Brain Microsomes Research Articles

Characterization of a O-fatty-acylated sulfatide from equine brain.

A sulfatide, O-fatty-acylated 3-sulfogalactosylceramide at C6-O on galactoside, was isolated from equine brain and the chemical structure was characterized by proton NMR and MS. The O-acylation site of the acylated sulfatide was determined by the down-field shift of protons attached to a carbon having an O-acyl group in the NMR spectrum and by analysis of a partially methylated derivative before and after acetalization of the intact sulfatide using GC-MS. The O-acyl chain length was determined by GLC, revealing that it exclusively had palmitoyl and stearoyl residues as the major fatty acids. The enzymatic conversion to the O-acyl sulfatide was further examined using equine brain microsomes as an enzyme source and different lipid substrates, resulting in O-acylation of 3-sulfogalactosylceramide from stearoyl CoA, while 6-O-acyl galactosylceramide was not O-sulfated from phosphoadenosine phosphosulfate. The results were supported by the comparably different N-linked fatty acid components between two lipid substrates, in which the component of 6-O-acyl sulfatide was mostly similar to that of sulfatide, but not to 6-O-acyl galactosylceramide.

Occurrence of an Unusual Phospholipid, Phosphatidyl-l-threonine, in Cultured Hippocampal Neurons: EXOGENOUS l-SERINE IS REQUIRED FOR THE SYNTHESIS OF NEURONAL PHOSPHATIDYL-l-SERINE AND SPHINGOLIPIDS

We have recently reported that L-serine released from astroglial cells supports the survival and neuritogenesis of hippocampal neurons under a serum- and glia-free culture condition (Mitoma, J., Furuya, S., and Hirabayashi, Y. (1998) Neurosci. Res. 30, 195-199). In this study, we show that exogenous L-serine is required for the synthesis of phosphatidyl-L-serine (PS) and sphingolipids in hippocampal neurons. When hippocampal neurons were maintained under an astroglial cell-free condition, the levels of sphingolipids and phosphatidyl-L-serine in the neurons were greatly reduced in the absence of external L-serine or glycine. Instead, a novel phospholipid appeared just ahead of PS on TLC. This novel lipid was determined to be phosphatidyl-L-threonine by TLC blotting/negative secondary ion mass spectrometry and amino acid analysis. Biochemical studies on rat brain microsomes have indicated that phosphatidyl-L-threonine is synthesized by the base exchange enzyme that is involved in PS synthesis with much lower affinity, that is, approximately (1)/(150) of L-serine. Addition of L-serine or glycine to the culture medium restored the synthesis of PS and sphingolipids in the neurons. These observations show that hippocampal neurons require exogenous L-serine for the synthesis of PS and sphingolipids in the absence of astroglial cells and suggested that astroglial cells contribute to neuronal lipid synthesis through the supply of L-serine.

Purification and Characterization of a Polyisoprenyl Phosphate Phosphatase from Pig Brain: POSSIBLE DUAL SPECIFICITY

Microsomal fractions from pig and calf brain catalyze the enzymatic dephosphorylation of endogenous and exogenous dolichyl monophosphate (Dol-P) (Sumbilla, C. A., and Waechter, C. J. (1985) Methods Enzymol. 111, 471-482). The Dol-P phosphatase (EC 3.1.3.51) has been solubilized by extracting pig brain microsomes with the nonionic detergent Nonidet P-40 and purified approximately 1,107-fold by a combination of anion exchange chromatography, polyethylene glycol fractionation, dye-ligand chromatography, and wheat germ agglutinin affinity chromatography. Treatment of the enzyme with neuraminidase prevented binding to wheat germ agglutinin-Sepharose, indicating the presence of one or more N-acetylneuraminyl residues per molecule of enzyme. When the highly purified polyisoprenyl phosphate phosphatase was analyzed by SDS-polyacrylamide gel electrophoresis, a major 33-kDa polypeptide was observed. Enzymatic dephosphorylation of Dol-P by the purified phosphatase was 1) optimal at pH 7; 2) potently inhibited by F-, orthovanadate, and Zn2+ > Co2+ > Mn2+ but unaffected by Mg2+; 3) exhibited an approximate Km for C95-Dol-P of 45 microM; and 4) was sensitive to N-ethylmaleimide, phenylglyoxal, and diethylpyrocarbonate. The pig brain phosphatase did not dephosphorylate glucose 6-phosphate, mannose 6-phosphate, 5'-AMP, or p-nitrophenylphosphate, but it dephosphorylated dioleoyl-phosphatidic acid at initial rates similar to those determined for Dol-P. Based on the virtually identical sensitivity of Dol-P and phosphatidic acid dephosphorylation by the highly purified enzyme to N-ethylmaleimide, F-, phenylglyoxal, and diethylpyrocarbonate, both substrates appear to be hydrolyzed by a single enzyme with an apparent dual specificity. This is the first report of the purification of a neutral Dol-P phosphatase from mammalian tissues. Although the enzyme is Mg2+-independent and capable of dephosphorylating Dol-P and PA, several enzymological properties distinguish this lipid phosphomonoesterase from PAP2.

Properties of specific binding site of myotoxin a, a powerful convulsant, in brain microsomes

Myotoxin a, a small basic polypeptide from prairie rattlesnakes (Crotalus viridis viridis), induces myonecrosis and binds to a single class of binding sites in skeletal muscle sarcoplasmic reticulum. In the present study, [125I]myotoxin a with a high specific activity was prepared and it was shown to bind mainly to microsomes in rat whole brain. [125I]Myotoxin a was further shown to bind to microsomes prepared from all regions tested in brain. Its specific binding to whole brain microsomes was of approximately 1.9 times lower affinity (KD = 0.76 microM; Bmax = 13.1 nmol/mg) than that to skeletal muscle sarcoplasmic reticulum. [125I]Myotoxin a binding to brain microsomes was displaced by unlabeled myotoxin a with an IC50 value of 4.5 microM. [125I]Myotoxin a binding was markedly reduced by treatment of microsomes with trypsin, suggesting that the binding site of [125I]myotoxin a is partially proteins. The binding was significantly inhibited by Mg2+ at concentrations above 1 mM. Having looked at several drugs, we noted that [125I]myotoxin a binding was noncompetitively inhibited by spermine, whereas it was enhanced by heparin. On the other hand, the i.c.v. injection of myotoxin a in mice induced potent convulsive effects at 0.05 nmol/mouse or more. This paper is the first to show that the specific binding site of myotoxin a is present in mouse brain and that myotoxin a is a novel peptidic convulsant in mice.

Imidazo[1,2- c]quinazolines with lipid peroxidation inhibitory effect

A series of imidazo[1,2- c]quinazolines of different lipophilic character was prepared. According to their antioxidant (cyclic voltammetry) properties they all should be potent inhibitors of lipid peroxidation. Under the given circumstances (NADPH-induced lipid peroxidation in rat brain microsomes and Fe 2+-induced lipid peroxidation in rat brain homogenate), however, their lipid peroxidation inhibitory activity was strongly dependent on their lipophilicity.

Quantification of concanavalin A binding to rat brain microsomal membranes detected by fluorescence polarization technique

A high sensitive method for detecting the change of microsomal membrane surface oligosaccharides was developed to study the regulatory role of lipid- or peptide-linked mannoside of endoplasmic reticulum in synaptic functions. The binding of concanavalin A to the microsomal membrane surface was measured quantitatively using a microgram-order of rat brain microsomal proteins. The fluorescence polarization of concanavalin A (Con A)-fluorescein isothiocyanate (FITC) conjugate bound to the membrane was analyzed to quantitate the change of binding constant and the number of binding sites. As a control, the non-specific binding of bovine serum albumin–FITC conjugate was measured by the same technique. We measured the change of fluorescence intensity of membrane-bound FITC conjugates by the flow cytometry and found that the intensity of FITC conjugate bound to the membrane increased more than that of free form of the probe. We observed that the alpha-mannosidase-treatment of rat brain microsomes resulted in the increase of binding constant of Con A to the microsomal surface without significant loss of binding sites.

Deleterious actions of chronic ethanol treatment on the glycosylation of rat brain clusterin

Clusterin is a N-glycosylated sialoglycoprotein present in rat brain cells. Clusterin, which elicits aggregation in a wide variety of cells, has been suggested to play an important role in synaptic remodeling through its cell adhesion property or lipid transport capacity in the brain. Sialic acid residues in clusterin may be responsible for its structural conformation, stability and functional ability. Maturation of clusterin is governed by the relative actions of sialyltransferases and sialidases that are present in brain microsomes, golgi bodies, cytosol and plasma membranes. We have earlier reported that chronic ethanol treatment in rats has a damaging effect on the hepatic glycosylation machinery. Others have reported increased hydrolysis of brain sialoconjugates in rats following chronic ethanol administration. Specificity of the effects of chronic ethanol treatment in the brain in relation to the glycosylation process, is still obscure. Therefore, in this investigation, we have studied the specific effects of chronic ethanol treatment on the glycosylation of rat brain clusterin and the causes that may lead to any possible defects in the glycosylation process. We have determined the effects of chronic ethanol treatment on (i) the incorporation of labeled leucine and N-acetylmannosamine into immunoprecipitable clusterin in whole brain homogenate, microsomes, golgi, cytosol, plasma membrane and synaptosomes, (ii) enzymatic activities of sialyltransferases in golgi and synaptosomes, and sialidase in brain cytosol and plasma membranes, and (iii) de novo synthetic rate of rat brain cytosolic sialidase. Our results showed that chronic ethanol treatment in rats resulted in (1) a decreased sialation index of brain clusterin by 47.2% ( p<0.001), 56.7% ( p<0.05), 51.7% ( p<0.05), 64.8% ( p<0.001), and 54.5% ( p<0.05), respectively, in whole brain homogenate, golgi, cytosol, plasma membranes, and synaptosomes; (2) a 46.1% ( p<0.05) and 12.5% ( p<0.05) decreased activities of brain sialyltransferases, respectively, in the golgi and the synaptosomal fractions; (3) a 70.1% ( p<0.05) and 42.6% ( p<0.05) increased activities of sialidases, respectively, in the cytosol and plasma membrane fractions; and (4) a 22.2%–64.3% ( p<0.001) increased incorporation of labeled leucine into brain cytosolic sialidase. Our findings have clearly established that long-term ethanol treatment in rats leads to a marked impairment in the glycosylation of rat brain clusterin as a result of altered activities of brain sialation and desialation enzymes. In particular, the specific increase noted in brain sialidase activity was due to concomitant increases in its synthetic rate. These defects in the glycosylation of brain clusterin may lead to changes in the molecular conformation of clusterin, and thus, may result in its structural instability and/or functional impairment.

Distribution and characterization of anandamide amidohydrolase in mouse brain and liver

Anandamide (N-Arachidonoylethanolamine) amidohydrolase catalyzing hydrolysis of anandamide was characterized in mice. The enzymatic activity was highest in the liver, followed by the brain and testis. Negligible activity was found in heart, lung and spleen. The activity in brain and liver was mainly localized in the microsomal fractions. Kinetic experiments demonstrated that Km (μM) and Vmax (nmol/min/mg protein) for the brain microsomes were 9.3 and 2.58, respectively, while those for the hepatic microsomes were 180 and 18.9, respectively. The activity in the microsomes from the liver and brain was markedly inhibited by Cu 2+, Hg 2+, Se 4+, phenylmethylsulfonylfluoride and sodium dodecylsulfate. Brain but not hepatic microsomal enzyme activity was inhibited by Δ 9-tetrahydrocannabinol, cannabidiol and cannabinol. Kinetic parameters demonstrated that the inhibition by the cannabinoids was competitive in nature. Relatively high distribution of the enzyme activity in brain suggests an importance of the enzyme in the central nervous system to regulate the neuromodulatory fatty-acid amides.

Luminal Ca2+ Protects against Thapsigargin Inhibition in Neuronal Endoplasmic Reticulum

Thapsigargin is a specific and potent inhibitor of sarco/endoplasmic reticulum Ca2+-ATPases. However, in whole rat brain microsomes, 1 microM thapsigargin had no significant effect on the 10-min time course of ATP-dependent Ca2+ uptake in the absence of the luminal Ca2+ chelator oxalate. In contrast, 50 mM oxalate resolved a thapsigargin-sensitive Ca2+ uptake rate (IC50 approximately 1 nM thapsigargin) five times that of a thapsigargin-insensitive rate. This remaining approximately 20% of the total ATP-dependent accumulation was insensitive to thapsigargin (up to 10 microM), slightly less sensitive to vanadate inhibition, and unresponsive to 5 microM inositol 1,4,5-trisphosphate or 10 mM caffeine. Measuring both 12-min Ca2+ uptake and initial Ca2+ uptake rates, the apparent thapsigargin sensitivity increased as oxalate concentrations increased from 10 to 50 mM, corresponding to a range of luminal free Ca2+ concentrations of approximately 300 down to 60 nM. Addition of oxalate during steady-state 45Ca accumulation rapidly resolved the aforementioned thapsigargin sensitivity. These results strongly suggest that luminal Ca2+ may protect a large portion of neuronal endoplasmic reticulum Ca2+ pumps against thapsigargin inhibition. Although high [Ca2+] has been previously shown to protect against thapsigargin inhibition in several reticular membrane preparations, our results suggest that luminal Ca2+ alone is responsible for mediating this effect in neurons.

Evidence for O-dealkylation of 7-pentoxyresorufin by cytochrome P450 2B1/2B2 isoenzymes in brain.

O-dealkylation of 7-pentoxyresorufin (PR) was studied in rat brain to characterise the functional activity specific for cytochrome P450 2B1/2B2 isoenzymes in brain microsomes. Brain microsomes catalyzed the O-dealkylation of PR in the presence of NADPH. Pretreatment with phenobarbital (PB; 80 mg/kg body wt, i.p. x 5 days) resulted in 3-4 fold induction of pentoxyresorufin-O-dealkylase (PROD) activity while 3-methylcholanthrene (MC; 30 mg/kg body wt, i.p. x 5 days) did not produce any significant increase in enzyme activity. Kinetic studies revealed that the rate of velocity (Vmax) for the O-dealkylation of PR was significantly increased to 2.9 times higher in brain microsomes isolated from PB pretreated rats. In vitro studies using metyrapone, an inhibitor of P450 2B1/2B2 catalyzed reactions and antibody for hepatic PB inducible P450s (P450 2B1/2B2) significantly inhibited the activity of PROD in cerebral microsomes prepared from PB pretreated animals. These studies suggest that PB inducible isoenzymes of P450, i.e. P450 2B1/2B2 specifically catalyze the O-dealkylation of PR in brain microsomes.

Luminol and lucigenin amplified chemiluminescence andlipidperoxidation with brain microsomes from rats during ontogenetic development

The chemiluminescence (CL) amplifiers luminol (LM) and lucigenin (LC) react with different reactive oxygen species (ROS) in dependence on the ROS generating system used. With liver microsomes LMCL indicates predominantly superoxide anion radicals, whereas LCCL is mainly a measure for hydroxyl radical formation or of reactive organic radicals. With brain microsomes only LCCL, but not LMCL could be measured. For both brain microsomes from newborn (both sexes) and 60 day-old (male) rats LCCL is dependent on protein and NADPH concentration, activity in newborns being only 15% compared with young adult rats. As compared with liver microsomes 10-fold higher protein concentrations are needed to obtain comparable LCCL, whereas the NADPH demand is the same as with liver. A distinct ontogenetic development was demonstrated: low activities in the fetus, in newborn and 10-day-old rat are followed by higher activities with increasing age, after a maximum at an age of 60 days a decline was observed. Microsomal lipidperoxidation (LPO) was measured as formation of thiobarbituric acid reactive substances (TBARS) and was also dependent on protein and NADPH concentration. Unexpectedly, LPO with brain microsomes from newborn rats did not show any developmental variation.

Effect of docosahexaenoic acid on the synthesis of phosphatidylserine in rat brain in microsomes and C6 glioma cells.

Docosahexaenoic acid (22:6n-3) is the major polyunsaturated fatty acid (PUFA) in the CNS and accumulates particularly in phosphatidylserine (PS). We have investigated the effect of the 22:6n-3 compositional status on the synthesis of PS. The fatty acid composition of brain microsomes from offspring of rats artificially reared on an n-3-deficient diet showed a dramatic reduction of 22:6n-3 content (1.7 +/- 0.1%) when compared with control animals (15.0 +/- 0.2%). The decrease was accompanied by an increase in docosapentaenoic acid (22:5n-6) content, which replaced the 22:6n-3 phospholipids with 22:5n-6 molecular species, as demonstrated using HPLC/electrospray mass spectrometry. The n-3 deficiency did not affect the total amount of polyunsaturated phospholipids in brain microsomes; however, it was associated with a decrease in the total polyunsaturated PS content and with increased levels of 1-stearoyl-2-docosapentanoyl (18:0/22:5n-6) species, particularly in phosphatidylcholine. Incorporation of [3H]serine into PS in rat brain microsomes from n-3-deficient animals was slightly but significantly less than that of the control animals. Similarly, C6 glioma cells cultured for 24 h in 22:6n-3-supplemented media (10-40 microM) showed a significant increase in the synthesis of [3H]PS when compared with unsupplemented cells. Our data show that neuronal and glial PS synthesis is sensitive to changes in the docosahexaenoate levels of phospholipids and suggest that 22:6n-3 may be a modulator of PS synthesis.

Properties of specific binding site of myotoxin &lt;i&gt;a&lt;/i&gt;, a powerful convulsant, in brain microsomes

Properties of specific binding site of myotoxin &lt;i&gt;a&lt;/i&gt;, a powerful convulsant, in brain microsomes

High protection by grape seed proanthocyanidins (GSPC) of polyunsaturated fatty acids against UV-C induced peroxidation

The antioxidative effects of grape seed proanthocyanidins (GSPC) were studied in three in-vitro models in which polyunsaturated fatty acids (PUFAs) in aqueous solution and mice liver or brain microsomes were used as oxidative substrates, and UVC irradiation as the pro-oxidant system. Analysis of UV-C induced lipid peroxidation was carried out by two methods: gas liquid chromatography of residual PUFAs and release of thiobarbituric acid-reactive substances (TBARs) measured by TBA reaction. Results indicate that PUFAs are more radiosensitive when incorporated in single component micelles than in mixed component micelles or microsomes. In every case, PUFA peroxidation was inhibited by low concentrations of GSPC (2 rng/L) while epigallocatecin (EGC) and epigallocatechin gallate (EGCG) monomers, at an equivalent level of epicatechin, exhibited no efficacy in our experimental conditions. This latter effect might be explained by a synergistic action of flavan-3-ol monomers, dimers and oligomers contained in the grape seed extract.

Paraquat-induced free radical reaction in mouse brain microsomes.

Paraquat has been implicated as an environmental toxin which may induce the syndrome of Parkinson's disease after exposure to this agent. However, the biochemical mechanism by which paraquat causes cell death and neurodegeneration has not been extensively studied. Paraquat was rapidly taken up by nerve terminals isolated from mouse cerebral cortices. It induced lipid peroxidation in a concentration dependent manner in the presence of NADPH and ferrous ion. The maximal stimulation effect was obtained at a paraquat concentration around 100 microM and the Km value for paraquat was 46.7 microM. The lipid peroxidation required microsomal enzymes. Antioxidants, such as superoxide dismutase, catalase and promethazine significantly inhibited paraquat-induced lipid peroxidation. Due to its structural similarity to the pyridinium compound MPP+ (N-methyl-4-phenyl pyridium ion), it may be taken up by dopamine neurons and cause lipid peroxidation and cell death resulting in the manifestation of Parkinsonian syndrome.

P-545 - Increase of Ca2+-ATPase Activity in the Brain Microsomes of Aged Rats: Involvement of Protein Kinase C

P-545 - Increase of Ca2+-ATPase Activity in the Brain Microsomes of Aged Rats: Involvement of Protein Kinase C

ＤＨＡと脂質膜表面構造変化による神経機能調節

Rats were fed either linoleate-rich safflower oil or α-linolenate-rich perilla oil diets. The brightness-discrimination learning performance was significantly higher in the perilla oil group than in the safflower oil group. In synapses of hippocampus CA1, the density of synaptic vesicles was significantly higher in the perilla oil-fed rats than the safflower oil-fed rats. The number of terminals with low density of synaptic vesicles was increased in the safflower oil group after the learning. Fourier transform infrared spectroscopy of rat brain microsomes revealed that the second derivative spectrum at 1050-1250 cm-1showed difference between perilla and safflower oil groups after the learning. The infrared band at 1727 cm-1 in the safflower oil group moved to the higher wavenumber position at 1731 cm-1in the perilla oil group after the learning. The results suggested that the hydrogen bond of fatty acid ester (sn-2) with water was weakened and moved inward of the membrane, and resulted in a lowered hydration. This was confirmed by the weakened reactivity of the membrane phospholipids to phospholipase A2 in the perilla oil group after the learning. These results suggested that n-3 polyunsaturated fatty acid-deficiency may affect the turnover of synaptic vesicles in hippocampus with loss of learning ability.

Dehydroepiandrosterone 7alpha- and 7beta-hydroxylation in mouse brain microsomes. Effects of cytochrome P450 inhibitors and structure-specific inhibition by steroid hormones.

Presently, several works question the effects of dehydroepiandrosterone (DHEA) reported in vivo and designate its 7-hydroxylated metabolites as native antiglucocorticoids and potent mediators in the triggering of immune response. Among mouse tissues and organs, and second to liver, the largest production of 7alpha-and 7beta-hydroxylated derivatives of DHEA takes place in brain microsomes. To contribute to identification of cytochromes P450 (CYPs) responsible for 7alpha- and 7beta-hydroxy-DHEA production, effects of CYP inhibitors and of several steroid hormones on DHEA 7-hydroxylation were examined. Using mouse brain microsomes as a source of enzyme, we report now that strong and smaller inhibitions of DHEA 7alpha-hydroxylation were obtained with ketoconazole and alpha-naphthoflavone, respectively, and that neither changed DHEA 7beta-hydroxylation. Metyrapone and antipyrine also inhibited 7alpha-hydroxylation, but by contrast, significantly increased 7beta-hydroxylation of DHEA. This indicated that at least, two different CYPs were responsible for 7alpha- and 7beta-hydroxylation of DHEA. Steroids sharing a 3beta-hydroxylated structure with DHEA, namely pregnenolone, 5-androstene-3beta,17beta-diol and 3beta-hydroxy-5alpha-androstan-17-one, were strong inhibitors of DHEA 7alpha-hydroxylation (non-competitive inhibition with pregnenolone, Ki=2.0 +/- 0.3 microM). In contrast, 7beta-hydroxylation yields were not decreased by the 3beta-hydroxysteroids tested. Moderate inhibition of 7alpha- and 7beta-hydroxylation was obtained with 3-oxosteroids, namely testosterone, progesterone, corticosterone and 4-androsten-3,17-dione. Taken together, these data indicate specific inhibition patterns of DHEA 7alpha- and 7beta-hydroxylation by CYP inhibitors and steroid hormones in mouse brain microsomes and may be used as criteria necessary for identification of the responsible CYP species.

Effects of cytochrome P450 inhibitors and of steroid hormones on the formation of 7-hydroxylated metabolites of pregnenolone in mouse brain microsomes.

Hydroxylations of pregnenolone (PREG) at the 7 alpha- and 7 beta-positions have been reported in numerous murine tissues and organs and responsible cytochrome P450 (CYP) species await identification. Using thin layer chromatography and gas chromatography-mass spectrometry, we report identification of 7 alpha-hydroxy-PREG and 7 beta-hydroxy-PREG metabolites produced in mouse brain microsome digests and kinetic studies of their production with apparent KM values of 0.5 +/- 0.1 microM and 5.1 +/- 0.6 microM for 7 alpha- and 7 beta-hydroxylation respectively. Investigation of CYP inhibitors and of steroid hormone effects on both 7 alpha- and 7 beta-hydroxylations of PREG showed that: (i) different CYP were involved in 7 alpha- and 7 beta-hydroxylation of PREG because solely 7 alpha-hydroxylation was extensively inhibited by metyrapone, alpha-naphthoflavone, ketoconazole and 3 beta-hydroxysteroids, (ii) CYP 1A2, 2D6, 2B1 and 2B11 were not responsible for 7 alpha- and 7 beta-hydroxylation of PREG because respective specific inhibitors furafylline, quinidine and chloramphenicol triggered no inhibition, (iii) CYP 1A1 was responsible for only part of the 7 beta-hydroxylation of PREG because use of alpha-naphthoflavone, which inhibits specifically CYP 1A1, did not suppress entirely 7 beta-hydroxylation, while ketoconazole, metyrapone and antipyrine, which do not inhibit CYP 1A1, decreased part of the 7 beta-hydroxylation, (iv) 7 alpha-hydroxylation of PREG may be shared with other 3 beta-hydroxysteroids such as isoandrosterone and 5-androstene-3 beta,17 beta-diol which were strong inhibitors, but not with dehydroepiandrosterone which was a non-competitive inhibitor as weak as 3-oxosteroids, and (v) 7 beta-hydroxylation of PREG was not markedly changed by other steroids. Taken together, these findings will be of use for identification of the CYP species responsible for 7 alpha- and 7 beta-hydroxylation of PREG and for studies of their activities in brain.

Importance of a Novel Oxidative Mechanism for Elimination of Brain Cholesterol: TURNOVER OF CHOLESTEROL AND 24(S)-HYDROXYCHOLESTEROL IN RAT BRAIN AS MEASURED WITH 18O2TECHNIQUES IN VIVO AND IN VITRO

The brain is the most cholesterol-rich organ in the body. Brain cholesterol is characterized by a very low turnover with very little exchange with lipoproteins in the circulation. Very recently we showed that there is a continuous age-dependent flux of 24(S)-hydroxycholesterol from the human brain into the circulation (Lütjohann, D., Breuer, O., Ahlborg, G., Nennesmo, I., Sidén, A., Diczfalusy, U., and Björkhem, I. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 9799-9804). Here we measured the rate of synthesis of cholesterol as well as the conversion of cholesterol into 24(S)-hydroxycholesterol in rat brain in vivo with use of an 18O2 inhalation technique and mass isotopomer distribution analysis. Cholesterol synthesis was found to correspond to 0.03 +/- 0.01% of the pool per h. Conversion of cholesterol into 24(S)-hydroxycholesterol was of a similar magnitude, about 0.02% of the pool per h. Brain microsomes converted endogenous cholesterol into 24(S)-hydroxycholesterol at a similar rate when incubated in the presence of NADPH. When incubated with whole homogenate and subcellular fractions of rat brain, there was no significant conversion of tritium-labeled 24-hydroxycholesterol into more polar products. Plasma from 18O2-exposed rats contained 24(S)-hydroxycholesterol with an enrichment of 18O similar to that in 24(S)-hydroxycholesterol in the brain. The results suggest that the present 24(S)-hydroxylase mediated mechanism is most important for elimination of cholesterol from the brain of rats. There is a slow conversion of brain cholesterol into 24(S)-hydroxycholesterol with a rapid turnover of the small pool of the latter oxysterol due to leakage to the circulation (half-life of brain 24(S)-hydroxycholesterol is about 0.5 days as compared with 2-4 months for brain cholesterol). It is evident that the 24(S)-hydroxylation greatly facilitates transfer of cholesterol over the blood-brain barrier and that this hydroxylation may be critical for cholesterol homeostasis in the brain.