Armando Karara

Experimental and/or genetically controlled alterations of the renal microsomal cytochrome P450 epoxygenase induce hypertension in rats fed a high salt diet.

Excess dietary salt induces a cytochrome P450 arachidonic acid epoxygenase isoform in rat kidneys (Capdevila, J. H., S. Wei, J. Yang, A. Karara, H. R. Jacobson, J. R. Falck, F. P. Guengerich, and R. N. Dubois. 1992. J. Biol. Chem. 267:21720-21726). Treatment of rats on a high salt diet with the epoxygenase inhibitor, clotrimazole, produces significant increases in mean arterial blood pressure (122 +/- 2 and 145 +/- 4 mmHg for salt and salt- and clotrimazole-treated rats, respectively). The salt- and clotrimazole-dependent hypertension is accompanied by reductions in the urinary excretion of epoxygenase metabolites and by a selective inhibition of the renal microsomal epoxygenase reaction. The prohypertensive effects of clotrimazole are readily reversed when either the salt or clotrimazole treatment is discontinued. The indication that a salt-inducible renal epoxygenase protects against hypertension, are supported by studies with the Dahl rat model of genetic salt-sensitive hypertension. Dahl resistant animals responded to excess dietary salt by inducing the activity of their kidney microsomal epoxygenase(s) (0.102 +/- 0.01 and 0.240 +/- 0.04 nmol of products formed/min per mg of microsomal protein for control and salt-treated rats, respectively). Despite severe hypertension during excess dietary salt intake (200 +/- 20 mmHg), Dahl salt-sensitive rats demonstrated no increase in renal epoxygenase activity. These studies indicate that acquired or inherited abnormalities in renal epoxygenase activities and/or regulation can be related to salt-sensitive hypertension in rodents. Studies on the human renal epoxygenase and its relationship to salt hypertension may prove useful.

Arachidonic acid epoxygenase: Structural characterization and quantification of epoxyeicosatrienoates in plasma

Gas chromatographic/mass spectroscopic and chiral analysis showed the presence of enzymatically derived 8,9-, 11,12- and 14,15-EET in rat plasma (2.8:1:3.4 molar ratio, respectively; 10.2 +/- 0.4 ng total EET/ml plasma). Greater than 90% of the plasma EETs was esterified to the phospholipids of circulating lipoproteins. The lipoprotein fraction with the highest EET concentration was LDL (8.1 +/- 0.9 ng/mg of protein) followed by HDL and VLDL (3.5 +/- 0.1 and 1.9 +/- 0.3 ng/mg of protein, respectively). In light of the biological activities of the EETs, these results suggest a potential systemic function for the cytochrome P-450 epoxygenase.

Cytochrome P-450 arachidonate oxygenase.

Publisher Summary The chapter presents a study on cytochrome P -450 arachidonate oxygenase. Cytochrome P -450, the oxygen-activating component of the microsomal electron transport system, is an efficient catalyst for the oxygenated metabolism of arachidonic acid (AA). The chapter mentions that the reaction has an absolute requirement for NADPH and states the procedure of their stoichiometric relationship. The chapter describes methodology utilized in laboratory for the biochemical characterization of the hepatic microsomal AA cytochrome P -450 oxidase as well as its reconstitution utilizing solubilized and purified components of the microsomal electron transport system. In addition to the biochemical advantages of working with a defined system, reconstituted systems are excellent tools for the study of the regioselectivity of AA oxidation by different cytochrome P -450 enzymes. The chapter includes experiment that illustrates the reconstitution of AA oxygenase, utilizing either the major phenobarbital-inducible form of rat liver microsomal cytochrome P -450 or, alternatively, the major ciprofibrate- inducible form. It is shown by the radiochromatograms that the phenobarbital-inducible enzyme is an active epoxygenase, generating four regioisomeric EETs in an enantioselective manner. On the other hand, the ciprofibrate-inducible form catalyzes, almost exclusively, the ω-oxidation of the fatty acid to form 20-hydroxyeicosatetraenoic acid.

Cytochrome P450 arachidonic acid epoxygenase: Stereochemical characterization of epoxyeicosatrienoic acids

Publisher Summary This chapter discusses the cytochrome P450 arachidonic acid epoxygenase. The epoxygenase regio- and stereoselectivity of oxygenation is under the control of the cytochrome P450 protein catalyst. The potential functional significance of this metabolic pathway has been highlighted by (a) the potent biological activities of the epoxyeicosatrienoic acids (EETs), and (b) the demonstration of a role for the hemoprotein in the in vivo metabolism of endogenous arachidonic acid. The enantioselective nature of the endogenous EET pools present in rat liver and human kidney established the epoxygenase as an additional member of the “arachidonate metabolic cascade.” This chapter discusses the methodology developed in laboratories for the regio- and stereochemical characterization of the cytochrome P450 epoxygenase metabolites generated in vitro by enzymatic incubations or present in vivo in extracts isolated from whole organs.

Arachidonic acid epoxygenase Stereochemical analysis of the endogenous epoxyeicosatrienoic acids of human kidney cortex

Mass spectral and Chromatographic analysis demonstrates the presence of 14,15‐, 11,12‐ and 8,9‐epoxyeicosatrienoic acids (44%, 33% and 23% of the total, respectively) in human kidney cortex. Chiral analysis of the human renal expoxyeicosatrienoic acids shows the formation of 8,9‐, 11,12‐ and 14,15‐epoxyeicosatrienoic acids in a 1:1, 4:1 and 2:1 ratio of antipodes, respectively. These results demonstrate the biosynthetic origin of the human kidney 11,12‐ and 14,15‐epoxyeicosatrienoic acids and suggest a role for renal cytochrome P‐450 in the bioactivation of endogenous pools of arachidonic acid.

Cytochrome P450 and the Metabolism of Arachidonic Acid and Oxygenated Eicosanoids

Eukaryotic cells contain substantial amounts of arachidonic acid (AA; 5,8,11,14-eicosatetraenoic acid) esterified predominantly to the sn-2 position of cellular glycerophospholipids. As with many lipid-derived mediators, e.g., cholesterol, phosphoinositides, diglycerides, AA serves a structural role, as a component of cellular membranes, and an important functional role, as a participant in a variety of receptor/agonist-mediated signaling cascades.1–4 In the absence of stimuli, the intracellular levels of nonesterified AA are nearly undetectable. However, most organ cells possess an elaborate enzymatic machinery that, in response to a variety of stimuli, catalyzes: (1) the hydrolytic cleavage of the AA molecule form selected, hormonally sensitive phospholipid pools, (2) the transduction of chemical information into the fatty acid molecular template by means of regio- and stereospecific oxygenation reactions, and (3) the decoding of that chemical information either by receptor-mediated processes or, alternatively, by the direct effects of these oxygenated metabolites on metabolic pathways1–4 (Fig. 1). As a net result, these processes provide cells with a rapid and versatile on/off molecular switch for the intra- or intercellular transduction and/or amplification of functionally meaningful information.

Palladium mediated allylic Mitsunobu displacement : stereocontrolled synthesis of hepoxilin A3 and trioxilin A3 methyl esters

A regio- and stereoselective palladium mediated allylic displacement under Mitsunobu conditions was exploited for the preparation of several hepoxilin A3 and trioxilin A3 stereoisomers.

Enantiospecific synthesis of 17- and 18-hydroxyeicosatetraenoic acids, cytochrome P450 arachidonate metabolites

Abstract The title bioactive eicosanoids were prepared from dimethyl L -malate by a convergent strategy exploiting the differential reactivity of ethereal dialkylcuprates towards tosylates versus bromides.

Total synthesis of 5(S),12(S)- and 5(S),12(R)-dihydroxyeicosa-6(Z),8(E),14(Z)-trienoic acids, metabolites of leukotriene B4

Abstract The recently identified dihydro-leukotriene B 4 metabolite 1 and its C(12)-epi analogue 2 were prepared by Wittig coupling of segments derived from 2-deoxy- D -ribose and L -glutamic acid.

Eicosanoid Metabolism and Bioactivation by Microsomal Cytochrome P450

Publisher Summary This chapter discusses the eicosanoid metabolism and bioactivation by microsomal cytochrome P450. Microsomal cytochrome P450 participates in the metabolic transformation of several eicosanoids by catalyzing both, nicotinamide adenine dinucleotide phosphate (NADPH)-independent and NADPH-dependent reactions. This distinction reflects the marked differences in types of oxygen chemistry involved in these reactions—that is, the NADPH dependent activation of atmospheric oxygen or the NADPH-independent isomerization of arachidonic acid hydroperoxides. Microsomal P450 oxidizes a variety of eicosanoids that, in addition to arachidonic acid, includes prostanoids and leukotrienes. For the most part, these reactions result in the hydroxylation of the eicosanoid ultimate or penultimate carbon atoms. While it has been generally accepted that these reactions result in an attenuation of the biological activities of their substrates and that they may be important in eicosanoid catabolism, recent studies have indicated that some ω/ω-1 oxidized prostanoids have shown unique and potent biological properties.