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Ingrid Fleming - One of the best experts on this subject based on the ideXlab platform.
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Role of Cytochrome P450 2C epoxygenases in hypoxia-induced cell migration and angiogenesis in retinal endothelial cells.
Investigative ophthalmology & visual science, 2008Co-Authors: U. Ruth Michaelis, Ning Xia, Eduardo Barbosa-sicard, John R. Falck, Ingrid FlemingAbstract:PURPOSE. Cytochrome P450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) elicit cell proliferation and promote angiogenesis. The aim of this study was to determine the expression of CYP epoxygenases in the bovine retina and the potential role of EETs in hypoxia-induced angiogenesis in bovine retinal endothelial cells. METHODS. Bovine retinal endothelial cells were cultured under normoxic (21% O 2 ) or hypoxic (1% O 2 ) conditions, and CYP2C expression was determined by Western blot analysis. The effect of hypoxia on EET levels was determined by LC-MS/MS. Cell migration (Transwell filter assays) and endothelial cell tube formation (on basement membrane matrix) were assessed in vitro in the absence and presence of pharmacologic inhibitors and CYP2C antisense oligonucleotides. RESULTS. Bovine retinal endothelial cells expressed CYP2C protein in culture and generated detectable levels of EETs under basal conditions. Hypoxia (6 - 48 hours) enhanced CYP2C protein expression (2-fold) and EET formation (1.5-fold). Moreover, endothelial cells preexposed to hypoxia demonstrated an increase in serum-induced cell migration that was sensitive to the CYP2C inhibitors sulfaphenazole and MS-PPOH and the EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid. Furthermore, preventing the hypoxia-induced expression of CYP2C (antisense oligonucleotides) suppressed hypoxia-induced cell migration. In an in vitro angiogenesis model, the preexposure of endothelial cells to hypoxia increased CYP2C expression and enhanced endothelial tube formation, which was blocked by the EET antagonist and by the CYP2C antisense oligonucleotides. CONCLUSIONS. Taken together, these data indicate that CYP2C-derived EETs are implicated in angiogenesis by retinal endothelial cells, especially under hypoxic conditions.
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Cytochrome P450 2C expression and EDHF-mediated relaxation in porcine coronary arteries is increased by cortisol.
Cardiovascular research, 2002Co-Authors: Johann Bauersachs, Beate Fisslthaler, Rudi Busse, Michael Christ, Georg Ertl, U.r. Michaelis, Ingrid FlemingAbstract:Objectives/methods: In addition to nitric oxide (NO) and prostacyclin, endothelium-dependent dilation is mediated by the endothelium-derived hyperpolarizing factor (EDHF) which, in the coronary circulation, has been characterised as a metabolite of arachidonic acid synthesised by an Cytochrome P450 (CYP) epoxygenase homologous to CYP 2C8/9. As the promotor regions of CYP 2C8 and 2C9 contain consensus sequences for glucocorticoid response elements, we determined the effect of cortisol on EDHF-mediated relaxations as well as on the expression of CYP 2C in isolated segments of porcine coronary artery. Results: Bradykinin-induced NO-mediated relaxation of KCl-constricted arterial rings was slightly attenuated following exposure to cortisol. However, EDHF-mediated relaxations of U46619-constricted arterial rings assessed in the presence of the cyclo-oxygenase inhibitor diclofenac and the NO synthase inhibitor Nωnitro-l-arginine (0.3 mM), were significantly enhanced (maximum relaxation: 66±7%, P
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Cytochrome P450 2C expression and edhf mediated relaxation in porcine coronary arteries is increased by cortisol
Cardiovascular Research, 2002Co-Authors: Johann Bauersachs, Beate Fisslthaler, Rudi Busse, Michael Christ, Georg Ertl, U.r. Michaelis, Ingrid FlemingAbstract:Objectives/methods: In addition to nitric oxide (NO) and prostacyclin, endothelium-dependent dilation is mediated by the endothelium-derived hyperpolarizing factor (EDHF) which, in the coronary circulation, has been characterised as a metabolite of arachidonic acid synthesised by an Cytochrome P450 (CYP) epoxygenase homologous to CYP 2C8/9. As the promotor regions of CYP 2C8 and 2C9 contain consensus sequences for glucocorticoid response elements, we determined the effect of cortisol on EDHF-mediated relaxations as well as on the expression of CYP 2C in isolated segments of porcine coronary artery. Results: Bradykinin-induced NO-mediated relaxation of KCl-constricted arterial rings was slightly attenuated following exposure to cortisol. However, EDHF-mediated relaxations of U46619-constricted arterial rings assessed in the presence of the cyclo-oxygenase inhibitor diclofenac and the NO synthase inhibitor Nωnitro-l-arginine (0.3 mM), were significantly enhanced (maximum relaxation: 66±7%, P <0.05 vs. control rings: 36±8%). Cortisol treatment (0.1 μM, 24 h) did not affect the endothelium-independent relaxation elicited by sodium nitroprusside and acute incubation with cortisol (0.1 μM, 30 min) did not alter either NO- or EDHF-mediated responses. The expression of CYP 2C (quantified by RT-PCR, Western blot analysis and confocal microscopy) was enhanced in porcine coronary endothelial cells following incubation with cortisol for 18–24 h. Conclusions: These results demonstrate the concomitant upregulation of EDHF-mediated relaxations and CYP 2C expression by long-term treatment with cortisol. These observations support the concept that an epoxygenase homologous to CYP 2C8/9 plays a crucial role in the generation of EDHF-mediated responses in the coronary endothelium.
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Cytochrome P450 2C is an EDHF synthase in coronary arteries.
Trends in cardiovascular medicine, 2000Co-Authors: Ingrid FlemingAbstract:Studies designed to elucidate the identity of the potent vasodilator autacoid endothelium-derived hyperpolarizing factor (EDHF) in the coronary vascular bed have highlighted a role for vascular Cytochrome P450 (CYP) enzymes in cardiovascular homeostasis. Not only is there strong evidence suggesting that the putative coronary EDHF synthase is an endothelially expressed CYP epoxygenase, but CYP products such as epoxyeicosatrienoic acids and reactive oxygen species have been implicated in the regulation of intracellular signalling cascades and vascular cell proliferation.
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Cytochrome P450 2C is an EDHF synthase in coronary arteries
Nature, 1999Co-Authors: Beate Fisslthaler, Ingrid Fleming, Rüdiger Popp, Ladislau Kiss, Michael Potente, David R. Harder, Rudi BusseAbstract:In most arterial beds a significant endothelium-dependent dilation to various stimuli persists even after inhibition of nitric oxide synthase and cyclo-oxygenase. This dilator response is preceded by an endothelium-dependent hyperpolarization of vascular smooth muscle cells, which is sensitive to a combination of the calcium-dependent potassium-channel inhibitors charybdotoxin and apamin, and is assumed to be mediated by an unidentified endothelium-derived hyperpolarizing factor (EDHF). Here we show that the induction of Cytochrome P450 (CYP) 2C8/34 in native porcine coronary artery endothelial cells by beta-naphthoflavone enhances the formation of 11,12-epoxyeicosatrienoic acid, as well as EDHF-mediated hyperpolarization and relaxation. Transfection of coronary arteries with CYP 2C8/34 antisense oligonucleotides results in decreased levels of CYP 2C and attenuates EDHF-mediated vascular responses. Thus, a CYP-epoxygenase product is an essential component of EDHF-mediated relaxation in the porcine coronary artery, and CYP 2C8/34 fulfils the criteria for the coronary EDHF synthase.
Matthew E. Wilson - One of the best experts on this subject based on the ideXlab platform.
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Effect of Cytochrome P450 and aldo-keto reductase inhibitors on progesterone inactivation in primary bovine hepatic cell cultures
Journal of Dairy Science, 2010Co-Authors: C O Lemley, Matthew E. WilsonAbstract:Abstract Progesterone is required for maintenance of pregnancy, and peripheral concentrations of progesterone are affected by both production and inactivation. Hepatic Cytochrome P450 (EC 1.14.14.1) and aldo-keto reductase (EC 1.1.1.145–151) enzymes play a pivotal role in the first step of steroid inactivation, which involves the addition of hydroxyl groups to various sites of the cyclopentanoperhydrophenanthrene nucleus. The current objective was to discern the proportional involvement of hepatic progesterone inactivating enzymes on progesterone decay using specific enzyme inhibitors. Ticlopidine, diltiazem, curcumin, dicumarol, and naproxen were used because of their selective inhibition of Cytochrome P450s, aldo-keto reductases, and glucuronosyltransferases. Liver biopsies were collected from 6 lactating Holstein dairy cows, and cells were dissociated using a nonperfusion technique. Confluent wells were preincubated for 4h with enzyme inhibitor and then challenged with progesterone for 1h. Cell viability was unaffected by inhibitor treatment and averaged 84±1%. In control wells, 50% of the progesterone had been inactivated after a 1-h challenge with 5ng/mL of progesterone. Preincubation with curcumin, ticlopidine, or naproxen caused the greatest reduction in progesterone inactivation compared with controls and averaged 77, 39, or 37%, respectively. Hydroxylation of 4-nitrophenol to 4-nitrocatechol in intact cells was inhibited by approximately 65% after treatment with curcumin or ticlopidine. Glucuronidation of phenol red or 4-nitrocatechol in intact cells was inhibited by treatment with curcumin, dicumarol, or naproxen. In cytoplasmic preparations, aldo-keto reductase 1C activity was inhibited by curcumin, dicumarol, or naproxen treatment. Microsomal Cytochrome P450 2C activity was inhibited by treatment with curcumin or ticlopidine, whereas Cytochrome P450 3A activity was inhibited by treatment with curcumin or diltiazem. The contribution of Cytochrome P450 2C and Cytochrome P450 3A enzymes to progesterone inactivation in bovine hepatic cell cultures was 40 and 15%, respectively. Depending on the inhibitor used, it would appear that the aldo-keto reductase enzymes contribute approximately 40% to the observed progesterone inactivation, although a portion of this inactivation may be attributed to the loss of glucuronosyltransferase activity. Future work focusing on decreasing the activity of these enzymes in vivo could lead to an increase in the bioavailability of progesterone.
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Diet-induced alterations in hepatic progesterone (P4) catabolic enzyme activity and P4 clearance rate in lactating dairy cows
The Journal of endocrinology, 2010Co-Authors: C O Lemley, K. M. Krause, L.r. Tager, Kimberly A. Vonnahme, Matthew E. WilsonAbstract:Elevated rates of steroid clearance may lead to lower reproductive success in several mammalian species. Cytochrome P450 (EC 1.14.14.1) and aldo-keto reductases (AKR; EC 1.1.1.145–151) are involved in the first phase of steroid inactivation, before second phase conjugation and excretion of the steroid metabolite. The current objectives were to determine liver blood flow (LBF), hepatic enzyme activity, and metabolic clearance rate (MCR) of progesterone (P4) in dairy cows consuming isoenergetic and isonitrogenous diets formulated to cause divergent insulin secretion. Insulin concentrations increased by 22% in cows fed the high cornstarch diet, and both Cytochrome P450 2C and Cytochrome P450 3A activities were decreased (P!0.05) by w50%, while AKR1C tended (P!0.10) to be lower in
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Effect of a high cornstarch diet on hepatic Cytochrome P450 2C and 3A activity and progesterone half-life in dairy cows.
Journal of dairy science, 2010Co-Authors: C O Lemley, K. M. Krause, T. A. Wilmoth, L.r. Tager, Matthew E. WilsonAbstract:In the cow, inadequate concentrations of progesterone during gestation may lead to an abrupt termination of pregnancy. The primary organ involved in progesterone catabolism is the liver, which contains an abundance of Cytochrome P450 isozymes (EC 1.14.14.1; mixed-function monooxygenases). The objectives of the current experiment were to determine the effect of feeding 2 isoenergetic and isonitrogenous diets, formulated to cause divergent insulin secretion, on hepatic Cytochrome P450 2C (CYP2C) and 3A (CYP3A) activity as well as the resulting biological half-life of progesterone. Twenty-two Holstein cows averaging 80 ± 7 d in milk were randomly assigned to either a high cornstarch diet or a high fiber diet in a crossover experimental design consisting of two 14-d periods. Dry matter intake, milk yield, milk lactose yield, and milk lactose percentage were similar between the 2 diets. Milk fat yield and milk fat percentage were higher in cows fed the high fiber diet, whereas milk protein yield tended to be higher and milk protein percentage was higher in cows fed the high cornstarch diet. Energy balance tended to be improved by 57% in cows consuming the high cornstarch diet. Insulin concentrations at the time of liver biopsy (3.16 ± 0.04 h post-feeding) were increased by 44% in cows consuming the high cornstarch diet compared with cows consuming the high fiber diet. Cytochrome P450 2C activity was decreased by 45%, whereas CYP3A activity tended to be lowered by 34% in cows consuming the high cornstarch diet. Cytochrome P450 2C mRNA expression tended to be decreased by 21% in cows fed the high cornstarch diet, whereas CYP3A mRNA expression was not different between the dietary treatments. The fractional rate constant of progesterone decay was not different between the 2 diets; however, the half-life of progesterone tended to be longer in cows fed the high cornstarch diet compared with cows fed the high fiber diet (85 vs. 64 min, respectively). In summary, cows consuming the high cornstarch diet had increased insulin concentrations and lower hepatic CYP2C and CYP3A activity and tended to have a longer progesterone half-life compared with cows consuming the high fiber diet. Feeding diets that stimulate insulin secretion could alter progesterone clearance during lactation, when dairy cows have increased rates of progesterone inactivation because of high energy demands and increased DMI.
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Concomitant changes in progesterone catabolic enzymes, Cytochrome P450 2C and 3A, with plasma insulin concentrations in ewes supplemented with sodium acetate or sodium propionate
Animal : an international journal of animal bioscience, 2008Co-Authors: C O Lemley, J. M. Koch, Kenneth P. Blemings, K. M. Krause, Matthew E. WilsonAbstract:Progesterone is essential for maintaining pregnancy, and several authors have suggested that low peripheral concentrations of progesterone may be responsible for high rates of embryonic loss. The primary organ involved in the catabolism of progesterone is the liver, and Cytochrome P450 2C and 3A sub-families account for a large proportion of this catabolism. Elucidating a mechanism to decrease progesterone catabolism, thereby increasing embryonic and uterine exposure to progesterone, seems a logical approach to ameliorate high rates of embryonic loss. The objectives of the current experiment were to determine the pattern of insulin secretion after supplementing feed with either sodium acetate or sodium propionate and to determine any association between the differential patterns of insulin secretion with the hepatic activity of Cytochrome P450 2C and 3A and progesterone clearance. Sixteen ovariectomized ewes were fed 3 kg/day for 10 days of a diet consisting of 50% corn silage, 38% triticale haylage, 12% soybean meal and 600 ml of 3.5 M sodium acetate (energy control; n = 8) or 2.0 M sodium propionate (gluconeogenic substrate; n = 8). Equal portions of the ration (1 kg as-fed basis along with 200 ml of 3.5 M sodium acetate or 2.0 M sodium propionate) were offered three times daily at 0600, 1400 and 2200 h. Concentrations of insulin in plasma were determined immediately before feeding and at 15, 30, 60, 90, 120, 180, 240 and 300 min after feeding. Progesterone clearance from peripheral circulation (ng/ml per min) was measured by giving a 5 mg injection of progesterone into the left jugular vein and collecting blood via the right jugular vein at 0, 2, 4, 6, 8, 10, 15, 20 and 30 min afterwards. Liver biopsies were taken 1 h after feeding to determine Cytochrome P450 2C and 3A activities. Insulin concentrations in ewes supplemented with sodium propionate were elevated at 15, 30 and 60 min after feeding compared to the sodium acetate group. Cytochrome P450 2C and 3A activities were decreased 1 h after feeding in the sodium propionate-treated ewes relative to sodium acetate. Insulin appears to down-regulate Cytochrome P450 activity, which could be used to decrease the catabolism of progesterone during early gestation, thereby increasing peripheral concentrations of progesterone and, consequently, embryonic exposure to progesterone.
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Short communication: insulin alters hepatic progesterone catabolic enzymes Cytochrome P450 2C and 3A in dairy cows.
Journal of dairy science, 2008Co-Authors: C O Lemley, Stephen T. Butler, W.r. Butler, Matthew E. WilsonAbstract:High proportions of embryonic and early fetal losses in dairy cattle are associated with low peripheral concentrations of progesterone, which could result from increased catabolism, decreased production, or both. Progesterone catabolism occurs primarily in the liver via the Cytochrome P450 2C (CYP2C) and Cytochrome P450 3A (CYP3A) subfamilies (EC 1.14.14.1; unspecific monooxygenases). Recent observations from our laboratory have shown that the fractional rate constant of progesterone decay can be dramatically reduced by insulin because of a decrease in hepatic CYP2C and CYP3A activity. Little information exists on the regulation of progesterone catabolic enzymes in dairy cows. We hypothesized that elevated insulin concentrations would down-regulate hepatic CYP2C and CYP3A mRNA; therefore, our objectives were to determine the relative abundance of hepatic CYP2C and CYP3A mRNA in dairy cows in response to elevated concentrations of insulin. In the first experiment, 17 mature Holstein cows were drenched daily with 500 mL of water (n = 10) or propylene glycol (a gluconeogenic substrate; n = 7) from 10 d before their expected calving date until d 25 postpartum. Cows drenched with propylene glycol had a 30% increase in peripheral concentrations of insulin. Liver biopsies were collected on d 25 postpartum to determine the relative abundance of CYP2C and CYP3A mRNA. In the second experiment, 19 mature, lactating Holstein cows were randomly assigned to a hyperinsulinemic-euglycemic clamp (0.3 or 1.0 microg of insulin/kg of BW per h; n = 6 each) or remained as controls (saline infused; n = 7) for 96 h beginning on d 10 postpartum. Insulin infusion resulted in a 2.6- or 8- fold increase in peripheral concentrations of insulin, respectively. On d 14 postpartum, a liver biopsy was collected to determine CYP2C and CYP3A mRNA abundance. In experiment 1, the relative abundance of CYP2C mRNA in cows treated with propylene glycol did not differ from controls; however, the relative abundance of CYP3A mRNA in the propylene glycol group was 63% that of controls. For experiment 2, there was a dose-dependent decrease in the relative abundance of both CYP2C and CYP3A mRNA with increasing dosage of insulin. In conclusion, this study demonstrated that, in the cow, either providing a gluconeogenic feed-stuff or treatment with insulin decreased the abundance of mRNA for enzymes responsible for hepatic progesterone catabolism.
C O Lemley - One of the best experts on this subject based on the ideXlab platform.
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Effect of Cytochrome P450 and aldo-keto reductase inhibitors on progesterone inactivation in primary bovine hepatic cell cultures
Journal of Dairy Science, 2010Co-Authors: C O Lemley, Matthew E. WilsonAbstract:Abstract Progesterone is required for maintenance of pregnancy, and peripheral concentrations of progesterone are affected by both production and inactivation. Hepatic Cytochrome P450 (EC 1.14.14.1) and aldo-keto reductase (EC 1.1.1.145–151) enzymes play a pivotal role in the first step of steroid inactivation, which involves the addition of hydroxyl groups to various sites of the cyclopentanoperhydrophenanthrene nucleus. The current objective was to discern the proportional involvement of hepatic progesterone inactivating enzymes on progesterone decay using specific enzyme inhibitors. Ticlopidine, diltiazem, curcumin, dicumarol, and naproxen were used because of their selective inhibition of Cytochrome P450s, aldo-keto reductases, and glucuronosyltransferases. Liver biopsies were collected from 6 lactating Holstein dairy cows, and cells were dissociated using a nonperfusion technique. Confluent wells were preincubated for 4h with enzyme inhibitor and then challenged with progesterone for 1h. Cell viability was unaffected by inhibitor treatment and averaged 84±1%. In control wells, 50% of the progesterone had been inactivated after a 1-h challenge with 5ng/mL of progesterone. Preincubation with curcumin, ticlopidine, or naproxen caused the greatest reduction in progesterone inactivation compared with controls and averaged 77, 39, or 37%, respectively. Hydroxylation of 4-nitrophenol to 4-nitrocatechol in intact cells was inhibited by approximately 65% after treatment with curcumin or ticlopidine. Glucuronidation of phenol red or 4-nitrocatechol in intact cells was inhibited by treatment with curcumin, dicumarol, or naproxen. In cytoplasmic preparations, aldo-keto reductase 1C activity was inhibited by curcumin, dicumarol, or naproxen treatment. Microsomal Cytochrome P450 2C activity was inhibited by treatment with curcumin or ticlopidine, whereas Cytochrome P450 3A activity was inhibited by treatment with curcumin or diltiazem. The contribution of Cytochrome P450 2C and Cytochrome P450 3A enzymes to progesterone inactivation in bovine hepatic cell cultures was 40 and 15%, respectively. Depending on the inhibitor used, it would appear that the aldo-keto reductase enzymes contribute approximately 40% to the observed progesterone inactivation, although a portion of this inactivation may be attributed to the loss of glucuronosyltransferase activity. Future work focusing on decreasing the activity of these enzymes in vivo could lead to an increase in the bioavailability of progesterone.
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Diet-induced alterations in hepatic progesterone (P4) catabolic enzyme activity and P4 clearance rate in lactating dairy cows
The Journal of endocrinology, 2010Co-Authors: C O Lemley, K. M. Krause, L.r. Tager, Kimberly A. Vonnahme, Matthew E. WilsonAbstract:Elevated rates of steroid clearance may lead to lower reproductive success in several mammalian species. Cytochrome P450 (EC 1.14.14.1) and aldo-keto reductases (AKR; EC 1.1.1.145–151) are involved in the first phase of steroid inactivation, before second phase conjugation and excretion of the steroid metabolite. The current objectives were to determine liver blood flow (LBF), hepatic enzyme activity, and metabolic clearance rate (MCR) of progesterone (P4) in dairy cows consuming isoenergetic and isonitrogenous diets formulated to cause divergent insulin secretion. Insulin concentrations increased by 22% in cows fed the high cornstarch diet, and both Cytochrome P450 2C and Cytochrome P450 3A activities were decreased (P!0.05) by w50%, while AKR1C tended (P!0.10) to be lower in
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Effect of a high cornstarch diet on hepatic Cytochrome P450 2C and 3A activity and progesterone half-life in dairy cows.
Journal of dairy science, 2010Co-Authors: C O Lemley, K. M. Krause, T. A. Wilmoth, L.r. Tager, Matthew E. WilsonAbstract:In the cow, inadequate concentrations of progesterone during gestation may lead to an abrupt termination of pregnancy. The primary organ involved in progesterone catabolism is the liver, which contains an abundance of Cytochrome P450 isozymes (EC 1.14.14.1; mixed-function monooxygenases). The objectives of the current experiment were to determine the effect of feeding 2 isoenergetic and isonitrogenous diets, formulated to cause divergent insulin secretion, on hepatic Cytochrome P450 2C (CYP2C) and 3A (CYP3A) activity as well as the resulting biological half-life of progesterone. Twenty-two Holstein cows averaging 80 ± 7 d in milk were randomly assigned to either a high cornstarch diet or a high fiber diet in a crossover experimental design consisting of two 14-d periods. Dry matter intake, milk yield, milk lactose yield, and milk lactose percentage were similar between the 2 diets. Milk fat yield and milk fat percentage were higher in cows fed the high fiber diet, whereas milk protein yield tended to be higher and milk protein percentage was higher in cows fed the high cornstarch diet. Energy balance tended to be improved by 57% in cows consuming the high cornstarch diet. Insulin concentrations at the time of liver biopsy (3.16 ± 0.04 h post-feeding) were increased by 44% in cows consuming the high cornstarch diet compared with cows consuming the high fiber diet. Cytochrome P450 2C activity was decreased by 45%, whereas CYP3A activity tended to be lowered by 34% in cows consuming the high cornstarch diet. Cytochrome P450 2C mRNA expression tended to be decreased by 21% in cows fed the high cornstarch diet, whereas CYP3A mRNA expression was not different between the dietary treatments. The fractional rate constant of progesterone decay was not different between the 2 diets; however, the half-life of progesterone tended to be longer in cows fed the high cornstarch diet compared with cows fed the high fiber diet (85 vs. 64 min, respectively). In summary, cows consuming the high cornstarch diet had increased insulin concentrations and lower hepatic CYP2C and CYP3A activity and tended to have a longer progesterone half-life compared with cows consuming the high fiber diet. Feeding diets that stimulate insulin secretion could alter progesterone clearance during lactation, when dairy cows have increased rates of progesterone inactivation because of high energy demands and increased DMI.
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Concomitant changes in progesterone catabolic enzymes, Cytochrome P450 2C and 3A, with plasma insulin concentrations in ewes supplemented with sodium acetate or sodium propionate
Animal : an international journal of animal bioscience, 2008Co-Authors: C O Lemley, J. M. Koch, Kenneth P. Blemings, K. M. Krause, Matthew E. WilsonAbstract:Progesterone is essential for maintaining pregnancy, and several authors have suggested that low peripheral concentrations of progesterone may be responsible for high rates of embryonic loss. The primary organ involved in the catabolism of progesterone is the liver, and Cytochrome P450 2C and 3A sub-families account for a large proportion of this catabolism. Elucidating a mechanism to decrease progesterone catabolism, thereby increasing embryonic and uterine exposure to progesterone, seems a logical approach to ameliorate high rates of embryonic loss. The objectives of the current experiment were to determine the pattern of insulin secretion after supplementing feed with either sodium acetate or sodium propionate and to determine any association between the differential patterns of insulin secretion with the hepatic activity of Cytochrome P450 2C and 3A and progesterone clearance. Sixteen ovariectomized ewes were fed 3 kg/day for 10 days of a diet consisting of 50% corn silage, 38% triticale haylage, 12% soybean meal and 600 ml of 3.5 M sodium acetate (energy control; n = 8) or 2.0 M sodium propionate (gluconeogenic substrate; n = 8). Equal portions of the ration (1 kg as-fed basis along with 200 ml of 3.5 M sodium acetate or 2.0 M sodium propionate) were offered three times daily at 0600, 1400 and 2200 h. Concentrations of insulin in plasma were determined immediately before feeding and at 15, 30, 60, 90, 120, 180, 240 and 300 min after feeding. Progesterone clearance from peripheral circulation (ng/ml per min) was measured by giving a 5 mg injection of progesterone into the left jugular vein and collecting blood via the right jugular vein at 0, 2, 4, 6, 8, 10, 15, 20 and 30 min afterwards. Liver biopsies were taken 1 h after feeding to determine Cytochrome P450 2C and 3A activities. Insulin concentrations in ewes supplemented with sodium propionate were elevated at 15, 30 and 60 min after feeding compared to the sodium acetate group. Cytochrome P450 2C and 3A activities were decreased 1 h after feeding in the sodium propionate-treated ewes relative to sodium acetate. Insulin appears to down-regulate Cytochrome P450 activity, which could be used to decrease the catabolism of progesterone during early gestation, thereby increasing peripheral concentrations of progesterone and, consequently, embryonic exposure to progesterone.
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Short communication: insulin alters hepatic progesterone catabolic enzymes Cytochrome P450 2C and 3A in dairy cows.
Journal of dairy science, 2008Co-Authors: C O Lemley, Stephen T. Butler, W.r. Butler, Matthew E. WilsonAbstract:High proportions of embryonic and early fetal losses in dairy cattle are associated with low peripheral concentrations of progesterone, which could result from increased catabolism, decreased production, or both. Progesterone catabolism occurs primarily in the liver via the Cytochrome P450 2C (CYP2C) and Cytochrome P450 3A (CYP3A) subfamilies (EC 1.14.14.1; unspecific monooxygenases). Recent observations from our laboratory have shown that the fractional rate constant of progesterone decay can be dramatically reduced by insulin because of a decrease in hepatic CYP2C and CYP3A activity. Little information exists on the regulation of progesterone catabolic enzymes in dairy cows. We hypothesized that elevated insulin concentrations would down-regulate hepatic CYP2C and CYP3A mRNA; therefore, our objectives were to determine the relative abundance of hepatic CYP2C and CYP3A mRNA in dairy cows in response to elevated concentrations of insulin. In the first experiment, 17 mature Holstein cows were drenched daily with 500 mL of water (n = 10) or propylene glycol (a gluconeogenic substrate; n = 7) from 10 d before their expected calving date until d 25 postpartum. Cows drenched with propylene glycol had a 30% increase in peripheral concentrations of insulin. Liver biopsies were collected on d 25 postpartum to determine the relative abundance of CYP2C and CYP3A mRNA. In the second experiment, 19 mature, lactating Holstein cows were randomly assigned to a hyperinsulinemic-euglycemic clamp (0.3 or 1.0 microg of insulin/kg of BW per h; n = 6 each) or remained as controls (saline infused; n = 7) for 96 h beginning on d 10 postpartum. Insulin infusion resulted in a 2.6- or 8- fold increase in peripheral concentrations of insulin, respectively. On d 14 postpartum, a liver biopsy was collected to determine CYP2C and CYP3A mRNA abundance. In experiment 1, the relative abundance of CYP2C mRNA in cows treated with propylene glycol did not differ from controls; however, the relative abundance of CYP3A mRNA in the propylene glycol group was 63% that of controls. For experiment 2, there was a dose-dependent decrease in the relative abundance of both CYP2C and CYP3A mRNA with increasing dosage of insulin. In conclusion, this study demonstrated that, in the cow, either providing a gluconeogenic feed-stuff or treatment with insulin decreased the abundance of mRNA for enzymes responsible for hepatic progesterone catabolism.
Rudi Busse - One of the best experts on this subject based on the ideXlab platform.
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Cytochrome P450 2C expression and edhf mediated relaxation in porcine coronary arteries is increased by cortisol
Cardiovascular Research, 2002Co-Authors: Johann Bauersachs, Beate Fisslthaler, Rudi Busse, Michael Christ, Georg Ertl, U.r. Michaelis, Ingrid FlemingAbstract:Objectives/methods: In addition to nitric oxide (NO) and prostacyclin, endothelium-dependent dilation is mediated by the endothelium-derived hyperpolarizing factor (EDHF) which, in the coronary circulation, has been characterised as a metabolite of arachidonic acid synthesised by an Cytochrome P450 (CYP) epoxygenase homologous to CYP 2C8/9. As the promotor regions of CYP 2C8 and 2C9 contain consensus sequences for glucocorticoid response elements, we determined the effect of cortisol on EDHF-mediated relaxations as well as on the expression of CYP 2C in isolated segments of porcine coronary artery. Results: Bradykinin-induced NO-mediated relaxation of KCl-constricted arterial rings was slightly attenuated following exposure to cortisol. However, EDHF-mediated relaxations of U46619-constricted arterial rings assessed in the presence of the cyclo-oxygenase inhibitor diclofenac and the NO synthase inhibitor Nωnitro-l-arginine (0.3 mM), were significantly enhanced (maximum relaxation: 66±7%, P <0.05 vs. control rings: 36±8%). Cortisol treatment (0.1 μM, 24 h) did not affect the endothelium-independent relaxation elicited by sodium nitroprusside and acute incubation with cortisol (0.1 μM, 30 min) did not alter either NO- or EDHF-mediated responses. The expression of CYP 2C (quantified by RT-PCR, Western blot analysis and confocal microscopy) was enhanced in porcine coronary endothelial cells following incubation with cortisol for 18–24 h. Conclusions: These results demonstrate the concomitant upregulation of EDHF-mediated relaxations and CYP 2C expression by long-term treatment with cortisol. These observations support the concept that an epoxygenase homologous to CYP 2C8/9 plays a crucial role in the generation of EDHF-mediated responses in the coronary endothelium.
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Cytochrome P450 2C expression and EDHF-mediated relaxation in porcine coronary arteries is increased by cortisol.
Cardiovascular research, 2002Co-Authors: Johann Bauersachs, Beate Fisslthaler, Rudi Busse, Michael Christ, Georg Ertl, U.r. Michaelis, Ingrid FlemingAbstract:Objectives/methods: In addition to nitric oxide (NO) and prostacyclin, endothelium-dependent dilation is mediated by the endothelium-derived hyperpolarizing factor (EDHF) which, in the coronary circulation, has been characterised as a metabolite of arachidonic acid synthesised by an Cytochrome P450 (CYP) epoxygenase homologous to CYP 2C8/9. As the promotor regions of CYP 2C8 and 2C9 contain consensus sequences for glucocorticoid response elements, we determined the effect of cortisol on EDHF-mediated relaxations as well as on the expression of CYP 2C in isolated segments of porcine coronary artery. Results: Bradykinin-induced NO-mediated relaxation of KCl-constricted arterial rings was slightly attenuated following exposure to cortisol. However, EDHF-mediated relaxations of U46619-constricted arterial rings assessed in the presence of the cyclo-oxygenase inhibitor diclofenac and the NO synthase inhibitor Nωnitro-l-arginine (0.3 mM), were significantly enhanced (maximum relaxation: 66±7%, P
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Cytochrome P450 2C is an EDHF synthase in coronary arteries
Nature, 1999Co-Authors: Beate Fisslthaler, Ingrid Fleming, Rüdiger Popp, Ladislau Kiss, Michael Potente, David R. Harder, Rudi BusseAbstract:In most arterial beds a significant endothelium-dependent dilation to various stimuli persists even after inhibition of nitric oxide synthase and cyclo-oxygenase. This dilator response is preceded by an endothelium-dependent hyperpolarization of vascular smooth muscle cells, which is sensitive to a combination of the calcium-dependent potassium-channel inhibitors charybdotoxin and apamin, and is assumed to be mediated by an unidentified endothelium-derived hyperpolarizing factor (EDHF). Here we show that the induction of Cytochrome P450 (CYP) 2C8/34 in native porcine coronary artery endothelial cells by beta-naphthoflavone enhances the formation of 11,12-epoxyeicosatrienoic acid, as well as EDHF-mediated hyperpolarization and relaxation. Transfection of coronary arteries with CYP 2C8/34 antisense oligonucleotides results in decreased levels of CYP 2C and attenuates EDHF-mediated vascular responses. Thus, a CYP-epoxygenase product is an essential component of EDHF-mediated relaxation in the porcine coronary artery, and CYP 2C8/34 fulfils the criteria for the coronary EDHF synthase.
Beate Fisslthaler - One of the best experts on this subject based on the ideXlab platform.
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Cytochrome P450 2C expression and edhf mediated relaxation in porcine coronary arteries is increased by cortisol
Cardiovascular Research, 2002Co-Authors: Johann Bauersachs, Beate Fisslthaler, Rudi Busse, Michael Christ, Georg Ertl, U.r. Michaelis, Ingrid FlemingAbstract:Objectives/methods: In addition to nitric oxide (NO) and prostacyclin, endothelium-dependent dilation is mediated by the endothelium-derived hyperpolarizing factor (EDHF) which, in the coronary circulation, has been characterised as a metabolite of arachidonic acid synthesised by an Cytochrome P450 (CYP) epoxygenase homologous to CYP 2C8/9. As the promotor regions of CYP 2C8 and 2C9 contain consensus sequences for glucocorticoid response elements, we determined the effect of cortisol on EDHF-mediated relaxations as well as on the expression of CYP 2C in isolated segments of porcine coronary artery. Results: Bradykinin-induced NO-mediated relaxation of KCl-constricted arterial rings was slightly attenuated following exposure to cortisol. However, EDHF-mediated relaxations of U46619-constricted arterial rings assessed in the presence of the cyclo-oxygenase inhibitor diclofenac and the NO synthase inhibitor Nωnitro-l-arginine (0.3 mM), were significantly enhanced (maximum relaxation: 66±7%, P <0.05 vs. control rings: 36±8%). Cortisol treatment (0.1 μM, 24 h) did not affect the endothelium-independent relaxation elicited by sodium nitroprusside and acute incubation with cortisol (0.1 μM, 30 min) did not alter either NO- or EDHF-mediated responses. The expression of CYP 2C (quantified by RT-PCR, Western blot analysis and confocal microscopy) was enhanced in porcine coronary endothelial cells following incubation with cortisol for 18–24 h. Conclusions: These results demonstrate the concomitant upregulation of EDHF-mediated relaxations and CYP 2C expression by long-term treatment with cortisol. These observations support the concept that an epoxygenase homologous to CYP 2C8/9 plays a crucial role in the generation of EDHF-mediated responses in the coronary endothelium.
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Cytochrome P450 2C expression and EDHF-mediated relaxation in porcine coronary arteries is increased by cortisol.
Cardiovascular research, 2002Co-Authors: Johann Bauersachs, Beate Fisslthaler, Rudi Busse, Michael Christ, Georg Ertl, U.r. Michaelis, Ingrid FlemingAbstract:Objectives/methods: In addition to nitric oxide (NO) and prostacyclin, endothelium-dependent dilation is mediated by the endothelium-derived hyperpolarizing factor (EDHF) which, in the coronary circulation, has been characterised as a metabolite of arachidonic acid synthesised by an Cytochrome P450 (CYP) epoxygenase homologous to CYP 2C8/9. As the promotor regions of CYP 2C8 and 2C9 contain consensus sequences for glucocorticoid response elements, we determined the effect of cortisol on EDHF-mediated relaxations as well as on the expression of CYP 2C in isolated segments of porcine coronary artery. Results: Bradykinin-induced NO-mediated relaxation of KCl-constricted arterial rings was slightly attenuated following exposure to cortisol. However, EDHF-mediated relaxations of U46619-constricted arterial rings assessed in the presence of the cyclo-oxygenase inhibitor diclofenac and the NO synthase inhibitor Nωnitro-l-arginine (0.3 mM), were significantly enhanced (maximum relaxation: 66±7%, P
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Cytochrome P450 2C is an EDHF synthase in coronary arteries
Nature, 1999Co-Authors: Beate Fisslthaler, Ingrid Fleming, Rüdiger Popp, Ladislau Kiss, Michael Potente, David R. Harder, Rudi BusseAbstract:In most arterial beds a significant endothelium-dependent dilation to various stimuli persists even after inhibition of nitric oxide synthase and cyclo-oxygenase. This dilator response is preceded by an endothelium-dependent hyperpolarization of vascular smooth muscle cells, which is sensitive to a combination of the calcium-dependent potassium-channel inhibitors charybdotoxin and apamin, and is assumed to be mediated by an unidentified endothelium-derived hyperpolarizing factor (EDHF). Here we show that the induction of Cytochrome P450 (CYP) 2C8/34 in native porcine coronary artery endothelial cells by beta-naphthoflavone enhances the formation of 11,12-epoxyeicosatrienoic acid, as well as EDHF-mediated hyperpolarization and relaxation. Transfection of coronary arteries with CYP 2C8/34 antisense oligonucleotides results in decreased levels of CYP 2C and attenuates EDHF-mediated vascular responses. Thus, a CYP-epoxygenase product is an essential component of EDHF-mediated relaxation in the porcine coronary artery, and CYP 2C8/34 fulfils the criteria for the coronary EDHF synthase.
