Binding of prostacyclin by plasma glycoproteins

The binding of prostacyclin (PGI2) to plasma proteins and the resulting increase in PGI2 stability was investigated. Using gel filtration to separate bound and free PGI2, we have found that Cohn Fraction VI can bind PGI2, and retard its hydrolysis to 6-keto-PGF1 alpha (6KPGF1 alpha). The biological activity of the bound PGI2 correlated well with the quantity of bound PGI2, measured as 6KPGF1 alpha by RIA. Fraction VI bound a greater percentage of PGI2 than the other eicosanoids tested (i.e., PGI2 greater than TXB2 greater than LTB4 greater than PGE1 greater than PGF2 alpha). The PGI2 binding activity of Fraction VI was lost after neuraminidase treatment. Our data suggest that Fraction VI glycoproteins may play an important role in the binding and stabilization of PGI2 by plasma proteins.     

Urinary levels of 2,3-dinor-6-oxo-PGF1 alpha: a reliable index of the production of PGI2 in the spontaneously hypertensive rat

The urinary levels of 2,3-dinor-6-oxo-PGF1 alpha (PGI2-M), a major metabolite of PGI2, are determined by the balance between the amount of PGI2 synthesized and the extent of its further metabolic oxidation. The purpose of the present study was to determine if the urinary excretion of PGI2-M can be used as a reliable index of the in vivo production of PGI2 in both normal Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). This involved the exclusion of differences in metabolism between these two strains of rats. In order to do so, we monitored the urinary excretion of PGI2-M during paired intravenous infusions of 6-oxo-PGF1 alpha (the stable product of the spontaneous hydrolysis of PGI2) in conscious, unrestrained SHR and WKY rats aged 12-15 weeks, in doses ranging from 250 to 700 ng. In one experiment, PGI2 was infused instead of 6-oxo-PGF1 alpha. The results of these experiments indicate that SHR and WKY rats are equal with regard to the transformation of 6-oxo-PGF1 alpha and PGI2 into PGI2-M. For both groups, there is a good correlation between the amount of 6-oxo-PGF1 alpha infused and the amount of PGI2-M excreted in urine. These observations confirm the validity of using the urinary levels of 2,3-dinor-6-oxo-PGF1 alpha as an index of PGI2 production in both WKY and SHR. In addition, they support the conclusions drawn from our previous studies, namely that SHR do not produce more PGI2 than WKY rats in vivo, contrary to the situation prevailing in vitro.     

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

A direct comparison of the relative potencies of the two antiaggregatory 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-PGI2 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 thatn 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 conversin of PGI2 and/or 6-keto-PGF1 alpha to 6-keto-PGE1.     

IL-1-induced prostacyclin production by cerebral vascular endothelial cells inhibits myelin basic protein-specific lymphocyte proliferation.

Addition of cerebral vascular endothelial cells (EC) to myelin basic protein (MBP) immune lymph node cells (LNC) cultured in the presence of MBP resulted in the inhibition of MBP-specific proliferative responses. Proliferation was not inhibited in cultures containing indomethacin (IM), suggesting a possible role for prostaglandins. Significant levels of 6-KPGF1 alpha, the stable hydrolysate product of PGI2, but not PGE2 were observed in culture (LNC + EC) supernatants but not in supernatants from cultures containing only LNC or EC. The levels of PGI2 release were proportional to the concentration of exogenous EC present in culture and synthesis of PGI2 could be blocked by IM. These results indicate the requirement for coculture in the generation PGI2. Additional experiments indicated that EC were required for the generation of PGI2 and that either macrophages (M phi), or recombinant murine IL-1 were able to replace LNC in cocultures with EC in order to generate PGI2. The ability of IL-1 to stimulate EC-derived PGI2 synthesis was dose dependent with maximal stimulation observed at 50 U/ml IL-1. The IL-1-induced production of PGI2 by EC as well as PGI2 production in cultures containing EC and either LNC or M phi was inhibited by treatment with anti-IL-1 antibody. These results indicate that EC are capable of inhibiting antigen-specific lymphocyte proliferation by producing PGI2, which can be induced by the lymphokine, IL-1.     

Antimitogenic effects of prostacyclin on the G1 phase cyclin-dependent kinases

The prostanoid prostacyclin (PGI2) inhibits proliferation of cultured vascular SMCs by inhibiting cell cycle progression from G1 to S phase. Progression through G1 phase is regulated by the sequential activation of the G1 phase cyclin-dependent kinases (cdks). Recent studies have shown that PGI2-dependent activation of its receptor, IP, inhibits G1 phase progression by blocking the degradation of p27 and the activation of cyclin E-cdk2. High Density Lipoproteins (HDL) and its associated apolipoprotein, ApoE, also inhibit S phase entry of vascular SMCs, and the effects of HDL and ApoE are, at least in part, also mediated by the production of PGI2. The antimitogenic effects of hyaluronan may also be controlled by PGI2. This review summarizes the effects of PGI2 on the G1 phase cyclin-cdks and discusses the potential role of PGI2 as a common component of multiple extracellular signals that attenuate the proliferation of vascular SMCs.     

Colocalization prostacyclin (PGI2) synthase--caveolin-1 in endothelial cells and new roles for PGI2 in angiogenesis

In vascular cells, prostacyclin (PGI2) synthase (PGI2s) has been localized in the endoplasmic reticulum of endothelial cells and in the nuclear and plasma membrane of smooth muscle cells. In human umbilical vein endothelial (HUVE) cells, we detected the enzyme in abundant cytoplasmic vesicles apparently originating from the plasma membrane and similar to those stained by gold-albumin, which interacts with a caveolar receptor. This prompted us to try a direct confocal microscopy approach aimed at colocalizing gold-albumin, caveolin-1, and PGI2 synthase. Moreover, the staining of HUVE cells with an anti-BiP7Grp78 antibody (a marker of endoplasmic reticulum) shows a perinuclear localization, sharply separated from PGI2 synthase localization. The results indicate that more than 80% of the enzyme resides in cellular sites costaining with caveolin-1 antibody and gold-albumin. This evidence was confirmed by the demonstration that PGI2 synthase and caveolin-1 coimmunoprecipitate in HUVE cell lysates and that they are associated to detergent-insoluble membrane domains in the same low-density fractions of a sucrose gradient. In addition, depletion of cellular cholesterol by mevalonate and methyl-beta-cyclodextrin leads to the shift of PGI2 synthase and caveolin-1 to higher density fractions of the gradient. Biochemical evidence about colocalization was supported by the use of a fusion protein glutathione S-transferase (GST)/caveolin-1, which retained either PGI2s purified from ram seminal vesicles or PGI2s present in HUVE cell lysates. Binding of PGI2s to caveolin "scaffolding domain" and to C-terminal region was deduced by using full-length GST--Cav-1, GST--Cav 61--101, and GST C- and N-terminal fusion proteins. A double approach based on the usage of filipin as a specific caveolae-disrupting agent and antisense oligonucleotides targeting PGI2 synthase mRNA suggests that the production of PGI2 in caveolae is likely to be connected to the regulation of angiogenesis, at least in vitro.     

Phosphoglucose isomerase from bananas: partial characterization and relation to main changes in carbohydrate composition during ripening.

Some characteristics of phosphoglucose isomerase (PGI, EC 5.3.1.9) from banana were measured during fruit ripening of three banana cultivars. In banana, PGI was present as two dimeric isoenzymes, named PGI1 and PGI2, which had similar native molecular masses but differed in relation to heat stability and isoelectric point. Total PGI activity showed a distinct two-step change during fruit ripening. Before the climacteric period, PGI activity gradually decreased with the starch content, then its activity began to increase with sucrose accumulation. The ratio of PGI1, and PGI2 was constant, indicating that both enzymes would be involved in starch degradation and sucrose synthesis. PGI activity and changes in carbohydrate composition suggests the existence of some control to fit the requirements of the intense carbon flow from starch to sucrose.     

12-Hydroxyeicosatetraenoic acid reduces prostacyclin production by endothelial cells

12-Hydroxyeicosatetraenoic acid (12-HETE), a lipoxygenase product released by activated platelets and macrophages, reduced prostacyclin (PGI2) formation in bovine aortic endothelial cultures by as much as 70%. Maximal inhibition required 1 to 2 h to occur and after 2 hr, a concentration of 1 microM 12-HETE produced 80% of the maximum inhibitory effect. 5-HETE and 15-HETE also inhibited PGI2 formation. The inhibition was not specific for PGI2; 12-HETE reduced the formation of all of the radioactive eicosanoids synthesized from [1-14C]arachidonic acid by human umbilical vein endothelial cultures. Inhibition occurred in the human cultures when PGI2 formation was elicited with arachidonic acid, ionophore A23187 or thrombin. These findings suggest that prolonged exposure to HETEs may compromise the antithrombotic and vasodilator properties of the endothelium by reducing its capacity to produce eicosanoids, including PGI2.     

Metabolism of prostacyclin in blood vessels.

The activity of 15-hydroxyprostaglandin dehydrogenase has been shown to be high in both mesenteric arteries and veins; the present study suggests that it may be responsible for the inactivation of prostacyclin (PGI2). The cytoplasmic fractions of bovine mesenteric arteries and veins were incubated with radiolabeled PGI2 in the presence of NAD+ or NADP+. The substrate was rapidly converted to a product, which was isolated and identified as 6,15-diketo prostaglandin F1alpha, (6,15-diketo-PGF1alpha) by thin layer chromatography and gas chromatography-mass spectrometry. The initial reaction rate began to level off after less than 1 min of incubation at 37 degrees C. When radiolabeled 6-keto-PGF1alpha, the stable hydrolysis product of PGI2, was used as substrate under the same conditions, 97% was recovered unmetabolized after 2 min of incubation. Catabolism of PGI2 may be a major determinant of its levels in blood vessels and, therefore, may be of crucial importance to regulating the action of PGI2. Further, estimation of PGI2 generation by either tissues or organs may be misleading if only 6-keto-PGF1alpha is measured.     

Effects of TRK-100, a stable prostacyclin analogue, on regulation of cyclic AMP metabolism in platelets

TRK-100 is a chemically stable analogue of prostacyclin and effective in inhibiting platelet aggregation when orally administered in experimental animals. In the present study we compared the potency of TRK-100 with those of PGI2 and PGE1 to cause an activation of adenylate cyclase activity in rat and human platelet membranes. TRK-100 was half as effective as PGI2, and 10 times more effective than PGE1 in both platelet membranes. TRK-100 also induced an activation of phosphodiesterase activity when directly added to intact platelets probably as a feedback mechanism of intracellular cAMP level like PGI2 did. TRK-100 would mimic PGI2 in the regulation of cAMP metabolism.     

Bronchoconstrictor and antibronchoconstrictor properties of inhaled prostacyclin in asthma

Prostacyclin (PGI2) is generated in appreciable amounts during allergic reactions in human lung tissue. To define its activity on human airways we have studied the effects of doubling concentrations of inhaled PGI2 and its hydrolysis product 6-oxoprostaglandin F1 alpha (6-oxo-PGF1 alpha) on specific airway conductance (sGaw), maximum expiratory flow at 30% vital capacity (Vmax30), forced expiratory volume in 1 s (FEV1), and static lung volumes in subjects with mild allergic asthma. In a second study the effect of inhaled PGI2 on bronchoconstriction provoked by increasing concentrations of inhaled prostaglandin (PG) D2 and methacholine was observed. Inhalation of PGI2 up to a concentration of 500 micrograms/ml had no significant effect on sGaw but produced a concentration-related decrease in FEV1 and Vmax30 in all subjects. In two of four subjects inhalation of PGI2 also increased residual volume and decreased vital capacity but had no effect on total lung capacity. PGI2, but not 6-oxo-PGF1 alpha, protected against bronchoconstriction provoked by either PGD2 or methacholine whether airway caliber was measured as sGaw, FEV1, or Vmax30. The apparent disparity between the bronchoconstrictor and antibronchoconstrictor effects of PGI2 might be explained by its potent vasodilator effect in causing airway narrowing through mucosal engorgement and reducing the spasmogenic effects of other inhaled mediators by increasing their clearance from the airways.     

Myocardial prostacyclin and thromboxane A2 synthetase activities during ischemia and reperfusion in isolated rabbit heart

The aim of the study was to determine the prostacyclin (PGI2) and thromboxane A2 (TXA2) synthetase activities of myocardial tissue and their variation during ischemia and reperfusion. Regional ischemia was induced by 10 min occlusion of the left anterior descending coronary artery in isolated Langendorff rabbit hearts. Biosynthesis of PGI2 and TXA2 were carried out by using arachidonic acid as substrate and left ventricle microsomes (LVM) from ischemic and non-ischemic areas as sources of PGI2 and TXA2 synthetase. 6-keto-PGF1 alpha and TXB2, stable metabolites of PGI2 and TXA2 respectively, were determined by radioimmunoassay. Experiments carried out under the adopted conditions showed that LVM were able to synthetise PGI2 as well as TXA2 from arachidonic acid. On the other hand, ischemia depressed both PGI2 and TXA2 synthetase activities of cardiac tissue: the depression was more pronounced on TXA2 synthetase than on PGI2 synthetase with no significant difference between ischemic and non-ischemic regions. Moreover, ischemia increased the ratio 6-keto-PGF1 alpha/TXB2 indicating therefore that it can facilitate the formation of PGI2. The post ischemic reperfusion of the heart counteracted the decrease in PGI2 synthetase induced by ischemia which returned to the normal level: reperfusion also slightly reversed the decrease in TXA2 the decrease in TXA2 synthetase. However, the diminution in TXA2 synthetase of non-ischemic myocardium was attenuated but it remained lower than the normal level. These results suggested that the whole left ventricle is affected by regional ischemia. Furthermore it appears that myocardial TXA2 synthetase is more vulnerable than PGI2 synthetase to a lack of oxygen and nutrients.     

A selective inhibitor of thromboxane synthetase activity of rabbit heart tissue: a pyridazinic derivative

The effects of 2-(2 dimethylaminoethyl) 5-benzylidene 6-methyl (2H,4H)-3-pyridazinone (III) were studied on the biosynthesis of TXA2 and PGI2 in vitro the TXA2 and PGI2 synthetase activity of heart tissue. Biosyntheses of TXA2 and PGI2 were carried out using arachidonic acid as a substrate and horse platelet and aorta microsomes as sources of TXA2 and PGI2 synthetases respectively. TXB2 and 6-keto PGF1 alpha were determined by RIA. III--did not significantly modify either the biosynthesis of PGI2 in vitro or the PGI2 synthetase activity of heart tissue. did not significantly inhibit TXA2 biosynthesis in vitro but markedly reduced the TXA2 synthetase activity of heart tissue: for a microsomal fraction concentration of 100 micrograms protein, the ID50 was 6.37 X 10(-5) M +/- 1.29 X 10(-8) M. Thus III behaves as a specific inhibitor of the TXA2 synthetase activity of heart tissue and could have a beneficial use in therapeutics.     

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

In vitro 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-PGF1 alpha per microgram 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.     

Production of prostacyclin in mice following intraperitoneal injection of acetic acid, phenylbenzoquinone and zymosan: its role in the writhing response

The magnitude and temporal production of PGI2, PGE2 and LTB4 were measured in the mouse peritoneal cavity for a 15 min period following the intraperitoneal injection of either acetic acid, phenyl-p-benzoquinone (PBQ) or zymosan. For each algogenic substance, PGI2 (assayed as the stable metabolite, 6-keto-PGF1 alpha) represented the major eicosanoid with lower levels of PGE2 also detected. Zymosan induced the greatest 6-keto-PGF1 alpha production among the three algogenic agents, but only a weak writhing response was observed. LTB4 was detected in the peritoneal lavage only after zymosan. The magnitude of eicosanoid production did not correlate with the writhing response induced by the algogenic agents, even though the inhibition of both 6-keto-PGF1 alpha and writhing by several peripheral analgesics was positively correlated. PGI2, (100 ng), 6-keto-PGF1 alpha (1 microgram) and PGE2 (100 ng) did not induce writhing. However, only PGI2 acted synergistically with acetic acid to produce writhing. Presumably due to the short biological lifetime of PGI2, this synergism was noted only when PGI2 was administered after the acetic acid. These results suggest that PGI2 acts to sensitize the animal for the writhing response.     

Effects of prostacyclin on the canine isolated basilar artery.

Prostacyclin (PGI2) produced a biphasic response in canine isolated basilar arteries. In low doses (1 X 10(-8)M-1 X 10(-7)M) PGI2 caused a slight but consistent relaxation of resting muscle tone. In low concentrations (1 X 10(-8)M-1 X 10(-6)M) PGI2 antagonized muscle contractions caused by serotonin or prostaglandin (PG) F2 alpha. This relaxant effect with low doses of PGI2 on the isolated cerebral artery contrasts with findings obtained with other PGs and supports the hypothesis that PGI2 is a mediator of vasodilatation. However, in 1 X 10(-5)M concentrations PGI2 contracted the arterial muscle and did not antagonize contractions induced by serotonin or PGF2 alpha.     

Role of cyclooxygenase-1-mediated prostacyclin synthesis in endothelium-dependent vasoconstrictor activity of porcine interlobular renal arteries

This study aimed to determine whether PGI(2) would be evoked by the endogenous endothelial B(2) receptor agonist bradykinin (BK) in the porcine interlobular renal artery and, if so, to determine how it would influence the vasomotor reaction, and the specific cyclooxygenase (COX) isoform(s) involved in its synthesis. The production of the PGI(2) metabolite 6-keto-PGF(1α) was analyzed with HPLC-mass spectroscopy, while vasomotor reaction to PGI(2) or BK was determined with isometric force measurement. Results showed that BK evoked an increase in the production of 6-keto-PGF(1α), which was abolished by endothelial denudation that removed COX-1 expression, or was reduced by COX-1 inhibition. Interestingly, PGI(2) evoked a potent contraction, which was prevented by antagonizing thromboxane-prostanoid (TP) receptors and was not enhanced by antagonizing the vasodilator PGI(2) (IP) receptors. The IP receptor agonists MRE-269 and iloprost did not induce any relaxation. Moreover, iloprost, which is also a PGI(2) analog, caused a contraction, which was sensitive to TP receptor antagonism, but was to a significantly lesser extent than that of PGI(2). Indeed, IP receptors were not detected by RT-PCR or Western blotting in the vessel. Following nitric oxide synthase (NOS) inhibition, BK also evoked an endothelium-dependent contraction, which was blocked by TP receptor antagonism. In addition, inhibition of COX-1 (but not COX-2) impeded the vasoconstrictor activity of BK and expedited the relaxation induced by the agonist in NOS-intact vessels. These results demonstrate that in the porcine interlobular renal artery BK evokes endothelial COX-1-mediated PGI(2) synthesis, which mainly leads to the activation of TP receptors and a vasoconstrictor response, possibly due to a scarcity of vasodilator activity mediated by IP receptors. Also, our data suggested that the effect of a PGI(2) analog on TP receptors could be reduced compared with that of PGI(2) due to modified structure as with iloprost.     

Nitric oxide enhances PGI(2)production by human pulmonary artery smooth muscle cells

To evaluate the effect of exogenous nitric oxide (NO) and endogenous NO on the production of prostacyclin (PGI(2)) by cultured human pulmonary artery smooth muscle cells (HPASMC) treated with lipopolysaccharide (LPS), interleukin-1(beta)(IL-1(beta)), tumor necrosis factor alpha (TNF(alpha)) or interferon gamma (IFN(gamma)), HPASMC were treated with LPS and cytokines together with or without sodium nitroprusside (SNP), NO donor, N(G)-monomethyl-L-arginine (L-NMMA), NO synthetase inhibitor, and methylene blue (MeB), an inhibitor of the soluble guanylate cyclase. After incubation for 24 h, the postculture media were collected for the assay of nitrite by chemiluminescence method and the assay of PGI(2)by radioimmunoassay. The incubation of HPASMC with various concentrations of LPS, IL-1(beta)or TNF(alpha)for 24 h caused a significant increase in nitrite release and PGI(2)production. However, IFN(gamma)slightly increased the release of nitrite and had little effect on PGI(2)production. Although the incubation of these cells for 24 h with SNP did not cause a significant increase in PGI(2)production, the incubation of HPASMC with SNP and 10 microg/ml LPS, or with SNP and 100 U/ml IL-1(beta)further increase PGI(2)production and this enhancement was closely related to the concentration of SNP. However, stimulatory effect of SNP on PGI(2)production was not found in TNF(alpha)- and IFN(gamma)- treated HPASMC. Addition of L-NMMA to a medium containing LPS or IL-1(beta)reduced nitrite release and attenuated the stimulatory effect of those agents on PGI(2)production. MeB significantly suppressed the production of PGI(2)by HPASMC treated with or without LPS or IL-1(beta). The addition of SNP partly reversed the inhibitory effect of MeB on PGI(2)production by HPASMC. These experimental results suggest that NO might stimulate PGI(2)production by HPASMC. Exogenous NO together with endogenous NO induced by LPS or cytokines from smooth muscle cells might synergetically enhance PGI(2)production by these cells, possibly in clinical disorders such as sepsis and acute respiratory distress syndrome.     

Prostacyclin and thromboxane formation in human umbilical arteries following stimulation with vasoactive autacoids

The formation of prostacyclin (PGI2) and thromboxane A2 (TXA2) (measured as the stable metabolites 6-keto-PGF1 alpha and TXB2) during stimulation with vasoactive autacoids was registered in human umbilical arteries perfused in vitro. Responses were registered within 3-4 minutes after addition of the substances. Both angiotensin I and II were found to increase the formation of PGI2 while depressing that of TXA2. Serotonin increased the formation of TXA2 but not that of PGI2. Both PGE2 and PGF2 alpha stimulated the PGI2 formation. The TXA2 mimetic U46619, increased PGI2 production, whereas PGI2 slightly increased the formation of TXA2. All responses were found to be completely inhibited by indomethacin.