Cholesterol 7-alpha-hydroxylase - Evidence For Transcriptional Regulation By Cholesterol Or Metabolic Products Of Cholesterol In The Rat
bile-acid synthesis; bile fistula; bile-acid synthesis; Biochemistry & Molecular Biology; biosynthesis; cloning; enterohepatic circulation; enzyme; hepatic cholesterol; hepatocytes; hmg-coa reductase; liver microsomes; lovastatin; messenger-rna; mevalonate; stimulation
Cholesterol 7alpha-hydroxylase, the rate-determining enzyme in the bile acid biosynthesis pathway, is regulated in a negative feedback manner by hydrophobic bile salts returning to the liver via the portal circulation. The role of cholesterol in the regulation of cholesterol 7alpha-hydroxylase and the interrelationship between the cholesterol and bile acid biosynthesis pathways remain controversial. The objective of the present study was to define the role of cholesterol in the regulation of cholesterol 7alpha-hydroxylase and determine the molecular level of its control. In order to avoid intestinal or intravenous administration of cholesterol, we manipulated the flow of cholesterol within the hepatocytes by decreasing cholesterol synthesis with lovastatin in bile fistula rats (bile acid synthesis is up-regulated), or by increasing cholesterol supply by administering mevalonate, a precursor of cholesterol, to rats with intact enterohepatic circulation (bile acid synthesis is normal). In the first series of studies, lovastatin was administered as a single intravenous bolus (10 mg/kg) to rats with chronic bile fistula and to rats with intact enterohepatic circulation (cholesterol and bile acid synthesis is normal). Three hours after lovastatin administration, cholesterol 7alpha-hydroxylase specific activity, enzyme mass, mRNA, and gene transcriptional activity were decreased by 35%, 32%, 56%, and 34%, respectively, in rats with chronic bile fistula. In rats with intact enterohepatic circulation, lovastatin administration resulted in a similar decrease (34%) of cholesterol 7alpha-hydroxylase specific activity. In the second group of experiments, rats with intact enterohepatic circulation were administered a 180 mum bolus of mevalonate followed by a continuous infusion of 180 mumol/h for 1.5, 3, 4.5, and 24 h prior to being killed. Continuous infusion of mevalonate increased cholesterol 7-alpha-hydroxylase specific activity, mRNA levels, and transcriptional activity by an average of 2- to 3-fold at all time intervals. We conclude that under circumstances in which cholesterol is present in excess, cholesterol 7alpha-hydroxylase transcriptional activity is up-regulated and removal of cholesterol from the hepatocytes is facilitated by an increase of bile acid synthesis. When cholesterol availability is decreased, cholesterol 7alpha-hydroxylase transcriptional activity is down-regulated leading to a decreased elimination of cholesterol via bile acid synthesis. In both instances, hepatic cholesterol homeostasis is effectively maintained.
Jones M P; Pandak W M; Heuman D M; Chiang J Y L; Hylemon P B; Vlahcevic Z R
Journal of Lipid Research
1993
1993-06
Journal Article or Conference Abstract Publication
n/a
Novel Approaches To Mitigating Parathion Toxicity: Targeting Cytochrome P450-mediated Metabolism With Menadione
acetylcholinesterase; agents; chlorcyclizine; cytotoxicity; enzyme; inhibition; menadione; organophosphate exposure; organophosphorus; paraoxon; parathion; pesticides; reactivators; redox cycling; reductase; Super oxide
Accidental or intentional exposures to parathion, an organophosphorus (OP) pesticide, can cause severe poisoning in humans. Parathion toxicity is dependent on its metabolism by the cytochrome P450 (CYP) system to paraoxon (diethyl 4-nitrophenyl phosphate), a highly poisonous nerve agent and potent inhibitor of acetylcholinesterase. We have been investigating inhibitors of CYP-mediated bioactivation of OPs as a method of preventing or reversing progressive parathion toxicity. It is well recognized that NADPH-cytochrome P450 reductase, an enzyme required for the transfer of electrons to CYPs, mediates chemical redox cycling. In this process, the enzyme diverts electrons from CYPs to support chemical redox cycling, which results in inhibition of CYP-mediated biotransformation. Using menadione as the redox-cycling chemical, we discovered that this enzymatic reaction blocks metabolic activation of parathion in rat and human liver microsomes and in recombinant CYPs important to parathion metabolism, including CYP1A2, CYP2B6, and CYP3A4. Administration of menadione to rats reduces metabolism of parathion, as well as parathion-induced inhibition of brain cholinesterase activity. This resulted in inhibition of parathion neurotoxicity. Menadione has relatively low toxicity and is approved by the Food and Drug Administration for other indications. Its ability to block parathion metabolism makes it an attractive therapeutic candidate to mitigate parathion-induced neurotoxicity.
Jan Y H; Richardson J R; Baker A A; Mishin V; Heck D E; Laskin D L; Laskin J D
Countermeasures against Chemical Threats Ii
2016
2016
Book Chapter
<a href="http://doi.org/10.1111/nyas.13156" target="_blank" rel="noreferrer noopener">10.1111/nyas.13156</a>
Hormonal-regulation Of Cholesterol-7-alpha-hydroxylase Messenger-rna Levels And Transcriptional Activity In Primary Rat Hepatocyte Cultures
bile-acid synthesis; Biochemistry & Molecular Biology; chick-embryo hepatocytes; cholesterol 7-alpha-hydroxylase activity; circadian-rhythm; cyclic-amp; enzyme; gene; liver; malic; metabolism; monolayer-cultures
In primary cultures of adult rat hepatocytes the level of cholesterol 7-alpha-hydroxylase steady-state mRNA markedly decreased by 72 h. However, the addition of L-thyroxine (T4) and dexamethasone synergistically returned cholesterol 7-alpha-hydroxylase steady-state mRNA levels near to that of cholestyramine-fed animals. The maximal responses to T4 and dexamethasone in serum-free medium were at 1.0 and 0.1-mu-M, respectively. The addition of T4 in combination with dexamethasone resulted in an 11-fold increase in transcriptional activity of the cholesterol 7-alpha-hydroxylase gene as compared to no addition controls. The specific activities of cholesterol 7-alpha-hydroxylase in microsomes prepared from cultures treated with dexamethasone and T4 were 1.56 +/- 1.17 nmol/h/mg protein which is similar to that of intact liver (1.70 +/- 0.062 nmol/h/mg protein), but lower than cholestyramine-fed animals. Cholesterol 7-alpha-hydroxylase activity was not detectable (<0.020 nmol/h/mg protein) at 72 h in cultures without the addition of both dexamethasone and T4. In the presence of optimal concentrations of dexamethasone and T4, glucagon (0.2-mu-M), or dibutyryl cAMP (50-mu-M) decreased (90%) cholesterol 7-alpha-hydroxylase mRNA within 6 h. Transcriptional activity decreased (62%) in 6 h following the addition of glucagon (0.2-mu-M) to the culture medium. The results reported in this paper suggest an important role for multiple hormones in the regulation of cholesterol 7-alpha-hydroxylase in the liver.
Hylemon P B; Gurley E C; Stravitz R T; Litz J S; Pandak W M; Chiang J Y L; Vlahcevic Z R
Journal of Biological Chemistry
1992
1992-08
Journal Article or Conference Abstract Publication
n/a
A Splicing Mutation In The Cytochrome B(5) Gene From A Patient With Congenital Methemoglobinemia And Pseudohermaphrodism
deficiency; enzyme; Genetics & Heredity; hereditary methemoglobinemia; human-liver; microsomes; rna
Giordano S J; Kaftory A; Steggles A W
Human Genetics
1994
1994-05
Journal Article or Conference Abstract Publication
n/a
REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY ASSAY OF CHOLESTEROL 7-ALPHA-HYDROXYLASE
Biochemistry & Molecular Biology; cloning; enzyme; purification; rat-liver microsomes; cholesterol 7-alpha-hydroxylase
Chiang J Y L
Methods in Enzymology
1991
1991
Journal Article or Conference Abstract Publication
n/a
Novel role of aminopeptidase-A in angiotensin-(1-7) metabolism post myocardial infarction
ace2; Aminopeptidase A; angiotensin peptides; antihypertensive agents; carboxypeptidase; Cardiovascular System & Cardiology; enzyme; inhibitors; kidney; MALDI-imaging; mass-spectrometry; Myocardial infarction; Physiology; renal damage; renin-angiotensin system; spontaneously hypertensive-rats; system
Aminopeptidase-A (APA) is a less well-studied enzyme of the renin-angiotensin system. We propose that it is involved in cardiac angiotensin (ANG) metabolism and its pathologies. ANG-(1-7) can ameliorate remodeling after myocardial injury. The aims of this study are to 1) develop mass spectrometric (MS) approaches for the assessment of ANG processing by APA within the myocardium; and 2) investigate the role of APA in cardiac ANG-(1-7) metabolism after myocardial infarction (MI) using sensitive MS techniques. MI was induced in C57Bl/6 male mice by ligating the left anterior descending (LAD) artery. Frozen mouse heart sections (in situ assay) or myocardial homogenates (in vitro assay) were incubated with the endogenous APA substrate, ANG II. Results showed concentration-and time-dependent cardiac formation of ANG III from ANG II, which was inhibited by the specific APA inhibitor, 4-amino-4-phosphonobutyric acid. Myocardial APA activity was significantly increased 24 h after LAD ligation (0.82 +/- 0.02 vs. 0.32 +/- 0.02 rho mol.min(-1).mu g(-1), MI vs. sham, P < 0.01). Both MS enzyme assays identified the presence of a new peptide, ANG-(2-7), m/z 784, which accumulated in the MI (146.45 +/- 6.4 vs. 72.96 +/- 7.0%, MI vs. sham, P < 0.05). Use of recombinant APA enzyme revealed that APA is responsible for ANG-(2-7) formation from ANG-(1-7). APA exhibited similar substrate affinity for ANG-(1-7) compared with ANG II {K-m (ANG II) = 14.67 +/- 1.6 vs. K-m [ANG-(1-7)] = 6.07 +/- 1.12 mu mol/l, P < 0.05}. Results demonstrate a novel role of APA in ANG-(1-7) metabolism and suggest that the upregulation of APA, which occurs after MI, may deprive the heart of cardioprotective ANG-(1-7). Thus APA may serve as a potentially novel therapeutic target for management of tissue remodeling after MI.
Alghamri M S; Morris M; Meszaros J G; Elased K M; Grobe N
American Journal of Physiology-Heart and Circulatory Physiology
2014
2014-04
Journal Article or Conference Abstract Publication
<a href="http://doi.org/10.1152/ajpheart.00911.2013" target="_blank" rel="noreferrer noopener">10.1152/ajpheart.00911.2013</a>
BILE-ACID SYNTHESIS .6. REGULATION OF CHOLESTEROL 7-ALPHA-HYDROXYLASE BY TAUROCHOLATE AND MEVALONATE
3-hydroxy-3-methylglutaryl; 7-alpha-hydroxylase; bile acids; Biochemistry & Molecular Biology; biosynthesis; circadian-rhythm; cloning; coenzyme; enzyme; hepatocytes; hmg-coa reductase; liver; messenger-rna; rat-liver microsomes; reductase; substrate
Taurocholate, a relatively hydrophobic bile salt, is a potent down-regulator of HMG-CoA reductase and cholesterol 7-alpha-hydroxylase (C7-alpha-H), the rate-determining enzymes of the cholesterol and bile acid biosynthetic pathways, respectively. Inhibition of cholesterol synthesis with a bolus dose of mevinolin (lovastatin) a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, profoundly decreases the specific activity of cholesterol 7-alpha-hydroxylase and rate of bile acid synthesis in rats with complete biliary diversion. It is therefore conceivable that taurocholate may suppress cholesterol 7-alpha-hydroxylase primarily by down-regulating the activity of HMG-CoA reductase. To test this hypothesis, taurocholate was coinfused simultaneously to rats with chronic bile fistula with mevalonate (administered as mevalonolactone), an intermediate in the cholesterol biosynthetic pathway. Mevalonolactone was administered to provide a constant supply of newly synthesized cholesterol to cholesterol 7-alpha-hydroxylase, in order to overcome any inhibitory effect of taurocholate on HMG-Coa reductase. Infusions were started 72 h after biliary diversion, and carried out for an additional 48 h. Complete biliary diversion resulted in an increase in C7-alpha-H specific activity (510%), protein mass (550%), steady-state mRNA levels (1430%), and transcriptional activities (330%) as compared to control rats with intact enterohepatic circulations. When rats with biliary diversion were infused intraduodenally with taurocholate, the specific activities of HMG-CoA reductase and cholesterol 7-alpha-hydroxylase activities decreased by 75% (P < 0.001) and 73% (P < 0.001), respectively. Cholesterol 7-alpha-hydroxylase mass, mRNA, and transcriptional activity decreased after intraduodenal infusion of taurocholate to levels similar to those of rats with an intact enterohepatic circulation. The combination of constant infusion of mevalonate and taurocholate failed to reverse the inhibitory effects of taurocholate on cholesterol 7-alpha-hydroxylase activity, mRNA levels, and in vitro transcriptional rates. These data provide evidence that taurocholate represses cholesterol 7-alpha-hydroxylase at the level of gene transcription, and not via down-regulation of HMG-CoA reductase. Infusion of mevalonate alone to biliary diverted rats did not alter cholesterol 7-alpha-hydroxylase activity or mRNA levels, while leading to a 57% decrease in C7-alpha-H gene transcription. This latter finding suggests that mevalonate or its metabolites may be capable of stabilizing C7-alpha-H mRNA levels while down-regulating transcriptional activity.
Pandak W M; Vlahcevic Z R; Chiang J Y L; Heuman D M; Hylemon P B
Journal of Lipid Research
1992
1992-05
Journal Article
n/a
Cholesterol 7 alpha-hydroxylase activities from human and rat liver are modulated in vitro posttranslationally by phosphorylation/dephosphorylation
bile-acid synthesis; cloning; dietary-cholesterol; enzyme; Escherichia coli; expression; Gastroenterology & Hepatology; hmg-coa reductase; messenger-rna levels; Phosphatase; purification
Purified cholesterol 7 alpha-hydroxylases (C7 alpha H) from human and rat liver microsomes, and from transformed Escherichia coli expression systems, were incubated with 0.3 mmol/L [gamma-P-32] adenosine triphosphate (ATP) in the presence and absence of bacterial alkaline phosphatase (AP) or rabbit muscle adenosine 3',5'-cyclic monophosphate (cAMP) dependent protein kinase. The amounts of P-32 incorporation after separation of human and rat C7 alpha H proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were related to C7 alpha H catalytic activities (determined by a radioisotope incorporation method) and enzyme protein mass (determined by Western blotting and laser densitometry). Both human and rat C7 alpha H activities significantly decreased after dephosphorylation by AP (-57%--72%) and increased up to twofold with phosphorylation by rabbit muscle cAMP-dependent protein kinase. The increases in C7 alpha H activities were proportional to the amounts of cAMP-dependent protein kinase used, and were coupled to P-32 incorporation into the purified enzymes, Both the activation of C7 alpha H and the amounts of P-32 incorporation were time-dependent and reached a maximum after 1 hour of incubation with 5 U of cAMP-dependent protein kinase. In a second set of experiments, purified human and rat Liver C7 alpha H were dephosphorylated by 30-minute incubation with AP, followed by inactivation of the phosphatase by the inhibitor NaF, and rephosphorylation of C7 alpha H by 30-minute incubation with rabbit muscle cAMP-dependent protein kinase or bovine heart cAMP-independent protein kinase. Rephosphorylation of the dephosphorylated C7 alpha H proteins by cAMP-dependent protein kinase increased C7 alpha H catalytic activities up to fourfold, and the stimulation in catalytic activities paralleled the increases in P-32 incorporation into the purified enzymes. Bovine heart protein kinase was as potent as rabbit muscle cAMP-dependent protein kinase in stimulating catalytic activity and P-32 incorporation into the human C7 alpha H protein. Because the protein mass of these purified enzymes did not change, the short-term regulation or catalytic efficiency of C7 alpha H (activity per protein mass unit) is modulated, in vitro, posttranslationally by a phosphorylation/ dephosphorylation mechanism in both the human and the rat enzymes.
Nguyen L B; Shefer S; Salen G; Chiang J Y L; Patel M
Hepatology
1996
1996-12
Journal Article
<a href="http://doi.org/10.1053/jhep.1996.v24.pm0008938182" target="_blank" rel="noreferrer noopener">10.1053/jhep.1996.v24.pm0008938182</a>