6-Keto-prostaglandin E1 is not equipotent to prostacyclin (PGI2) as an antiaggregatory agent

A direct comparison of the relative potencies of the two anti-aggregatory prostaglandins PGI₂ and 6-keto-PGE₁ showed PGI₂ was at least 20 times more potent than 6-keto-PGE₁ when tested against ADP-induced human platelet aggregation. This marked difference in potency was even more evident when the ability of PGI₂ and 6-keto-PGE₁ to stimulate platelet cyclic AMP levels was determined. When cyclic AMP levels were measured direct comparisons were difficult because the respective dose response curves were not parallel, but 10 ng of PGI₂ was equivalent to 300 ng of 6-keto-PGE₁.PGI₂ was also more potent (10–20 times) than 6-keto-PGE₁ as a disaggregatory agent, and the disaggregatory activity of both prostaglandins was enhanced by the phosphodiesterase inhibitor 1-methyl-3-isobutylmethylxanthine.PGI₂ was also more active than 6-keto-PGE₁ as an inhibitor of thrombus formation in dog coronary arteries in vivo. In vivo, 6-keto-PGE₁ was at least 10 times less potent than PGI₂, the exact difference could not be determined because 6-keto-PGE₁ caused significant falls in blood pressure before anti-platelet activity could be detected.PGI₂ is an intrinsically more potent anti-aggregatory molecule than 6-keto-PGE₁, but these data do not rule out the possibility that some of the activities attributed to PGI₂ could be the result of the conversion of PGI₂ and/or 6-keto-PGF_1α to 6-keto-PGE₁.     

A comparison of the effects of prostacyclin and 6-keto-prostaglandin E1 on renin release in the isolated rat and rabbit kidney

A direct comparison of the relative potencies of the prostaglandins PGI₂ and 6-kto-PGE₁ to induce renin release was made in the isolated rat kidney, which was perfused with a synthetic medium at constant perfusion pressure.Both prostaglandins stimulated renin release in a dose-dependent manner (0.01 to 1 μM) and with equal potency.Also in the isolated rabbit kidney, PGI₂ and 6-keto-PGE₁ had the same potency to induce renin release at 1 μM final concentration.Following infusion of 6-keto-PGE₁ a small increase of vascular resistance in the rat kidney was observed, whereas in the rabbit kidney no constrictor effect was seen.When perfusate of PGI₂ or 6-keto-PGE₁-infused rat kidneys were tested for antiaggregatory activity in the ADP induced aggregation of human platelets and compared with authentic standards, the results showed 6-keto-PGE₁ passes the kidney essentially unchanged, whereas only 25–40% of the infused PGI₂ appear in the venous perfusates, as judged from the recovery of antiaggregatory activity.Analysis of venous perfusates from ³H-PGI₂ infused kidneys by high performance liquid chromatography indicates that about 25% of the infused PGI₂ remains intact, a major portion of the perfused radioactivity was identified as 6-keto-PGF_1α by combined gaschromatography-mass-spectrometry (19).We conclude that the renin-stimulating effect of PGI₂ is not secondary to its metabolism to 6-keto-PGE₁, as has been suggested in the literature (8).     

Inhibition of vasoconstrictor responses by 6-keto-PGE1 in the feline mesenteric vascular bed

The effects of 6-keto-PGE₁ on vascular resistance and vascular responses to sympathetic nerve stimulation and vasoconstrictor hormones were investigated in the feline mesenteric vascular bed. Infusions of 6-keto-PGE₁ into the superior mesenteric artery dilated the mesenteric vascular bed and markedly inhibited vasoconstrictor responses to sympathetic nerve stimulation, norepinephrine and angiotensin II. The effects of 6-keto-PGE₁ and PGE₁ on vascular resistance and vasoconstrictor responses were quite similar and both substances inhibited responses to nerve stimulation and pressor hormones in a reversible manner. Responses to nerve stimulation, norepinephrine and angiotensin II were inhibited to a similar extent during infusion of 6-keto-PGE₁ and PGE₁. Results of these studies suggest that 6-keto-PGE₁, a newly identified prostaglandin metabolite, and PGE₁ possess the ability to inhibit the vasconstrictor effects of sympathetic nerve stimulation and pressor hormones by a nonspecific action on vascular smooth muscle in the feline small intestine.     

Protective action of 6-keto-prostaglandin E1 in traumatic shock

Since prostaglandin E₁ (PGE₁) is known to have a beneficial effect in hemorrhagic shock, a biologically active derivative of PGE₁, 6-keto-PGE₁, was examined for its effect on traumatic shock in rats. In sham-operated rats, infusion of 6-keto-PGE₁, at a rate of 250 ng/kg/min intravenously decreased arterial blood pressure by 23 mm Hg at 5 hr. In rats subjected to Noble-Collip drum trauma, infusion of 6-keto-PGE₁, starting 15 min after the trauma, significantly improved the survival time from 1.0 ± 0.1 hr to 2.6 ± 0.3 hr compared to rats given only the vehicle (i.e., Tris buffer). The improved survival was accompanied by a diminished plasma accumulation of the cardiotoxic peptide, myocardial depressant factor (MDF), and the lysosomal protease cathepsin D. 6-keto-PGE₁ also exerted a direct lysosomal stabilizing effect in isolated cat liver lysosomes, as well as reducing cardiac afterload in rats. It is concluded that 6-keto-PGE₁ protects in traumatic shock by hemodynamic as well as cytoprotective actions.     

Determination of cyclooxygenase products and prostaglandin metabolites using high-pressure liquid chromatography and radioimmunoassay.

A method is described for measurement of the cyclooxygenase products, thromboxane,prostacyclin, and prostaglandins (PG), and several prostaglandin metabolites. The procedure involves separation of the compounds by high-pressure liquid chromatography combined with identification and estimation by serologic analysis. These combined procedures have been used to identify and estimate five such products, PGE₂, PGE₁ PGF2α, PGF_1α, and 6-keto-PGF_1α, in the culture fluids of dog kidney cells stimulated by a tumor-promoting phorbol diester. The prostaglandin metabolites, 13,14-dihydro-15-keto-PGE₂, 13,14-dihydro-15-keto-PF2_2α, 13,14-dihydro-PGE₂, and 13,14-dihydro-PGF_2α, were not found in these culture fluids.     

Prostacyclin induces a surprising long-lasting motility in quiescent uterine strips (indomethacin-treated) isolated from ovariectomized rats

Dose-response curves for several prostaglandins (PGI₂; PGD₂; PGF₂ and PGE₂); BaCl₂ or prostaglandin metabolites (15-keto-PGF_2α; 13, 14-diOH-15-keto-PGF_2α; 6-keto-PGF_1α and 6-keto-PGE₁ in quiescent (indomethacin-treated) uterine strips from ovariectomized rats, were constructed. All PGs tested as well as BaCl₂, triggered at different concentrations, evident phasic contractions. Within the range of concentrations tested the portion of the curves for the metabolites of PGF_2α was shifted to the right of that for PGF_2α itself; the curve for 6-keto-PGF_1α was displaced to the right of the curve for PGI₂ and that for 6-keto-PGE₁ to the left.It was also demonstrated that the uterine motility elicited by 10⁻⁵ M PGF_2α and its metabolites was long lasting (more than 3 hours) and so it was the activity evoked by PGI₂; 6-keto-PGF_1α and BaCl₂, but not the contractions following 6-keto-PGE₁, which disappeared much earlier. The contractile tension after PGF_2α; 15-keto-PGF_2α; 13, 14-diOH-15-keto-PGF_2α and PGI₂, increased as time progressed whilst that evoked by 6-keto-PGF_2α or BaCl₂ fluctuated during the same period around more constant levels.The surprising sustained and gradually increasing contractile activity after a single dose of an unstable prostaglandin such as PGI₂, on the isolated rat uterus rendered quiescent by indomethacin, is discussed in terms of an effect associated to its transformation into more stable metabolites (6-keto-PGF_1α, or another not tested) or as a consequence of a factor which might protects prostacyclin from inactivation.     

Inhibition of platelet aggregation by 6-keto-PGE1; lack of an effect on cyclic GMP levels

The effects of 6-keto-PGE₁ on aggregatory responses to arachidonic acid (AA), adenosine diphosphate (ADP) and collagen were studied in human platelet-rich plasma (PRP). In addition, experiments were carried out to determine if these effects correlate with changes in platelet cyclic AMP and cyclic GMP levels. 6-Keto-PGE₁ incubated in PRP produced dose-related increases in platelet cyclic AMP levels whereas platelet cyclic GMP levels were unchanged. Control aggregations induced by AA and ADP did not alter cyclic AMP and cyclic GMP levels whereas control aggregations induced by collagen elevated cyclic GMP levels while cyclic AMP levels were unchanged. 6-Keto-PGE₁ produced a dose-dependent inhibition of platelet aggregation induced by AA, ADP and collagen and this inhibition correlated with a dose-related increase in cyclic AMP levels. Since 6-keto-PGE₁ does not consistently alter cyclic GMP levels in human PRP, the present data support previous studies suggesting that 6-keto-PGE₁ produces inhibition of platelet aggregation through the stimulation of cyclic AMP accumulation.     

Hepatic metabolism of prostacyclin (PGI2) in the rabbit: Formation of a potent novel inhibitor of platelet aggregation

Metabolism of [9-³H]-PGI₂ was studied in the isolated Tyrode's perfused rabbit liver. Five products, four radioactive and one non-radioactive, were identified in the perfusate: 19-hydroxy-6-keto-PGF_1α, 6-keto-PGF_1α, dinor-6-keto-PGF_1α, pentanor PGF_1α and a 6-keto-PGE₁-like substance. The first two, 19-hydroxy-6-keto-PGF_1α and 6-keto-PGF_1α, represented 5% and 45% respectively, of the total radioactivity; the last two accounted for 39%. The presence of dinor and pentanor derivatives of 6-keto-PGF_1α indicated that β -oxidation and oxidative-decarboxylation occurs in the liver as the major metabolic pathway of PGI₂. One non-radioactive metabolite which co-migrated with authentic 6-keto-PGE₁ was found to inhibit platelet aggregation, having a potency similar to authentic 6-keto-PGE₁, and its effect can be eliminated by boiling and by alkali treatment. This metabolite, having similar Rf value on TLC and biological behavior as 6-keto-PGE₁, may arise from oxidation of 6-keto-PGF_1α via the 9-hydroxyprostaglandin dehydrogenase pathway, as suggested by recovery of tritiated water in the aqueous phase of the perfusate. This material, a potent inhibitor of platelet aggregation, may arise from PGI₂ or its hydrolysis product, 6-keto-PGF_1α.     

Plasma prostaglandin levels and circulating fuel levels in rats with diabetic ketoacidosis: Effects of cyclooxygenase inhibitors and of alpha and beta adrenergic blockade

We studied the effects of two structurally unrelated inhibitors of the fatty acid cyclooxygenase and of alpha and beta adrenergic blockade on the elevated plasma levels of 13,14-dihydro-15-keto-prostaglandin (PG)E₂, 6-keto-PGF_1α and thromboxane(TX)B₂, the stable derivatives of PGE₂, PGI₂ (prostacyclin) and TXA₂, respectively, in rats with streptozotocin-induced diabetic ketoacidosis (DKA). Meclofenamic acid and indomethacin each produced a significant decrease in the elevated plasma levels of 13,14-dihydro-15-keto-PGE₂, 6-keto-PGF_1α and TXB₂. Phentolamine significantly reduced the plasma level of TXB₂ but had no effect on the elevated circulating levels of glucose, free fatty acids, total ketones, 13,14,-dihydro-15-keto-PGE₂ or 6-keto-PGF_1α. Propranolol significantly reduced the elevated circulating levels of glucose, free fatty acids and total ketones but had no effect on the levels of the three prostaglandin derivatives. The ability of meclofenamic acid and indomethacin to reduce the plasma levels of 13,14-dihydro-15-keto-PGE₂, 6-keto-PGF_1α and TXB₂ confirms that the plasma levels of these three derivatives are elevated in rats with DKA. Since abnormalities in the production of PGI₂ and perhaps other cyclooxygenase derivatives may contribute to the pathogenesis of certain important hemodynamic and gastrointestinal features of DKA, cyclooxygenase inhibitors may play a role in the management of selected patients with this disorder. Alpha adrenergic activity is essential for the maintenance of the elevated plasma TXB₂ level in rats with DKA. The fall in the plasma TXB₂ level during alpha adrenergic blockade appears to reflect inhibition of platelet aggregation and platelet TXA₂ production, but other sources of the elevated plasma TXB₂ level in DKA are not excluded. Beta adrenergic activity contributes to the maintenance of elevated circulating levels of glucose, free fatty acids and total ketones in experimental DKA but not to the elevated plasma levels of the prostaglandin derivatives.     

Fluorescent/ultraviolet absorbing ester derivative formation and analysis of eicosanoids by high-pressure liquid chromatography

A method is described for the quantitative analysis of eicosanoids (arachidonic acid metabolites, nee, prostaglandins) by reverse-phase high-pressure liquid chromatography following formation of the ester derivative with p-(9-anthroyloxy)phenacyl bromide. The lower limit of detection of the eicosanoid ester is 280 pg (ultraviolet—254 nm) and approximately 50 pg (fluorescence 249 emission, 413-nm cutoff). We separated the esters of seven common eicosanoids by reverse-phase chromatography with acetonitrile and water. Thromboxane B₂ chromatographs as two species and coelutes with PGF_2α. Separation of all others is adequate, including the three metabolites of prostacyclin (6-keto-PGF_1α, 6-keto-PGE₁, 13,14-dihydro-6,15-diketo-PGF_1α). We obtained good correlation between radioimmunoassay and derivative analysis of standard 6-keto-PGF_1α extracted from lactated Ringer's solution with standard technique, as well as 6-keto-PGF_1α quantitation from tissue culture medium that had contained pulmonary endothelial cells. This method should be applicable to analysis of eicosanoids extracted from biological matrices.     

Analysis by high performance liquid chromatography of cyclooxygenase products of arachidonic acid metabolism in plasma of rabbits bearing the VX2 carcinoma

Cyclooxygenase products of arachidonic acid metabolism in the plasma of normal rabbits and animals bearing the VX₂ carcinoma were separated by high performance liquid chromatography and the effluent fractions assayed by serologic methods. The products measured were 6-keto-PGF_lα, thromboxane B₂, PGE₂, PGF_2α, 13,14-dihydro-PGE₂, 13,14-dihydro-15-keto-PGE₂, 15-keto-PGE₂, and 13,4-dihydro-15-keto-PGF_2α. In hypercalcemic, tumor-bearing rabbts, the plasma concentrations o 13,14-dihydro-15-keto-PGE₂ and 13,14-dihydro-15-keto-PGF_2α were markedly elevated (in the range of 0.5 to 16 ng/ml). Previously unmeasured 6-keto-PGF_1α, thromboxape B₂, 13,14-dihydro-PGE₂ and 15-keto-PGE₂ were not found in high concentrations in the plasma of tumor-bearing rabbits. These results add further support to our conclusion that the VX₂ tumor produces hypercalcemia in the host by a mechanism which utilizes PGE₂, rather than a subsequent metabolite of this prostaglandin, as the mediator between the neoplasm and bone.     

Accumulation of Cyclooxygenase Products of Arachidonic Acid Metabolism in Gerbil Brain During Reperfusion After Bilateral Common Carotid Artery Occlusion

: Several of the cyclooxygenase products of arachidonic acid were measured in the cerebral hemispheres of gerbils subjected to transient interruption of the cerebral circulation. The levels of PGD₂, PGF₂α, PGE₂, TXB₂, 13,14-H₂-15-keto-PGE₂, and the stable nonenzymic product of prostacyclin, 6-keto-PGF₁α, were not altered at the end of a 5-min period of ischemia. However, the onset of reperfusion was accompanied by a rapid accumulation of these products. Levels were highest during the initial period of reperfusion, then decreased to approach control levels after 120 min. PGD₂, PGF₂α, and PGE₂ were the predominant metabolites detected. This postischemic accumulation of arachidonic acid metabolites could be blocked by prior administration of inhibitors of cyclooxygenase activity.     

Hypoxia stimulates prostacyclin generation by dug lung in vitro

We have investigated the possibility that pulmonary biosynthesis of prostacyclin and thramboxane A₂ (TXA₂) may be affected by variations in PO₂. Fresh lung ha ogenates from 8 dogs were incubated for 1 hour at 37°C in Krebs-Ringer solution, at high (492 mnHg) or low (53 mmHg) PO₂. After incubation, the stable metabolites of prostacyclin (6-keto-PGF₁) and of TXA₂(TXB₂) were measured by radioimunoassay. The basal (“pre-incubtion”) levels of these metabolites, measured in tubes in which biosynthesis was arrested by the addition of indomethacin (10 g/ml), were 0.76 ± 0.15 and 0.76 ± 0.11 ng/mg wet wt. for 6-keto-PGF_1α, in high and low PO₂ respectively, and 0.97 ± 0.09 and 0.82 ± 0.15 × 10⁻¹ ng/mg wet wt. for TXB₂, in high and low P0₂ respectively. Synthesis during incubation in other tubes was estimated by subtracting basal values from those measured at the end of incubation. More 6-keto-PGF_1α was formed in homogenates exposed to low PO₂ (3.01 ± 0.45) than in those exposed kept at high PO₂ (1.89 ± 0.37, p< 0.001), but equal amounts of TXB₂ were found under both conditions. These results suggest that hypoxia may stimulate pulmonary prostacyclin synthesis; a pulmonary vasodilator, prostacyclin may help modulate hypoxic vasocontriction in the lung.     

Prostaglandin reductase-3 negatively modulates adipogenesis through regulation of PPARγ activity

Adipocyte differentiation is a multistep program under regulation by several factors. Peroxisome proliferator-activated receptor γ (PPARγ) serves as a master regulator of adipogenesis. However, the endogenous ligand for PPARγ remained elusive until 15-keto-PGE₂ was identified recently as an endogenous PPARγ ligand. In this study, we demonstrate that zinc-containing alcohol dehydrogenase 2 (ZADH2; here termed prostaglandin reductase-3, PTGR-3) is a new member of prostaglandin reductase family that converts 15-keto-PGE₂ to 13,14-dihydro-15-keto-PGE₂. Adipogenesis is accelerated when endogenous PTGR-3 is silenced in 3T3-L1 preadipocytes, whereas forced expression of PTGR-3 significantly decreases adipogenesis. PTGR-3 expression decreased during adipocyte differentiation, accompanied by an increased level of 15-keto-PGE₂. 15-keto-PGE₂ exerts a potent proadipogenic effect by enhancing PPARγ activity, whereas overexpression of PTGR-3 in 3T3-L1 preadipocytes markedly suppressed the proadipogenic effect of 15-keto-PGE₂ by repressing PPARγ activity. Taken together, these findings demonstrate for the first time that PTGR-3 is a novel 15-oxoprostaglandin-Δ¹³-reductase and plays a critical role in modulation of normal adipocyte differentiation via regulation of PPARγ activity. Thus, modulation of PTGR-3 might provide a novel avenue for treating obesity and related metabolic disorders.     

Biosynthesis of prostaglandins in human ovarian tissues

Experiments in vitro demonstrate, that there are different patterns of PG-biosynthesis in the corpus luteum and in the follicles containing cortical substance of the human ovary. In the follicles 6-keto-F₁α. the transformation product of prostacyclin, is the main fraction; prostaglandins F₂α and E 2 being of inferior importance with regard to their amounts. The formation of the corpus luteum is in close correlation with a strongly increased prostaglandin E₂ and a diminished prostacyclin biosynthesis; prostaglandin F₂α hardly seems to be involved in this process.By means of indomethacin the formation of all three examined prostaglandins can be prevented almost completely, in the cortical substance as well as in the corpus luteum. LH (or HCG) at concentrations ranging from 2 ng to 20 μg per ml homogenate produce no stimulating effect of statistical significance on the rate of biosynthesis in both tissues.     

6-Keto-PGE1 is a potent relaxant of the fetal ductus arteriosus

6-Keto-PGE₁ is nearly as potent as PGE₂ in relaxing the ductus arteriosus of fetal lambs. This finding raises the possibility that 6-keto-PGE₁, if occuring naturally as a by-product of PGI₂ transformations, may contribute to prenatal patency of the vessel.     

Pulmonary and systemic vascular responses to 6-keto-PGE1 in the conscious lamb

6-Keto-PGE₁ is a potent direct dilator of the pulmonary and systemic circulations of the newborn lamb under both normoxic and hypoxic conditions. Its threshold dose is similar to that of PGI₂ and PGE₁. Under hypoxia, 6-keto-PGE₁ appears equally effective on the pulmonary and systemic circulations, while under normoxia it predominantly affects the systemic circulation.     

Effect of mercaptoethanol in the radioactive thin layer chromatography assay of NAD-15-hydroxyprostagland in dehydrogenase

The use of mercaptoethanol in the assay of rat kidney 15-hydroxyprostaglandin dehydrogenase (PGDH) was found to have minimal effect on activity assayed with the spectrophotometric and substrate loss assays. However, mercaptoethanol appeared to inhibit PGDH when assayed by thin-layer chromatography, based upon conversion of ³H-PGE₁ to 15-keto-³H-PGE₁. Mercaptoethanol reacted with 15-keto-PGE₁ to alter its chromatographic mobility and to suppress the U.V. absorption spectrum of 15-keto-PGE₁. The implication of the use of ME in radiometric assays is discussed.     

Agonist specific desensitization of leukotriene C4-stimulated PGI2 biosynthesis in human endothelial cells

Leukotriene C₄ (LTC₄) and, to a lesser extent, leukotriene D₄ (LTD₄) concentration dependently stimulate prostacyclin (PGI₂) biosynthesis in cultured human umbilical vein endothelial cells. PGI₂ biosynthesis was quantitated by radioimmunoassay and its structure confirmed by gas chromatography/mass spectrometry. Preincubation of endothelial cells with LTC₄ resulted in desensitization to subsequent LTC₄ stimulation. However, PGI₂ biosynthesis in response to thrombin, PGH₂ and arachidonic acid was not inhibited by preincubation with LTC₄. The C-6-sulfidopeptide leukotriene receptor level antagonist FPL-55712 attenuates LTC₄, but not thrombin-stimulated PGI₂ biosynthesis. These data suggest that human umbilical vein endothelial cells have a C-6-sulfidopeptide leukotriene receptor, and that stimulation of this receptor results in PGI₂ biosynthesis.     

Phorbol myristate acetate-stimulated release of cyclooxygenase products in rat pleural cells: Derivatization of prostaglandins with 9-anthryldiazomethane for fluorometric determination by high performance liquid chromatography

White cells were collected from the wash of rat pleural cavity after exsanguination. The incubation mixture of the pleural cells with 1 μM phorbol myristate acetate (PMA) was extracted with acidified ethanol and purified with a Sep-pak C18. The resultant fraction containing prostaglandins (PG) and thromboxane (TX) was allowed to react with 9-anthryldiazomethane (ADAM). After removing contaminants and degraded reagent by silica gel Sep-pak, samples were applied to reversed phase high performance liquid chromatography of octadecylsilyl silica gel and monitored by a fluorescent detector. ADAM derivatives of the authentic PGD₂, PGE₂, PGF_2α, 6-keto-PGF_1α, 6-keto-PGE₁, TXB₂, 15-keo-PGE₂, 13, 14-dihydro-15-keto-PGF_2α and 13, 14-dihydro-15-keto-PGE₂ showed linear regression lines of peak heights within a range of 0.5–25 ng.By using this method PGD₂, 6-keto-PGF_1α and TXB₂ were detected in the incubation mixture of the rat pleural cells with PMA. The result clarified the origin of these PGs and TX found in the exudate of rat pleurisy induced by PMA.ADAM method for HPLC with a help of clean-up by Sep-pak could be a useful tool for detection of a series of arachidonate metabolites in biological materials.