Prostanoids and hemostasis in chickens: Anti-aggregating activity of the prostaglandins E1 and E2, but not of prostacyclin and prostaglandin D2

Aggregation of chicken thrombocytes was studied in whole blood using an electronic aggregometer. Serotonin (5-hydroxytryptamine, 5HT), arachidonic acid (AA) and collagen, but not adenosinediphosphate (ADP) induced aggregation. Prostaglandin (PG) endoperoxides were essential for arachidonic acid-induced aggregation, but were not involved in 5HT-induced aggregation, as indicated by inhibitory studies with indomethacin. Similar experiments indicated that biosynthesis of endogenous PG endoperoxides contributed to the aggregation induced by low concentrations of collagen, but was of little importance when high collagen doses were employed. PGE₁ and PGE₂ could abolish all types of aggregation studied, whereas prostacyclin (PGI₂) and PGD₂ were without any anti-aggregatory activity at 1 μg/ml. Between 1 and 100 ng/ml PGE₁ and PGE₂ inhibited arachidonic acid- and 5HT-induced aggregation dose-dependently.The lack of any hemostatic function of PGI₂ in chickens was also indicated by the absence of biosynthesis of endogenous PGI₂ in chicken aorta. PGI₂ was assessed as anti-aggregating activity, released by aortic fragments stirred in rabbit platelet rich plasma. Still, the presence of chicken aortic tissue i chicken whole blood inhibited 5HT-, but not arachidonic acid-induced aggregation. This inhibition was not affected by pretreatment of the aortic fragments with indomethacin or pargyline.     

Limited effect of anti-rheumatic treatment on 15-prostaglandin dehydrogenase in rheumatoid arthritis synovial tissue

Introduction

Rheumatoid arthritis (RA) is a chronic inflammatory disease in which prostaglandin E₂ (PGE₂) displays an important pathogenic role. The enzymes involved in its synthesis are highly expressed in the inflamed synovium, while little is known about 15- prostaglandin dehydrogenase (15-PGDH) that metabolizes PGE₂. Here we aimed to evaluate the localization of 15-PGDH in the synovial tissue of healthy individuals or patients with inflammatory arthritis and determine the influence of common RA therapy on its expression.

Methods

Synovial tissue specimens from healthy individuals, psoriatic arthritis, ostheoarthritis and RA patients were immunohistochemically stained to describe the expression pattern of 15-PGDH. In addition, the degree of enzyme staining was evaluated by computer analysis on stained synovial biopsies from two groups of RA patients, before and after RA specific treatment with either intra-articular glucocorticoids or oral methotrexate therapy. Prostaglandins derived from the cyclooxygenase (COX) pathway were determined by liquid-chromatography mass spectrometry in supernatants from interleukin (IL) 1β-activated fibroblast-like synoviocytes (FLS) treated with methotrexate.

Results

15-PGDH was present in healthy and inflamed synovial tissue, mainly in lining macrophages, fibroblasts and vessels. Intra-articular glucocorticoids showed a trend towards reduced 15-PGDH expression in RA synovium (p = 0.08) while methotrexate treatment left the PGE₂ pathway unaltered both in biopsies ex vivo and in cultured FLS.

Conclusions

Early methotrexate therapy has little influence on the expression of 15-PGDH and on any of the PGE₂ synthesizing enzymes or COX-derived metabolites. Thus therapeutic strategies involving blocking induced PGE₂ synthesis may find a rationale in additionally reducing local inflammatory mediators.     

Prostaglandin E2 synthesis in cartilage explants under compression: mPGES-1 is a mechanosensitive gene

Knee osteoarthritis (OA) results, at least in part, from overloading and inflammation leading to cartilage degradation. Prostaglandin E2 (PGE₂) is one of the main catabolic factors involved in OA. Its synthesis is the result of cyclooxygenase (COX) and prostaglandin E synthase (PGES) activities whereas NAD+-dependent 15 hydroxy prostaglandin dehydrogenase (15-PGDH) is the key enzyme implicated in the catabolism of PGE₂. For both COX and PGES, three isoforms have been described: in cartilage, COX-1 and cytosolic PGES are constitutively expressed whereas COX-2 and microsomal PGES type 1 (mPGES-1) are inducible in an inflammatory context. COX-3 (a variant of COX-1) and mPGES-2 have been recently cloned but little is known about their expression and regulation in cartilage, as is also the case for 15-PGDH. We investigated the regulation of the genes encoding COX and PGES isoforms during mechanical stress applied to cartilage explants. Mouse cartilage explants were subjected to compression (0.5 Hz, 1 MPa) for 2 to 24 hours. After determination of the amount of PGE₂ released in the media (enzyme immunoassay), mRNA and proteins were extracted directly from the cartilage explants and analyzed by real-time RT-PCR and western blotting respectively. Mechanical compression of cartilage explants significantly increased PGE₂ production in a time-dependent manner. This was not due to the synthesis of IL-1, since pretreatment with interleukin 1 receptor antagonist (IL1-Ra) did not alter the PGE₂ synthesis. Interestingly, COX-2 and mPGES-1 mRNA expression significantly increased after 2 hours, in parallel with protein expression, whereas COX-3 and mPGES-2 mRNA expression was not modified. Moreover, we observed a delayed overexpression of 15-PGDH just before the decline of PGE₂ synthesis after 18 hours, suggesting that PGE₂ synthesis could be altered by the induction of 15-PGDH expression. We conclude that, along with COX-2, dynamic compression induces mPGES-1 mRNA and protein expression in cartilage explants. Thus, the mechanosensitive mPGES-1 enzyme represents a potential therapeutic target in osteoarthritis.     

Enhanced formation of PGI2, a potent hypotensive substance, by aortic rings and homogenates of the spontaneously hypertensive rat

Intact rings and homogenates of aorta from spontaneously hypertensive rats (SHR) contain enhanced capacity over normal rats (NR) to convert arachidonic acid into PGI₂. The PGI₂ synthetic system in SHR is stimulated to a greater extent than NR by norepinephrine. Indomethacin blocks this stimulation. PGE₂ and PGF_2α were detected in much smaller amounts in homogenates (undetected in rings) but their formation was not enhanced by the hypertensive tissue. The identity of PGI₂ was based on 1) direct pharmacological assay on the rat blood pressure. In this system identical vasodepressor responses to PGI₂ are observed after intracarotid and intrajugular administration 2) indirectly as 6-keto PGF_1α isolated after incubation of aortic homogenates with tritiated arachidonic acid and 3) indirectly by GC-MS assay of PGE₂, PGF_2α and 6-keto PGF_1α formed during incubation of aortic homogenates with excess unlabeled arachidonic acid. These results provide additional support to our recent hypothesis that PGI₂, of aortic origin, might actively participate in the regulation of systemic blood pressure. Its enhanced formation by intact hypertensive vascular tissue reflects an increase in the number of enzyme molecules immediately available to the substrate. This could probably be an adaptive response to the elevated levels of catecholamines in the circulation.     

A comparison of the pulmonary, renal and hepatic extractions of PGI2 and PGE2 - PGI2 a potential circulating hormone.

Prostaglandin (PG) I₂ and PGE₂ were infused into the aortic arch, femoral vein, renal artery and portal vein in anesthetized dogs over a dose range to produce a steady decrease in systemic blood pressure after 10 mins infusion. Parallel log dose-response relationships were observed with both PGI₂ and PGE₂. PGE₂ was a more potent depressor than PGI₂ when infused into the aortic arch. The doses to reduce blood pressure by 5 mm Hg were used to calculate the extraction of the compounds by the lungs, kidney and liver. The pulmonary extraction of PGE₂ was 96 ± 2% and was essentially complete following combined pulmonary and renal or pulmonary and hepatic extraction. In contrast, there was no significant pulmonary extraction of PGI₂. Combined renal and pulmonary extraction was 43 ± 11% and combined hepatic and pulmonary extraction 87 ± 5%. These results indicate a marked difference in the organ metabolising capacity for PGE₂ and PGI₂. Since PGI₂ has been shown to be produced both in the kidney and stomach it is possible that PGI₂ produced endogenously could pass into the circulation and exert systemic pharmacological effects.     

Prostaglandin I2 has more potent hypotensive properties than prostaglandin E2 in the normal and spontaneously hypertensive rat

The blood pressure lowering effects of PGI₂ in the normal and spontaneously hypertensive rat are described. Comparison of dose response curves for PGI₂ and PGE₂ indicate that PGI₂ is twice as potent as PGE₂ in the normal rat and 3–4 times more active in the spontaneously hypertensive rat. Furthermore PGI₂ is equiactive through intracarotid and intrajugular administration indicative of the complete lack of pulmonary inactivation. These findings supported by evidence of enhanced PGI₂ synthesis in aorta during hypertension support the notion that PGI₂ could participate in blood pressure control mechanisms.     

Increased production of prostacyclin (PGI2) by vascular intimal smooth muscle cells from atherosclerotic rabbit aorta

PGI₂ synthesis by aortic strips obtained from thoracic aorta of rabbits fed a high cholesterol diet was examined and compared with that of control rabbits fed a normal diet. In this report, the amounts of PGI₂ produced were shown as 6-keto-PGF_1α per μg of aortic tissue DNA instead of per mg wet weight. We also investigated PGI₂ synthesis by cultured smooth muscle cells (SMC) obtained from atherosclerotic intima.Basal PGI₂ production by aortic strips from atherosclerotic rabbit aorta was significantly augmented compared with that of controls. Arachidonic acid (AA)-induced PGI₂ production by atherosclerotic aorta was also significantly higher than that of controls. PGI₂ producing capacities of intimal and medial layers, separated from atherosclerotic aorta, were examined and the intimal layer was found to elicit a significantly greater PGI₂ production than the medial layer.Furthermore, cultured intimal SMC obtained from atherosclerotic rabbit aorta produced a greater amount of PGI₂ than medial SMC from normal rabbit aorta at various cultured conditions. These results suggest that the possibility of enhanced PGI₂ production by atherosclerotic aorta may well be considered as a defence mechanism of the vessel wall against damaging stimuli.     

Prostacyclin production of rat aorta “in vitro” is increased by the combined action of dipyridamole plus pentoxifylline

The present study evaluates the effect of dipyridamole and pentoxifylline, individually and in combination, on PGI₂-like production and arachidonic acid metabolism of rat aorta “in vitro”. Pentoxifylline 100 μM and dipyridamole 92 and 184 μM increased PGI₂-like activity, as measured by the platelet aggregation inhibitory capacity of the aortic ring incubates, by 71%, 46% and 60% respectively; a greater increase in PGI₂-like activity was observed with the combination of the drugs than when they were used separately. This effect was observed even at the lowest doses assayed. In fact, dipyridamole 9.2 μM plus pentoxifylline 1 μM increased the PGI₂-like activity by 30% while the individual increase was 4.5% and 10.6% respectively. To obtain more information on the effect of the dipyridamole-pentoxifylline combination on arachidonic acid metabolism, arteries were incubated with (1-¹⁴C) arachidonic acid, and the 6-keto-PGF_1α and PGE₂ quantified. Dipyridamole 92 μM plus pentoxifulline 1 and 10 μM increased 6-keto-PGF_1α and PGE₂ production by about 30% and 48% respectively while combination with pentoxifylline 100 μM increased the 6-keto-PGF_1α 76.5% and the PGE₂ 50%. The possible biological effect and therapeutic implications of increased PGI₂ production by the arteries due to the dipyridamole-pentoxifylline combination remains to be ascertained.     

Effect of 7-fluoro prostacyclin,a stable prostacyclin analogue,on cAMP accumulation and prostaglandin binding in mastocytoma P-815 cells

The effect of 7-fluoro proscyclilin (PGI₂-F), a chemically stable analogue of prostacyclin, on cAMP accumulation in and [³H]PGE binding to mastocytoma P-815 cells was compared with those of the Na salt and methyl ester of prostacyclin (PGI₂Na or PGI₂Me), which are rapidly inactivated in aqueous solution or metabolized in the tissue.PGIF was as effective as PGI₂Me, and slightly less effective than PGI₂Na in stimulating cAMP accumulation in mastocytoma cells and rabbit platelets. PGI₂F was also more stable than PGI₂Me or PGI₂Na, and retained its original cAMP elevating activity even after incubation with or without cells for 4 h at 37°C. Cells which had been exposed to PGI₂F and then washed free of unbound reagent continued to produced cAMP for more than 3 h. PGI₂F was also as effective as PGE₁ or PGE₂ in displacing [³H]PGE₂ bound to the cells. Non-competitive inhibition by PGI₂F or PGI₂Me of [³H]PGE₂ binding to the cells, with apparent K_is of 1.29 μM and 1.13 μM, respectively, indicates the presence of different receptors for PGE₂ and for PGI₂F or PGI₂Me in mastocytoma P-815 cells.     

The wound-healing effect of 7,3′,4′-trimethoxyflavone through increased levels of prostaglandin E<Subscript>2</Subscript> by 15-hydroxyprostaglandin dehydrogenase inhibition

Objective

To find an inhibitor of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) that rapidly metabolises Prostaglandin E₂ (PGE₂) as a mediator of wound healing, we examined seven flavonoids for this role.

Results

7,3′,4′-Trimethoxyflavone (TMF) had the lowest IC₅₀ value of 0.34 µM for 15-PGDH inhibition but >400 µM for cytotoxicity, indicating a high therapeutic index. TMF elevated PGE₂ levels in a concentration-dependent manner in both A549 lung cancer and HaCaT cells. It also significantly increased mRNA expression of multidrug resistance-associated protein 4 (MRP4) and of prostaglandin transporter (PGT) slightly in HaCaT cells. In addition, TMF facilitated in vitro wound healing in a HaCaT scratch model, which was completely inhibited by adding both 15-PGDH and NAD⁺ as cofactor, confirming the involvement of PGE₂ in its wound healing effect.

Conclusion

TMF with a high therapeutic index can facilitate wound healing through PGE₂ elevation by 15-PGDH inhibition.

     

The effects of prostacyclin (PGI2) on tissues which detect prostaglandins (PG'S)

The effects of prostacyclin (PGI₂) and its stable metabolite 6-oxo-PGF_1α on various bioassay tissues are compared with those of PGE₂ and PGF_2α, using the cascade superfusion method. On vascular smooth muscle, PGI₂ caused relaxation of all tissues tested except the rabbit aorta. PGE₂ relaxed rabbit coeliac and mesenteric artery but contracted bovine coronary artery and had no effect on rabbit aorta. 6-oxo-PGF_1α was ineffective at the concentrations tested.On gastro-intestinal smooth muscle, PGI₂ contracted strips of rat and hamster stomach and the chick rectum. It was less potent than PGE₂ or PGF_2α. None of these substances contracted that cat terminal ileum. 6-oxo-PGF_1α was inactive on these tissues at the doses tested.PGI₂ was less active than PGE₂ or PGF_2α in contracting guinea-pig trachea and rat uterus; 6-oxo-PGF_1α was active only on the rat uterus. Thus, PGI₂ can be distinguished from the other stable prostaglandins using the cascade method of superfusion.     

On the multiplicity of platelet prostaglandin receptors I. Evaluation of competitive antagonism by aggregometry

Methods for the evaluation of competitive interactions at receptors associated with platelet activation and inhibition using aggregometry of human PRP have been developed. The evidence supports the suggestion that PGE₁ and PGI₂ share a common receptor for inhibition of platelet reactivity, but only a portion (if any) of the aggregation stimulation associated with PGE₂ is the result of PGE₂ binding (without efficacy) to this receptor. PGE₂ (@.3–20 μ ) is an effective antagonist of PGE₁, PGI₂, producing a shift of about one order of magnitude in the IC₅₀-values obtained from complete aggregation inhibition dose response curves. The antagonism of PGD₂ inhibition is particularly notable, 80 n PGE₂ levels are detectable. This and other actions of PGE₂ indicate another platelet receptor for PGE₂. PGE₁ acts at both the PGE₂ and PGI₂ receptor. Other substances showing PGI₂-like actions only at high doses (1–30 μ ), display additive responses with PGI₂ indicative of decreased affinity for the I₂/E₁ receptor and the absence of PGE₂-like aggregation stimulation activity.PGI₂ methyl ester has intrinsic inhibitory action not associated with in situ ester hydrolysis. The methyl ester is dissaggregatory showing particular specificity for inhibition of release and second wave aggregation.     

Metabolism of prostacyclin. III. Urinary metabolite profile of 6-keto PGF1α in rat

The in vivo metabolism of 6-keto PGF_1α was investigated in rats. Following continuous intravenous infusion for 14 days the urinary metabolites were isolated and identified. A substantial amount of unchanged 6-keto PGF_1α was recovered in the urine. The metabolic pattern very closely resembles that of PGI₂ in rats. Metabolites were found which represented 15-dehydrogenation, β-oxidation, ω and ω-1-hydroxylation and oxidation.Previous work showed that 6-keto PGF_1α is very poorly oxidized by 15-PGDH. We administered 15-[H³]-PGI₂ and 15-[H³]-6-keto PGF_1α to rats and measured urinary tritiated water as an index for in vivo 15-PGDH activity. The results showed that PGI₂ and 6-keto PGF_1α were both oxidized to the 15-keto product, although the rate of oxidation of PGI₂ was greater than that of 6-keto PGF_1α. We concluded that the administered PGI₂ was oxidized by 15-PGDH before hydrolysis to 6-keto PGF_1α. A portion of the dose is probably hydrolyzed before 15-dehydrogenation.     

Reversal of indomethacin-induced inhibition of tubuloglomerular feedback by prostaglandin infusion

Experiments were performed in rats to study the effect of infusion of PGI₂, PGE₂, and PGF_2α on tubuloglomerular feedback responses (i.e. the change of SNGFR in response to a change of loop of Henle flow rate) in the presence and absence of simultaneous inhibition of endogenous PG synthesis with indomethacin. Infusion of PGI₂ or PGE₂ at rates that did not alter arterial blood pressure did not significantly modify the magnitude of feedback responses (PGI₂) 8.5 μg/hr, PGE₂ 85 μg/hr). Some inhibition of feedback responses was seen when PGI₂ and PGE₂ were administered at higher rates were associated with a reduction of blood pressure (PGI₂ 20 μg/hr, PGE₂ 200 μg/hr). PGI₂ (8.5 μg/hr) and PGE₂ (85 μg/hr) largely prevented feedback inhibition induced by indomethacin. When given subsequent to indomethacin PGI₂ and PGE₂ restored feedback responsiveness almost to normal. In contrast, PGF_2α did not influence feedback inhibition caused by indomethacin. Infusion of PGI₂ induced partial restoration of feedback responses in DOCA-salt treated animals in which the feedback system is virtually completely inactive. Our results indicate that availability of PGI₂ or PGE₂ is necessary for the normal operation of the tubuloglomerular feedback mechanism for control of nephron filtration rate.     

Regulation of pancreatic β-cell function and mass dynamics by prostaglandin signaling

Prostaglandins (PGs) are signaling lipids derived from arachidonic acid (AA), which is metabolized by cyclooxygenase (COX)-1 or 2 and class-specific synthases to generate PGD₂, PGE₂, PGF_2α, PGI₂ (prostacyclin), and thromboxane A₂. PGs signal through G-protein coupled receptors (GPCRs) and are important modulators of an array of physiological functions, including systemic inflammation and insulin secretion from pancreatic islets. The role of PGs in β-cell function has been an active area of interest, beginning in the 1970s. Early studies demonstrated that PGE₂ inhibits glucose-stimulated insulin secretion (GSIS), although more recent studies have questioned this inhibitory action of PGE₂. The PGE₂ receptor EP3 and one of the G-proteins that couples to EP3, Gα_Z, have been identified as negative regulators of β-cell proliferation and survival. Conversely, PGI₂ and its receptor, IP, play a positive role in the β-cell by enhancing GSIS and preserving β-cell mass in response to the β-cell toxin streptozotocin (STZ). In comparison to PGE₂ and PGI₂, little is known about the function of the remaining PGs within islets. In this review, we discuss the roles of PGs, particularly PGE₂ and PGI₂, PG receptors, and downstream signaling events that alter β-cell function and regulation of β-cell mass.     

Control of the intracellular levels of prostaglandin E2 through inhibition of the 15-hydroxyprostaglandin dehydrogenase for wound healing

Excessive scar formation is an aberrant form of wound healing and is an indication of an exaggerated function of fibroblasts and excess accumulation of extracellular matrix during wound healing. Much experimental data suggests that prostaglandin E₂ (PGE₂) plays a role in the prevention of excessive scarring. However, it has a very short half-live in blood, its oxidization to 15-ketoprostaglandins is catalyzed by 15-hydroxyprostaglandin dehydrogenase (15-PGDH). Previously, we reported that 15-PGDH inhibitors significantly increased PGE₂ levels in A549 cells. In our continuing attempts to develop highly potent 15-PGDH inhibitors, we newly synthesized various thiazolidine-2,4-dione derivatives. Compound 27, 28, 29, and 30 demonstrated IC₅₀ values of 0.048, 0.020, 0.038 and 0.048 μM, respectively. They also increased levels of PGE₂ in A549 cells. Especially, compound 28 significantly increased level of PGE₂ at 260 pg/mL, which was approximately fivefold higher than that of control. Scratch wounds were analyzed in confluent monolayers of HaCaT cells. Cells exposed to compound 28 showed significantly improved wound healing with respect to control.     

Endogenous factors affecting arachidonic acid metabolism: I. Biosynthesis of prostacyclin and prostaglandins by carrageenin granulomas of rats

Arachidonic acid was converted by incubated slices of the rat carrageenin granuloma to prostacyclin (PGI₂), prostaglandins (PGs) E₂ and F_2∞ as detected by bioassay and radiochemical assay. PGI₂ was the major product of arachidonic acid metabolism in the granuloma slices. PGI₂ and PGE₂ formation was dependent on the concentration of the substrate and on the age of the granuloma. Slices obtained from 5-day old granulomas produced significantly more PGI₂ than slices prepared from 3-day old or 8- to 9-day old granulomas while PGE₂ generation was not dependent on the stage of the development of the granuloma. Homogenates of granuloma tissue hardly converted arachidonic acid to PGI₂ at all. This was probably due to the presence of an non- dialysable and heat labile material which, when partially isolated, inhibited PGI₂ production by bovine aortic microsomes.     

Prostaglandin I2 stimulation of granulosa cell cyclic AMP production

The ability of prostaglandin I₂ (PGI₂) to stimulate cyclic AMP production by granulosa cells, isolated from intact immature rats, has been demonstrated in vitro. The minimal effective dose was 15 ng/ml, which was comparable to the minimal effective dose for PGE₂. However, a concentration of 15 μg/ml PGI₂ was required to stimulate cyclic AMP production maximally, compared to a concentration of 1 μg/ml PGE₂, which produced the maximum response. It therefore appears that PGI₂ is not more effective than PGE₂ in stimulating cyclic AMP production in granulosa cells, and is possibly less effective. Submaximal concentrations of PGI₂ appeared to be able to modify the stimulation of cyclic AMP production by follicle- stimulating hormone (FSH), but whether or not PGI₂ plays any role in follicular function remains to be established.