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PubMed1983

Microsomal glutathione transferase. Purification in unactivated form and further characterization of the activation process, substrate specificity and amino acid composition.

The procedure developed for purification of the N-ethylmaleimide-activated microsomal glutathione transferase was applied successfully to isolation of this same enzyme in unactivated form. The microsomal glutathione transferases, the unactivated and activated forms, were shown to be identical in terms of molecular weight, immunochemical properties, and amino acid composition. In addition the microsomal glutathione transferase purified in unactivated form could be activated 15-fold with N-ethylmaleimide to give the same specific activity with 1-chloro-2,4-dinitrobenzene as that observed for the enzyme isolated in activated form. This activation involved the binding of one molecule N-ethylmaleimide to the single cysteine residue present in each polypeptide chain of the enzyme, as shown by amino acid analysis, determination of sulfhydryl groups by 2,2'-dithiopyridyl and binding of radioactive N-ethylmaleimide. Except for the presence of only a single cysteine residue and the total absence of tryptophan, the amino acid composition of the microsomal glutathione transferase is not remarkable. The contents of aspartic acid/asparagine + glutamic acid/glutamine, of basic amino acids, and of hydrophobic amino acids are 15%, 12% and 54% respectively. The isoelectric point of the enzyme is 10.1. Microsomal glutathione transferase conjugates a wide range of substrates with glutathione and also demonstrates glutathione peroxidase activity with cumene hydroperoxide, suggesting that it may be involved in preventing lipid peroxidation. Of the nine substrates identified here, the enzymatic activity towards only two, 1-chloro-2,4-dinitrobenzene and cumene hydroperoxide, could be increased by treatment with N-ethylmaleimide. This treatment results in increases in both the apparent Km values and V values for 1-chloro-2,4-dinitrobenzene and cumene hydroperoxide. Thus, although clearly distinct from the cytosolic glutathione transferases, the microsomal enzyme shares certain properties with these soluble enzymes, including a relative abundance, a high isoelectric point and a broad substrate specificity. The exact role of the microsomal glutathione transferase in drug metabolism, as well as other possible functions, remains to be established.

Amino Acids↗

PubMed1996

Bile acids produce a generalized reduction of the catalytic activity of cytochromes P450 and other hepatic microsomal enzymes in vitro: relevance to drug metabolism in experimental cholestasis.

In bile duct-ligated male rats, there is a reduction of total hepatic microsomal cytochrome P450 (P450) levels and of NADPH-cytochrome P450 reductase (P450-reductase) activity, but the changes in activity of individual microsomal enzymes are nonuniform. We have proposed that the initial effect of cholestasis on microsomal proteins is a non-specific reduction caused by bile acid-mediated destruction, whereas the disproportionate lowering of male-specific P450 enzymes results from secondary down-regulation of some cytochrome P450 (CYP) genes. We report herein the results of experiments to test the first part of this hypothesis, at least as indicated by enzyme inhibition. Hepatic microsomal fractions from normal male rats were incubated at 37 degrees C with increasing concentrations of a range of bile acids selected for their varying physicochemical properties. The endpoints were catalytic activity of three individual CYP proteins, CYP 2A1 (measured as testosterone 7 alpha-hydroxylase activity), 2C11 (testosterone 2 alpha-hydroxylase and 16 alpha-hydroxylase) and 3A2 (testosterone 6 beta-hydroxylase), and the non-CYP enzymes, steroid 17 beta-dehydrogenase and P450-reductase. With 0.25 mmol/L cholic acid, a concentration exceeded in serum following bile duct ligation, there was a significant reduction in the activity of all enzymes at 4 h. Cholic acid-mediated inhibition was dose-dependent and there was no difference in inhibitory activity towards the male sex-dependent CYP 2C11 and 3A2 and the non-sex-dependent CYP 2A1 and other microsomal enzymes. Taurocholic acid was twice as potent an inhibitor as unconjugated cholic acid, the respective apparent I50 values being approximately 0.6 mmol/L compared with approximately 1.2 mmol/L. The dihydroxy bile acids, chenodeoxycholic acid and deoxycholic acid, were also more potent inhibitors than cholic acid, exhibiting I50 values in the range of 0.3-0.5 mmol/L, but the monohydroxy bile acid, lithocholic acid, was the most potent inhibitor (I50 approximately 0.2 mmol/L). Thus, the inhibitory potential of bile acids towards microsomal enzymes was inversely related to their extent of hydroxylation, while taurine conjugation enhanced the inhibitory potential of cholic acid. These data confirm the potential of bile acids to inhibit the activity of microsomal enzymes in livers of bile ductligated rats and indicate that such changes can occur with concentrations of bile acids that are physiologically relevant. Further, the results are consistent with the proposal that the disproportionately greater reduction of the male sex-dependent CYP, 2C11 and 3A2, is not explained by a destructive mechanism.

Animals↗

PubMed1990

Generation of reactive oxygen species and reduction of ferric chelates by microsomes in the presence of a reconstituted system containing ethanol, NAD+ and alcohol dehydrogenase.

Many of the toxic metabolic actions of ethanol on the liver have been ascribed to the enhanced cellular production of NADH, which arises as a consequence of the oxidation of ethanol by alcohol dehydrogenase (ADH). Experiments were conducted to evaluate whether NADH generated from a reconstituted system containing ethanol plus NAD+ plus ADH could interact with ferric chelates to promote microsomal lipid peroxidation and generation of a hydroxyl radical (OH)-like species. In the presence of the reconstituted system and iron, microsomes produced.OH as assessed by the oxidation of .OH scavenging agents. This oxidation was inhibited by catalase and competitive.OH scavengers but not by superoxide dismutase. The ADH-dependent microsomal production of.OH was effectively catalyzed by ferric-EDTA and -diethylenetriamine pentaacetic acid (-DTPA), but not by ferric-ATP or -citrate. However, all these ferric chelates were reduced by the microsomes in the presence of the reconstituted system. Hydrogen peroxide (H2O2) was produced in the presence of ADH and appeared to be a limiting factor for the production of.OH. The reconstituted system also catalyzed microsomal lipid peroxidation, and the pattern of effectiveness of ferric chelates was opposite that of catalysis of.OH production. There was little effect by catalase, superoxide dismutase or dimethyl sulfoxide (DMSO) on the ADH-dependent microsomal lipid peroxidation. The reconstituted system was characterized with respect to dependence on NAD+ and ADH; ethanol could be replaced by other alcohols, which are substrates for ADH. Pyrazole, a potent inhibitor of ADH, blocked the ability of the reconstituted system to interact with iron and microsomes to produce reactive oxygen species.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol Dehydrogenase↗

PubMed1977

Evidence for two catalytically different binding sites of liver microsomal cytochrome P-450: importance for species and sex differences in oxidation pattern of lidocaine.

When the local anaesthetic drug lidocaine is added to liver microsomes biphasic type I spectral change titration curves can be observed. A high-affinity and a low-affinity phase is observed. In the present study we have found that microsomes from female rats have a dominant high-affinity phase, which can hardly be observed within microsomes from female guinea pigs. Male rats showed an intermediate phase. On incubation of lidocaine at concentrations of 1 micron or less with female rat liver microsomes a larger fraction of the drug was aromatically hydroxylated than deethylated. The opposite was true for guinea pig liver microsomes, and microsomes from male rats were intermediate. The ratio between the formation of deethylated and hydroxylated metabolites increased with the lidocaine concentration and at a lidocaine concentration of 10(-4)M deethylation was the dominant oxidation type in all microsomes. The data suggest that the two spectral phases represent two binding sites of cytochrome P-450 each having a certain "catalytic specificity" - the high affinity catalyzing aromatic hydroxylation and the "low-affinity site" deethylation. This hypothesis is further supported by the observed differential effects of pH and MgCl2 concentration on the two types of oxidation.

Animals↗

PubMed2005

Direct determination of unbound intrinsic drug clearance in the microsomal stability assay.

The microsomal stability assay is commonly used to rank compounds according to their metabolic stability. Determination of the unbound intrinsic clearance (CL(in,u)) is essential for the accurate comparison of compounds, since nonspecific binding to microsomes can lead to an underestimation of the microsomal clearance. In this study, a new method (linear extrapolation in the stability assay, LESA) was established, which allows direct calculation of CL(in,u) from microsomal stability data, without the need to independently determine the fraction of free (unbound) drug. The method was validated using nine drugs with different chemical structures and physicochemical properties. The CL(in,u) of these compounds was extrapolated from the intrinsic clearance values obtained at different concentrations of human liver microsomes and compared with that calculated by the conventional method, using microsomal intrinsic clearance values and the free fraction of drug determined by equilibrium dialysis, ultracentrifugation, or ultrafiltration. A good agreement was observed between the data generated by the LESA method and those determined by conventional procedures. The method was further evaluated using a published dataset for 10 additional drugs and found to yield intrinsic clearance data comparable to the previously reported values. LESA provides a convenient and rapid method to determine the influence of microsome binding on intrinsic clearance in a single assay.

Biological Assay↗

PubMed2002

Involvement of multiple UDP-glucuronosyltransferase 1A isoforms in glucuronidation of 5-(4'-hydroxyphenyl)-5-phenylhydantoin in human liver microsomes.

In humans, orally administered phenytoin, 5,5-diphenylhydantoin, is mainly excreted as 5-(4'-hydroxyphenyl)-5-phenylhydantoin (4'-HPPH) O-glucuronide. Phenytoin is oxidized to 4'-HPPH by CYP2C9 and to a minor extent by CYP2C19, and then 4'-HPPH is metabolized to 4'-HPPH O-glucuronide by UDP-glucuronosyltransferase (UGT). In the present study, 4'-HPPH O-glucuronidation in human liver microsomes was investigated. The metabolite formed by incubation with human liver microsomes, 4'-HPPH, and UDP-glucuronic acid was identified as 4'-HPPH O-glucuronide by liquid chromatography-tandem mass spectrometry analysis. The 4'-HPPH O-glucuronosyltransferase activity in human liver microsomes was not saturated at concentrations up to 500 microM of 4'-HPPH. Any commercially available recombinant human UGTs (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B7, and UGT2B15) expressed in baculovirus-infected insect cells did not show detectable 4'-HPPH O-glucuronide. The 4'-HPPH O-glucuronidation in pooled human liver microsomes was inhibited by beta-estradiol as a typical substrate for UGT1A1 (IC(50) = 21.1 microM) and imipramine as a typical substrate for UGT1A4 (IC(50) = 57.7 microM). The inhibitory effects of propofol as a specific substrate for UGT1A9 (IC(50) = 167.1 microM) and emodin as a substrate for UGT1A8 and UGT1A10 (IC(50) = 287.6 microM) were not prominent. The interindividual difference in the 4'-HPPH O-glucuronidation in 14 human liver microsomes was 28.5-fold (0.023-0.656 nmol/min/mg of protein). The 4'-HPPH O-glucuronosyltransferase activity in 11 human liver microsomes was significantly (r = 0.609, P < 0.05) correlated with the 4-nitrophenol glucuronosyltransferase activity, which is catalyzed by UGT1A6 and UGT1A9. These results suggest that multiple UGT1As such as UGT1A1, UGT1A4, UGT1A6, and UGT1A9 are involved in 4'-HPPH O-glucuronidation in human liver microsomes, although the percentage contribution of each UGT1A could not be estimated. Large interindividual differences in the glucuronidation of 4'-HPPH might be responsible for the nonlinearity of the phenytoin plasma concentration or adverse reactions in humans.

Biotransformation↗

PubMed2002

Characterization of nicotine and cotinine N-glucuronidations in human liver microsomes.

The nicotine and cotinine N-glucuronidations in human liver microsomes were characterized. The Eadie-Hofstee plots of nicotine N-glucuronidation in human liver microsomes were clearly biphasic, indicating the involvement of multiple enzymes. The apparent K(m) and V(max) values were 33.1 +/- 28.1 micro M and 60.0 +/- 21.0 pmol/min/mg and 284.7 +/- 122.0 micro M and 124.0 +/- 44.0 pmol/min/mg for the high- and low-affinity components, respectively, in human liver microsomes (n = 4). However, the Eadie-Hofstee plots of cotinine N-glucuronidation in human liver microsomes were monophasic (apparent K(m) = 1.9 +/- 0.3 mM, V(max) = 655.6 +/- 312.3 pmol/min/mg). The nicotine and cotinine N-glucuronidations in the recombinant human UDP-glucuronosyltransferases (UGTs) (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B15) expressed in baculovirus-infected insect cells or human B-lymphoblastoid cells that are commercially available were determined. However, no recombinant UGT isoforms showed detectable nicotine and cotinine N-glucuronides (the concentrations of nicotine and cotinine were 0.5 and 2 mM, respectively). Nicotine and cotinine N-glucuronidations in pooled human liver microsomes were competitively inhibited by bilirubin as a substrate for UGT1A1 (K(i) = 3.9 and 3.3 micro M), imipramine as a substrate for UGT1A4 (K(i) = 6.1 and 2.7 micro M), and propofol as a substrate for UGT1A9 (K(i) = 6.0 and 12.0 micro M). The nicotine N-glucuronidation (50 micro M nicotine) in 14 human liver microsomes was significantly (r = 0.950, P < 0.0001) correlated with the cotinine N-glucuronidation (0.2 mM cotinine), indicating that the same isoform(s) is involved in both glucuronidations. Furthermore, weak correlations between imipramine N-glucuronidation and nicotine N-glucuronidation (r = 0.425) or cotinine N-glucuronidation (r = 0.517) were observed. In conclusion, the involvement of UGT1A1 and UGT1A9 as well as UGT1A4 in nicotine and cotinine N-glucuronidations in human liver microsomes was suggested, although the contributions of each UGT isoform could not be determined conclusively.

Cotinine↗

PubMed2006

Mouse-adapted scrapie infection of SN56 cells: greater efficiency with microsome-associated versus purified PrP-res.

The process by which transmissible spongiform encephalopathy agents, or prions, infect cells is unknown. We employed a new differentiable cell line (SN56) susceptible to infection with three mouse-adapted scrapie strains to gain insight into the cellular infection process. The effect of disease-associated PrP (PrP-res) association with microsomal membranes on infection efficiency was examined by comparing sustained PrP-res production in cells treated with either scrapie brain microsomes or purified, detergent-extracted PrP-res. When normalized for quantity of input PrP-res, scrapie brain microsomes induced dramatically enhanced persistent PrP-res formation compared to purified PrP-res. Infected SN56 cells released low levels of PrP-res into the culture supernatant, which also efficiently initiated infection in recipient cells. Interestingly, microsomes labeled with a fluorescent marker were internalized by SN56 cells in small vesicles, which were subsequently found in neuritic processes. When bound to culture wells to reduce internalization during the infection process, scrapie microsomes induced less long-term PrP-res production than suspended microsomes. Long-term differentiation of infected SN56 cells was accompanied by a decrease in PrP-res formation. Our observations provide evidence that infection of cells is aided by the association of PrP-res with membranes and/or other microsomal constituents.

Animals↗

PubMed1980

Mitochondrial importation of lipids and liponucleotides from microsomes independent of and facilitated by purified cytosol proteins.

The mitochondrial importation of microsomal lipids and liponucleotides in the presence and in the absence of partially purified cytosol protein(s) isolated from guinea pig liver was studied by the aid of isomeric (5-, 12-, and 16-(N-oxyl-4',4'-dimethyloxazolidine)stearoyl) spin-labelled radioactive phosphatide acid, phosphatidylcholine, neutral lipids, and CDP-diglycerides. Using a conventional procedure for the protein purification, cytosol protein(s) was purified approximately 1000-fold in respect to its ability to catalyze the translocation of isomeric spin-labelled lipids and liponucleotides from the microsomal to mitochondrial membranes. The highest activity of this protein was exhibited with biosynthesized spin-labelled lipids and liponucleotides bound to the microsomal membranes as substrates and the lowest, with the synthetic liponucleotides and derived lipids bound to the microsomal membranes. The partially purified protein was active in catalyzing the mitochondrial import of phospholipids from microsomes after heat treatment up to 90 degrees C. In addition to the cytosol protein catalyzing mechanism of mitochondrial import of lipids and liponucleotides from microsomal membranes, another cytosol protein independent mechanism of the mitochondrial importation of the same lipids and liponucleotides was also demonstrated in an agreement with our previous reports on the existence of cytosol protein independent intermembranous translocation of phospholipids. These experimental findings are discussed in terms of possible physiological significance and reaction mechanisms involved in the mitochondrial import of lipids and liponucleotides from the microsomal membranes of guinea pig liver.

Animals↗

PubMed1983

Phosphatidylinositol movement between isolated microsomal and mitochondrial membranes.

Transfer of membrane-bound phosphatidyl-[2'-3H]inositol from microsomal to unlabelled mitochondrial and from mitochondrial to unlabelled microsomal membranes was studied using partially purified cytosol proteins isolated from guinea pig liver cytosol. In the absence and presence of these proteins the amounts of phosphatidylinositol transfer from microsomal to mitochondrial membranes were approximately 21 and 33%, respectively, and the amounts from mitochondrial to microsomal membranes were approximately 31 and 39%, respectively. The release of phosphatidyl-[2'-3H]inositol from microsomal membranes in the absence of mitochondria was dependent on concentration of cytosol proteins. Two mechanisms for movement between membranes are proposed. In cytosol-protein-independent movement of phosphatidyl-[2'-3H]inositol from microsomal to mitochondrial membranes, a direct contact between membranes is required, since phosphatidyl-[2'-3H]inositol was not detected in the reaction medium. In the cytosol-protein-catalyzed transfer, formation of phosphatidyl-[2'-3H]inositol - cytosol protein complex is postulated, since phosphatidyl-[2'-3H]inositol was released into the reaction medium and its movement proceeded from mitochondrial to microsomal membranes in the presence of partially purified cytosol proteins. Thus, contact between the two membranes is probably not necessary for this transfer. Implications for the movement of phospholipids between biological membranes are discussed.

Animals↗

PubMed1985

Effect of nonsteroidal anti-inflammatory drugs on the microsomal monooxygenase system of rat liver.

The effect of acetylsalicylic acid, ibuprofen, indomethacin, ketoprofen, naproxen, phenylbutazone, and salicylic acid on the microsomal oxidative drug metabolism of rat liver was studied. Pretreatment of the rats with pharmacologic doses of acetylsalicylic acid, indomethacin, and ketoprofen decreased both the demethylase and hydroxylase activities of rat liver microsomes. These effects were paralleled by decreases in microsomal cytochrome P-450 content. The rate of the microsomal reactions was increased after pretreatment with ibuprofen and naproxen but only the former increased the concentration of cytochrome P-450. Phenylbutazone and salicylic acid had no in vivo effect on the hepatic monooxygenase. The addition of 1 mM of ibuprofen, indomethacin, ketoprofen, naproxen, and phenylbutazone to rat liver microsomes inhibit both the aminopyrine N-demethylase and p-nitro-anisole O-demethylase activities. The extent of the inhibition varied between 21 and 73% of the control incubation. Indomethacin, naproxen, and phenylbutazone also decreased the aniline hydroxylase activity to roughly 60% of the control value. Acetylsalicylic acid and salicylic acid had no in vitro effect on the microsomal monooxygenase. The nonsteroidal anti-inflammatory drugs produced a reverse type I binding spectrum with oxidized cytochrome P-450; indomethacin and phenylbutazone were the strongest ligands. There is no correlation between the effect of addition of nonsteroidal anti-inflammatory drugs to the hepatic microsomal homogenate and their in vivo effect on the monooxygenase activity.

Animals↗

PubMed1996

Effects of the intraluminal Ca load on the kinetics of 45Ca uptake and efflux in brain microsomes.

Effects of increasing intraluminal Ca ([Ca]i) on the kinetics of rat brain microsomal uptake and efflux are reported here. Isolated rat brain microsomes accumulated 45Ca in an extravesicular free Ca ([Ca]o)- and ATP-dependent manner. Increased microsomal Ca load resulted in a decreased initial rate of 45Ca uptake and an increased tau, time to reach 63% of steady-state accumulation. Isolated rate brain microsomes lost 45Ca in a temperature- and [Ca]i-dependent manner. Whether preloaded with tracer 45Ca and either < or = 0.5 or 25 microM [Ca]o, the time constant of efflux was larger at 4 degrees C as compared with 37 degrees C. Additionally, increased microsomal Ca load resulted in a decreased time constant of 45Ca efflux. This shorter efflux time constant cannot explain the effect of [Ca]i on tau during uptake which was in fact longer for preloaded microsomes. Rather, these data suggest that, as Ca accumulates into unloaded microsomes, a steadily increasing [Ca]i slows unidirectional Ca influx (presumably by inhibiting the endoplasmic reticulum Ca pump) and enhances unidirectional Ca efflux, and that these combined effects ultimately shorten the time needed to achieve steady-state luminal [Ca]i.

Adenosine Triphosphate↗

PubMed1995

Ryanodine-induced calcium release from hepatic microsomes and permeabilized hepatocytes.

Inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] is a second messenger that releases Ca2+ from hepatocyte microsomes. The toxic alkaloid ryanodine modulates Ca2+ release via a receptor (RyR) identified in a variety of cell systems, but its regulation and functional significance in liver are undefined. Similarly, the role in hepatocyte Ca2+ regulation of adenosine 5'-cyclic diphosphate-ribose (cADPR), which is the putative endogenous ligand for RyR in other cell systems, has not been defined. Utilizing microsomes and permeabilized cells, we have investigated Ca2+ regulation in hepatocytes and, in particular, effects of ryanodine, cADPR, and other putative modulators on Ca2+ release and compared these with Ins(1,4,5)P3-induced Ca2+ release. Ryanodine at > or = 50 microM released 20% of microsomal Ca2+, and, in contrast to Ins(1,4,5)P3, no potentiation was observed with guanosine 5'-triphosphate and polyethylene glycol. Ins(1,4,5)P3-induced Ca2+ release was demonstrable after maximal ryanodine-induced Ca2+ release, suggesting that distinct Ca2+ stores are involved. cADPR (5 microM) did not induce Ca2+ release, alone or in combination with calmodulin or hepatic cytosol, nor did it influence ryanodine-induced release, in microsomes or permeabilized hepatocytes (in which ryanodine released 25% of the sequestered Ca2+). Ryanodine-induced Ca2+ release in microsomes was not influenced by 20 mM caffeine, which itself did not mobilize Ca2+, but was prevented by 500 microM tetracaine, which was shown to induce Ca2+ release. We conclude that ryanodine is capable of mobilizing Ca2+ in the hepatocyte from microsomal stores that are distinct from those that can be regulated by Ins(1,4,5)P3 but that cADPR has no such effect. These data suggest that cADPR does not serve as the endogenous ligand for RyR in liver cells or that the site of action of ryanodine in hepatocyte microsomes is distinct from that in other cell types.

Adenosine Diphosphate Ribose↗

PubMed1994

Properties of a superoxide anion-generating microsomal NADH oxidoreductase, a potential pulmonary artery PO2 sensor.

In this study, we describe properties of a microsomal NADH oxidoreductase that is a potential PO2-dependent source of vasoactive reactive O2 species in the calf pulmonary artery. Microsomes show an NADH-dependent production of superoxide anion (O2-.), as detected by lucigenin-elicited chemiluminescence, a superoxide dismutase inhibited reduction of nitro blue tetrazolium (NBT) and 2,6-dichlorophenol-indophenol, and O2 consumption. The microsomal production of O2-. was modulated by physiologically relevant levels of NADH and PO2, and O2-. production was reduced by inhibitors of NADH-dependent microsomal electron transport. Microsomes catalyzed an NADH-mediated reduction of several electron acceptor dyes, cytochrome c (rotenone insensitive) and methemoglobin. On reduction with dithionite, a cytochrome with an absorbance at approximately 558 nm was observed. Arterial O2-. levels (chemiluminescence) were also reduced by NBT and microsomal electron transport inhibitors. In pulmonary arteries, NBT selectively inhibited PO2 and lactate elicited changes in force generation, presumably by trapping O2-. and preventing H2O2 formation. Thus these studies are consistent with an involvement of O2-.-derived H2O2 generation via a microsomal NADH-cytochrome b558 electron transport system in calf pulmonary artery smooth muscle PO2 and lactate-elicited tone responses.

Animals↗

PubMed1981

Role of changes in microsomal calcium uptake in the effects of reperfusion of Ca2+-deprived rat hearts.

This study was designed to investigate changes in contractile force, resting tension, and microsomal Ca2+ uptake in isolated rat hearts perfused under conditions associated with reversible and irreversible stages of the calcium paradox phenomenon. Five minutes of reperfusion with normal medium containing 1.25 mM calcium after 5 minutes of Ca2+-free perfusion produced a marked rise in resting tension, no recovery of contractile force, and a 63% depression in microsomal Ca2+ uptake. When reperfusion was carried out after 5 minutes of perfusion with 0.025 mM or greater concentrations of Ca2+, after less than 5 minutes of Ca2+-free exposure or after 5 minutes of varying degrees of hypothermic Ca2+-free perfusion, the increase in resting tension and decrease in contractile force development as well as microsomal Ca2+ accumulation were either absent or reduced. Furthermore, reperfusion-induced increases in resting tension and decreases in microsomal Ca2+ uptake also were found to be dependent on the duration of reperfusion as well as on the calcium concentration of the reperfusion medium. Microsomes isolated from control, Ca2+-free perfused or reperfused hearts were found to have similar phospholipid composition, protein profiles (SDS-polyacrylamide gel electrophoresis), and electron microscopic appearance. Whereas Ca2+-free perfusion alone had no effect on any of the parameters studied, reperfusion also depressed microsomal Ca2+-binding, Mg2+-ATPase, and Ca2+-stimulated ATPase activities. Changes in microsomal Ca2+ uptake exhibited sigmoidal relationships with the ability of Ca2+-depleted hearts to recover their contractile force or increase their resting tension upon reperfusion. Our findings suggest that reperfusion-induced contracture and intracellular calcium overload may be associated in part with a defect in the ability of sarcoplasmic reticulum to regulate calcium.

Animals↗

PubMed1989

Effects of hyperoxia and vitamin E on the fatty acid composition of rat lung microsomes and mitochondria.

Peroxidation of lung membrane lipids in vitro produces very specific changes in lung membrane fatty acid content with some fatty acids being affected more than others. We performed a series of experiments to determine the changes occurring in fatty acid composition in lung microsomes and mitochondria during an in vivo hyperoxic exposure. Hyperoxia did produce specific changes in the relative content of fatty acids present in lung microsomes and mitochondria of both vitamin E-supplemented and vitamin E-deficient rats. Changes were noted to occur in saturated and polyunsaturated fatty acids. The total amount of lung lipids extractable in the microsomal fractions decreased after hyperoxia in both the vitamin E-supplemented and the vitamin E-deficient animals with no changes occurring in extraction of lung mitochondrial total lipids. Decreases in lung mitochondrial fatty acids caused by hyperoxia occurred in the same fatty acids in both the vitamin E-supplemented and the vitamin E-deficient animals with few polyunsaturated fatty acids (PUFA) being affected. Decreases in lung microsomal fatty acids occurring during hyperoxia were different in the vitamin E-supplemented animals from those in the vitamin E-deficient animals with many more PUFA decreasing in the vitamin E-deficient group. The greatest number of PUFA found to decrease after hyperoxia when comparing all the different groups occurred in the microsomal fraction of the vitamin E-deficient rats. These data suggest that vitamin E-deficient animals have increased peroxidation of lung microsomal PUFA or a decrease in production of lung microsomal PUFA in vivo during a hyperoxic exposure.

Animals↗

PubMed1993

Alterations in the pulmonary microsomal cytochrome P-450 system after exposure of rats to silica.

Because some evidence indicates that there is an increased incidence of lung cancer in silicosis, we studied the effects of exposing rats to silica on the pulmonary microsomal cytochrome P-450 system. Rats were exposed to silica by intratracheal administration, lung microsomes were obtained from untreated and silica-treated animals, and the amount of microsomal tissue, the level of total cytochromes P-450 (all isozymes), the activity of NADPH cytochrome P-450 reductase, the metabolism of two xenobiotics, and the relative amounts of cytochrome P-4502B1 and P-4501A1 were measured. Lungs from silica-treated rats were almost 2-fold heavier and contained more than 10 times more alveolar phospholipids than lungs from untreated animals, indicating that acute silicosis had been produced. In lungs from silica-treated animals, the concentration of microsomal tissue, expressed as milligrams of microsomal protein per gram of lung, was increased by more than 2-fold, and total microsomal protein content was increased by almost 5-fold relative to untreated animals. When expressed as activity or amount per milligram of protein, the microsomal concentrations of NADPH cytochrome P-450 reductase, total cytochromes P-450, 7-ethoxycoumarin (EC)-0-deethylase, and cytochrome P-4502B1 are reduced by approximately 50% in silica-treated rats. However, when expressed as total activity or amount in the lungs, all are increased by approximately 1.5- to 2.5-fold in silica-treated lungs. On the other hand, total lung 7-ethoxyresorufin (ER)-0-deethylase activity and cytochrome P-4501A1 are increased by 4- to 5-fold in silica-treated lungs.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1975

Mechanism of secretion of biliary lipids. I. Role of bile canalicular and microsomal membranes in the synthesis and transport of biliary lecithin and cholesterol.

The role of bile canalicular and microsomal membranes in the synthesis and transport of biliary lipids was investigated by using the isolated perfused rat liver model. Labeled lecithin precursors ((3H)-palmitic acid, (14C)linoleic acid, (3H)choline, and 32PO4) and a cholesterol precursor ((3H)mevalonic acid) were administered with and without sodium taurocholate. The incorporation pattern of these labeled precursors into linoleyl and arachidonyl lecithins and cholesterol fractions of microsomes, bile canaliculi, and bile were examined at 30-min intervals up to 90 min. Marker enzymes and electron microscopy indicated that isolated subfractions of plasma membranes were enriched with bile canaliculi (less than 10 percent microsomal contamination). Taurocholate significantly stimulated the incorporation of 32PO4, (3H)choline, (3H)palmitic acid, and (14C)linoleic acid into linoleyl and arachidonyl lecithin with parallel incorporation curves for microsomal and bile canalicular membranes throughout the 90-min study period. During the 30-60-min period, however, these same lecithin fractions in bile significantly exceeded the specific activity of the membrane lecithins. The enzyme CDP-choline diglyceride transferase was virtually absent from canaliculi relative to microsomes, indicating that canaliculi lack the capacity for de novo lecithin synthesis. Incorporation of (3H)mevalonic acid into membranous and biliary cholesterol followed a pattern similar to that for lecithin. These data provide evidence that (a) biliary lecithin and cholesterol are derived from a microsomal subpool regulated by the flux of enterohepatic bile acids, (b) the role of the bile canalicular membranes with respect to biliary lipids is primarily transport rather than synthesis, and (c) lecithin and cholesterol are transported together from microsomes to bile. The findings are consistent with the existence of a cytoplasmic lipid complex within the hepatocyte which is actively involved in the intermembrane transport of biliary lipid.

Animals↗