Prostacyclin contributes to inhibition of hypoxic pulmonary vasoconstriction by alkalosis

The mechanism by which extracellular alkalosis inhibits hypoxic pulmonary vasoconstriction is unknown. We investigated whether the inhibition was due to intrapulmonary production of a vasodilator prostaglandin such as prostacyclin (PGI₂). Hypoxic vasoconstriction in isolated salt-solution-perfused rat lungs was blunted by both hypocapnic and NaHCO₃_induced alkalosis (perfusate pH increased from 7.3 to 7.7). The NaHCO₃-induced alkalosis was accompanied by a significant increase in the perfusate level of 6-keto-prostaglandin F_1α (6-keto-PGF_1α), an hydrolysis product of PGI₁. Meclofenamate, an inhibitor of cyclooxygenase, counteracted both the blunting of hypoxic vasoconstriction and the increased level of 6-keto-PGF_1α. In intact anesthetized dogs, hypocapnic alkalosis (blood pH increased from 7.4 to 7.5) blunted hypoxic pulmonary vasoconstriction before but not after administration of meclofenamate. In separate cultures of bovine pulmonary artery endothelial and smooth muscle cells stimulated by bradykinin, the incubation medium levels of 6-keto-PGF_1α were increased by both hypocapnia and NaHCO₃-induced alkalosis (medium pH increased from 7.4 to 7.7). These results suggest that inhibition of hypoxic pulmonary vasoconstriction by alkalosis is mediated at least partly by PGI_2.     

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.     

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.     

Effect of alpha-adrenergic stimulation on prostacyclin and renin release in the rat kidney

The relationship between renin secretion and PGI₂ production, in response to intrarenal infusion of norepinephrine, was examined in the isolated perfused rat kidney. Infusion of norepinephrine in a dose which caused substantial vasoconstriction (100 ng/min), markedly increased urinary excretion of 6-keto PGF_1α, the stable derivative of PGI₂, without significantly altering renin secretion measured in the effluent perfusate. No change in urinary 6-keto PGF_1α occurred when vasoconstriction was prevented by infusing the alpha-adrenoceptor blocking drug phenoxybenzamine (2 × 10³ ng/min) alongside norepinephrine 100 ng/min). However, under these conditions there was marked stimulation of renin secretion which, as has been demonstrated previously, is mediated by a beta-adrenoceptor. There were significant increase in urine flow rates during both vasoconstrictor and non-vasoconstrictor infusions. These findings clearly indicate that in the rat kidney prostacyclin production and renin release in response to norepinephrine are dissociated.     

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).     

Influence of type of dietary fat on the prostaglandin release from isolated rabbit and rat hearts and from rat aortas

It has previously been found (1) that feeding rats a diet containing a high amount of sunflowerseed oil results in a higher coronary flow and left ventricular work of their isolated hearts as compared to hearts of rats fed hydrogenated coconut oil or lard. It was hypothesized that this phenomenon can be explained by an influence of dietary linoleic acid on prostaglandin synthesis in the heart. To verify this hypothesis rabbits and rats were fed for four weeks sunflowerseed oil (SSO), hydrogenated coconut oil (HCO) or lard (L) to a maximum of 30 to 40 per cent of the total digestable energy, and the prostaglandin release from the isolated perfused hearts and rat aortas was determined by gas chromatography and bio-assay (PGI₂).For the isolated hearts of rabbits fed SSO, the release of PGE₂, PGF_2α and 6-oxo-PGF_1α was 1.7, 0.7 and 3.0 ng min⁻¹ g⁻¹ dry weight respectively; when fed L, these values were 2.9, 1.1 and 5.6 ng min⁻¹ g⁻¹. For the isolated hearts of rats fed SSO, HCO or L, the total release of PGE₂, PGD₂, PGF_2α and thromboxane B₂ (TXB₂) was 5.9, 5.8 and 5.6 ng min⁻¹ g⁻¹ respectively; the release of 6-oxo-PGF_1α was 3.4, 5.7 and 6.4 ng min⁻¹ g⁻¹ respectively. Relatively, 26% PGE₂, 13% PGD₂, 8% PGF_2α, 6% TXB₂ and 47% 6-oxo-PGF_1α were released. For the isolated aortas of rats fed SSO or HCO, the release of PGI₂-like activity was 0.37 ± 0.05 and 0.49 ± 0.05 ng min⁻¹ cm⁻². The release of PGI₂-like activity from hearts of EFA-deficient rats was about 20% of that from control hearts.We conclude that, although feeding sunflowerseed oil, with respect to feeding hydrogenated coconut oil or lard, does increase coronary flow and left ventricular work, it does not increase the basal prostaglandin production in the isolated rat or rabbit heart; instead there is a tendency for a lower PGI₂ synthesis.     

Prostanoid synthesis by human umbilical artery

A discrepancy between published values of PGI₂ production by human umbilical artery measured by platelet bioassay, compared with values of 6-oxo-PGF_1α by radioimmunoassay, raised the possibility that another anti-aggregatory prostanoid was produced by this tissue. To test this hypothesis, umbilical artery rings were incubated in buffer and PGI₂ determined by platelet bioassay and by a more specific radioimmunoassay based on comparison of 6-oxo-PGF_1α in hydrolysed and non-hydrolysed samples. 6-oxo-PGF_1a, PGF_2α and TXB₂ were also measured by gas chromatography negative ion chemical ionisation mass spectrometry. PGI₂ concentrations by radioimmunoassay and bioassay were significantly correlated (r = 0.92, p < 0.01). There was no difference between them, disproving the presence of an additional antiaggregatory substance. PGI₂ production determined by bioassay (mean 1.21 ng/mg wet weight/h, range 0.59–1.53 ng/mg/h) differed from previously reported values (range 70–325 ng/mg/h). 6-oxo-PGF_1α concentrations were confirmed by gas chromatography negative ion chemical ionisation mass spectrometry. Previous determinations of PGI₂ production by this tissue overestimated it by approximately 100 times.     

Role of prostacyclin on steroidogenesis by frog interrenal gland

The role of prostacyclin (PGI₂) on amphibian adrenal steroidogenesis was studied in perifused interrenal fragments from adult male frogs. Exogenous PGI₂ (3×10⁻⁸ M to 3×10⁻⁵ M) and, in a lesser extent, 6-keto-PGF_1α increased both corticosterone and aldosterone production in a dose-related manner. Short pulses (20 min) of 0.88 μM PGI₂ administered at 90 min intervals within the same experiment did not induce any desensitization phenomenon. A prolonged administration (6 h) of PGI₂ gave rise to an important increase in steroid production followed by a decline of corticosteroidogenesis. Indomethacin (IDM, 5 μM) induced a marked reduction of the spontaneous secretion of corticosteroid which confirmed the involvement of endogenous PGs in the process of corticosteroid biosynthesis. The IDM-induced blockade of corticosterone and aldosterone secretion was totally reversed by administration of exogenous PGI₂ in our model. Angiotensin II (AII) induced a massive release of 6-keto-PGF_1α, the stable metabolite of PGI₂. The increase of 6-keto-PGF_1α preceded the stimulation of corticosterone and aldosterone secretions. In contrast, the administration of ACTH did not modify the release of 6-keto-PGF_1α. These results indicate that PGI₂ might be an important mediator of adrenal steroidogenesis in frog. They confirm that the corticosteroidogenic actions of ACTH and AII are mediated by different mechanisms.     

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.     

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.     

Effect of the protein kinase inhibitors, staurosporine and K-252a, on PGI2 production by rat liver cells (the C-9 cell line)

Staurosporine and K-252a, known inhibitors of several protein kinases, stimulated PGI₂ production (measured as 6-keto-PGF_1α in rat liver cells (the C-9 cell line). Preincubation of the rat liver cells with staurosporine or K-252a enhanced the PGI₂ production stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA), platelet activating factor (PAF) and the Ca²⁺-ionophore a-23187, but not the PGI₂ synthesis stimulated by exogeneous arachidonic acid. These results suggest that phosphorylation of some proteins or certain amino acids on a protein can regulate arachidonic acid metabolism probably in the pathway leading to deesterification of phospholipids.     

Estrogen increases male rat aortic endothelial cell (RAEC) PGI2 release

Estrogen has been proposed as a negative risk factor for development of peripheral vascular disease yet mechanisms of this protection are not known. This study examines the hypothesis that estrogen stimulates rat aortic endothelial cell (RAEC) release of PGI₂. Male Sprague-Dawley rat abdominal aortic 1-mm rings were placed on 35 mm matrigel plates, and incubated for 1 week. The cells were transferred to a Primaria 60-mm dish and maintained from passage 3 in RAEC complete media and experiments performed between passages 4–10. Cells were incubated with Krebs-Henseleit buffer (pH 7.4) containing carrier or increasing concentrations of β-estradiol or testosterone for 60 min. The effluent was analyzed for eicosanoid release of 6-keto-PGF_1α (6-keto, PGI₂ metabolite), PGE₂ and thromboxane B₂ (TXB₂) by EIA (hormone stimulated — basal). Cells were analyzed for total protein by the Bradford method and for cyclooxygenase-1 (COX-1) and prostacyclin synthase (PS) content by Western blot analysis and densitometry. Testosterone did not alter RAEC 6-keto-PGF_1α release, whereas estrogen increased RAEC 6-keto-PGF_1α release in a dose-related manner. Estrogen preincubation (10 ng/ml) decreased COX-1 and PS content by 40% suggesting that the estrogen-induced increase in male RAEC PGI₂ release was not due to increased synthesis of COX-1 or PS. These data support the hypothesis that estrogen stimulation can increase endogenous male RAEC release of PGI₂.     

Prostacyclin and blood glucose levels in humans and rabbits

In patients with peripheral vascular disease and in healthy rabbits, infusion of PGI₂ but not of 6-keto PGF_1α induced a rise in blood glucose level and a pathological deviation in glucose tolerance test. In experiments , the increased concentrations of glucose produced dose-dependent inhibition of PGI₂ release from isolated rat aortic rings. The link between PGI₂ and carbohydrate metabolism is discussed.     

Studies of the mechanisms involved in the fate of prostacyclin (PGI2) and 06-keto-PGF1α in the pulmonary circulation

We have investigated the metabolism of [3]H-prostaglandin (PG)I₂ and its non-enzymatic breakdown product [3]H-6-keto-PGF_1α by rat pulmonary tissue and their possible uptake and metabolism upon passage through the isolated perfused rat lung. When incubated with rat lung homogenate in the presence of β-NAD, [3]H-PGI₂ was extensively degraded into at least one metabolite, while [3]H-6-keto-PGF_1α was only minimally metabolized. However, on passage through isolated perfused rat lungs, neither [3]H-PGI₂ nor [3]H-6-keto-PGF_1α were removed from the circulation into the lung or degraded. This demonstration that PGI₂ is not a substrate for the transport system for the removal of PGs from the circulation into the lung further illustrates that this system is a critical determinant for the pulmonary inactivation of circulating prostaglandins. The experimental findings are discussed in reference to the structure-activity requirements necessary for pulmonary transport and subsequent metabolism.     

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.     

Effects of orally administered ethyl ester of eicosapentaenoic acid (EPA; C20:5, ω-3) on PGI2-like substance production by rat aorta

A highly purified ethyl ester of EPA (EPAEE) (74%) was manufactured from sardine oil. Sixty mg/kg/day of EPAEE was given orally to male Wishar rats for 8 weeks. No side effect or toxicity from the administration of EPAEE was observed. Plasma EPA concentration and the ratio of EPA to arachidonic acid were significantly increased, compared with control Wistar rats. An enhancement of PGI₂-like substance production by aortas obtained from rats fed EPAEE was noted. Conversion of EPA to Λ¹⁷-6-keto-PGF_1α, a stable metabolite of PGI₃, could not be detected by an incubation study of ¹⁴C-EPA and aortas either from rats fed EPAEE or from control rats. Therefore, PGI₂-like substance produced by rat aorta is most likely to be PGI₂. itself and not PGI₃.     

Metabolism of PGI2 by 15-hydroxyprostaglandin-dehydrogenase and Δ-reductase in different vascular beds of the cat in vivo

The metabolism of endogenous PGI₂ (released by angiotensin II or bradykinin) and exogenous PGI₂ by 15-hydroxy-PG-dehydrogenase and Δ¹³-reductase was studied in five different vascular beds of the anaesthetized cat. Plasma concentrations of 6-keto-PGF_1α (the product of spontaneous hydrolysis of PGI₂) and 6,15-diketo-13,14-dihydro-PGF_1α (the metabolite formed from PGI₂ by 15-hydroxy-PG-dehydrogenase and Δ¹³-reductase) were determined in the efferent vessels of the respective vascular beds by specific radioimmunoassays.No major metabolism of PGI₂ by 15-hydroxy-PG-dehydrogenase and Δ¹³-reductase was detected in the head and the hindlimbs of the cat. In the lung exogenous (circulating) PGI₂ was not metabolized, whereas PGI₂ synthetized in the lung itself was converted to 6,15-diketo-13,14-dihydor-PGF_1α. No significant amounts of 6,15-diketo-13,14-dihydro-PGF_1α-immunoreactivity were detected in hepatic venous blood after infusion of PGI₂ into the portal vein. However as also no 6-keto-PGF_1α was found, the liver seems to efficiently extract PGI₂ from the circulation. The cat kidney had the highest capacity of all vascular beds investigated to release endogenous and exogenous PGI₂ as 6-15-diketo-13,14-dihydro-PGF_1α. In other organs (vascular beds) investigated PGI₂ is either metabolized less efficiently by the 15-hydroxy-PG-dehydrogenase or further transformed to other metabolites.     

The use of stable prostaglandins to investigate prostacyclin (PGI2)-binding sites and PGI2-sensitive adenylate cyclase in human platelet membranes

Prostacyclin, (PGI₂) is a potent but unstable inhibitor of platelet aggregation, probably acting through stimulation of adenylate cyclase.A stable analogue of prostacyclin with antiaggregatory properties, 5,6-dihydro-PGI₂ (6β-PGI), and PGE₁ can compete for the binding sites labelled by ³H-PGI₂ in human platelet membranes (the affinity being PGI₂ > PGE₁ > 6β -PGI₁). Both 6β-PGI₁ and PGE₁, as well as PGI₂, bind to two classes of binding sites. 6β -PGI₁ and PGE₁ activate adenylate cyclase to the same extent as PGI₂,with a rank order of potency which parallels that observed in binding experiments. The stimulation of this enzyme is brought about by interaction of each these prostanoids with two different classes of components. The comparison of binding and adenylate cyclase data suggests that the sites to which PGI₂, 6β -PGI₁ and PGE₁ bind might be coupled to the activation of adenylate cyclase. Since 6β-PGI₁ seems to act through the same molecular mechanisms as PGI₂, because of its stability it is an useful tool to investigate the mode of action of prostacyclin in platelets.     

Lack of effect of ischemia and dipyridamole on prostacyclin production in arteriosclerosis obliterans

Six patients with advanced arteriosclerosis obliterans in the lower extremities were subjected to an exercise test on a tread mill with and without dipyridamole treatment. Prostacyclin (PGI₂) release was measured by the concentration of its stable metabolite, 6-keto prostaglandin F_1α in plasma. All the patients suffered from ischemic pain during both tests, but no changes were seen in plasma 6-keto-PGF_1α. Dipyridamole did not affect the physical performance. Our results suggest that atherosclerotic vessels do not increase PGI₂ production in response to ischemia and that a single dose of dipyridamole does not change PGI₂ production.     

Prostacyclin and thromboxane A2 release in isolated rat lungs

The influence of platelets and platelet membranes on the generation of prostacyclin (PGI₂) and thromboxane A₂(TXA₂) by isolated rat lung and porcine aortic endothelial cell, as measured by RIA of their stable end-producs, 6-oxo-PGF_1α and TXB₂ respectively, was studied. After introduction of either aspirin-treated platelets or membranes from aspirin-treated platelets to the perfusate, 1 5-fold increase in the amount of 6-oxo-PGF_1α and TXB₂ in the perfusate was observed. Treatment of the lung with aspirin produced a 50% reduction in the platelet-stimulated release of PGI₂ and TXA₂. Treatment of the lung with the phospholipase inhibitor, mepacrine, significantly reduced the platelet-stimulated release of PGI₂ and TXA₂. Incubation of endothelial cells with untreated platelet membranes did not alter the generation of PGI₂. These results suggest that platelet-stimulated release of PGI₂ and TXA₂ occurs via mechanical stimulation of phospholipase A₂, liberating arachidonic acid.