Platelet-neutrophil interactions. (12S)-hydroxyeicosatetraen-1,20-dioic acid: a new eicosanoid synthesized by unstimulated neutrophils from (12S)-20-dihydroxyeicosatetraenoic acid

In the course of a cell-cell interaction, 12-HETE (12-hydroxy-5,8,10,14-eicosatetraenoic acid), the arachidonic acid lipoxygenase product released from stimulated platelets, is metabolized by a cytochrome P-450 enzyme system in unstimulated neutrophils to 12,20-DiHETE (12,20-dihydroxy-5,8,10,14-eicosatetraenoic acid). This report describes time-dependent formation of a new eicosanoid by unstimulated neutrophils exposed to 12-HETE, which is more polar than 12,20-DiHETE (reversed-phase high performance liquid chromatography). Time course studies indicated that the precursor compound of this new eicosanoid was 12,20-DiHETE. This was determined by incubation of purified 12,20-DiHETE with neutrophils, which resulted in a progressive decrease in 12,20-DiHETE as formation of the polar metabolite increased. In the absence of neutrophils, 12,20-DiHETE was quantitatively unchanged. The new metabolite of 12,20-DiHETE was identified as 12-hydroxyeicosatetraen-1,20-dioic acid, based upon its UV spectrum, co-chromatography with a chemically synthesized standard in both high performance liquid chromatography and thin layer chromatography systems, and gas chromatography-mass spectrometry. Formation of 12-HETE-1,20-dioic acid was partially inhibited by 20-hydroxy-LTB4. This indicated that the neutrophil dehydrogenase responsible for further metabolism of 12,20-DiHETE may also be involved in conversion of 20-hydroxy-LTB4 to 20-carboxy-LTB4. The 12,20-DiHETE dehydrogenase enzyme system specifically requires NAD as cofactor and has subcellular components in both cytosolic and microsomal fractions which are synergistic in their activity. These results provide additional evidence for the occurrence of multicellular metabolic events during hemostasis, thrombosis, and the inflammatory response.     

5,6-Epoxyeicosatrienoic acid mobilizes Ca2+ in anterior pituitary cells

Luteinizing hormone releasing hormone stimulates the concomitant release of luteinizing hormone and 45Ca2+ from prelabeled anterior pituitary cells. Indomethacin (10 microM) and nordihydroguaiaretic acid (10 microM) had no effect on the luteinizing hormone releasing hormone-stimulated release of either luteinizing hormone or 45Ca2+. Eicosatetraynoic acid (10 microM) blocked both luteinizing hormone releasing hormone-stimulated luteinizing hormone secretion and luteinizing hormone releasing hormone-stimulated 45Ca2+ efflux. 5,6-Epoxyeicosatrienoic acid stimulated both luteinizing hormone secretion and 45Ca2+ efflux from anterior pituitary cells. Additionally, 5,6-epoxyeicosatrienoic acid closely mimics the ability of luteinizing hormone releasing hormone to increase intracellular free calcium. These results are consistent with the hypothesis that 5,6-EET alters calcium homeostasis in a manner similar to that observed during luteinizing hormone releasing hormone stimulation of luteinizing hormone release.     

Influence of a fibric acid type of hypolipidemic agent on the oxidative metabolism of arachidonic acid by liver microsomal cytochrome P-450

The regiospecificity of arachidonic acid oxygenation, catalyzed by rat liver microsomal fractions in the presence of NADPH, can be altered by animal pretreatment with a fibric acid type of hypolipidemic drug, ciprofibrate. While microsomal fractions isolated from either control or phenobarbital-treated animals oxygenate arachidonic acid to mainly epoxyeicosatrienoic acids (EETs), animal pretreatment with ciprofibrate results in an eightfold stimulation of omega and omega-1 oxidation, concomitant with a net decrease in the formation of both HETEs and EETs. The isomeric composition of the EETs and of the omega and omega-1 oxidation products formed is also dependent on the type of animal pretreatment. Associated decreases in the amounts of HETEs and the rate of hydrogen peroxide formation suggests a modification of the "uncoupler action" of arachidonic acid during the function of different cytochromes P-450.     

Adrenergic nerves in the pars intermedia of the pituitary in the toad,Bufo arenarum

Summary By means of a highly sensitive and specific histochemical method for the demonstration of certain biogenic monoamines a plexus of nerves containing a primary catecholamine has been demonstrated in the pars intermedia of the toad, Bufo arenarum. These nerves are restricted in distribution to the part of the gland which contains colloid vesicles (stored MSH ?) in the cells. The view is put forward, based on the results of pharmacological and surgical experiments, that the adrenergic nerves inhibit the release of the MSH from the pars intermedia. The origin of the nerves in the brain is unknown, but experiments with lesions have shown that it is not to be found in the nucleus periventricularis arcuatus.The research reported in this document has been sponsored by the Swedish Medical Research Council and by the Swedish Natural Science Research Council.Research fellow of the Commission of Scientific Research of the Province of Buenos Aires, Argentina.     

Metabolism of 20-hydroxyeicosatetraenoic acid by cyclooxygenase. Formation and identification of novel endothelium-dependent vasoconstrictor metabolites

We recently demonstrated that 20-hydroxyeicosatetraenoic acid (20-HETE) constricts rat aortic rings. The contractile response was partially dependent on the presence of endothelium and was abolished by pretreatment of the rings with either indomethacin or the endoperoxide/thromboxane receptor antagonist, SQ29548. Addition of GSH or SnCl2 to the organ bath diminished the contractile response of 20-HETE, whereas preincubation of the rings with a thromboxane synthase inhibitor did not affect the 20-HETE induced contractions. Short time incubation (2 min) of 20-HETE with ram seminal vesicle microsomes in the presence of p-hydroxymercurybenzoate yielded metabolites which migrated similarly on thin layer chromatography to the known arachidonate endoperoxides prostaglandin (PG) G2 and PGH2 and possess vasoconstrictory properties. The vasoconstriction was dose-dependent with a half-life of approximately 6.3 +/- 0.6 min. Addition of SQ29548 to the aortic ring bath 1 min after metabolite elicited vasoconstriction produced immediate relaxation. Furthermore, pretreatment of the rings with SQ29548 totally abolished the contraction. SnCl2 reduction of the metabolites produced in incubation of rat seminal vesicles with 20-HETE and p-hydroxymercurybenzoate resulted in a single radioactive peak which was further identified by gas chromatography/mass spectrometry as 20-hydroxy-PGF2 alpha. The inhibitory effect of SQ29548, the appearance of labile metabolites with a half-life of approximately 6 min and the production of 20-hydroxy-PGF2 alpha by SnCl2 reduction clearly indicate that the vasoconstrictor metabolites of 20-HETE are the labile endoperoxides of 20-HETE, 20-hydroxy-PGG2, and 20-hydroxy-PGH2.     

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.     

Patterns of sequence variation in the mitochondrial D-loop region of shrews

Direct sequencing of the mitochondrial displacement loop (D-loop) of shrews (genus Sorex) for the region between the tRNA(Pro) and the conserved sequence block-F revealed variable numbers of 79-bp tandem repeats. These repeats were found in all 19 individuals sequenced, representing three subspecies and one closely related species of the masked shrew group (Sorex cinereus cinereus, S. c. miscix, S. c. acadicus, and S. haydeni) and an outgroup, the pygmy shrew (S. hoyi). Each specimen also possessed an adjacent 76-bp imperfect copy of the tandem repeats. One individual was heteroplasmic for length variants consisting of five and seven copies of the 79-bp tandem repeat. The sequence of the repeats is conducive to the formation of secondary structure. A termination-associated sequence is present in each of the repeats and in a unique sequence region 5' to the tandem array as well. Mean genetic distance between the masked shrew taxa and the pygmy shrew was calculated separately for the unique sequence region, one of the tandem repeats, the imperfect repeat, and these three regions combined. The unique sequence region evolved more rapidly than the tandem repeats or the imperfect repeat. The small genetic distance between pairs of tandem repeats within an individual is consistent with a model of concerted evolution. Repeats are apparently duplicated and lost at a high rate, which tends to homogenize the tandem array. The rate of D- loop sequence divergence between the masked and pygmy shrews is estimated to be 15%-20%/Myr, the highest rate observed in D-loops of mammals. Rapid sequence evolution in shrews may be due either to their high metabolic rate and short generation time or to the presence of variable numbers of tandem repeats.