RESUMEN
Studies of the cytochrome P450 arachidonic acid (AA) monooxygenase, now established as a major pathway for the bioactivation of this physiological important fatty acid, have uncovered new and important roles for this enzyme system in the regulation of kidney function, including renal hemodynamics and tubular ion transport. Associations between genetically controlled alterations in blood pressure and the activity and/or transcriptional regulation of the kidney Cyp2c AA epoxygenases and Cyp4a omega-hydroxylases revealed a role for these enzymes in the pathophysiology of hypertension, a leading cause of cardiovascular, cerebral, and renal morbidity and mortality. Furthermore, analysis of associations between genetic variants of human CYP4A11 and hypertension suggest a potential role for this gene as a determinant of polygenic blood pressure control in humans. These results are providing conceptually novel approaches for studies of the molecular basis of human hypertension that could lead to new strategies for the early diagnosis and clinical management of this devastating disease.
Asunto(s)
Ácido Araquidónico/metabolismo , Presión Sanguínea/fisiología , Sistema Enzimático del Citocromo P-450/metabolismo , Hipertensión/metabolismo , Hipertensión/fisiopatología , Animales , Sistema Enzimático del Citocromo P-450/genética , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones MutantesRESUMEN
The demonstration of a role for microsomal P450 in the metabolism of endogenous pools of arachidonic acid established this enzyme system as a member of the arachidonic acid cascade and characterized a new an important metabolic function for this enzyme system. Studies from several laboratories documenting the powerful biological activities of the P450-derived eicosanoids have suggested important roles for the P450 arachidonic acid monooxygenase in renal and vascular physiology, and in the pathophysiology of experimental hypertension. These studies provide significant evidence to indicate that in addition to its recognized traditional toxicological and pharmacological roles, microsomal P450s also play important physiological roles in the control of tissue and body homeostasis.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Eicosanoides/metabolismo , Microsomas/enzimología , Animales , Homeostasis , Humanos , Microsomas Hepáticos/enzimología , NADP/metabolismo , Oxidación-ReducciónRESUMEN
The studies of the cytochrome P450 (P450) arachidonic acid (AA) monooxygenase, now established as a major pathway for the bioactivation of AA, have uncovered new and important functional roles for this enzyme system in cell and organ physiology, and in the metabolism of endogenous substrate. Past and present advances in P450 biochemistry and molecular biology are beginning to provide a description of the P450 isoform specificity of AA bioactivation, and the mechanisms of action and physiological relevance of the P450 metabolites. Associations between genetically controlled alterations in P450 function, expression, or regulation and functionally meaningful phenotypes point to the critical roles played by the AA monooxygenase in the control of systemic blood pressure and the pathophysiology of hypertension.
Asunto(s)
Ácido Araquidónico/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Hipertensión/fisiopatología , Transducción de Señal , Animales , Presión Sanguínea , Compuestos Epoxi/metabolismo , Humanos , Isoenzimas/metabolismo , Oxigenasas de Función Mixta/metabolismo , Oxidación-Reducción , Especificidad por SustratoRESUMEN
In the kidney, epoxyeicosatrienoic acids (EETs) have been suggested to be endothelium-derived hyperpolarizing factors (EDHFs). The aim of the present study was to determine the contribution of EETs to the preglomerular vasodilation elicited by bradykinin. Sprague-Dawley rats were studied utilizing an in vitro perfused juxtamedullary nephron preparation. The afferent arteriolar diameter was determined and the diameter averaged 19 +/- 1 microm (n = 26) at a renal perfusion pressure of 100 mm Hg. Addition of 1, 10 and 100 nM bradykinin to the perfusate dose-dependently increased afferent arteriolar diameter by 5 +/- 1, 12 +/- 2 and 17 +/- 2%, respectively. The nitric oxide inhibitor N(omega)-nitro-L-arginine reduced bradykinin-induced afferent arteriolar vasodilation by 50%, and the diameter increased by 9 +/- 2% in response to 100 nM bradykinin. Epoxygenase inhibitors N-methylsulphonyl-6-(2-propargyloxyphenyl)hexanamide or miconazole greatly attenuated the nitric oxide-independent component of the vasodilation elicited by bradykinin. Cyclooxygenase (COX) inhibition attenuated the nitric oxide-independent vasodilation elicited by 1 nM bradykinin but did not significantly affect the vascular response to 100 nM bradykinin. Combined inhibition of nitric oxide, COX and epoxygenase pathways completely abolished bradykinin-mediated afferent arteriolar vasodilation. In additional studies, renal microvessels were isolated and incubated with bradykinin and samples were analyzed by NICI/GC/MS. Under control conditions, renal microvascular EET levels averaged 49 +/- 9 pg/mg/20 min (n = 7). In the presence of bradykinin, EET levels were significantly higher and averaged 81 +/- 11 pg/mg/20 min (n = 7). These data support the concept that EETs are EDHFs and contribute to the nitric oxide-independent afferent arteriolar vasodilation elicited by bradykinin.
Asunto(s)
Arteriolas/fisiología , Bradiquinina/farmacología , Sistema Enzimático del Citocromo P-450/fisiología , Riñón/irrigación sanguínea , Óxido Nítrico/fisiología , Oxigenasas/fisiología , Vasodilatación/efectos de los fármacos , Ácido 8,11,14-Eicosatrienoico/sangre , Amidas/farmacología , Animales , Arteriolas/efectos de los fármacos , Técnicas de Cultivo , Inhibidores de la Ciclooxigenasa/farmacología , Citocromo P-450 CYP2J2 , Inhibidores Enzimáticos del Citocromo P-450 , Inhibidores Enzimáticos/farmacología , Indometacina/farmacología , Riñón/enzimología , Cinética , Masculino , Microcirculación/efectos de los fármacos , Nitroarginina/farmacología , Oxigenasas/antagonistas & inhibidores , Prostaglandina-Endoperóxido Sintasas/fisiología , Ratas , Ratas Sprague-Dawley , Circulación Renal/efectos de los fármacosRESUMEN
OBJECTIVES: Epoxygenase metabolites produced by the kidney affect renal blood flow and tubular transport function and 11,12-epoxyeicosatrienoic acid (11,12-EET) has been putatively identified as an endothelium-derived hyperpolarizing factor. The current studies were performed to determine the influence of 11,12-EET on the regulation of afferent arteriolar diameter in angiotensin II-infused hypertensive rats. MATERIALS AND METHODS: Male Sprague-Dawley rats received angiotensin II (60 ng/min) or vehicle via an osmotic minipump. Angiotensin II-infused hypertensive and vehicle-infused normotensive rats were studied for 2 weeks following implantation of the minipump. Renal microvascular responses to the sulfonimide analog of 11,12-EET (11,12-EET-SI) and angiotensin II were observed utilizing the in-vitro juxtamedullary nephron preparation. Renal cortical epoxygenase enzyme protein levels were quantified by Western blot analysis. Renal microvessels were also isolated and epoxygenase metabolite levels measured by negative ion chemical ionization (NICI)/gas chromatography-mass spectroscopy. RESULTS: Systolic blood pressure averaged 118 +/- 2 mmHg prior to pump implantation and increased to 185 +/- 7 mmHg in rats infused with angiotensin II for 2 weeks. Afferent arteriolar diameters of 2-week normotensive animals averaged 22 +/- 1 microm. Diameters of the afferent arterioles were 17% smaller in hypertensive rats (P< 0.05); however, arterioles from both groups responded to 11,12-EET-SI (100 nmol) with similar 15-17% increases in diameter. As we previously demonstrated, the afferent arteriolar reactivity to angiotensin II was enhanced in angiotensin II-infused animals. Interestingly, elevation of 11,12-EET-SI levels to 100 nmol reversed the enhanced vascular reactivity to angiotensin II associated with angiotensin II hypertension. Renal microvascular EET levels were not different between groups and averaged 81 +/- 9 and 87 +/- 13 pg/mg per 30 min in normotensive and hypertensive animals, respectively. Renal cortical microsomal levels of the epoxygenase CYP2C23 and CYP2C11 proteins were also similar in normotensive and angiotensin II hypertensive rats. CONCLUSIONS: Taken together, these data support the concept that renal microvascular 11,12-EET activity and levels may not properly offset the enhanced angiotensin II renal vasoconstriction during angiotensin II hypertension.
Asunto(s)
Angiotensina II/farmacología , Circulación Renal/efectos de los fármacos , Sulfonamidas/farmacología , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/sangre , Ácido 8,11,14-Eicosatrienoico/metabolismo , Animales , Arteriolas/fisiopatología , Citocromo P-450 CYP2J2 , Sistema Enzimático del Citocromo P-450/metabolismo , Sinergismo Farmacológico , Hipertensión/inducido químicamente , Hipertensión/fisiopatología , Corteza Renal/metabolismo , Masculino , Microcirculación/efectos de los fármacos , Microsomas/metabolismo , Ratas , Ratas Sprague-Dawley , Sulfonamidas/sangre , VasoconstricciónRESUMEN
The determinants of the regio- and stereoselective oxidation of fatty acids by cytochrome P450 BM-3 were examined by mutagenesis of residues postulated to anchor the fatty acid or to determine its active site substrate-accessible volume. R47, Y51, and F87 were targeted separately and in combination in order to assess their contributions to arachidonic, palmitoleic, and lauric acid binding affinities, catalytic rates, and regio- and stereoselective oxidation. For all three fatty acids, mutation of the anchoring residues decreased substrate binding affinity and catalytic rates and, for lauric acid, caused a significant increase in the enzyme's NADPH oxidase activity. These changes in catalytic efficiency were accompanied by decreases in the regioselectivity of oxygen insertion, suggesting an increased freedom of substrate movement within the active site of the mutant proteins. The formation of significant amounts of 19-hydroxy AA by the Y51A mutant and of 11,12-EET by the R47A/Y51A/F87V triple mutant, suggest that wild-type BM-3 shields these carbon atoms from the heme bound reactive oxygen by restricting the freedom of AA displacement along the substrate channel, and active site accessibility. These results indicate that binding affinity and catalytic turnover are fatty acid carbon-chain length dependent, and that the catalytic efficiency and the regioselectivity of fatty acid metabolism by BM-3 are determined by active site binding coordinates that control acceptor carbon orientation and proximity to the heme iron.
Asunto(s)
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Proteínas Bacterianas , Dominio Catalítico , Sistema Enzimático del Citocromo P-450/metabolismo , Ácidos Grasos/metabolismo , Oxigenasas de Función Mixta/metabolismo , Ácido 8,11,14-Eicosatrienoico/metabolismo , Ácido Araquidónico/metabolismo , Dominio Catalítico/genética , Sistema Enzimático del Citocromo P-450/genética , Ácidos Grasos Monoinsaturados/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Ácidos Láuricos/metabolismo , Oxigenasas de Función Mixta/genética , Mutagénesis Sitio-Dirigida , NADPH Oxidasas/metabolismo , NADPH-Ferrihemoproteína Reductasa , Oxidación-Reducción , Estereoisomerismo , Especificidad por SustratoRESUMEN
Hypertension is a leading cause of cardiovascular, cerebral, and renal disease morbidity and mortality. Here we show that disruption of the Cyp 4a14 gene causes hypertension, which is, like most human hypertension, more severe in males. Male Cyp 4a14 (-/-) mice show increases in plasma androgens, kidney Cyp 4a12 expression, and the formation of prohypertensive 20-hydroxyarachidonate. Castration normalizes the blood pressure of Cyp 4a14 (-/-) mice and minimizes Cyp 4a12 expression and arachidonate omega-hydroxylation. Androgen replacement restores hypertensive phenotype, Cyp 4a12 expression, and 20-hydroxy-arachidonate formation. We conclude that the androgen-mediated regulation of Cyp 4a arachidonate monooxygenases is an important component of the renal mechanisms that control systemic blood pressures. These results provide direct evidence for a role of Cyp 4a isoforms in cardiovascular physiology, establish Cyp 4a14 (-/-) mice as a monogenic model for the study of cause/effect relationships between blood pressure, sex hormones, and P450 omega-hydroxylases, and suggest the human CYP 4A homologues as candidate genes for the analysis of the genetic and molecular basis of human hypertension.
Asunto(s)
Andrógenos/farmacología , Sistema Enzimático del Citocromo P-450/metabolismo , Hipertensión/enzimología , Oxigenasas de Función Mixta/metabolismo , Andrógenos/sangre , Animales , Ácido Araquidónico/metabolismo , Presión Sanguínea , Castración , Citocromo P-450 CYP4A , Sistema Enzimático del Citocromo P-450/genética , Dihidrotestosterona/farmacología , Inducción Enzimática/efectos de los fármacos , Femenino , Eliminación de Gen , Ácidos Hidroxieicosatetraenoicos/metabolismo , Hipertensión/inducido químicamente , Hipertensión/genética , Riñón/irrigación sanguínea , Riñón/efectos de los fármacos , Riñón/enzimología , Riñón/metabolismo , Masculino , Ratones , Ratones Noqueados , Microsomas/efectos de los fármacos , Microsomas/enzimología , Oxigenasas de Función Mixta/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Circulación Renal/fisiología , Caracteres Sexuales , Testosterona/farmacología , Resistencia VascularRESUMEN
Practical, asymmetric total syntheses of the title phospholipids from a readily available myo-inositol derivative as well as short chain and cross-linkable aminoether analogues are described.
Asunto(s)
Fosfatidilinositoles/síntesis química , Fosfatidilinositoles/química , FosforilaciónRESUMEN
The function of cytochrome P450 (P450) in the mammalian brain is not well understood. In an effort to further this understanding, this study identifies two endogenous substrates for P450 2D18. Previous reports have shown that this isoform is expressed in the rat brain, and the recombinant enzyme catalyzes the N-demethylation of the antidepressants imipramine and desipramine. By further examining the substrate profile of P450 2D18, inferences can be made as to potential endogenous P450 substrates. Herein we demonstrate the metabolism of the central nervous system-acting compounds chlorpromazine and chlorzoxazone with turnover numbers of 1.8 and 0. 9 nmol/min/nmol, respectively. Because the four aforementioned pharmaceutical substrates work by binding to neurotransmitter receptors, binding assays and oxidation reactions were performed to test whether dopamine is a substrate for P450 2D18. These data indicate a K(S) value of 678 microM and that P450 2D18 can support the oxidation of dopamine to aminochrome through a peroxide-shunt mechanism. We also report the P450 2D18-mediated omega-hydroxylation and epoxygenation of arachidonic acid, primarily leading to the formation of 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids, compounds that have been shown to have vasoactive properties in brain, kidney, and heart tissues. The data presented herein suggest a possible role for P450 involvement in membrane and receptor regulation via epoxyeicosatrienoic acid formation and a potential involvement of P450 in the oxidation of dopamine to reactive oxygen species under aberrant physiological conditions where the sequestering of dopamine becomes compromised, such as in Parkinson's disease.
Asunto(s)
Ácido Araquidónico/química , Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/química , Dopamina/química , Indolquinonas , Animales , Antipsicóticos/química , Ácido Araquidónico/metabolismo , Encéfalo/metabolismo , Clorpromazina/química , Clorzoxazona/química , Cromatografía Líquida de Alta Presión , Sistema Enzimático del Citocromo P-450/metabolismo , Glutatión/química , Hidroxilación , Técnicas In Vitro , Indoles/análisis , Cinética , Peróxidos Lipídicos/química , Relajantes Musculares Centrales/química , Oxidación-Reducción , Unión Proteica , Ratas , Proteínas Recombinantes/químicaAsunto(s)
Ácido Araquidónico/metabolismo , Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/metabolismo , Eicosanoides/metabolismo , Oxigenasas de Función Mixta , Oxigenasas/química , Oxigenasas/metabolismo , Animales , Sistema Enzimático del Citocromo P-450/genética , Familia 2 del Citocromo P450 , Hemoproteínas , Humanos , Conformación Molecular , NADP , Oxigenasas/genética , RatasRESUMEN
cDNAs coding for rat P450 2C11 fused to either a bacterial (the NADPH-cytochrome P450 BM3 reductase domain of P450 BM3) or a truncated form of rat NADPH-P450 reductases were expressed in Escherichia coli and characterized enzymatically. Measurements of NADPH cytochrome c reductase activity showed fusion-dependent increases in the rates of cytochrome c reduction by the bacterial or the mammalian flavoprotein (21 and 48%, respectively, of the rates observed with nonfused enzymes). Neither the bacterial flavoprotein nor the truncated rat reductase supported arachidonic acid metabolism by P450 2C11. In contrast, fusion of P450 2C11 to either reductase yielded proteins that metabolized arachidonic acid to products similar to those obtained with reconstituted systems containing P450 2C11 and native rat P450 reductase. Addition of a 10-fold molar excess of rat P450 reductase markedly increased the rates of metabolism by both fused and nonfused P450s 2C11. These increases occurred with preservation of the regioselectivity of arachidonic acid metabolism. The fusion-independent reduction of P450 2C11 by bacterial P450 BM3 reductase was shown by measurements of NADPH-dependent H(2)O(2) formation [73 +/- 10 and 10 +/- 1 nmol of H(2)O(2) formed min(-)(1) (nmol of P450)(-)(1) for the reconstituted and fused protein systems, respectively]. These studies demonstrate that (a) a self-sufficient, catalytically active arachidonate epoxygenase can be constructed by fusing P450 2C11 to mammalian or bacterial P450 reductases and (b) the P450 BM3 reductase interacts efficiently with mammalian P450 2C11 and catalyzes the reduction of the heme iron. However, fusion is required for metabolism and product formation.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/genética , NADH NADPH Oxidorreductasas/química , NADH NADPH Oxidorreductasas/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Esteroide 16-alfa-Hidroxilasa , Esteroide Hidroxilasas/química , Esteroide Hidroxilasas/genética , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Catálisis , Sistema Enzimático del Citocromo P-450/metabolismo , Escherichia coli , NADH NADPH Oxidorreductasas/metabolismo , NADPH-Ferrihemoproteína Reductasa , Ingeniería de Proteínas , Pliegue de Proteína , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Esteroide Hidroxilasas/metabolismoRESUMEN
The demonstration of in vivo arachidonic acid epoxidation and omega-hydroxylation established the cytochrome P450 epoxygenase and omega/omega-1 hydroxylase as formal metabolic pathways and as members of the arachidonate metabolic cascade. The characterization of the potent biological activities associated with several of the cytochrome P450-derived eicosanoids suggested new and important functional roles for these enzymes in cellular, organ, and body physiology, including the control of vascular reactivity and systemic blood pressures. Past and current advances in cytochrome P450 biochemistry and molecular biology facilitate the characterization of cytochrome P450 isoforms responsible for tissue/organ specific arachidonic acid epoxidation and omega/omega-1 hydroxylation, and thus, the analysis of cDNA and/or gene specific functional phenotypes. The combined application of physiological, biochemical, molecular, and genetic approaches is beginning to provide new insights into the physiological and/or pathophysiological significance of these enzymes, their endogenous substrates, and products.
Asunto(s)
Ácido Araquidónico/metabolismo , Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/metabolismo , Oxigenasas de Función Mixta , Oxigenasas/metabolismo , Animales , Citocromo P-450 CYP2J2 , Sistema Enzimático del Citocromo P-450/genética , Familia 2 del Citocromo P450 , Modelos Animales de Enfermedad , Eicosanoides/metabolismo , Humanos , Hipertensión/etiología , Hipertensión/metabolismo , NADP/metabolismo , Oxigenasas/genética , Especificidad por SustratoRESUMEN
Excess dietary salt intake induces the activity of the kidney arachidonate epoxygenase and markedly increases the urinary excretion of its metabolites. The epoxyeicosatrienoic acids, products of the kidney P-450 arachidonate epoxygenase, inhibit distal nephron Na(+) reabsorption. Nucleic acid hybridization studies demonstrated the expression of P-450s 2C23, 2C24, and 2C11 as the predominant kidney 2C isoforms and the lack of significant dietary salt-dependent transcriptional regulation of these proteins. Recombinant P-450s 2C11, 2C23, and 2C24 catalyze arachidonate metabolism to mixtures of epoxy- and monohydroxylated acids. Whereas the arachidonate 11,12-olefin was the preferred target for epoxidation by P-450 2C23 (57% of total products), P-450s 2C11 and 2C24 epoxidized the 11,12-olefins and 14,15-olefins with nearly equal efficiency. Stereochemical comparisons demonstrated that the regiochemical and enantiofacial selectivity of P-450 2C23 matched that of the kidney microsomal epoxygenase and that excess dietary salt does not alter the regiochemical or stereochemical selectivity of the kidney arachidonate epoxygenase. Inhibition and immunoelectrophoresis experiments using antibodies raised against recombinant P-450s 2C11 and 2C23 demonstrated that P-450 2C23 is the major 2C arachidonic acid epoxygenase in the rat kidney and the renal P-450 isoform regulated by excess dietary salt intake.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/biosíntesis , Riñón/enzimología , Oxigenasas/biosíntesis , Cloruro de Sodio Dietético/farmacología , Esteroide 16-alfa-Hidroxilasa , Animales , Ácido Araquidónico/metabolismo , Clonación Molecular , Citocromo P-450 CYP2J2 , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Masculino , Oxigenasas/genética , Oxigenasas/aislamiento & purificación , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Esteroide Hidroxilasas/biosíntesis , Regulación hacia ArribaRESUMEN
Epoxyeicosatrienoic acids (EETs), products of the cytochrome P-450 monooxygenase metabolism of arachidonic acid, can regulate the activity of ion channels. We examined the effects of EETs on cardiac L-type Ca2+ channels that play important roles in regulating cardiac contractility, controlling heart rate, and mediating slow conduction in normal nodal cells and ischemic myocardium. Our experimental approach was to reconstitute porcine L-type Ca2+ channels into planar lipid bilayers where we could control the aqueous and lipid environments of the channels and the regulatory pathways that change channel properties. We found that 20 to 125 nM EETs inhibited the open probability of reconstituted L-type Ca2+ channels, accelerated the inactivation of the channels, and reduced the unitary current amplitude of open channels. There was no selectivity among different EET regioisomers or stereoisomers. When 11,12-EET was esterified to the sn-2 position of phosphatidylcholine, restricting it to the hydrophobic phase of the planar lipid bilayer, the reconstituted channels were similarly inhibited, suggesting that the EET interacts directly with Ca2+ channels through the lipid phase. The inhibitory effects of EET persisted in the presence of microcystin, an inhibitor of protein phosphatases 1 and 2A, suggesting that dephosphorylation was not the mechanism through which these eicosanoids down-regulate channel activity. This inhibition may be an important protective mechanism in the setting of cardiac ischemia where arachidonic acid levels are dramatically increased and EETs have been shown to manifest preconditioning-like effects.
Asunto(s)
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Canales de Calcio/efectos de los fármacos , Miocardio/metabolismo , Ácido 8,11,14-Eicosatrienoico/química , Ácido 8,11,14-Eicosatrienoico/farmacología , Animales , Canales de Calcio/metabolismo , Relación Dosis-Respuesta a Droga , Conductividad Eléctrica , Inhibidores Enzimáticos/farmacología , Membrana Dobles de Lípidos/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Microcistinas , Péptidos Cíclicos/farmacología , Fosfatidilcolinas/química , Fosfatidilcolinas/farmacología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , PorcinosRESUMEN
The novel cytochrome P450, CYP2B19, is a specific cellular marker of late differentiation in skin keratinocytes. CYP2B19 was discovered in fetal mouse skin where its onset of expression coincides spatially (upper cell layer) and temporally (day 15.5) with the appearance of loricrin-expressing keratinocytes during the stratification stage of fetal epidermis. CYP2B19 is also present postnatally in the differentiated keratinocytes of the epidermis, sebaceous glands, and hair follicles. CYP2B19 mRNA is tightly coupled to the differentiated (granular cell) keratinocyte phenotype in vivo and in vitro. In primary mouse epidermal keratinocytes, it is specifically up-regulated and correlated temporally with calcium-induced differentiation and expression of the late differentiation genes loricrin and profilaggrin. Recombinant CYP2B19 metabolizes arachidonic acid and generates 14,15- and 11, 12-epoxyeicosatrienoic (EET) acids, and 11-, 12-, and 15-hydroxyeicosatetraenoic (HETE) acids (20, 35, 18, 7, and 7% of total metabolites, respectively). Arachidonic acid metabolism was stereoselective for 11S,12R- and 14S,15R-EET, and 11S-, 12R-, and 15R-HETE. The CYP2B19 metabolites 11,12- and 14,15-EET are endogenous constituents of murine epidermis and are present in similar proportions to that generated by the enzyme in vitro, suggesting that CYP2B19 might be the primary enzymatic source of these EETs in murine epidermis.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Regulación Enzimológica de la Expresión Génica , Queratinocitos/enzimología , Oxigenasas de Función Mixta , Oxigenasas/genética , Transcripción Genética , Secuencia de Aminoácidos , Animales , Ácidos Araquidónicos/metabolismo , Secuencia de Bases , Biomarcadores , Diferenciación Celular , Células Cultivadas , Familia 2 del Citocromo P450 , Células Epidérmicas , Epidermis/enzimología , Feto , Cabello/citología , Cabello/enzimología , Queratinocitos/citología , Queratinas/genética , Proteínas de la Membrana/genética , Ratones , Datos de Secuencia Molecular , Oxigenasas/química , Oxigenasas/metabolismo , ARN Mensajero/genética , Ratas , Glándulas Sebáceas/citología , Glándulas Sebáceas/enzimología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Piel/citología , Piel/enzimología , Especificidad por SustratoRESUMEN
The cDNAs encoding cytochromes CYP 4A2 and 4A3 were cloned by RT-PCR amplification of male rat kidney and liver RNAs, respectively. Sequence analysis demonstrated that these cDNAs were nearly identical to the published sequences for CYPs 4A2 and 4A3. CYP 4A2 and 4A3 share extensive sequence homology that extends into their 3'- and 5'-untranslated segments ( approximately 97% overall nucleotide identity). Analysis of cDNA and genomic DNA sequences shows that a sequence of 123 bp, recognized as an intron during the processing of CYP 4A2 transcripts, is conserved in the 4A3 mRNAs and that these otherwise highly homologous genes show different exon-intron distributions. The CYP 4A2 and 4A3 cDNAs were expressed in a baculovirus-insect cell expression system. Purified recombinant CYP 4A2 oxidized arachidonic acid to a mixture of 19- and 20-hydroxyeicosatetraenoic acids (20 and 80% of the total products, respectively). Reaction rates were maximal when CYP 4A2 was reconstituted in the presence of an equimolar concentration of cytochrome b5 and a 10-fold molar excess of NADPH-cytochrome P450 reductase. Studies using microsomal fractions isolated from noninfected insect cells and from cells infected with CYP 4A3 recombinant baculoviruses showed (a) the presence of an endogenous lauric acid omega-hydroxylase and arachidonic acid epoxygenase in the noninfected cells, (b) the CYP 4A3-dependent oxidation of lauric acid to 11- and 12-hydroxylaurate (24 and 76% of the total products, respectively), and (c) the lack of arachidonic acid metabolism by microsomal recombinant CYP 4A3. Nucleic acid hybridization and immunoelectrophoresis studies demonstrated that (a) CYP 4A2 transcripts are abundantly expressed in the female kidney and that CYP 4A3 is expressed in female but not in male liver, (b) anti-CYP 4A2 immunoreactive material was detected only in the male kidney, (c) male and female livers or kidneys support only low levels of CYP 4A3 translation, and (d) excess dietary salt does not alter the kidney levels of mRNA transcripts encoding CYP 4A1, 4A2, or 4A3 or change the levels of microsomal anti-4A1 or -4A2 immunoreactive proteins. Finally, no significant differences were observed between Dahl salt resistant or Dahl salt sensitive rats in the levels and/or salt regulation of mRNA transcripts enecoding CYP 4A1, 4A2, or 4A3 or the in levels of the corresponding proteins.
Asunto(s)
Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/genética , Riñón/enzimología , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Citocromo P-450 CYP4A , Sistema Enzimático del Citocromo P-450/metabolismo , ADN Complementario/aislamiento & purificación , Femenino , Regulación de la Expresión Génica , Hipertensión/enzimología , Hipertensión/genética , Masculino , Oxigenasas de Función Mixta/metabolismo , Datos de Secuencia Molecular , Especificidad de Órganos/genética , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Compuestos de Estaño/efectos adversos , Transcripción GenéticaRESUMEN
Cytochrome P450 BM-3 catalyzes the high turnover regio- and stereoselective metabolism of arachidonic and eicosapentaenoic acids. To map structural determinants of productive active site fatty acid binding, we mutated two amino acid residues, arginine 47 and phenylalanine 87, which flank the surface and heme ends of the enzyme's substrate access channel, respectively. Replacement of arginine 47 with glutamic acid resulted in a catalytically inactive mutant. Replacement of arginine 47 with alanine yielded a protein with reduced substrate binding affinity and arachidonate sp3 carbon hydroxylation activity (72% of control wild type). On the other hand, arachidonic and eicosapentaenoic acid epoxidation was significantly enhanced (154 and 137%, of control wild type, respectively). As with wild type, the alanine 47 mutant generated (18R)-hydroxyeicosatetraenoic, (14S,15R)-epoxyeicosatrienoic, and (17S,18R)-epoxyeicosatetraenoic acids nearly enantiomerically pure. Replacement of phenylalanine 87 with valine converted cytochrome P450 BM-3 into a regio- and stereoselective arachidonic acid epoxygenase ((14S,15R)-epoxyeicosatrienoic acid, 99% of total products). Conversely, metabolism of eicosapentaenoic acid by the valine 87 mutant yielded a mixture of (14S,15R)- and (17S,18R)-epoxyeicosatetraenoic acids (26 and 69% of total, 94 and 96% optical purity, respectively). Finally, replacement of phenylalanine 87 with tyrosine yielded an inactive protein. We propose that: (a) fatty acid oxidation by P450 BM-3 is incompatible with the presence of residues with negatively charged side chains at the surface opening of the substrate access channel or a polar aromatic side chain in the vicinity of the heme iron; (b) the high turnover regio- and stereoselective metabolism of arachidonic and eicosapentaenoic acids involves charge-dependent anchoring of the fatty acids at the mouth of the access channel by arginine 47, as well as steric gating of the heme-bound oxidant by phenylalanine 87; and (c) substrate binding coordinates, as opposed to oxygen chemistries, are the determining factors responsible for reaction rates, product chemistry, and, thus, catalytic outcome.
Asunto(s)
Proteínas Bacterianas , Sistema Enzimático del Citocromo P-450/metabolismo , Oxigenasas de Función Mixta/metabolismo , Oxigenasas/metabolismo , Animales , Sitios de Unión , Citocromo P-450 CYP2J2 , Cromatografía de Gases y Espectrometría de Masas , Modelos Moleculares , Mutagénesis Sitio-Dirigida , NADP/metabolismo , NADPH-Ferrihemoproteína Reductasa , Fenilalanina , Estereoisomerismo , ValinaRESUMEN
As a participant of the endogenous arachidonic acid metabolic cascade, microsomal cytochrome P450 metabolizes the fatty acid to biologically active hydroxyeicosatetraenoic and epoxyeicosatrienoic acids. Studies from several laboratories have documented the powerful vasoactive properties of these P450-derived eicosanoids. Associated changes in cell membrane ion permeability and fluxes may provide the molecular basis underlining their vasoactivity. Furthermore, a role for the P450 arachidonic acid monooxygenase in renal physiology and pathophysiology has been suggested by: 1) an association between the activities of the arachidonic acid omega/omega-1 oxygenase and the development of hypertension in spontaneously hypertensive rats, and 2) a relationship between acquired or inherited abnormalities in the renal epoxygenase activities and/or regulation and salt-sensitive hypertension in Dahl rats. These studies provide significant evidence to indicate that microsomal P450, in addition to its recognized traditional toxicological and pharmacological roles, may also play an important physiological role in the control of tissue and body homeostasis.