chromatography-mass spectrometry. Apparent Km values of cytosol and microsomal 12-lipoxygenase for arachidonic acid were 5.0 microM and 6.2 microM respectively. Apparent Vmax values were 14 pmol/min/mg protein for the cytosol enzyme and 32 pmol/min/mg protein for the microsomal enzyme. Net activities of cytosol and microsomal 12-lipoxygenase were 214 and 109 pmol/min/g wet tissue, respectively. Both cytosol and microsomal lipoxygenase activities were neither dependent on calcium nor ATP. Carbon monoxide failed to affect these enzyme activities. There were considerable differences either in the effect of glutathione or in the sensitivities toward several lipoxygenase inhibitors, indicating that the cytosol and the microsomal 12-lipoxygenase activities are derived from two different enzymes. Alternatively, the differences could be attributable to the different microenvironments of these enzymes.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1992

Metabolism of [3-14C]coumarin to polar and covalently bound products by hepatic microsomes from the rat, Syrian hamster, gerbil and humans.

The metabolism of 0.19 and 2.0 mM-[3-14C]coumarin to polar products and covalently bound metabolites has been studied with hepatic microsomes from the rat, Syrian hamster, Mongolian gerbil and humans. [3-14C]Coumarin was metabolized by liver microsomes from all species to a number of polar products and to metabolite(s) that became covalently bound to microsomal proteins. The polar products included 3-, 5- and 7-hydroxycoumarins, o-hydroxyphenylacetaldehyde and o-hydroxyphenylacetic acid. Coumarin 7-hydroxylation was observed in all species except the rat. With 0.19 mM-[3-14C]coumarin, 7-hydroxycoumarin was the major metabolite in human liver microsomes, whereas in the other species with 0.19 mM substrate and in all species with 2.0 mM substrate o-hydroxyphenylacetaldehyde was the major metabolite. Of the three animal species studied the gerbil most resembled humans as this species also had a high coumarin 7-hydroxylase activity. The administration of Aroclor 1254 to the rat and Syrian hamster induced both microsomal cytochrome P-450 content and [3-14C]coumarin metabolism. With liver microsomes from all species a good correlation between rates of [3-14C]coumarin metabolism and covalent binding was observed at both substrate concentrations. However, in view of the known species difference between the rat and Syrian hamster in coumarin-induced hepatotoxicity, the present data are not consistent with microsomal coumarin metabolite covalent binding being an indicator of potential liver damage.

Acetaldehyde↗
PubMed1987

Stimulation of cyclophosphamide-induced pulmonary microsomal lipid peroxidation by oxygen.

Cyclophosphamide (CP) causes lung toxicity in a wide variety of animals including humans. Recent reports suggest that CP increases lipid peroxide formation in the lung, and that oxygen (O2) potentiates CP-induced lung toxicity. We hypothesized that CP, or one of its toxic metabolites, acrolein, stimulates lung lipid peroxide formation in the presence of high O2 tensions. To test this, rat lung microsomes were treated in vitro with CP or acrolein in the presence of NADPH and 0-100% O2 with and without superoxide dismutase (SOD), glutathione (GSH), dithiothreitol (DTT), and EDTA (agents which scavenge reactive O2 species and/or detoxify reactive metabolites). Lipid peroxide formation in untreated microsomes was increased 40, 39, and 37% in 60, 80 and 100% O2 respectively (P less than 0.02 vs. 21% O2 air). Lipid peroxide formation in microsomes treated with CP increased 2-3-fold under 21% O2 (P less than 0.05 vs. untreated under 21% O2). However, increases in lipid peroxide formation were 3-4 fold in CP treated microsomes under 40-100% O2 (P less than 0.001 vs. untreated at same % O2). CP and acrolein-stimulated lipid peroxidation with and without O2 exposure was significantly (P less than 0.05) reduced by prior addition of SOD, GSH, DTT, or EDTA to the lung microsomal suspension. These results indicate that lipid peroxide formation increases in CP and acrolein-treated lung microsomes, and high O2 tensions stimulate CP-induced lipid peroxidation. Stimulation of CP-induced microsomal lipid peroxidation appears to be mediated by reactive O2 species or metabolites.

Acrolein↗
PubMed1983

Effects of lipid peroxidation on adrenal microsomal monooxygenases.

Incubation of guinea pig adrenal microsomes with 10(-6) M ferrous (Fe2+) ion and adrenal cytosol initiated high levels of lipid peroxidation as measured by the production of malonaldehyde. Cytosol or Fe2+ alone had little effect on microsomal malonaldehyde formation. When microsomes were incubated in the presence of Fe2+ and cytosol, malonaldehyde levels continued to increase for at least 60 min. Accompanying the lipid peroxidation was a decline in adrenal microsomal monooxygenase activities. The rates of metabolism of xenobiotics (benzphetamine demethylase, benzo[a]pyrene hydroxylase) as well as steroids (21-hydroxylation) decreased as malonaldehyde levels increased. In addition, cytochrome P-450 levels, NADPH- and NADH-cytochrome c reductase activities, and substrate interactions with cytochrome(s) P-450 decreased as lipid peroxidation progressed. Inhibition of lipid peroxidation by increasing microsomal protein concentrations during the incubation period prevented the changes in microsomal metabolism. Malonaldehyde had no direct effects on adrenal microsomal enzyme activities. The results indicate that lipid peroxidation may have significant effects on adrenocortical function, diminishing the capacity for both xenobiotic and steroid metabolism.

Adrenal Glands↗
PubMed1988

Examination of differences between benzo[a]pyrene and steroid hydroxylases in guinea pig adrenal microsomes.

The effects of antibodies to cytochromes P-45017 alpha,lyase and P-450C21 on benzo[a]pyrene hydroxylase activity were measured in microsomes from guinea pig adrenals. Anti-cytochrome P-45017 alpha,lyase IgG inhibited about 30% of the benzo[a]pyrene hydroxylase activity of the microsomes in the presence of excess amounts of the IgG, but anti-cytochrome P-450C21 IgG did not affect the activity. In a reconstituted system, consisting of cytochrome P-450, NADPH-cytochrome-P-450 reductase and dilauroylphosphatidylcholine, cytochrome P-45017 alpha,lyase catalyzed, in addition to steroid hydroxylation, benzo[a]pyrene hydroxylation, but cytochrome P-450C21 did not hydroxylate benzo[a]pyrene. Since anti-cytochrome P-45017 alpha,lyase IgG inhibited benzo[a]pyrene hydroxylase activity completely in the reconstituted system with cytochrome P-45017 alpha,lyase, the presence of non-inhibited benzo[a]pyrene hydroxylase in the microsomes suggests that the residual activity in the microsomes may be due to some enzyme other than cytochrome P-45017 alpha,lyase. Benzo[a]pyrene hydroxylase activity was detected in detergent-solubilized microsomes from which cytochrome P-45017 alpha,lyase was removed by using an immobilized anti-cytochrome P-45017 alpha,lyase IgG-Sepharose column. This shows the existence in microsomes of a benzo[a]pyrene hydroxylase other than cytochrome P-45017 alpha,lyase. Benzo[a]pyrene hydroxylase in the solubilized microsomes required O2, NADPH and NADPH-cytochrome-P-450 reductase for its activity and was inhibited by CO. This suggests that benzo[a]pyrene hydroxylase is a cytochrome P-450-dependent monooxygenase. This novel enzyme was also active in xenobiotic metabolism, such as 2-nitropropane denitrification and aminopyrine demethylation.

Adrenal Glands↗
PubMed1990

High-affinity binding sites for oxygenated sterols in rat liver microsomes: possible identity with antiestrogen binding sites.

Oxygenated derivatives of cholesterol are known to exhibit a number of biological activities including the inhibition of cholesterol biosynthesis and of cell proliferation, but their mechanism of action remains unclear. Previous studies have identified a cytosolic protein which binds 25-hydroxycholesterol, as well as several other oxysterols, with high affinity, possibly mediating some of their effects. We now report the existence of a high-affinity oxysterol binding site in rat liver microsomes which is distinct from the cytosolic binding protein. Among the oxygenated sterols examined, 5 alpha-cholestan-3 beta-ol-7-one (7-ketocholestanol) had the highest affinity for this microsomal binding site (Kd = 2.7 nM). Using 7-keto[3H]cholestanol as the radioactive ligand, we found that binding of this oxysterol to the microsomal binding site was saturable and reversible and was displaceable by the following oxysterols in descending order of potency: 7-ketocholestanol greater than 6-ketocholestanol greater than 7 beta-hydroxycholesterol = 7-ketocholesterol greater than cholesten-3 beta,5 alpha, 6 beta-triol = 7 alpha-hydroxycholesterol greater than 4-cholesten-3-one. All other sterols studied, including, notably, 25-hydroxycholesterol, had little or no inhibitory effect on 7-keto[3H]cholestanol binding. Additional studies revealed that the microsomal oxysterol binding site was probably identical to the antiestrogen binding site described by other workers. First, saturation analysis and kinetic studies demonstrated that the antiestrogen tamoxifen competed directly with 7-keto[3H]cholestanol for the same binding site. Second, the ability of different oxysterols and antiestrogens to inhibit 7-keto[3H]cholestanol binding to the microsomal binding site paralleled their ability to inhibit [3H]tamoxifen binding to the antiestrogen binding site. Third, the tissue distribution of binding sites for 7-keto[3H]cholestanol was similar to that of the antiestrogen binding site. We conclude that: (1) in rat liver microsomes there are high-affinity oxysterol binding sites whose ligand specificity is different from that of the cytosolic oxysterol binding protein; and (2) the microsomal oxysterol binding site is probably identical to the antiestrogen binding site. The biological significance of these observations remains to be explored.

Animals↗
PubMed1981

In vivo and in vitro effects of 3-methylcholanthrene on the microsome-mediated in vitro mutagenicity of 2-acetylaminofluorene.

Pretreatment of rat, hamster or mouse by 3-methylcholanthrene (3-MC) largely induces the liver microsomal N-hydroxylase activity. The same pretreatment given simultaneously with 2-acetylaminofluorene (2-AAF) inhibits the hepatocarcinogenicity in the rat but not in the hamster. The present report compared the in vivo and in vitro effects of 3-MC on liver microsomal N-hydroxylation and liver microsome-mediated mutagenicity of 2-AAF in hamster, rat and mouse. The induction of hamster or mouse liver microsomal N-hydroxylase activity correlated well with the increase in the microsome-mediated mutagenicity of 2-AAF. With rat, however, even though the N-hydroxylase activity is largely enhanced, microsome-mediated mutagenicity is significantly reduced after pretreatment with 3-MC. Such a reduction parallels a decrease in enzyme affinity. Added in vitro to the incubation medium, 3-MC (microM concentration) inhibits both the N-hydroxylase activity and the microsome-mediated mutagenicity of 2-AAF. Those data are discussed in relationship with the biological interactions between 3-MC and 2-AAF.

2-Acetylaminofluorene↗
PubMed1994

Regioselective metabolism of benzo[a]pyrene and 7-chlorobenz[a]anthracene by fish liver microsomes.

Many polycyclic aromatic hydrocarbons (PAHs) and chlorinated PAHs in the environment are potent mutagens and carcinogens. Using benzo[a]pyrene (BaP) and 7-chlorobenz[a]anthracene (7-Cl-BA) as representatives of PAHs and chlorinated PAHs, respectively, we studied the metabolism of these compounds in liver microsomes of Tilapia (Oreochromis hybrid), one of the most common fish in south Asia. The regioselective metabolism of BaP and 7-Cl-BA by the fish liver microsomes resulted in the formation of hydroxylated and trans-dihydrodiol metabolites of both BaP and 7-Cl-BA. The metabolites were purified by HPLC and identified by both UV/VIS and mass spectroscopic methods. The fish liver microsomes metabolized BaP to form BaP-7,8-dihydrodiol (11%), 3-hydroxy-BaP (17%), and 9-hydroxy-BaP (22%) as the major products and metabolized 7-Cl-BA to form 7-Cl-BA trans-8,9-dihydrodiol as the major metabolite (40%). The Tilapia liver microsomal P-450 enzyme activities were inducible by pretreatment with 3-methylcholanthrene (3-MC), which increased microsomal aryl hydrocarbon hydroxylase and 7-ethoxyresorufin O-deethylase activities by 74- and 360-fold, respectively. The induction of these enzymes by 3-MC was greater in fish microsomes than in rat liver. This study is the first to demonstrate the regioselective metabolism of BaP and 7-Cl-BA by fish liver microsomes.

Animals↗
PubMed1996

Increase of lipid peroxidation in rat liver microsomes by dehydroepiandrosterone feeding.

Oral administration of the adrenal steroid dehydroepiandrosterone (DHEA), a peroxisome proliferator and hepatocarcinogen in the rat, caused an increase in NADPH-dependent lipid peroxidation in microsomes isolated from rat liver and kidney cortex, but not from brain. The increase of liver microsomal lipid peroxidation was greater in male than in female rats. the effect of DHEA on lipid peroxidation became discernible after feeding steroid-containing diet (0.6%) to male and female rats for 2 and 3 days and reached maximal levels at 1 and 2 weeks, respectively. The increase of microsomal lipid peroxidation reached a plateau stimulation at 0.05% in the diet. The addition of DHEA in the concentration range 0.1-100 microM to microsomes isolated from control rats had no effect on lipid peroxidation. Furthermore, a significant increase of the endogenous concentration of thiobarbituric acid reactive substances was found in microsomes after DHEA-administration at 0.05% in the diet. These results provide in vivo evidence that DHEA can cause lipid peroxidation in rat liver. Administration of DHEA at 0.6% in the diet for 7 consecutive days also significantly enhanced NADH- and ascorbate-dependent lipid peroxidation in liver microsomes. The DHEA-stimulated rat liver microsomal lipid peroxidation was completely inhibited by EDTA but not by superoxide dismutase, catalase or mannitol applied as OH-radical scavenger. The findings indicate that membrane lipid peroxidation is an early effect of DHEA, and that this process may be involved in the steroid-induced carcinogenesis in rats.

Adenosine Diphosphate↗
PubMed1991

Microsomal cytochrome P450-dependent steroid metabolism in male sheep liver. Quantitative importance of 6 beta-hydroxylation and evidence for the involvement of a P450 from the IIIA subfamily in the pathway.

The metabolism of testosterone (TEST), androstenedione (AD) and progesterone (PROG) was assessed in hepatic microsomal fractions from male sheep. Rates of total hydroxylation of each steroid were lower in sheep liver than in microsomes isolated from untreated male rat, guinea pig or human liver, 6 beta-Hydroxylation was the most important pathway of biotransformation of each of the three steroids (0.80, 0.89 and 0.43 nmol/min/mg protein for TEST, AD and PROG, respectively). Significant minor metabolites from TEST were the 2 beta-, 15 beta- and 15 alpha-alcohols (0.19, 0.22 and 0.17 nmol/min/mg microsomal protein, respectively). Apart from the 6 beta-hydroxysteroid, only the 21-hydroxy derivative was formed from PROG at a significant rate (0.27 nmol/min/mg protein). The 6 beta-alcohol was the only metabolite formed from AD at a rate greater than 0.1 nmol/min/mg protein. Antisera raised in rabbits to several rat hepatic microsomal P450s were assessed for their capacity to modulate sheep microsomal TEST hydroxylation. Anti-P450 IIIA isolated from phenobarbital-induced rat liver effectively inhibited TEST hydroxylation at the 2 beta-, 6 beta-, 15 alpha- and 15 beta-positions (by 31-56% when incubated with microsomes at a ratio of 5 mg IgG/mg protein). IgG raised against rat P450 IIC11 and IIB1 inhibited the formation of some of the minor hydroxysteroid metabolites but did not decrease the rate of TEST 6 beta-hydroxylation. Western immunoblot analysis confirmed the cross-reactivity of anti-rat P450 IIIA with an antigen in sheep hepatic microsomes; anti-IIC11 and anti-IIB1 exhibited only weak immunoreactivity with proteins in these fractions. Considered together, the present findings indicate that, as is the case in many mammalian species, 6 beta-hydroxylation is the principal steroid biotransformation pathway of male sheep liver. Evidence from immunoinhibition and Western immunoblot experiments strongly implicate the involvement of a P450 from the IIIA subfamily in ovine steroid 6 beta-hydroxylation.

Animals↗
PubMed2004

Inhibition of Na+/Ca2+ exchanger by peroxynitrite in microsomes of pulmonary smooth muscle: role of matrix metalloproteinase-2.

Treatment of bovine pulmonary artery smooth muscle microsomes with peroxynitrite (ONOO-) (100 microM) markedly stimulated matrix metalloproteinase-2 (MMP-2) activity and also enhanced Ca2+ATPase activity and ATP-dependent Ca2+ uptake. Pretreatment of the microsomes with vitamin E (1 mM) and TIMP-2 (50 microg/ml) preserved the increase in MMP-2 activity, Ca2+ATPase activity and also ATP-dependent Ca2+ uptake in the microsomes. In contrast, Na(+)-dependent Ca2+ uptake in the microsomes was inhibited by ONOO- and this was found to be reversed by vitamin E (1 mM) and TIMP-2 (50 microg/ml). However, changes caused by ONOO- in MMP-2 activity, ATP-dependent Ca2+ uptake and Na(+)-dependent Ca2+ uptake were not reversed upon pretreatment of the microsomes with a low concentration of 5 microg/ml of TIMP-2 which, on the contrary, reversed MMP-2 (1 microg/ml)-mediated alteration on these parameters. The inhibition of Na(+)-dependent Ca2+ uptake by ONOO- and MMP-2 overpowered the stimulation of ATP-dependent Ca2+ uptake in the microsomes. Treatment with ONOO- abolished the inhibitory effect of TIMP-2 (5 microg/ml) on MMP-2 (1 microg/ml) causing 14C-gelatin degradation. Overall, the present study suggests that ONOO- inactivated TIMP-2, the ambient inhibitor of MMP-2, leading to activation of the ambient proteinase, MMP-2, and subsequently stimulated Ca2+ATPase activity and ATP-dependent Ca2+ uptake, but inhibited Na(+)-dependent Ca2+ uptake, resulting in a marked decrease in Ca2+ uptake in microsomes of bovine pulmonary artery smooth muscle.

Adenosine Triphosphate↗
PubMed2006

Biotransformation of letrozole in rat liver microsomes: effects of gender and tamoxifen.

The in vitro metabolic kinetics of letrozole were investigated by incubating letrozole (10-500 microM) in female or male rat liver microsomes to assess the effect of gender and to predict the in vivo biotransformation characteristics of letrozole in rats. The effects of tamoxifen (TAM) on the metabolic kinetics of letrozole were also examined by incubating letrozole in female rat liver microsomes in the presence or absence of TAM. The effects of chronic pretreatment of female rats with TAM (0.5, 1.0, 5.0 mg/kg/day, i.p. for 7 consecutive days) on liver microsomal protein content and metabolic activity were also examined. The formation rate of the carbinol metabolite of letrozole, CGP44 645, was significantly higher (p<0.05) in male rat liver microsomes in comparison to female. The V(max)/K(m) ratio for letrozole metabolism in female rat liver microsomes did not change significantly (p>0.05) in the presence of TAM. After chronic pretreatment of female rats with TAM (up to a dose of 1.0mg/kg/day), the hepatic microsomal protein content was significantly increased but the formation rate of CGP44 645, when normalized for protein content, did not change significantly. These results suggest that there is a marked gender difference in letrozole metabolism in rats. It also appears that acute treatment of female rat liver microsomes with TAM produces negligible inhibitory effect on the CYP mediated metabolic clearance of letrozole. However, chronic pretreatment of female rats with TAM appear to induce CYPs, but does not significantly impact the metabolic activities of the enzymes associated with the formation of the carbinol metabolite of letrozole.

Animals↗
PubMed1997

Metabolism of rifabutin in human enterocyte and liver microsomes: kinetic parameters, identification of enzyme systems, and drug interactions with macrolides and antifungal agents.

Biotransformation of rifabutin, an antibiotic used for treatment of tuberculosis in patients infected with the human immunodeficiency virus (HIV), and its interactions with some macrolide and antifungal agents were studied in human intestinal and liver microsomes. Both liver and enterocyte microsomes metabolized rifabutin to 25-O-deacetylrifabutin, 27-O-demethylrifabutin, and 20-, 31-, and 32-hydroxyrifabutin. The same products (except 25-O-deacetylrifabutin) were formed by microsomes from lymphoblastoid cells that contained expressed CYP3A4. The apparent Michaelis-Menten constant (Km); approximately 10 to 12 mumol/L) and maximal velocity (Vmax; approximately 100 pmol/min/mg of protein) values for CYP-mediated metabolism were similar in liver and enterocyte microsomes. Deacetylation of rifabutin (Km approximately 16 to 20 mumol/L and Vmax approximately 50 to 100 pmol/min/mg of protein) was catalyzed by microsomal cholinesterase. Clarithromycin, ketoconazole, and fluconazole inhibited CYP-mediated metabolism of rifabutin in enterocyte microsomes equally or more potently than in liver microsomes but had no effect on cholinesterase activity. Azithromycin did not inhibit in vitro metabolism of rifabutin. This study provides evidence that CYP3A4 and cholinesterase are major enzymes that biotransform rifabutin in humans and that intestinal CYP3A4 contributes significantly to rifabutin presystemic first-pass metabolism and drug interactions with macrolide and antifungal agents.

Acetylation↗
PubMed2000

Effects of cAMP-dependent protein kinase and ATP on N1-oxidation of 9-benzyladenine by animal hepatic microsomes.

N1-Oxidation is a major metabolic pathway for 9-benzyladenine (BA) catalyzed by the cytochrome P450 system in animal hepatic microsomes. After normal hamster hepatic microsomes or phenobarbital induced rabbit hepatic microsomes were preincubated in the presence of cyclic AMP-dependent protein kinase catalytic subunit (PKA), MgCl2 and ATP, BA-N1-oxidation was significantly decreased. However, further investigation indicated that the decrease of BA-N1-oxidation seemed to be a combination of the effects of PKA and ATP, as ATP alone showed a biphasic regulatory effect on BA-N1-oxidation when microsomes were preincubated in the presence of various concentrations of ATP. In the lower ATP concentration range (0.5-2.5mM), BA-N1-oxidation increased along with the increase of ATP concentration; whereas BA-N1-oxidation decreased when the ATP concentration was higher (>5mM). The biphasic regulatory effects of ATP on BA-N1-oxidation seem dependent on the incubation process, as preincubation markedly strengthened the effects. When microsomes were incubated at 37 degrees C for different time lengths in the absence or presence of ATP (2.5 or 20mM), the activity of BA-N1-oxidase decreased at similar rates in all groups, but the activity levels of BA-N1-oxidase were different among the groups. The cytochrome P450 content was not changed parallel to the variation of BA-N1-oxidation when microsomes were incubated in the presence of ATP, indicating that the effects of ATP on BA-N1-oxidation were not mediated by affecting CYP stability. In addition, the activity of NADPH-cytochrome P450 reductase was not markedly affected by ATP without incubation. The result implied that ATP did not inhibit the reductase directly. After microsomes were incubated in the presence of low ATP concentration (2.5mM), the reductase was slightly inhibited, whilst high ATP concentration (20mM) showed marked inhibition (83% of control). This may partially contribute to the down-regulatory effect of ATP on BA-N1-oxidation. Furthermore, it was found that the presence of magnesium ions during preincubation weakened the up-regulatory effect of ATP (2.5mM) on BA-N1-oxidation, but showed no effect on the down-regulatory effect of ATP (20mM). Since these observed phenomena are not readily explained, a possible mechanism, i.e. phosphorylation and dephosphorylation of cytochrome P450, is suggested.

Adenine↗
PubMed1999

Existence of acyl-CoA hydrolase-mediated pathway supplying arachidonic acid for prostaglandin synthesis in microsomes from rabbit kidney medulla.

We have previously shown that acyl-coenzyme A (CoA) hydrolase that hydrolyzes arachidonoyl-CoA (AA-CoA) to arachidonic acid (AA) and CoA is present in the cytosol of rabbit kidney medulla and that this enzyme can supply AA for prostaglandin (PG) synthesis in this region. In the present study, the existence of the acyl-CoA hydrolase-mediated pathway that supplies AA available for PG synthesis in microsomes from the kidney medulla was examined. AA-CoA (20 microM) was preincubated with the 105,000 g pellet (microsomes, 0.5 mg of protein) from the medulla for 5 min at 37 degrees C followed by incubation with the medulla microsomes (0.5 mg of protein) (the source of PG synthesizing enzymes) in the presence of hydroquinone and reduced glutathione for 5 min at 37 degrees C. The PGs formed were measured by high-pressure liquid chromatography using 9-anthryldiazomethane for derivatization. The addition of the microsomal fraction from the medulla in the preincubation mixture increased total PG formation from 3.86 to 8.70 nmol, and this stimulatory effect was somewhat weaker than that of the cytosolic fraction. On the other hand, the microsomal fraction in the kidney cortex has an extremely lower capacity to supply AA for PG synthesis than do medulla microsomes. These results suggest that, in kidney medulla, the microsomes as well as the cytosol have the potential route that supplies AA from AA-CoA for PG synthesis and that this pathway is mediated by acyl-CoA hydrolase.

Acyl Coenzyme A↗
PubMed1980

CMP-N-acetylneuraminic acid: isolation from and penetration into mouse liver microsomes.

We present three lines of evidence for the existence of an intramicrosomal pool of CMP-NeuNAc in liver slices incubated with 3H-NeuNAc: first, enrichment in the microsomal fraction over the cell homogenate of CMP-3H-NeuNAc relative to CMP-14C-NeuNAc, the latter added as marker for sugar nucleotide trapped between and adsorbed onto vesicles; second, removal from microsomes by isotonic sucrose washing of only half of the CMP-3H-NeuNAc synthesized in vivo under conditions which remove all the CMP-14C-NeuNAc; and third, complete loss of both sugar nucleotides upon treatment of microsomes by hypotonic shock or with low concentrations of detergents. Incubation of CMP-NeuNAc with microsomes in vitro showed that the intact sugar nucleotide was penetrating the vesicles. When the sugar nucleotide is labeled in the sugar moiety, microsomal vesicles accumulate soluble radiolabeled products. This accumulation was saturable and dependent on time, temperature and concentration of sugar nucleotide, and was also inhibited by substrate analogues and by pretreatment of microsomes with proteases. Together, these results provide strong evidence for the existence of a specific transport system for CMP-NeuNAc in microsomes.

Animals↗
PubMed1998

Increase of Ca2+-ATPase activity in the brain microsomes of rats with increasing ages: involvement of protein kinase C.

A possible mechanism of aging-induced increase in brain microsomal Ca2+-adenosine triphosphatase (ATPase) activity of rats was investigated. Calcium content in the brain tissues and Ca2+-ATPase activity in the brain microsomes of aging rats (50 weeks of age) increased significantly as compared with those of young rats (5 weeks of age). Brain microsomal Ca2+-ATPase activity in aging rats was decreased significantly by treatment of ethyleneglycol-bis-(aminoethylether) N,N,N',N'-tetraacetic acid (EGTA) (2.7 mM) or digitonin (10(-3)%), while such decrease was not seen in the enzyme activity of young rats. Microsomal Ca2+-ATPase activity in aging rats was markedly decreased by the presence of staurosporine (10(-8) and 10(-7) M), an inhibitor of protein kinase C, in the enzyme reaction mixture, although the enzyme activity of young rats was not inhibited. Meanwhile, dibucaine (10(-6) and 10(-5) M), an inhibitor of Ca2+/calmodulin-dependent protein kinase, did not have an effect on Ca2+-ATPase activity in the brain microsomes of young and aging rats. The addition of protein kinase C (100 and 200 mU/ml) in the reaction mixture caused a significant increase in brain microsomal Ca2+-ATPase activity of young rats. These results suggest that protein kinase C is partly involved in the elevation of brain microsomal Ca2+-ATPase activity in rats with increasing ages.

Aging↗
PubMed1998

Ethylmorphine N-demethylase activity as a marker for cytochrome P450 CYP3A activity in rat hepatic microsomes.

The purpose of this study was to evaluate the selectivity and sensitivity of ethylmorphine N-demethylase (EMD) as an indicator of chemically-induced cytochrome P450 CYP3A activity in liver microsomes of rats following treatment with selective enzyme inducers. Male and female Sprague-Dawley (CD) rats were dosed with either pregnenolone-16alpha-carbonitrile (PCN; 50 mg/kg per day for 5 days), phenobarbital (PB; 100 mg/kg per day for 4 days), beta-naphthoflavone (betaNF; 100 mg/kg per day for 3 days), clofibrate (CF; 300 mg/kg per day for 14 days), isoniazid (ISO; 100 mg/kg per day for 3 days), or dexamethasone (DEX; 50 mg/kg per day for 4 days). Microsomes were isolated, frozen and subsequently assayed for protein, cytochrome P450 content and EMD activity. In males, significant elevations (P < 0.01) in EMD activity were observed in microsomes from PB-, DEX- and PCN-dosed animals compared with untreated controls. Microsomes from ISO- and betaNF-dosed males showed a reduction (P < 0.05) in EMD activity when compared with control microsomes, and CF was without effect. In females, EMD activities were significantly increased in microsomes from PCN, DEX and PB-dosed but not betaNF, ISO, or CF-dosed animals. As expected on the basis of sex-related differences in gene expression, EMD activities in untreated animals were considerably higher in males than females, attributable to constitutive CYP3A and CYP2C11 activities. The selectivity of EMD for induced CYP3A was confirmed on the basis of inhibition studies with selected steroid substrates of CYP3A, polyclonal anti-CYP3A1 antibodies and triacetyloleandomycin (TAO), a selective inhibitor of CYP3A. In conclusion, for both sexes, the greatest elevations (approximately 3-13-fold) in EMD activity were observed in microsomes from rats dosed with DEX, a potent archetypal inducer with lesser but significant increases noted for PCN and PB, indicating that EMD is a reliable indicator of induced rat hepatic cytochrome P450 CYP3A activity.

Animals↗