Eicosanoids最新文献

Phospholipase A2 (PLA2) activity of human term placenta is distributed about equally between cytosol and membranes. The latter activity was detached by treating membranes with EGTA, but this extraction also released inhibitory protein, which complicated the assay and has probably often led to underestimation of such PLA2. Varying the substrate concentration, we found that large amounts of liposome substrate relieve PLA2 suppression in the extract. This suggests substrate depletion by the inhibitory protein as the mechanism by which PLA2 enzymes are negatively controlled in placenta. Membrane-bound PLA2 was purified about 700-fold and appeared to be one enzyme species (PLA2-M). By contrast, cytosolic PLA2 activity could be fractionated into four separate fractions, one of which was further purified (PLA2-S1). As judged on the basis of a variety of biochemical properties, PLA2-M and PLA2-S1 seem to be identical enzyme forms. They are distinct from the class of pancreas/venom-type phospholipases A2.

The effect of carbocyclic thromboxane A2 (CTXA2) on short-circuit current (Isc) was studied in two preparations of the rat colon descendens, one with and one without the submucosal plexus. In both preparations, CTXA2 (10(-7)-5 x 10(-6) mol.l-1) increased Isc concentration-dependently. Its action was not inhibited by the neurotoxin, tetrodotoxin, or by indomethacin, indicating a direct action on the epithelium. The increase in Isc was dependent on the presence of Cl- and HCO3- anions in the medium, but it was not affected by inhibitors of Cl-secretion such as furosemide or a Cl- channel blocker. Measurements of the unidirectional fluxes of Na+ and Cl-revealed that the dominant action of CTXA2 was an inhibition of the mucosa to serosa flux of Cl-. The action of CTXA2 was prevented by pretreatment with the thromboxane receptor blocker, SK&F 88046. It was also inhibited by TMB-8, a substance preventing the release of Ca2+ from intracellular stores, but its effect was not dependent on the presence of extracellular Ca2+. The results indicate that thromboxanes can modulate Cl-transport through the colonic epithelium by a mechanism dependent on intracellular Ca2+.

Changes in eicosanoid generation have been examined in stimulated human peripheral leukocytes incubated with plasma lipoprotein fractions. Leukocytes (2.5 X 10(7) cells/ml, 90% neutrophils) were incubated with physiological concentrations of low density lipoprotein (LDL), high density lipoprotein (HDL) and very low density lipoprotein (VLDL). No release of leukotriene B4 (LTB4) or platelet activating factor (PAF) was noted prior to cell stimulation with either calcium ionophore, opsonized zymosan or FMLP. After stimulation with ionophore, LDL led to a 40% enhancement of LTB4 release compared to control incubations while there was no effect on PAF production. HDL caused a small but not significant increase in LTB4 while VLDL had no effect on the release of LTB4. The formation of the other major lipoxygenase product 5-hydroxy-eicosatetraenoic acid (5-HETE) was decreased by 20% following LDL incubation and by more than 50% after VLDL incubation compared to controls. LTB4 release was also enhanced by 27% after incubation with LDL and stimulation with opsonized zymosan. LDL did not cause any increase in superoxide production by leukocytes stimulated with opsonized zymosan or PMA. PGE2 release was stimulated directly in cells incubated with lipoproteins, particularly LDL and VLDL. Oxidised LDL enhanced LTB4 production to an even greater extent than native LDL. The observed enhancement of LTB4 by LDL is not the result of LDL oxidation during incubation, the provision of arachidonic acid substrate by the lipoprotein nor the uptake of cholesterol by the cell. The effect is most likely associated with the binding of LDL to the cell membrane, since LTB4 enhancement was partially blocked by dextran sulphate.(ABSTRACT TRUNCATED AT 250 WORDS)

Prostacyclin (PGI2) and taprostene (CG-4203) were studied in a highly lethal model of splanchnic artery occlusion (SAO) shock in pentobarbital anesthetized rats. Total occlusion of the superior mesenteric and celiac arteries for 40 min resulted in a severe shock state often resulting in a fatal outcome within 2 h following release of the occlusion. PGI2 or taprostene was infused at a rate of 100 ng/kg/min commencing at occlusion of the celiac and superior mesenteric arteries. Taprostene significantly improved survival time and taprostene treated animals maintained post-reperfusion mean arterial blood pressure (MABP) at significantly higher values compared to rats receiving taprostene vehicle (final MABP 96 +/- 3 vs 45 +/- 3.5 mmHg, p less than 0.001, respectively). In addition, taprostene significantly (p less than 0.05) attenuated the rise in hematocrit in SAO shock and the activity of plasma cathepsin D (p less than 0.005 from SAO vehicle). Taprostene also tended to decrease the accumulation of free amino-nitrogen compounds, but not significantly. In contrast, PGI2 neither improved survival time and the maintenance of post-reperfusion MABP, nor attenuated the rise in hematocrit, the plasma accumulation of free amino-nitrogen compounds, or plasma cathepsin D activity. These findings suggest that taprostene may possess greater cytoprotective properties than PGI2.

The effect of a selective LTD4 receptor antagonist SK & F104353 was studied in septic pigs anesthetized with isoflurane. Yorkshire pigs (25.2 +/- 2.3 kg) were instrumented and monitored for cardiac output (CO), mean arterial pressure (MAP), systemic vascular resistance (SVR), mean pulmonary arterial pressure (MPAP), pulmonary vascular resistance (PVR), renal artery blood flow (RABF), renal vascular resistance (RVR), arterial PO2, and extravascular lung water (EVLW). Blood samples were also collected for platelet, white blood cell and hematocrit determinations and plasma was assayed for thromboxane (TX) B2. Sepsis was induced by infusion of Pseudomonas aeruginosa (3 x 10(8) CFU%kg/h) for 2 h. Cardiovascular and hematologic data were determined at 30 min intervals for 4 h. Groups were infused with either SK & F104353 (3 mg/kg/h; n = 5) or drug vehicle (n = 6) beginning 15 min prior to infusion with the P. aeruginosa. In the vehicle group beginning at -90 min after sepsis induction, there was a 30 +/- 7% decrease of CO, a 27 +/- 5.0% decrease of MAP, and a 44 +/- 7% decrease of RABF, whereas, MPAP increased to 147 +/- 37% and plasma TXB2 increased from less than 200 pg/ml to 3,049 +/- 367 pg/ml (P less than 0.05). The EVLW and hematocrit increased (P less than 0.05), and the arterial PO2, white blood cell count, and platelet count decreased with the severity of the sepsis. In pigs pretreated with SK & F104353 the MAP and RABF were transiently improved (P less than 0.05), and the decrease in arterial PO2 was delayed.(ABSTRACT TRUNCATED AT 250 WORDS)

We evaluated whether defibrotide, a single-stranded polydeoxyribonucleotide that enhances prostacyclin (PGI2) release from various isolated organs, could also release PGI2 from the rabbit kidney and prove effective against renal ischemic injury. Isolated perfused kidneys responded to defibrotide (100, 250 and 500 micrograms ml-1 min-1) with a dose-dependent release of immunoreactive 6-keto-PGF1 alpha (4-fold increase at highest dose), which was prevented by indomethacin pre-treatment. In vivo, venous blood withdrawn from heparinized rabbits (and representative of renal outflow) was conveyed over a collagen matrix, onto which platelets adhered and aggregated. Recording the weight increase of the matrix was used as a bioassay to follow the time-course of released PGI2. We observed that renal outflowing blood from defibrotide treated animals (50 mgKg-1 i.v.) displayed lower (P less than 0.05 versus controls) platelet activation, consistent with enhanced PGI2 release from the kidneys. Furthermore, the duration of this effect was longer lasting than that predicted from the known plasma half-life of the drug. After transient (30 min) occlusion of the renal arteries, glomerular filtration rate (GFR) dropped by about 50% (P less than 0.01) during the first reperfusion hour in control animals, with only mild recovery having occurred 4 h later. Defibrotide (16 mgKg-1 bolus + 16 mgKg-1h-1, i.v.) could not antagonize the initial impairment (40% GFR reduction), but allowed full recovery at the end of the observation period (P less than 0.05 vs controls). Indomethacin, instead, caused a dramatic reduction of GFR (70%) during early reperfusion, with no subsequent recovery.(ABSTRACT TRUNCATED AT 250 WORDS)

Iloprost is a potent chemically stable PGI2-mimetic. Therapeutic efficacy was shown after i.v. infusion treatment in several states of peripheral vascular disease. For out-patient therapy an oral dosage form should be developed. Based upon dissolution profiles and in vivo data of a pig model, three different film-coated pellet formulations were selected for pharmacokinetic characterization in nine healthy volunteers. In the first part of the study groups of three test subjects were treated with increasing dosages (150-300 micrograms) of iloprost. At 300 micrograms flush and headache led to the discontinuation of those titration. All formulations exhibited dose-dependent serum level profiles. The cross-over characterization in all test subjects showed that one formulation, which exhibited a modified in vitro dissolution of 60% of the dose within 1 h in pH 7.4 phosphate buffer, was optimal from the pharmacokinetic profile. After oral administration of this formulation the bioavailable dose fraction was highest and half-maximal serum levels lasted for 2.4 h (mean); therapeutic serum levels were maintained for 2.1-5.0 h. This formulation was chosen for further investigation to imitate therapeutic serum level profiles as obtained after i.v. infusion for 4-6 h with a once-a-day dosage form.

Term decidual stromal cells metabolized extracellular arachidonic acid to cyclo-oxygenase, lipoxygenase and epoxygenase products. Prostaglandins were the major metabolites from extracellular arachidonic acid. In contrast, intracellular arachidonic acid was metabolized mainly to an epoxygenase product, together with some lipoxygenase products. Decidual macrophages showed similar results, though these cells had higher production rates per cell of most metabolites. The calcium ionophore A23187 increased the levels of arachidonic acid released from the intracellular stores of decidual stromal cells and had variable effects on the production of cyclo-oxygenase, lipoxygenase and epoxygenase metabolites. Less than 15% of the total metabolites released from A23187-stimulated cells were cyclo-oxygenase products, which suggested that the cyclo-oxygenase products released by decidual stromal cells or macrophages may be mainly derived from extracellular arachidonic acid. This implies that the regulation of decidual cyclo-oxygenase may have a major role in determining prostaglandin output from this tissue.

Platelet aggregation can be triggered by addition of exogenous arachidonate owing to its conversion to endoperoxides and thromboxane A2. The dose-response curve of arachidonate-induced platelet aggregation exhibited a very steep slope. Simultaneous addition of H2O2 (1-200 microM) significantly shifted this curve to the left. H2O2 alone did not induce aggregation up to a concentration of 1 mM; however, a reversible increase of cytoplasmic Ca2+ and a small increase of the thromboxane levels could be observed. In the presence of exogenous arachidonate H2O2 led to an increased formation of arachidonate metabolites. Our data demonstrate that at threshold levels of 20:4 H2O2 is able to promote conversion of 20:4 to proaggregatory prostaglandin endoperoxides, and subsequently platelet activation is facilitated. Thus we support the evidence for a role of H2O2 in the activation of cyclooxygenase possibly by providing an adequate peroxide tone.