Angiotensin II: A releaser for PGI2 from fetal and newborn rabbit lungs

Angiotensin II (AII) induces generation of prostacyclin (PGI₂) in rabbit lung in vitro at different ages, i.e., fetus, newborn and adult. Particularly, neonatal rabbit lungs display a pattern of sensitivity to AII in producing PGI₂, measured as 6-oxo-PGF_1α, which seems to be higher than that observed in fetal lungs. The PGI₂ release appears to be specific for AII stimulation since the vasoconstriction induced by noradrenaline and ergotamine was not associated with generation of this lipidic material. The inability of PGI₂ to relax the extralobar pulmonary artery in the newborn suggests that the lung microcirculation is the most likely site where the vasodilating and antiaggregatory functions of PGI₂ may have a physiological role.     

Atherosclerosis decreased prostacyclin formation in rabbit lungs and kidneys

Metabolism of arachidonic acid (AA) was studied in perfused lungs and kidneys of normal and atherosclerotic rabbits by determination of PGE₂, PGF_2α and the stable metabolites of PGI₂ (6-keto-PGF_1α) and TXA₂ (TXB₂). PGI₂ was the main AA metabolite formed by normal lungs and kidneys. Atherosclerosis reduced the formation of PGI₂ by about 50 % in both organs. TXA₂ formation was similarily decreased in lungs. In kidneys, the decrease in PGI₂ formation was accompanied by an increase in PGE₂ formation.     

Mass-spectrometric evidence for spontaneous and angiotensin-induced generation of prostacyclin by perfused cat lungs

Isolated perfused cat lungs secreted spontaneously a PGI₂-like substance that relaxed a strip of bovine coronary artery. The presence of PGI₂ was confirmed by the identification of 6-oxo-PGF_α by GC-MS. Both bioassay and mass fragmentography showed that PGI₂ was released at a rate of 4 – 12 ng/ml. Generation of PGI₂ by the perfused cat lungs was stimulated by angiotensin II (3 – 10 ng/ml). The above results along with our $\text{in vivo}$ experiments point to the lung as an important source of endogenous PGI₂ in the body.     

The effects of a 5-lipoxygenase inhibitor, AA-861, on PGI2-like substance production in guinea-pig lungs

To determine the effects of AA-861 on PGI₂ production in guinea-pig lungs, 3 g of guinea-pig lung was chopped in 4 ml of buffer (control group), in buffer with 4 μg/ml indomethacin (indomethacin group) and in buffer with 2.5 × 10⁻⁵M AA-861 (AA-861 group). The chopped lungs were incubated for 30 min. 250 μl of incubation medium from each group was assessed before and after 3, 5, 10, 15, 20, 25 and 30 min of incubation. The incubation medium was centrifuged and the supernatant was tested for a PGI₂-like substance (PGI₂) by platelet aggregation inhibition. PGI₂ was produced mainly during the initial 3–5 min of incubation and was decreased thereafter. PGI₂ production was almost completely inhibited in the indomethacin group at all of the incubation times and was partially inhibited in the AA-861 group during the initial 3–5 minutes. Endogenous 5-lipoxygenase products generated in the early stages of incubation seem to be involved in PGI₂ production in guinea-pig lungs.     

Low prostacyclin synthetase activity of fetal rat aorta. Progressive increase after birth.

Aortae from fetal or 3 weeks old rats produced very small amounts of PGI₂, prostacyclin. This production increased from 4 weeks on, reaching adult values at about ten weeks. This maturation seemed to be predominantly determined by a change in the PGI₂ synthetase system, rather than in arachidonic acid availability, phospholipase or cyclo-oxygenase activity. The anti-oxidant ascorbic acid stimulated prostacyclin production more strongly in adult than in young rat aortae. This finding suggests that the lower production of PGI₂ by young tissues is not due to an enhanced inhibition of prostacyclin synthetase by lipid peroxides.     

Regulation of histamine-mediated prostacyclin synthesis in cultured human vascular endothelial cells

Histamine stimulates the production of prostacyclin (PGI₂) in cultured human endothelial cells. We have examined the cell specificity of histamine-mediated PGI₂ synthesis in primary and subcultured human cells. Venous and arterial smooth muscle cells and skin fibroblasts synthesized PGI₂ from exogenous arachidonic acid, but they did not synthesize a significant amount of PGI₂ when treated with histamine. Endothelial cells, however, produced similar amounts of PGI₂ in response to histamine and arachidonic acid. Thrombin also stimulates PGI₂ production in endothelial cells. Histamine and thrombin yielded an additive production of PGI₂ when added simultaneously to endothelial cells. When histamine and thrombin were added sequentially, the amount of PGI₂ produced was not additive but equaled the amount characteristic of the first agonist alone. Following an initial treatment with histamine, endothelial cells were unable to respond to histamine for 3 hr, after which the PGI₂ biosynthetic response rapidly returned to normal by $\text{4}\text{1}\text{2}$ hr. When the initial histamine treatment was carried out under mildly alkaline conditions, the complete return of activity was delayed to 8 hr after treatment. The synthesis of PGI₂ from exogenous arachidonic acid was unaffected by prior treatment with histamine. Recovery of histamine-mediated PGI₂ production was not dependent on protein synthesis but required a component of fetal calf serum that is nondialyzable and moderately heat stable. Thus endothelial cell PGI₂ synthesis in response to a physiologic agonist is subject to several levels of regulation, reflecting not only intracellular events but also the extracellular environment.     

Prostaglandin synthesis by fetal rat bone in vitro: Evidence for a role of prostacyclin

Prostaglandin synthesis by fetal rat bones was examined by thin-layer chromatography of culture media after preincubation with labeled arachidonic acid. Cultures in rabbit complement (non-heat inactivated serum) were compared with cultures in heat-inactivated serum or cultures treated with indomethacin. The major complement-dependent products were PGE₂, PGF_2α and 6-keto-PGF_1α, the metabolite of prostacyclin (PGI₂). Since PGI₂ had not been previously identified in bone its ability to stimulate bone resorption was tested. Repeated addition of PGI₂ stimulated release of previously incorporated ⁴⁵Ca from fetal rat long bones in both short-term and long-term cultures at concentrations of 10⁻⁵ to 10⁻⁹M. Because of the short half life of PGI₂ in solution at neutral pH, we tested a sulfur analog, thiaprostacyclin (S-PGI₂) which was found to be a stimulator of bone resorption at concentrations of 10⁻⁵ to 10⁻⁶M. These studies suggest that endogenous PGI₂ production may play a role in bone metabolism. Since vessels produce PGI₂ it is possible that PGI₂ release may be responsible for the frequent association between vascular invasion and resorption of bone or calcified cartilage in physiologic remodeling and pathologic osteolysis.     

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.     

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.     

The metabolism of prostaglandin E2 in perinatal rabbit lungs

The inactivation of prostaglandin E₂ (PGE₂) was studied in isolated perfused lungs of fetal and neonatal rabbits. 200 nmol of ¹⁴C-PGE₂ was infused into the pulmonary circulation and the metabolites of PGE₂ were analysed from the nonrecirculating perfusion effluent. The amount of the main metabolite, 13,14-dihydro-15-keto-PGE₂, increased significantly between the 28th and 30th day of fetal life, remained relatively constant at the time of birth and increased again between 1st and 7th postnatal day. In contrast the amount of 15-keto-PGE₂ remained relatively stable during the studied period. The activity of NAD⁺-dependent 15-hydroxyprostaglandin dehydrogenase (15-OH-PGDH) was determined from the 100.000 g supernatant fraction of fetal, neonatal and maternal rabbit lungs using ¹⁴C-PGE₂ (20 μM) as the substrate. In the lungs of late fetal rabbits the activity of 15-OH-PGDH was significantly higher compared to the early postnatal period. Maternal rabbit lungs possessed, however, very high activities compared to the studied perinatal lungs. The results show, that the activity of the pulmonary 15-OH-PGDH is high already during the late fetal period. The inactivation of PGE₂ in isolated perfused lungs seems, however, to increase during the last prenatal days. Thus it seems possible that the uptake mechanism could be the rate limiting step in the metabolism of PGE₂ in rabbit lungs during the perinatal period.     

The metabolism of arachidonic acid to an antiaggregatory compound in isolated lungs of fetal and neonatal rabbits

The developmental pattern of fetal and neonatal rabbit lungs to generate an antiaggregatory compound from arachidonic acid (AA) was studied in isolated rabbit lungs, which were perfused with Krebs bicarbonate buffer. The antiaggregatory effect of the nonrecirculating perfussion effluent was tested by adding a small portion of the effluent to human platelet rich plasma (PRP) in a Born-type aggregometer before the aggregation was induced by ADP. The production of an antiaggregatory compound was minimal, when exogenous AA was not infused into the pulmonary circulation. When arachidonate (40 nmol/min) was infused into the pulmonary circulation of rabbits which were 1 day or 1 week old, the perfusion effluent significantly inhibited the ADP induced aggregation of PRP. Perfused lungs from fetal rabbits (gestation age 28–31 days) formed also an antiaggregatory compound fro AA, but the antiaggregatory effect was not as great as 1 day after birth. It seems that neonatal rabbit lungs metabolize AA more to an antiaggregatory compound than late fetal lungs. The fact that the AA induced production of an antiaggregatory compound is inhibited by simultaneous infusion of indomethacin favours the hypothesis that this antiaggregatory compound could he PGI₂.     

Effects of nitrogen dioxide exposure on prostacyclin synthesis in lung and thromboxane A2 synthesis in platelets in rats

Effects of nitrogen dioxide (NO₂) exposure on prostacyclin (PGIP₂) synthesis in the rat lung and thromboxane A₂ (TXA₂) synthesis in the platelets were studied. Male Wistar rats were exposed to 10 ppm NO₂ for 1, 3, 5, 7 and 14 days. PGI₂ synthesizing activity of homogenized lung decreased. The damage of PGI₂ synthesizing activity reaches its maximum at 3 days. At 14 days, PGI₂ synthesizing activity returned to the normal level. The activity of PGI₂ synthetase decreased significantly. The formation of lipid peroxides due to NO₂ exposure may cause the depression of PGI₂ synthesizing activity of lung. On the other hand, platelet TXA₂ synthesizing activity increased. This increased TXA₂ synthesizing activity lasted at least till 3 days. Then, it returned to the normal level. The counts of platelet were decreased significantly by 1, 3, 5 and 7 days NO₂ exposure. Then the decreased counts of platelet returned to the normal level at 14 days NO₂ exposure. These results indicate that the depression of PGI₂ synthesizing activity lung by NO₂ exposure cause an increase in TXA₂ synthesizing activity of platelets. It may contribute to induce platelet aggregation and to the observed decrease in the number of platelets during NO₂ exposure.     

Formation of prostacyclin (PGI2) by the ductus arteriosus of fetal lambs at different stages of gestation

Prostaglandins appear to play a role in maintaining patency of the ductus arteriosus during gestation. Prostacyclin (PGI₂) is the major product of prostaglandin biosynthesis in the lamb ductus arteriosus. This factor is both a vasodilator and a potent inhibitor of human platelet aggregation. We used inhibition of platelet aggregation as a sensitive bioassay to measure PGI₂ generation in rings of ductus arteriosus from fetal lambs. Mechanical manipulation accelerated the rate of PGI₂ released from the tissue 10 to 50 times. Tranylcypromine, an antagonist of prostacyclin synthetase, suppressed production of PGI₂ by rings of ductus arteriosus. Rings from immature animals (98–103 days gestation, term is 150 days) released significantly more PGI₂ (190 ± 28 ng/g wet weight/ 20 min, n=9) than did those from near term animals (136–146 days; 106 ± 23 ng/g wet weight/20 min, n=10). The capacity of the ductus arteriosus to generate more PGI₂ earlier in gestation is consistent with the observation that vessels from animals less than 110 days gestation have a significantly larger indomethacin induced contraction than do vessels near term.     

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.     

Comparison of gastric and intestinal antisecretory and protective effects of prostacyclin and its stable thia-amino-analogue (HOE 892) in consious rats

The effects of prostacyclin (PGI₂) and its stable thia-thimo-analogue (Hoe 892) on gastric and intestinal secretions and gastric mucosal lesions have been determined in conscious rats. Both PGI₂ and Hoe 892 given subcutaneously (s.c.) reduced dose-dependent gastric acid secretion, the ID₅₀ (dose producing 50% inhibition) being about 48.6 and 11.8 gmg/kg, respectively. In contrast, intragastric (i.g.) PGI₂ and Hoe 892 did not cause any change in gastric acid secretion at doses ranging from 1 to 100 gmg/kg. Both PGI₂ and Hoe 892 reduced significantly intestinal fluid secretion (antienteropooling activity). PGI₂ and Hoe 892 given i.g. or s.c. reduced dose-dependent gastric ulcer formation induced by acidified aspirin (ASA), Hoe 892 being somewhat less potent than PGI₂. Both PGI₂ and Hoe 892 were equally effective against mucosal necrosis induced by absolute ethanol and this effect was observed both after i.g. and s.c. administration of these agents. We conclude that stable thia-amino-PGI₂ analogue, Hoe 892, has similar gastric and intestinal antisecretory and protective activity as PGI₂ and may be useful in the prevention of gastric damage by various noxious agents.     

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.     

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.     

Enhancement of anaphylactic mediator release from guinea-pig perfused lungs by fatty acid hydroperoxides

Fragments of chopped lung from indomethacin treated guinea-pigs had an anti-aggregating effect when added to human platelet rich plasma (PRP), probably due to the production of prostacyclin (PGI₂) since the effect was inhibited by 15-hydroperoxy arachidonic acid (15-HPAA, 10 μg ml^?1). Both 15-HPAA (1–20 μg ml^?1 min^?1) and 13-hydroperoxy linoleic acid (13-HPLA, 20 μg ml^?1 min^?1) caused a marked enhancement of the anaphylactic release of histamine, slow-reacting substance of anaphylaxis (SRS-A) and rabbit aorta contracting substance (RCS) from guinea-pig isolated perfused lungs. This enhancement was not reversed by the concomitant infusion of either PGI₂ (5 μg ml^?1 min^?1) or 6-oxo-prostaglandin F_1α (6-oxo-PGF_1α, 5 μg ml^?1 min^?1). Anaphylactic release of histamine and SRS-A from guinea-pig perfused lungs was not inhibited by PGI₂ (10 ng - 10 μg ml^?1 min^?1) but was inhibited by PGE₂ (5 and 10 μg ml^?1 min^?1). Antiserum raised to 5,6-dihydro prostacyclin (PGI₁) in rabbits, which also binds PGI₂, had no effect on the release of anaphylactic mediators. The fatty acid hydroperoxides may enhance mediator release either indirectly by augmenting thromboxane production or by a direct effect on sensitized cells. Further experiments to distinguish between these alternatives are described in the accompanying paper (27).     

Effects of PGI2 on the inactivation of thrombin,factor Xa,and plasmin by antithrombin-III and hepartin

The influence of PGI₂ on the activity and on the inactivation of enzymes participating in blood coagulation /thrombin and Factor Xa/ and fibrinolysis /plasmin/ were investigated. According to the results PGI₂ has no effect on the activity of Factor Xa and plasmin nor on the inactivation of these enzymes by antithrombin-III in the absence and presence of heparin at a concentration of PGI₂ up to 400 μg/ml. An acceleration of the inactivation of thrombin by antithrombin-III was found in the presence of PGI₂ within a concentration of 100–400 μg/ml without any effect on the heparin-accelerated inactivation of thrombin by antithrombin. We got similar results using clotting tests for the assay and the application of synthetic substrate for thrombin. This inactivation-accelerating effect of PGI₂ on thrombin was only demonstratable at a concentration five magnitudes higher than that of the anti-aggregation effect on platelets.     

Beraprost sodium attenuates cigarette smoke extract-induced apoptosis in vascular endothelial cells

Apoptosis is now widely recognized as an important part of chronic obstructive pulmonary disease (COPD) pathogenesis. Our previous study demonstrated that a prostacyclin (PGI₂) analogue (beraprost sodium, BPS) prevented cigarette smoke extract (CSE) induced apoptosis of the pulmonary endothelium in rats. So we determined to clarify the apoptosis of vascular endothelial cells in COPD patient and the role of prostacyclin in the protection against apoptosis in vascular endothelial cells induced by CSE. Surgical specimens were obtained from 12 patients with COPD and 10 controls, and the level of apoptosis, prostacyclin synthase (PGI₂S) expression and 6-keto-PGF1α (a stable metabolite of PGI₂) were detected. The apoptotic index (AI), caspase-3 activity, expression of caspase-3 and 6-keto-PGF1α were examined in human umbilical vein endothelial cells (HUVECs) under exposure to varied concentrations of CSE for 24?h as well as under exposure to 2.5?% CSE for varied durations. Then, HUVECs under 2.5?% CSE were exposed to varied concentrations of BPS for 24?h and observed the alteration and the level of cAMP. Increased AI, decreased expression of PGI₂S and 6-keto-PGF1α, were found in the lungs of patients with COPD compared with controls. Moreover, CSE induced apoptosis in means of both dose-dependent and time-dependent manners, and reduced the level of 6-keto-PGF1α in HUVECs. And with the treatment of BPS, an enhanced level of cAMP and decreased apoptosis were detected. The deficiency of PGI₂ critically contributes to the COPD-associated endothelial dysfunction and apoptosis. And BPS protects against the apoptosis in the vascular endothelial cells induced by CSE.