Isolated rat liver cells catalyze the metabolism of benzo(alpha)pyrene (BP) with the resulting formation of phenols, dihydrodiols, and conjugates. The rate of the primary oxidative step in the process was similar to that catalyzed by... more
Isolated rat liver cells catalyze the metabolism of benzo(alpha)pyrene (BP) with the resulting formation of phenols, dihydrodiols, and conjugates. The rate of the primary oxidative step in the process was similar to that catalyzed by isolated rat liver microsomes in the presence of a reduced nicotinamide adenine dinucleotide phosphate-generating system and responded similarly to various inhibitors, including 2-diethylaminoethyl-2,2-diphenylvalerate, metyrapone, alpha-naphthoflavone, and hexobarbital. The level of cytoplasmic, reduced nicotinamide adenine dinucleotide phosphate was not rate limiting in liver cells isolated from either fed or fasted animals. The conjugates and dihydrodiols formed were readily excreted, whereas low concentrations of phenols accumulated intracellularly. The pattern of metabolites of BP was the same in isolated rat liver cells and in the isolated perfused rat liver. 3-Methylcholanthrene treatment of the rats caused a marked increase in cellular BP metabo...
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The mechanistic plurality of the microsomal cytochrome P-450 enzyme system is illustrated by studies of the oxidative metabolism of benzo[a]pyrene, 3-hydroxybenzo[a]pyrene and arachidonic acid. Rat liver microsomal metabolism of... more
The mechanistic plurality of the microsomal cytochrome P-450 enzyme system is illustrated by studies of the oxidative metabolism of benzo[a]pyrene, 3-hydroxybenzo[a]pyrene and arachidonic acid. Rat liver microsomal metabolism of benzo[a]pyrene or 3-hydroxy-benzo[a]pyrene, supported by cumene hydroperoxide, generates benzo[a]pyrene quinones via molecular oxygen-dependent and -independent pathways. Arachidonic acid is metabolized by rat liver microsomal fractions to a variety of oxygenated products, including cis-trans diene conjugated monohydroxy-acids, epoxy-acids as well as omega- and omega-1-oxidation products. The chemistry of the different reaction products is discussed in terms of the possible mechanisms responsible for their formation and the role of the haemoprotein during catalysis. An integrated view for the reaction cycle of cytochrome P-450 is presented.
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Purified synthetic products from the cytochrome P450 pathway of arachidonate metabolism were applied to the intestinal serosa. Arteriolar blood flow was calculated using video microscopy. After a steady-state baseline, a bolus containing... more
Purified synthetic products from the cytochrome P450 pathway of arachidonate metabolism were applied to the intestinal serosa. Arteriolar blood flow was calculated using video microscopy. After a steady-state baseline, a bolus containing 10-60 micrograms 14,15-epoxyeicosatrienoic acid/ml (14,15-EET) had no detectable effect on blood flow. However, 25 +/- 3 micrograms 11,12-EET/ml and 36 +/- 2 micrograms 8,9-EET/ml caused increases (134 +/- 8% and 127 +/- 6%) that were similar to those elicited by 8 +/- 2 micrograms adenosine/ml (138 +/- 12%). Furthermore, the increases (275 +/- 38%) produced by 32 +/- 6 micrograms 5,6-EET/ml exceeded those elicited (160 +/- 10%) by a similar concentration (27 +/- 3 micrograms/ml) of adenosine. Thus, a structure-activity relationship is suggested. Nevertheless, these values probably underestimate the potency of the EETs because the vasoactivity was reduced by contact with water. The activity of the cyclooxygenase pathway seemed to limit the formation...
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Publisher Summary The formation of an ultimate carcinogenic form of benzo(a)pyrene (B(a)P), the trans–7, 8–dihydro–7, 8–diol–9, 10–oxide, requires several metabolic steps. This realization has increased interest in the further metabolism... more
Publisher Summary The formation of an ultimate carcinogenic form of benzo(a)pyrene (B(a)P), the trans–7, 8–dihydro–7, 8–diol–9, 10–oxide, requires several metabolic steps. This realization has increased interest in the further metabolism of the various dihydrodiols, quinones, and phenols that are derived from polycyclic aromatic hydrocarbon model. Several studies have suggested that the 3– and 9–phenols can be metabolized to yield products that bind to cellular nucleophiles, such as DNA. This chapter describes the metabolite profiles obtained during the oxidative metabolism of 3– and 9–hydroxy–B(a)P by rat liver microsomes and the potential reactivity for certain of the metabolic products. The potential biological reactivity of B(a)P and its metabolites have been studied using assays that either measure the induction of mutation in prokaryotes or the alkylation of DNA. Results suggest that 3– and 9–hydroxy–B(a)P are further metabolized by the cytochrome P-450–dependent monooxygenase of liver microsomes and that in the case of the 9–phenol, a biologically reactive molecule is formed capable of inducing mutations in prokaryotes and alkylating DNA.
The 5,6-; 8,9-; 11,12- and 14,15-epoxyeicosatrienoic acids and their respective hydration products, the vic-diols, recently reported as metabolites of arachidonic acid in rat liver microsomes, were examined for effect on release of 45Ca... more
The 5,6-; 8,9-; 11,12- and 14,15-epoxyeicosatrienoic acids and their respective hydration products, the vic-diols, recently reported as metabolites of arachidonic acid in rat liver microsomes, were examined for effect on release of 45Ca from canine aortic smooth muscle microsomes. At 10(-6) M, the diols had no effect, but the 5,6-; 11,12- and 14,15-epoxyacids increased the loss of 45Ca. Further studies with the 14,15-epoxyacid demonstrated a dose-dependent decrease of Ca++ uptake (ATP present) in canine aortic microsomes in 0.03 mM Ca++, whereas Ca++ binding (ATP absent) was not affected. Ca++ uptake, binding and release in rat liver microsomes was similarly affected by the 14,15-epoxyacid, the major epoxyeicosatrienoic acid derivative produced by rat liver microsomal incubations. It is suggested that alterations in Ca++ metabolism might be a possible mechanism of action for these derivatives of arachidonic acid.
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Abstract 1. 1. The levels of DDT-dehydrochlorinase (E.C. 4.5.1.1) measured in the soluble fraction obtained from homogenates of Musca domestica L. are increased after treatment of the insect with DDT. 2. 2. The increase enzymatic activity... more
Abstract 1. 1. The levels of DDT-dehydrochlorinase (E.C. 4.5.1.1) measured in the soluble fraction obtained from homogenates of Musca domestica L. are increased after treatment of the insect with DDT. 2. 2. The increase enzymatic activity is shown to be paralleled by an increased de novo synthesis of the enzyme protein. 3. 3. Experiments using protein synthesis inhibitors suggested that this enhanced enzyme synthesis is dependent on RNA synthesis and gave further support to the idea of an inductive phenomenon.
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... 4. J. Capdevila, N. Chacos, JR Falck, S. Manna, A. Negro-Vilar, SR Ojeda, submitted for publication. ... EJ Corey, Sadahiko Iguchi, John 0. Albright, and Biswanath De Department of Chemistry, Harvard University, Cambridge,... more
... 4. J. Capdevila, N. Chacos, JR Falck, S. Manna, A. Negro-Vilar, SR Ojeda, submitted for publication. ... EJ Corey, Sadahiko Iguchi, John 0. Albright, and Biswanath De Department of Chemistry, Harvard University, Cambridge, Massachusetts 02138 Summary: 20 ...
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... 4. J. Capdevila, N. Chacos, JR Falck, S. Manna, A. Negro-Vilar, SR Ojeda, submitted for publication. ... EJ Corey, Sadahiko Iguchi, John 0. Albright, and Biswanath De Department of Chemistry, Harvard University, Cambridge,... more
... 4. J. Capdevila, N. Chacos, JR Falck, S. Manna, A. Negro-Vilar, SR Ojeda, submitted for publication. ... EJ Corey, Sadahiko Iguchi, John 0. Albright, and Biswanath De Department of Chemistry, Harvard University, Cambridge, Massachusetts 02138 Summary: 20 ...
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Abstract Cytochrome P-450 from rat lung microsomes has been solubilized and purified 8-fold by using affinity chromatography on an ω-amino- n -octyl derivative of Sepharose 4B. The purified fraction was free of cytochrome b 5 and... more
Abstract Cytochrome P-450 from rat lung microsomes has been solubilized and purified 8-fold by using affinity chromatography on an ω-amino- n -octyl derivative of Sepharose 4B. The purified fraction was free of cytochrome b 5 and NADPH-cytochrome c reductase and showed spectral characteristics similar to those of lung microsomal cytochrome P-450. When combined with NADPH-cytochrome c reductase partially purified from liver microsomes, the cytochrome P-450 fraction supported the hydroxylation of benzo (α)pyrene and the activity was proportional to the content of the hemoprotein. No absolute requirement for phosphatidylcholine was found.
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ABSTRACT Arachidonic acid is enzymatically oxidized by the rat liver microsomal mixed-function oxidase system, in the presence of NADPH and oxygen, to a wide variety of products. We report here, the identification of the major... more
ABSTRACT Arachidonic acid is enzymatically oxidized by the rat liver microsomal mixed-function oxidase system, in the presence of NADPH and oxygen, to a wide variety of products. We report here, the identification of the major organic-soluble metabolites. They are the 5,6-,8,9-,11,12-, and 14,15-epoxy-eicosatrienoic acid derivatives of arachidonic acid.
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ABSTRACT Studies of benzo(a)pyrene metabolism by liver microsomes have revealed a complex pattern of products formed. The distribution of products generated depends on the types of cytochromes P-450 present and the mechanism of oxidation... more
ABSTRACT Studies of benzo(a)pyrene metabolism by liver microsomes have revealed a complex pattern of products formed. The distribution of products generated depends on the types of cytochromes P-450 present and the mechanism of oxidation reactions catalyzed. It is proposed that cytochrome P-450 may function to catalyze the oxidation of benzo(a)pyrene not only by a NADPH-dependent monooxygenation type reaction, but also concomitantly by a peroxidatic type reaction. In particular, attention is directed to the role of the latter type of reaction for the formation of benzo(a)pyrene quinones. The pathways for the metabolic generation of quinones, as well as the reactivity of hydroquinones with oxygen, provide additional pathways for the formation of highly reactive, toxic products which may contribute to the alterations of cellular characteristics that result in either tumor growth or cell death.
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Research Interests: Kidney, Female, Animals, Temperature, Renal Function, and 11 moreGAS LIQUID CHROMATOGRAPHY, Rats, Gas Chromatography/mass Spectrometry, Mass Spectroscopy, Lipid, Biological activity, Spectroscopic Techniques, Oxidation-Reduction, Methyl Ester, Biochemistry and cell biology, and Medical biochemistry and metabolomics
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... METABOLISM OF ARACHIDONIC ACID BY PURIFIED CYTOCHROMES P450* Jorge Capdevila, Linda Parkhill, Nicholas Chacos, Richard Okita, Bettie Sue S. Masters ... 1) that the addition of cytochrome b5 to the reaction mixture, in a stoichiometric... more
... METABOLISM OF ARACHIDONIC ACID BY PURIFIED CYTOCHROMES P450* Jorge Capdevila, Linda Parkhill, Nicholas Chacos, Richard Okita, Bettie Sue S. Masters ... 1) that the addition of cytochrome b5 to the reaction mixture, in a stoichiometric ratio of 1 mole of cytochrome ...
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ABSTRACT Analysis of repetitive scan difference spectra of incubation mixtures containing rat liver microsomes, 3- or 9-hydroxybenzo(a)pyrene, oxygen, and NADPH shows the formation of products with absorbance in the 400–450 nm region.... more
ABSTRACT Analysis of repetitive scan difference spectra of incubation mixtures containing rat liver microsomes, 3- or 9-hydroxybenzo(a)pyrene, oxygen, and NADPH shows the formation of products with absorbance in the 400–450 nm region. Based on the chromatographic retention time, absorbance, and fluorescence spectra, the two major products of 9-hydroxybenzo(a)pyrene metabolism may be diphenols. The existence of spectral intermediates which resemble phenols rather than quinones during the steady-state metabolism of 3-hydroxybenzo(a)pyrene strongly indicates that either the major product is a diphenol which slowly oxidizes to yield 3,6-quinone and/or that an active quinone reductase exists in liver microsomes.
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... P-450-LINKED ACTIVATION OF 3-HYDROXYBENZO(~)PYRENEJorge Capdevila, Bengt Jernstr~m, Helena Vadi and Sten OrreniusDepartment of Forensic ... J. 9_~7, 712. Jerina, DM, Daly, JW, Witkop, B., Zaltman-Nirenberg, P.and Udenfriend, SJ (1970)... more
... P-450-LINKED ACTIVATION OF 3-HYDROXYBENZO(~)PYRENEJorge Capdevila, Bengt Jernstr~m, Helena Vadi and Sten OrreniusDepartment of Forensic ... J. 9_~7, 712. Jerina, DM, Daly, JW, Witkop, B., Zaltman-Nirenberg, P.and Udenfriend, SJ (1970) Biochemistry, 2, 14713. ...
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... Reinhard Renneberg , Jorge Capdevila , Nicholas Chacos , Ronald W. Estabrook and RussellA. Prough Corresponding Author Contact ... of thephysiological electron donor, NADPH, remains aviable explanation for the metabolic formation... more
... Reinhard Renneberg , Jorge Capdevila , Nicholas Chacos , Ronald W. Estabrook and RussellA. Prough Corresponding Author Contact ... of thephysiological electron donor, NADPH, remains aviable explanation for the metabolic formation ofthe B[a]P quinones.BENZO(o ...
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Arachidonic acid (AA) is oxidized via three pathways which result in several series of distinct metabolites. Cyclooxygenase produces prostaglandins (PGs), prostacyclins, and thromboxanes. Lipoxygenase produces... more
Arachidonic acid (AA) is oxidized via three pathways which result in several series of distinct metabolites. Cyclooxygenase produces prostaglandins (PGs), prostacyclins, and thromboxanes. Lipoxygenase produces hydroperoxy/hydroxyeicosatetraenoic acids (HPETE/HETEs) and leukotrienes. Epoxygenase, a recently uncovered pathway, results in epoxyeicosatrienoic acids (EETs). Based on reverse phase HPLC product analysis, this study establishes that all three pathways of AA metabolism are present in microsomal incubates of the neural lobe of the pituitary gland. Addition of PGE2 to incubated fragments of neural lobes of the rat pituitary stimulates secretion of both arginine vasopressin (AVP) and oxytocin in vitro. Inclusion of 5-HETE and 12-HETE in the incubation medium stimulates marginal release of AVP and oxytocin by 12-HETE only. The magnitude of AVP and oxytocin secretion stimulated by the epoxygenase metabolites 8,9-, 11,12-, and 14,15-EET is equal to that caused by PGE2. Maximal stimulation of secretion (3- to 4-fold) requires an EET concentration 10-15 times greater than that of PGE2. In contrast, 5,6-EET is inactive. These data suggest that oxygenated products of AA play a role in AVP and oxytocin secretion. Although PGs appear to be the dominant arachidonate metabolites involved in the release of AVP and oxytocin, the EETs probably have a contributing role.
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ABSTRACT
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20-hydroxyeicosatetraenoic acid (20-HETE) has been shown to positively correlate with body mass index, hyperglycemia and plasma insulin levels. This study seeks to identify a causal relationship between 20-HETE and obesity-driven insulin... more
20-hydroxyeicosatetraenoic acid (20-HETE) has been shown to positively correlate with body mass index, hyperglycemia and plasma insulin levels. This study seeks to identify a causal relationship between 20-HETE and obesity-driven insulin resistance. Cyp4a14 male mice, a model of 20-HETE overproduction, were fed a regular or high-fat diet (HFD) for 15 weeks. 20-SOLA, a 20-HETE antagonist, was administered from week 0 or week 7 of HFD. HFD-fed mice gained significant weight (16.7±3.2 vs. 3.8±0.35 g, p<0.05) and developed hyperglycemia (157±3 vs 121±7 mg/dl, p<0.05) and hyperinsulinemia (2.3±0.4 vs 0.5±0.1 ng/ml, p<0.05) compared to regular diet-fed mice. 20-SOLA attenuated HFD-induced weight gain (9.4±1 vs 16.7±3 g, p<0.05), and normalized the hyperglycemia (157±7 vs 102±5 mg/dl, p<0.05) and hyperinsulinemia (1.1±0.1 vs 2.3±0.4 ng/ml, p<0.05). The impaired glucose homeostasis and insulin resistance in HFD-fed mice evidenced by reduced insulin and glucose tolerance we...
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Increased expression of cytochrome P450 CYP2C9, together with elevated levels of its products epoxyeicosatrienoic acids (EETs), is associated with aggressiveness in cancer. Cytochrome P450 variants CYP2C9*2 and CYP2C9*3 encode proteins... more
Increased expression of cytochrome P450 CYP2C9, together with elevated levels of its products epoxyeicosatrienoic acids (EETs), is associated with aggressiveness in cancer. Cytochrome P450 variants CYP2C9*2 and CYP2C9*3 encode proteins with reduced enzymatic activity, and individuals carrying these variants metabolize drugs more slowly than individuals with wild-type CYP2C9*1, potentially impacting their response to drugs and altering their risk of disease. Although genetic differences in CYP2C9-dependent oxidation of arachidonic acid (AA) have been reported, the roles of CYP2C9*2 and CYP2C9*3 in EET biosynthesis and their relevance to disease are unknown. Here we report that CYP2C9*2 and CYP2C9*3 metabolize AA less efficiently than CYP2C9*1 and that they play a role in the progression of non-small cell lung cancer (NSCLC) via impaired EET biosynthesis. When injected into mice, NSCLC cells expressing CYP2C9*2 and CYP2C9*3 produced lower levels of EETs and developed fewer, smaller, a...
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We have recently demonstrated that disruption of the murine cytochrome P450 2c44 gene exacerbates chronic hypoxia-induced pulmonary artery remodeling and hypertension in mice. Subsequently, we serendipitously found that Cyp2c44 gene... more
We have recently demonstrated that disruption of the murine cytochrome P450 2c44 gene exacerbates chronic hypoxia-induced pulmonary artery remodeling and hypertension in mice. Subsequently, we serendipitously found that Cyp2c44 gene disruption also increases hematopoietic stem cell (HSC) number in bone marrow and blood. Therefore, the objective of this study was to investigate whether Cyp2c44 disruption regulates HSC phenotype and whether increases in differentiated HSCs contribute to chronic hypoxia-induced remodeling of pulmonary arteries. Our findings demonstrated that lack of a CYP2C44 epoxygenase, which produces epoxyeicosatrienoic acids and hydroxyeicosatetraenoic acids, increases: 1] HSC (CD34(+), CD117(+), and CD133(+)) numbers, 2] proangiogenic (CD34(+)CD133(+), CD34(+)CD117+CD133(+)) cells, and 3] immunogenic/inflammatory (CD34(+)CD11b(+), CD133(+)CD11b(+), F4/80(+), CD11b(+), and F4/80(+)CD11b(+)) monocytes and macrophages, in bone morrow and blood as compared to wild typ...
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Insulin resistance is frequently associated with hypertension and type 2 diabetes. The cytochrome P450 (CYP) arachidonic acid epoxygenases (CYP2C, CYP2J) and their epoxyeicosatrienoic acid (EET) products lower blood pressure and may also... more
Insulin resistance is frequently associated with hypertension and type 2 diabetes. The cytochrome P450 (CYP) arachidonic acid epoxygenases (CYP2C, CYP2J) and their epoxyeicosatrienoic acid (EET) products lower blood pressure and may also improve glucose homeostasis. However, the direct contribution of endogenous EET production on insulin sensitivity has not been previously investigated. In this study, we tested the hypothesis that endogenous CYP2C-derived EETs alter insulin sensitivity by analysing mice lacking CYP2C44, a major EET producing enzyme, and by testing the association of plasma EETs with insulin sensitivity in humans. We assessed insulin sensitivity in wild-type (WT) and Cyp2c44 (-/-) mice using hyperinsulinaemic-euglycaemic clamps and isolated skeletal muscle. Insulin secretory function was assessed using hyperglycaemic clamps and isolated islets. Vascular function was tested in isolated perfused mesenteric vessels. Insulin sensitivity and secretion were assessed in hum...
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Rationale: 20-Hydroxyeicosatetraenoic acid (20-HETE), one of the principle cytochrome P450 (CYP) eicosanoids, is a potent vasoactive lipid whose vascular effects include stimulation of smooth muscle contractility, migration and... more
Rationale: 20-Hydroxyeicosatetraenoic acid (20-HETE), one of the principle cytochrome P450 (CYP) eicosanoids, is a potent vasoactive lipid whose vascular effects include stimulation of smooth muscle contractility, migration and proliferation, as well as endothelial cell dysfunction and inflammation. Increased levels of 20-HETE in experimental animals and in humans are associated with hypertension, stroke, myocardial infarction and vascular diseases. Objective: To date, a receptor/binding site for 20-HETE has been implicated based on the use of specific agonists and antagonists. The present study was undertaken to identify a receptor to which 20-HETE binds and through which it activates a signaling cascade that culminates in many of the functional outcomes attributed to 20-HETE in vitro and in vivo. Methods and Results: Using crosslinking analogs, click chemistry, binding assays, and functional assays, we identified GPR75, currently an orphan G-protein coupled receptor (GPCR), as a s...
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... 136146,1981 The Metabolism of Benzo[a]pyrene Phenols by Rat Liver Microsomal Fractions RUSSELL A. PROUGH,1 YUKI SAEKI, AND JORGE CAPDEVILA Department of ... 4. THAKKER, DR, YAGI, H., Lu, AYH, LEVIN, W., CONNEY, AH, AND JERINA, DM... more
... 136146,1981 The Metabolism of Benzo[a]pyrene Phenols by Rat Liver Microsomal Fractions RUSSELL A. PROUGH,1 YUKI SAEKI, AND JORGE CAPDEVILA Department of ... 4. THAKKER, DR, YAGI, H., Lu, AYH, LEVIN, W., CONNEY, AH, AND JERINA, DM (1976) Proc. Nat. ...
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Male and female homozygous 129/Sv mice carrying four copies of the human cytochrome P450 4A11 gene (CYP4A11) under control of its native promoter (B-129/Sv-4A11+/+) develop hypertension (142±8 versus 113±7 mm Hg systolic blood pressure... more
Male and female homozygous 129/Sv mice carrying four copies of the human cytochrome P450 4A11 gene (CYP4A11) under control of its native promoter (B-129/Sv-4A11+/+) develop hypertension (142±8 versus 113±7 mm Hg systolic blood pressure (BP)), and exhibit increased 20-hydroxyeicosatetraenoic acid (20-HETE) in kidney and urine. The hypertension is reversible by a low-sodium diet and by the CYP4A inhibitor HET0016. B-129/Sv-4A11+/+ mice display an 18% increase of plasma potassium (p<0.02), but plasma aldosterone, angiotensin II (ANGII), and renin activities are unchanged. This phenotype resembles human genetic disorders with elevated activity of the sodium chloride co-transporter (NCC) and, accordingly, NCC abundance is increased by 50% in transgenic mice, and NCC levels are normalized by HET0016. ANGII is known to increase NCC abundance, and renal mRNA levels of its precursor angiotensinogen are increased 2-fold in B-129/Sv-4A11+/+, and blockade of the ANGII receptor type 1 with lo...
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... Christine A. Moustakis, Jacques Viala,2 Jorge Capdevila, and J. R. Falck* ... by silylation (t-BuPh,SiCI, KH, THF, 12h) and hydrolysis in AcOH/THF/H,O (5:2:2, 60-65 OC, 2-2.5 h). Condensation of 4 with the ylide (3.3 equiv) derived... more
... Christine A. Moustakis, Jacques Viala,2 Jorge Capdevila, and J. R. Falck* ... by silylation (t-BuPh,SiCI, KH, THF, 12h) and hydrolysis in AcOH/THF/H,O (5:2:2, 60-65 OC, 2-2.5 h). Condensation of 4 with the ylide (3.3 equiv) derived from [ 10-carbomethoxydeca-(Z,Z)-3,6-dien-...