Pendri Yadagiri

Inhibitors of cytochrome P-450-dependent arachidonic acid metabolism.

A new generation of heteroatom analogs of arachidonic acid are documented as powerful and selective inhibitors of the cytochrome P-450-dependent arachidonic acid oxygenase reaction (IC50, 5-10 microM) with little effect on either cyclooxygenase or soybean lipoxidase at 100 microM. The imidazole derivatives, ketoconazole and clotrimazole, are potent and selective inhibitors of the arachidonic acid epoxygenase and lipoxidase-like activities of phenobarbital-induced rat liver microsomal fractions (IC50, 2.0 and 0.3 microM, respectively). In contrast, the w/w-1 oxygenase activity of ciprofibrate-induced microsomal fractions was relatively resistant to inhibition by these compounds (IC50, 50 and 25 microM for ketoconazole and clotrimazole, respectively). Nordihydroguaiaretic acid (NDGA), eicosatetraynoic acid (ETYA), and indomethacin, extensively utilized inhibitors of the cyclooxygenase and lipoxygenase branches of the arachidonate cascade, also inhibit cytochrome P-450-dependent arachidonic acid metabolism. In decreasing order of potency, they were NDGA, ETYA, and indomethacin (IC50, 15, 40, and 70 microM, respectively).

Absolute configuration of the hydroxyeicosatetraenoic acids (HETEs) formed during catalytic oxygenation of arachidonic acid by microsomal cytochrome P-450

Rat liver microsomal cytochrome P-450 catalyzes the NADPH dependent metabolism of arachidonic acid to six regioisomeric cis,trans-dienols. The 12(R)-hydroxyeicosatetraenoic acid is generated with a high degree of enantioselectivity while the remaining regioisomers are produced as nearly racemic mixtures.

19(S)-hydroxyeicosatetraenoic acid is a potent stimulator of renal Na+-K+ATPase

Omega- and omega-1 hydroxylations are the major pathways by which arachidonic acid is metabolized in cortical and outer medullary microsomes of rat and rabbit kidneys. It is a cytochrome P450-dependent oxidation leading to the formation of 20-hydroxy- and 19-hydroxyeicosatetraenoic acids. In this study, we compared the effects of the synthetically prepared omega- and omega-1 metabolites of arachidonic acid on the activity of the renal Na+-K+-ATPase partially purified from rat renal cortical microsomes. 19(S)-hydroxyeicosatetraenoic acid caused a dose related stimulation of Na+-K+-ATPase activity with an EC50 of 3 x 10(-7) M. In contrast, neither 19(R)-hydroxyeicosatetraenoic acid, 20-hydroxyeicosatetraenoic acid nor arachidonic acid at 10(-6) M had any effect on Na+-K+-ATPase activity. In the same preparation, ouabain at 10(-3) M and 12(R)-hydroxyeicosatetraenoic acid at 10(-6) M inhibited the enzyme activity by 75% and 60%, respectively. We conclude that 19(S)-hydroxyeicosatetraenoic acid is a specific stimulator of renal Na+-K+-ATPase. Therefore, the formation of 19(S)-hydroxyeicosatetraenoic acid by renal cortical cytochrome P450 omega-1-hydroxylase may contribute to the regulation of renal function by regulating Na+-K+-ATPase which is essential for transtubular transport processes.

Synthesis of epoxyeicosatrienoic acids and heteroatom analogs.

Publisher Summary The chapter presents a study on synthesis of epoxyeicosatrienoic acids and heteroatom analogs. The chapter mentions the need for a comprehensive review of the current status of the epoxygenase pathway with a critical discussion of its implications. The most characteristic and extensively studied epoxygenase metabolites are the four regioisomeric cis -epoxyeicosatrienoic acids (EETs). They are relatively stable to hydrolysis at physiological pH [for example, leukotriene A 4 (LTA 4 ) and hepoxilin A 3 ]. The exception is 5,6-EET which, in its free-acid form, readily decomposes to 5,6-dihydroxyeicosatrienoic acid and/or the corresponding δ -1actone. The absolute configuration of the EETs has been determined using material obtained from in vitro incubation of arachidonic acid with the major phenobarbital-inducible form of rat liver microsomal cytochrome P -450. Recent results suggest that the enantiomeric composition is highly dependent on the identity of the cytochrome P -450 isozyme. This may be significant because in some instances biological activity is sensitive to EET stereochemistry. Chiral syntheses of all the EET enantiomers have been achieved. Most investigators have relied on racemic EETs prepared by Coreys site-specific oxidation methodology or, more conveniently, by nonselective peracid epoxidation of arachidonic acid. This chapter describes procedures based on the latter reaction that have proved reliable in laboratories for the production and purification of nanomole-millimole amounts of EETs and their methyl esters. In light of the increasing interest in EET heteroatom analogs, protocols for converting EETs to cis -thiiranes and cis -aziridines are also included.

12(R)-hydroxyeicosatrienoic acid, a potent chemotactic and angiogenic factor produced by the cornea

Human and bovine corneal epithelial cytochrome P450 convert arachidonic acid to compound D [12(R)-hydroxy-5,8,14(Z,Z,Z)-eicosatrienoic acid], a metabolite with inflammatory properties including vasodilatation and breakdown of the blood-aqueous barrier. Angiogenic properties of the endogenous compound D and the synthetic enantiomers DR and DS were examined using the corneal micropocket technique. The synthetic compound DR was as active as the endogenously formed compound D. Neovascularization of the cornea was found in all the implants containing as little as 0.5 micrograms of compound DR. In contrast, the stereoisomer DS at the same concentration (0.5 micrograms) was inactive. Since angiogenesis can be secondary to a local inflammatory response, we evaluated the effects of compound DR and its stereoisomer DS on human neutrophil chemotaxis by using a modified Boyden chamber technique. DR, but not DS, was found to be a potent chemotactic factor, exhibiting dose-dependent neutrophil chemotaxis with significant responses observed at doses as low as 10(-11) M, a concentration at which leukotriene B4 does not exhibit significant chemotactic activity. Therefore, compound D produced by the cornea may qualify as an intrinsic corneal angiogenic factor which, in association with other inflammatory mechanisms, account for the growth of new vessels in the cornea that appear in chronic inflammation or in the reparative stages of an acute process.

A stereoselective synthesis of coriolic acid and dimorphecolic acid

Abstract Described herein is a convenient synthesis of coriolic acid ( 1 ) and dimorphecolic acid ( 2 ), the two natural ionophores derived respectively from bovine heart mitochondria and also shown to be self defensive substances in rice plant against rice blast disease.

NADPH-dependent microsomal metabolism of 14,15-epoxyeicosatrienoic acid to diepoxides and epoxyalcohols

The arachidonic acid epoxygenase metabolite 14,15-epoxyeicosatrienoic acid is further metabolized by rat liver microsomal fractions to regioisomeric diepoxides and epoxyalcohols. Diepoxides result from epoxidation at the 5,6-, 8,9-, or 11,12-olefins. Hydroxylation leading to epoxyalcohols with a cis, trans-conjugated dienol occurs at carbons 5, 8, 9, or 12. Structural assignments were established by chromatographic and mass spectral comparisons with synthetic standards. The reaction requires NADPH and is inhibited by typical cytochrome P-450 inhibitors. Analysis of the time course of product formation during arachidonic acid oxidation by rat liver microsomal fractions indicated that all four regioisomeric epoxyeicosatrienoic acids can be further metabolized by the enzyme system.

Synthesis of 12(S),20-, 12(S),19(R)-, and 12(S),19(S)-dihydroxyeicosa-cis-5,8,14-trans-10-tetraenoic acids, metabolites of 12(S)-hete

Abstract Enantiospecific syntheses of the 20- and both 19-hydroxy metabolites of 12(S)-HETE were accomplished using readily available, chiral precursors.

Modified pyridinium chlorochromate oxidation of aldehydes to carbamoyl azides/acyl azides or carboxylic acids

Abstract Aldehydes are transformed into carbamoyl azides/acyl azides by pyridinium chlorochromate in the presence of sodium azide. Comparable oxidations modified with sodium cyanide generate carboxylic acids from simple aliphatic aldehydes whereas conjugated aldehydes are unreactive.

Cytochrome P450-dependent arachidonic acid metabolites, 19- and 20-hydroxyeicosatetraenoic acids, enhance sodium-potassium ATPase activity in vascular smooth muscle

Several cytochrome P450-dependent arachidonic acid metabolites have been shown to affect Na+,K+-ATPase activity. In the present study, we tested the effect of α and ω - 1-hydroxylated products, i.e., 19− and 20-hydroxyeicosatetraenoic acids (19− and 20-HETE), on the K-induced relaxation in rat aortic rings. 19-HETE and 20-HETE increased the magnitude of the potassium-induced relaxation in a dose-dependent fashion (10−7-10−5 M). The inhibitory effect of ouabain on the potassium-induced relaxation was reversed by both 19− and 20-HETE. In addition, indomethacin fully inhibited the stimulatory effect of 19− and 20-HETE on relaxation induced by potassium. Vascular ouabainsensitive 86Rb uptake was also increased by 19− and 20-HETE. These observations suggest that 19− and 20-HETE stimulate vascular Na+,K+-ATPase via their conversion by cyclooxygenase to prostaglandin-like material.