Prostacyclin biosynthesis in activated, stimulated and normal mouse peritoneal cell populations.

Nonspecific resistance to infectious and neoplastic disease can be enhanced by administration of "immunomodulators". The levels of enhancement can be monitored by following in vitro function of cells of the lympho-reticuloendothelial system. To gain a better understanding of the physiological and biochemical nature of this enhancement, the metabolism of prostaglandin endoperoxide PGH2 was followed in mouse peritoneal cells (PCs). Homogenates of PCs from normal, unstimulated mice yielded primarily prostacyclin (PGI2) when incubated with PGH2. Homogenates of PCs from mice injected with the immunomodulators C. parvum, levamisole HCl, pyran copolymer, or thioglycollate yielded less PGI2. Reductions ranged from 73% for C. parvum to 32% for levamisole. A statistically significant inverse correlation existed between the level of macrophage "activation" and ability of cellular homogenates to produce prostacyclin. The results suggest that prostacyclin may be involved in modulation of nonspecific resistance.     

Conversions of prostaglandin endoperoxides by prostacyclin synthase from pig aorta

Partially purified prostacyclin synthase from pig aorta converted the prostaglandin (PG) endoperoxide PGH₂ to prostacyclin (PGI₂), and PGH₁ to 12-hydroxy-8,10-heptadecadienoic acid (HHD). Both reactions were inhibited by 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HP) in a dose-dependent fashion. However, the reactions PGH₂ → PGI₂ and PGH₁ → HHD appeared to differ: substrate availability was rate limiting in the latter reaction, while the enzyme became rapidly saturated with PGH₂ and a steady rate of prostacyclin formation was observed at higher substrate levels.     

Prostacyclin stimulation of dog arterial cyclic AMP levels

Prostacyclin (PGI₂) dose-dependently increases the adenosine 3′,5′-cyclic monophosphate (cyclic AMP) levels in canine femoral, carotid, and canine and bovine coronary arteries. The prostacyclin-stimulation is enhanced by phosphodiesterase inhibitors, and is readily measurable after 60 sec incubation. The prostaglandin endoperoxide PGH₂, but not PGH₁, also elevates cAMP levels in femoral arteries. Inhibition of arterial prostacyclin synthetase with 28 μM 9,11-azoprosta-5,13-dienoic acid (azo analog I) blocks the PGH₂-stimulation of cAMP accumulation. Azo analog I does not attenuate a direct PGI₂ stimulation, indicating that the PGH₂ dependent elevation of cAMP is due to conversion of PGH₂ to PGI₂ by the artery. PGI₂ and PGE₁ increase cyclic AMP levels and relax dog femoral and bovine coronary arteries, while PGE₂, which actually contracts bovine coronary arteries, has no effect on arterial cyclic AMP levels. The significance of the PGI₂-stimulation of arterial cyclic AMP is not known, but it is probably related to relaxation of arterial strips.     

PGI2 production by rat blood vessels: Diminished prostacyclin formation in veins compared to arteries

Homogenates of eleven different blood vessels from normal Sprague-Dawley rats varied in their ability to produce PGI₂ (i.e., 6-keto-PGF_1α) from [1−¹⁴C]PGH₂. The most notable difference was seen between arteries and veins. Arterial tissues produced more 6-keto-PGF_1α from exogenous PGH₂ than veins at all enzyme (i.e., protein) concentrations tested. Similar results were obtained utilizing different homogenization techniques or arterial and venous rings, indicating this difference was real and not due to homogenization artifacts. In addition, the thoracic segment of the inferior vena cava was more active in converting added [1−¹⁴C]PGH₂ to 6-keto-PGF_1α than the abdominal segment of added inferior vena cava suggestive of a possible segmental distribution of the enzyme activity in blood vessels. These results may be interpreted as indicating that PGI₂ may have a vasomotor function for blood vessels in addition to its proposed antithrombotic role.     

The effect of radiation on prostacycliln (PG2) productnio by cultured endothelial cells

The effect of ionizing irradiation on the synthesis of prostacyclin (PGI₂) by cult8erd bovine aortic endothelial cells was detemined. PGI₂ was measuured in the culture medium by a radioimmunoassay for 6-Kto PGF_1α. Two phenomena were observed following irradiation: a) Cells which suffered an immediate radiation damage (1000–5000 rads) released high quantities of PGI₂ to the culture medium. This was due to a de novo synthesis of PGI₂ stimulated by radiation induced cellular damage, since pretreatment with aspirin of the endothelial cell monolayers resulted in a marked inhibition of PGI₂ release following irradiation. b) Metabolically active cells which remained confluent and firmly attaached to the culture dish following single, low and intermediate doses (200–1200 rads) radiation, exhibited a marked decreased in their capacity to synthesize PGI₂ upon exposure to various stimuli of the archidonic acid cascade (arachidonic acid, melittin, ionophore A23187 and PGH₂. Similar results were observed with cells treated with fractionated radiation.The quantities of PGI₂ produced by the endothelial cells decreased as a function of the dose of radiation and time interval between irradiation and subsequent stimulation. Radiation had a minimal effect on the nonthrombogenic properties of the endothelial cells, as evidenced by the small increase in the platelet adherence to the endothelial cells. The effect of radiation on PGI₂ pruction by the vascular endothelium may be relevant to the development of radiation induced capillary occlusions, and the enhancement of atherosclerotic lesions in large vesses.     

The major product of PGG2 metabolism by aortic microsomes is 6, 15-dioxo-PGF1α

Incubations of PGG₂ with aortic microsomes yielded two products which were not formed in boiled enzyme control, one of which was 6-oxo-PGF_1α. The major metabolite was identified by gas-liquid chromatography-mass spectrometry as 6,15-dioxo-PGF_1α. Thus, unlike PGH₂, PGG₂ is probably converted to 15-hydroperoxy PGI₂ which subsequently decomposes to 6,15-dioxo-PGF_1α.     

Differential effects of ibuprofen,indomethacin, and meclofenamate on prostaglandin endoperoxide H2 metabolism

Summary Previous studies have suggested the possibility that the non-steroidal antiflammatory drug (NSAID), ibuprofen, may inhibit thromboxane (TX) A₂ synthase activity in addition to inhibiting cyclooxygenae activity. Microsomal fractions isolated from the cat lung contain cyclooxygenase as well as prostacyclin (PGI₂) synthase, TX synthase, and a GSH-dependent prostaglandin (PG) E₂ isomerase activities. When [1-¹⁴C] PG endoperoxide H₂ (PGH₂) was used as substrate, ibuprofen, indomethacin, and meclofenamate exhibited differential effects on terminal enzyme activities. Ibuprofen, at concentrations up to 1 mM, had no effect on the activities of PGI₂ synthase, TXA₂ synthase of GSH-dependent PGE₂ isomerase, whereas indomethacin selectively inhibited PGI₂ synthase activity at 5 x 10^–4 M and 10^–3 M. Meclofenamate selectively inhibited TXA₂ synthase activity at 5 x 10^–4 M and 10^–3 M. At concentrations of 5 x 10^–3 M, this selectivity was not oberved, and indomethacin and meclofenamate decreased the formation of both 6-keto-PGF₁ and TXB₂. These data indicate that the choice of NSAID and the concentration employed may specifically alter PGH₂ metabolism. This action may affect the physiologic consequences of the exchange of PGH₂ between cells. The data further indicate that indomethacin has the potential for use as a tool to specifically attenuate PGI₂ synthase activity in vitro.     

A comparison of the effects of PGI2, PGE2 and PGH2 on the cyclic nucleotide levels in rat anterior pituitary glands in vitro

Rat anterior pituitary explants were incubated with PGI₂, PGH₂ and PGE₂ in the presence of theophylline (1mM) and the production of cyclic AMP was measured. PGE₂ was found to be about 20 times more potent than PGI₂ while PGH₂ was slightly more effective than PGI₂. The results suggest that PGI₂ does not play a physiological role in cyclic AMP mediated events in the rat anterior pituitary.     

A comparison of the effects of PGI2, PGE2 and PGH2 on the cyclic nucleotide levels in rat anterior pituitary glands

Rat anterior pituitary explants were incubated with PGI₂, PGH₂ and PGE₂ in the presence of theophylline (1mM) and the production of cyclic AMP was measured. PGE₂ was found to be about 20 times more potent than PGI₂ while PGH₂ was slightly more effective than PGI₂. The results suggest that PGI₂ does not play a physiological role in cyclic AMP mediated events in the rat anterior pituitary.     

Microsomal preparations of normal bovine iris-ciliary body generate prostacyclin-like but not thromboxane-A2-like activity

Indomethacin-treated bovine iris-ciliary body microsomes (IBIM) have been studied for their ability to convert PG endoperoxides into either thromboxance-A₂ (TxA₂)-like or prostacyclin (PGI₂)-like activity. The biological activity of the ocular tissue microsomes were compared with either indomethacin-treated human platelet microsomes (for TxA₂-like activity) or rabbit aorta microsomes (for PGI₂-like activity) under appropriate incubation conditions. No evidence could be found for the formation of TxA₂-like activity from PG endoperoxides by the IBIM. In contrasts, when the IBIM were incubated with PGH₂ for 1 min at 22°C without cofactors, PGI₂-like activity was produced, causing profound relaxation of the isolated dog coronary artery preparation without contracting the rabbit aorta and inhibiting arachidonic acid-induced platelet aggregation. Equivalent quantities of boiled IBIM failed to aleter the biological activity of PGH₂ under identical conditions. Tranylcypromine (500 μg/ml) completely abolished the appearance of PGI₂-like activity. Furthermore, the PGI₂-like activity found was stable for 10 min at 22°C at pH 8.5 but completely lost under similar conditions at pH 5.5. It is concluded than microsomal preparations of normal bovine iris-ciliary body can synthesize PGI₂-like activity in substantial amounts but not TxA₂-like activity.     

Increased platelet activity after termination of prostacyclin infusion into man

Infusion of PGI₂ at a dose of 5 or 10 ng/kg/min during 72 hours into patients with peripheral vascular disease was followed by increased susceptibility of platelets to proaggregatory action of ADP and collagen but not that of arachidonate. The above effects were observed 24 hours after termination of infusion of PGI₂. A tendency to an increased formation of TXA₂ in PRP aggregated by arachidonate was also noticed. Infusion of PGI₂ at a dose of 2 mg/kg/min during 72 hours into the patients caused the decreased platelt aggregability to ADP and arachidonate but not to collagen, and a decreased tendency of production of TXA₂ in PRP aggregated by arachidonate. The existence of a “rebound effect” in platelets after a long term PGI₂ therapy is suggested.     

Prostacycin production by arterialized autogenous venous grafts in dogs

Arteries are capable of producing significantly larger quantities of protacyclin than are veins. To test the hypothesis, whether prostacyclin production by the vessel wall is related to blood pressure and flow, we measured the amounts of PGI₂ released and synthesized by venous segments transplanted for 6 weeks into the arterial circulation. These results were compared with the production of prostacyclin by normal veins and arteries. In 20 dogs a segment of jugular vein was interposed into the carotid system; a sham dissection was done on the opposite side. “Arterialized” vein grafts showed prominent intima lined by endothelium, medial smooth muscle cell proliferation and fibrotic proliferation in adventitia. Spontaneous and arachidonic acid- stimulated prostacyclin production (measured by radioimmunoassay for 6-keto-PGF_1α) was not significantly different between arterialized venous autografts and jugular veins. Significantly larger amounts of prostacyclin were synthesized by the carotid artery. Thus, histologic changes and rheologic effects occurring in vein grafts transposed to the arterial site do not affect prostacyclin production.     

Thromboxane synthase inhibition: Implications for prostaglandin endoperoxide metabolism: I Characterisation of an acute intravenous challenge model to measure prostaglandin endoperoxide metabolism

It has been proposed that thromboxane synthase inhibition (TXSI) may be a useful form of anti-thrombotic therapy and that this is due, in part, to redirection of PGH₂ metabolism in favour of PGI₂, a potent vasodilator and anti-platelet agent. While redirection has been observed there are conflicting reports of its occurrence . We now describe the characterisation of an acute intravenous challenge model using thrombin, collagen, arachidonic acid (AA) and PGH₂ for the study of PGH₂ metabolism. Following challenge, plasma concentrations of TXB₂, 6-oxo-PGF_1α, alleged metabolites of PGI₂ (PGI₂m) and PGE₂ were measured by radioimmunoassay (RIA). Thrombin and collagen challenge resulted in a dose-related increase in plasma TXB₂ while AA and PGH₂, in addition, elevated 6-oxo-PGF_1α and PGI₂m. Injection of PGH₂ elevated 6-oxo-PGF_1α, PGI₂m, TXB₂ and PGE₂ levels. Experimental conditions were defined such that challenge with thrombin (40 NIH units kg⁻¹), collagen (100 kg⁻¹), AA (1mg kg⁻¹) and PGH₂ (5μg kg⁻¹) and measurement of eicosanoids 0.5min following challenge (5μg kg⁻¹) and measurement of eicosanoids 0.5min following challenge were optimal for detection of redirection of PGH₂ metabolism . The identity of immunoreactive TXB₂ and 6-oxo-PGF_1α was further supported by experiments in which the extracted immunoreactive eicosanoids co-eluted with authentic [³H]standards when subject to reverse phase high performance liquid chromatography (RPHPLC). Evidence is also presented that the levels of plasma eicosanoids measured in this model reflect biosynthesis.     

Effects of all CIS-5, 8,11,14,17 eicosapentaenoic acid and PGH3 on platelet aggregation

In human platelet-rich plasma (PRP) eicosapentaenoic acid (EPA) inhibited platelet aggregation induced by a stable analogue of PGH₂ (U46619), arachidonic acid, collagen or ADP. EPA was more potent than oleic, linoleic, α-linolenic or γ-linolenic acids. In aspirin-treated platelets, aggregation induced by U46619 was inhibited to a similar extent by arachidonic acid or by EPA over a range of concentrations of 0.05–0.3 mM. EPA incubated with PRP did not induce the generation of a thromboxane (TXA)-like activity; indeed it prevented the formation of TXA₂ induced by arachidonic acid or by collagen. The anti-aggregatory activity of EPA was not influenced by inhibitors of cyclo-oxygenase and lipoxygenase. The anti-aggregatory action of EPA may be caused by a rapid occupancy by EPA of TXA₂/PGH₂ “receptors” on platelet membrane as well as by a slower displacement of arachidonic acid from platelet phospholipids by chemically unchanged molecules of EPA.Not all samples of PRP were irreversibly aggregated by PGH₂, but in those that were, PGH₃ also induced an immediate dose-dependent but reversible aggregation. After a 4 min incubation of non-aggregating doses of PGH₂ or PGH₃ (100–300 nM) with PRP a stable anti-aggregatory compound was detected. The inhibitory activity produced from PGH₃ was apparently more potent (ca 10 times) than that obtained from PGH₂. The anti-aggregating compounds were identified by TLC and GLC-MS as PGD₂ and PGD₃. The apparent difference of potency between PGD₂ and PGD₃ was attributed to the concurrent production of PGE₂ and PGE₃. PGE₂ prevented the inhibitory effect of PGD₂ whereas PGE₃ did not affect the activity of PGD₃.It is concluded that one of the reasons for the low incidence of myocardial infarction in Eskimos could be that the pro-aggregatory arachidonic acid is replaced in their phospholipids by the anti-aggregatory EPA.     

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.     

Actions of prostacyclin (PGI2) and its product, 6-oxo-PGF1α on the rat gastric mucosa in vivo and in vitro

The effects of prostacyclin (PGI₂) and its breakdown product 6-oxo-PGF_1α on various aspects of gastric function were investigated in the rat. PGI₂ increased mucosal blood flow when infused intravenously. PGI₂ was a more potent inhibitor of gastric acid secretion in vivo than PGE₂. Like PGE₂, PGI₂ inhibited acid secretion from the rat stomach in vitro. PGI₂ had comparable activity to PGE₂ in inhibiting indomethacin-induced gastric erosions. Thus prostacyclin shares several of the activities of PGE₂, and may be involved in the regulation of gastric mucosal function.     

The human placental anti-aggregating factor is neither prostacyclin, nor a prostacyclin metabolite

Using PGH₂ as substrate, we have previously demonstrated that human placenta synthetizes mainly PGE₂, TxB₂ and PGD₂(1,2). Other reports have shown that placental tissue generates a substance which inhibits ADP-induced platelet aggregation and which was supposed to be PGI₂ (3). The present study indicates that the stability of that substance is different from the stability of prostacyclin (released by umbilical artery pieces). By GC-MS and multiple ion-monitoring, we have shown the presence of 6 keto-PGF_1α (the stable metabolite of PGI₂) in the umbilical artery incubation medium, while no trace of 6-keto-PGF_1α could be found in the placental medium. No conversion of AA to 6-keto-PGF_1α by placental microsomes was observed, even in the presence of antioxidants. The placenta possesses, in addition to the known 15-OH-PGDH and Δ-13 reductase activities, a weak 9 OH pGDH which is specific for PGF_2α (and not PGI₂ nor 6-keto-PGF_1α). GC-MS analysis is showed that the expected metabolites of PGI₂ through those three enzymes were not found in the placental medium, indicating that neither PGI₂ synthesis nor metabolism could be demonstrated in the placenta.     

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.     

Some new prospects in the mechanism of control of arachidonate metabolism in human placenta and amnion

The conversion of (1-¹⁴C) PGH₂ was studied in human placental and fetal membrane cellular preparations (tissue fragments, homogenate, cytosol, microsomes). Placental and amnion homogenates convert labelled PGH₂ into PGE₂ through a very active PGE₂ isomerase. However isolated placental microsomes do not metabolise PGH₂ into PGE₂ but into T×A₂ (identified as T×B₂ by GC-MS) and presumably 12-HHT. This microsomal T×A₂ synthetase is not active in the whole tissue nor in the homogenate. Placental cytosol gives mainly PGD₂. No conversion into PGI₂ (identofied as 6 keto PGF_1α) nor PGF_2α was observed in any fraction.Some aspects of PG synthesis regulation by the placental cytosol were studied: the cytosol contains a heat-stable factor that inhibits T×A₂ synthesis and shifts PGH₂ placental microsome metabolism towards PGE₂. In addition the placental cytosol inhibits human platelet-aggregation through a heat-labile factor which is not PGI₂ nor PGD₂. A multiple step regulation of the various PG metabolites synthetised from arachidonic acid in the placenta can be outlined and its physiological implications are discussed.     

Metabolism of prostaglandin endoperoxide in animal tissues

The endoperoxide PGH₂ serves as a common intermediate for the enzymatic production of prostaglandins (PGEs and PGFs), thromboxanes (Tx) and prostacyclin (PGI₂). These compounds have quite different physiological activities and apparently perform important regulatory functions in various tissues and organs. We have obtained information on the distribution of individual enzymes responsible for the bioconversion of PGH₂ into these compounds in various tissue preparations. [1-C¹⁴] PGH₂ was incubated with a membrane fraction from each tissue homogenate. The products were isolated and identified by radiometric TLC and gas chromatography-mass spectrometry. Short life intermediates were detected by their specific biological activities. With this approach, we have demonstrated the formation of thromboxanes in rhesus monkey platelets, spleen and bone marrow, guinea pig lung and spleen, rabbit lung, human platelets and thioglycollate stimulated peritoneal macrophage from rat. On the other hand, the membrane preparation of bovine and mare corpus luteum, uteri from rabbit, monkey and human, rat stomach and small intestine, and rabbit lung produced predominantly prostacyclin. In addition, a PGH₂ to PGD₂ isomerase was found in the homogenate of rat brain and polymorphonuclear leukocytes. In those tissues which possess more than one enzyme catalyzing the metabolism of prostaglandin endoperoxide, substrate availability appeared to be one factor controlling the metabolic fate of the endoperoxide. The wide occurrence of thromboxane and prostacyclin synthetases suggests that their biological roles are not limited to the cardiovascular system.