Epoxygenase products of arachidonic acid are endogenous constituents of the hypothalamus involved in D2 receptor-mediated, dopamine-induced release of somatostatin.

The epoxyeicosatrienoic acids (EETs) were discovered as products of a cyclooxygenase/lipoxygenase-independent, cytochrome P-450 catalyzed metabolism of arachidonic acid (AA) termed the "epoxygenase" pathway. The rat hypothalamus is able to synthesize EETs from exogenous AA, and 5,6-EET has been found to release the neuropeptide somatostatin (SRIF) from hypothalamic nerve terminals of the median eminence (ME). In the present study, hypothalami from male rats were examined for the presence of endogenous EETs, using chemical, chromatographic, and mass spectral analysis procedures. The samples were initially separated in a C18 Sepralyte column, fractionated on TLC plates, and purified by reverse phase HPLC. Thereafter, they were esterified (pentafluorobenzyl esters) and subjected to negative ion chemical ionization/gas chromatography (GC)/mass spectral (MS) analysis. The GC retention time and the MS fragmentation patterns revealed the presence of a mixture of 8,9-, 11,12- and 14,15-EETs; instability of 5,6-EET during the isolation protocol precluded its identification. Total hypothalamic EET concentration was estimated to be 120 ng/g wet tissue. The 8,9-regiosomer released SRIF from ME nerve terminals with an ED50 of 5 x 10(-12) M; Dopamine (DA) and the D2 receptor agonist PPHT, but not the D1 receptor agonist SKF-38393, induced SRIF release from the ME. This effect was blocked by clotrimazole and ketoconazole, two inhibitors of microsomal cytochrome P-450 function and AA epoxygenase in particular. In contrast, the inhibitors failed to affect the increase in SRIF release induced by 8,9-EET. These results indicate that: 1) in addition to cyclooxygenase and lipoxygenase products, epoxygenase metabolites of AA are endogenous compounds of the hypothalamus, and 2) EETs may mediate the increase in SRIF release from hypothalamic neurons induced by the interaction of DA with D2 receptors.

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 epoxyeicosatrienoic 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.

Molecular, enzymatic, and regulatory characterization of rat kidney cytochromes P450 4A2 and 4A3.

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.

A novel pool of rat liver inositol and ethanolamine phospholipids contains epoxyeicosatrienoic acids (EETs).

A pool of phosphatidylinositol and phosphatidylethanolamine containing epoxyeicosatrienoic acids esterified to the sn-2 position of the glycerol moiety has been identified in rat liver. The mole fraction of epoxide containing phosphatidylinositol is 7-8 times that of phosphatidylethanolamine.

Endogenous epoxyeicosatrienoic acids. Cytochrome P-450 controlled stereoselectivity of the hepatic arachidonic acid epoxygenase.

Chiral analysis of the endogenous rat liver epoxyeicosatrienoic acids shows the biosynthesis of 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids in a 4:1, 2:1, and 3:1 ratio of antipodes, respectively. Animal treatment with phenobarbital results in a 3.7-fold increase in microsomal cytochrome P-450 concentration and a concomitant, regioselective 6.8- and 3.4-fold increase in the liver concentration of 8,9- and 14,15-epoxyeicosatrienoic acids, respectively. Phenobarbital induces the in vivo synthesis of both regioisomers as nearly optically pure enantiomers. These results demonstrate the enzymatic origin of the epoxyeicosatrienoic acids present in rat liver and document a novel metabolic function for cytochrome P-450 in the regio- and enatioselective epoxygenation of endogenous pools of arachidonic acid.

Endogenous epoxyeicosatrienoyl-phospholipids. A novel class of cellular glycerolipids containing epoxidized arachidonate moieties.

By chromatographic and mass spectral techniques we document in rat liver the presence of new classes of glycerophospholipids which contain an epoxyeicosatrienoate moiety, esterified to the glycerol-sn-2 position. These novel lipids are formed in vivo from endogenous precursors and under physiological conditions. Chromatographic resolution followed by hydrolysis and regioisomeric analysis showed that they consist of the 8,9-, 11,12-, and 14,15-epoxyeicosatrienoyl derivatives of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. Their relative concentrations (micromoles of oxidized lipid/mol of phospholipid) were 70, 85, and 106 for phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol, respectively. Chiral analysis of the fatty acids at sn-2 revealed an enantioselective preference for 8(S),9(R)-, 11(S),12(R)-, and 14(R),15(S)-epoxyeicosatrienoates in all three lipid classes. The data suggest a multienzyme process initiated by cytochrome P-450 epoxidation of arachidonic acid followed by ATP-dependent activation to epoxyeicosatrienoyl-CoA derivatives and stereoselective lysolipid acylation. These results provide a molecular basis for a potential physiological role of cytochrome P-450 in the biosynthesis of unique cellular glycerolipids and, consequently, in the control of cell membrane structure and function.

Cytochrome P-450-dependent oxidation of arachidonic acid to 16-, 17-, and 18-hydroxyeicosatetraenoic acids.

Incubation of rat liver microsomal fractions with arachidonic acid in the presence of NADPH results in the formation of three novel monohydroxylated fatty acid metabolites. Utilizing chromatographic and mass spectral techniques, these metabolites have been identified as 16-, 17-, and 18-hydroxyeicosatetraenoic acids. The NADPH-dependent microsomal metabolism of arachidonic acid to 16-, 17-, 18-, and 19-hydroxyeicosatetraenoic acids is induced after animal treatment with beta-naphthoflavone. Reconstitution of the arachidonic acid oxygenase utilizing individual purified cytochrome P-450 enzymes demonstrates regioselectivity, controlled by the protein catalyst, for the hydroxylation of the sp3 carbon atoms adjacent to the methyl end of the fatty acid.