Thrombin-induced release of von Willebrand factor from endothelial cells is mediated by phospholipid methylation. Prostacyclin synthesis is independent of phospholipid methylation

The biochemical events that lead to thrombin-stimulated release of von Willebrand factor and prostacyclin synthesis in cultured endothelial cells are examined. Treatment of human umbilical vein endothelial cells with thrombin results in an instantaneous increase in phospholipid methylation which can be blocked by 3-deazaadenosine, a methyltransferase inhibitor. 3-Deazaadenosine also blocks the thrombin-induced Ca2+ influx into endothelial cells and the release of von Willebrand factor, indicating that these processes are coupled. The phorbol ester 4 beta-phorbol 12-myristate 13-acetate (PMA) and the Ca2+ ionophore A23187 both bypass the phospholipid methylation and directly stimulate Ca2+ influx and von Willebrand factor release. In contrast to the stimulus-induced von Willebrand factor release, the thrombin-induced prostacyclin synthesis cannot be blocked by 3-deazaadenosine. Similarly, incubation of endothelial cells with EDTA has no influence on the thrombin-induced prostacyclin synthesis, and PMA has no stimulatory effect on prostacyclin synthesis. These observations indicate that thrombin induces different metabolic responses in endothelial cells: phospholipid methylation followed by a Ca2+ influx, which subsequently leads to release of von Willebrand factor, and liberation of arachidonic acid from phospholipids for prostacyclin formation, which is independent of phospholipid methylation and Ca2+ influx.     

Kallikrein stimulates prostacyclin production in bovine vascular endothelial cells

Cultured endothelial cells isolated from bovine carotid aorta produce prostacyclin (prostaglandin I2) and a small amount of prostaglandin E2. The effects of kallikrein (EC 3.4.21.8) on the release of prostacyclin from the cells were studied with the radioimmunoassay technique. Kallikrein stimulated the release of prostacyclin in a dose-dependent manner. The maximal stimulation reached up to 9.2-fold at 0.1 micrograms/ml of kallikrein. The effect was not associated with the activation of the fatty acid cyclooxygenase, but with the stimulation of arachidonic acid release. But kallikrein itself did not have phospholipase activity. On the other hand, at the same doses, kallikrein failed to induce platelet aggregation or enhance platelet aggregation induced by collagen. Our findings suggest that the vasodilator effect of kallikrein is mediated in part by prostacyclin production. Furthermore, we investigated the possibility that the stimulatory effect of kallikrein on prostacyclin production in endothelial cells is associated with kinin formation. Bradykinin and lysylbradykinin (kallidin) also stimulated the release of prostacyclin, but the effects were far less than that of kallikrein. And the stimulation due to the addition of both kallikrein and bradykinin on prostacyclin and arachidonic acid release was not competitive or additive, but synergistic. Moreover, even if fetal calf serum was incubated with kallikrein, bradykinin was not detected at all. When kallikrein was pre-incubated with aporotinin, which is an inactivator of kallikrein, the effect of kallikrein was completely abolished. These findings suggest that the stimulatory effect of kallikrein on the release of prostacyclin from vascular cells is possibly not due to kinin formation, but to other substance(s) produced by this serine proteinase.     

Role of protein kinase C in the control of vascular prostacyclin: study of phorbol esters effect in bovine aortic endothelium and smooth muscle

In bovine aortic endothelial cells, phorbol 12-myristate, 13-acetate induced a smaller stimulation of prostacyclin release than ionophore A23187: the combination of both agents was highly synergistic. The responses of the bovine aortic smooth muscle were very different in the 2 preparations studied. In media explants cultured for short periods, neither phorbol 12-myristate, 13-acetate, nor A23187, alone or in combination, were able to increase prostacyclin release, whereas serotonin was an effective stimulus. In cultured smooth muscle cells, outgrown from the explants, phorbol 12-myristate, 13-acetate increased prostacyclin release to the same levels as A23187 or serotonin. It is concluded that increased cytosolic Ca++ level and protein kinase C activity induce a synergistic stimulation of endothelial prostacyclin. On the other hand, the phenotypic modulation of the arterial smooth muscle, from a contractile to a synthetic state, seems to be associated with a profound change in the control of prostacyclin.     

Role of protein kinase C in the control of vascular prostacyclin: Study of phorbol esters effect in bovine aortic endothelium and smooth muscle

In bovine aortic endothelial cells, phorbol 12-myristate, 13-acetate induced a smaller stimulation of prostacyclin release than ionophore A23187: the combination of both agents was highly synergistic. The responses of the bovine aortic smooth muscle were very different in the 2 preparations studied. In media explants cultured for short periods, neither phorbol 12-myristate, 13-acetate, nor A23187, alone or in combination, were able to increase prostacyclin release, whereas serotonin was an effective stimulus. In cultured smooth muscle cells, outgrown from the explants, phorbol 12-myristate, 13-acetate increased prostacyclin release to the same levels as A23187 or serotonin. It is concluded that increased cytosolic Ca⁺⁺ level and protein kinase C activity induce a synergistic stimulation of endothelial prostacyclin. On the other hand, the phenotypic modulation of the arterial smooth muscle, from a contractile to a synthetic state, seems to be associated with a profound change in the control of prostacyclin.     

ATP stimulates the release of prostacyclin from perfused veins isolated from the hamster hindlimb

ATP-stimulated prostacyclin release from veins was investigated using epigastric veins isolated from hamsters. Veins were perfused with MOPS-buffered physiological salt solution (PSS). ATP was administered into the perfusate, and the bath solution (MOPS-PSS) was collected and assayed for the presence of the stable prostacyclin metabolite 6-keto-PGF1alpha. ATP (100 microM) resulted in reproducible increases in bath concentration from 73 +/- 22 to 279 +/- 50 pg/ml (P < 0.05, n = 5). This response was abolished by indomethacin (10 microM, P < 0.05). To ascertain whether the endothelium was the source of prostacyclin, endothelium was disrupted using air (n = 10) or deoxycholic acid (n = 6). Perfusion with air significantly reduced (P < 0.05) but did not completely abolish ATP-stimulated release of prostacyclin, while deoxycholic acid totally abolished the response (P < 0.05). The nonselective P2 receptor antagonist reactive blue 2 (100 microM) attenuated ATP-mediated release of prostacyclin but did not significantly alter ACh-stimulated release of prostacyclin. The nonselective adenosine receptor antagonist xanthine amine congener (1 microM) had no effect on ATP-stimulated release, and adenosine did not stimulate the release of prostacyclin. These results show that increases in intraluminal concentration of ATP stimulate abluminal release of prostacyclin from the venous endothelium. This effect is mediated by P2 receptors while adenosine and its receptors are not involved in this response.     

Effects of prostacyclin and its stable analog, iloprost, upon insulin secretion in isolated pancreatic islets

We investigated the effects of prostacyclin in three concentrations of 2.7 nM, 53.8 nM and 267 nM on insulin release by isolated rat islets. The lowest concentration produced no significant effects. The middle concentration led to stimulation followed by inhibition of the reaction studied, while the highest concentration strongly depressed insulin release. These effects of prostacyclin appeared to be specific, because they were mimicked by its stable analog, iloprost, but not by its metabolite, 6-keto-PGF1 alpha. The results suggest that prostacyclin generated in islets might exert locally an influence upon insulin release.     

Arachidonic acid releasing systems in pig aorta endothelial cells

Endothelial cells synthesize prostacyclin both from platelet-derived endoperoxides and from the arachidonic acid released from its intracellular stores. The mechanisms controlling this release does not appear to be mediated through phospholipid methylation but by means of phosphoinositide hydrolysis. As yet two possible mechanisms have so far been proposed to regulate arachidonic acid release in a number of cellular systems: phospholipase C-controlled phospholipase A2 activity or phospholipase C-diglyceride lipase system. The results presented here show that using phospholipases inhibitors is not a reliable strategy to study arachidonic acid release in cultures of endothelial cells. Our data also strongly suggest that the release of prostacyclin may be accounted in these cells for by a phospholipase C-diglyceride lipase system.     

Role of endothelium, oxygen and ionic milieu in the prostacyclin and thromboxane production of rat aortic tissue slices.

The present study was executed in order to get further data on the role of vessel wall constituents in prostanoid synthesis and on the effect of anorganic constituents on it. Prostacyclin and tromboxane production of rat aortic tissue slices with intact endothelium and after mechanical as well as chemical endothelium removal were studied. The effects of hypoxia and changes in the ionic milieu on the release of these prostanoids were also examined. The tissue slices were incubated in normal or in modified Krebs-Ringer solution, bubbled with 95% O2 and 5% CO2 (with the exception of the studies in hypoxic conditions). Prostacyclin and thromboxane release was determined by specific radioimmunoassay of the stable metabolites, 6-keto-PGF1 alpha and TxB2, from the incubation medium. 174 tissue samples obtained from 164 rats were studied. Mechanical removal of the endothelium increased prostacyclin production of the aortic segments about fivefolds from a basal rate of 52.9 +/- 19.4 ng/gr/min, while it had no significant effect on thromboxane release (basal rate 0.83 +/- 0.13 ng/gr/min). Treating the endothelium with 1.0 M HCl almost totally suppressed prostacyclin release. Lowering the partial oxygen tension of the incubation medium significantly decreased the production of prostacyclin, while release of TxB2 somewhat increased. Increasing the Ca2+ concentration of the medium between 0-5 mM the release of prostacyclin was augmented and the release of thromboxane was diminished. Potassium free medium caused a very large increase in prostacyclin release of the tissue slices. The results show that release of vasoactive prostanoids from isolated rat aortic wall is dependent not exclusively on the endothelium and that various methods of endothelium removal may have distinct influences on prostacyclin and thromboxane productions. The changes in anorganic constituents of the surrounding medium could massively affect prostacyclin and thromboxane production of rat aortic tissue. The alternative effects of the above listed treatments on the release of prostacyclin and thromboxane from the rat aortic wall suggest the existence of different mechanisms in the control of the production of the two major prostanoids possessing opposite physiological effects.     

Endothelin-1 induces prostacyclin release from bovine aortic endothelial cells

The effects of endothelin-1 (ET-1) on the release of prostacyclin from cultured bovine aortic endothelial cells were studied. ET-1 induced a time- and dose-dependent release of 6-keto PGF1 alpha, the stable metabolite of prostacyclin, with an apparent EC50 value of 3.0 +/- 0.9 nM (n = 6). ET-1 up to a concentration of 500 nM did not affect cellular integrity. Preincubation of the cells for 30 min with 10 microM indomethacin inhibited ET-1 (100 nM) - induced prostacyclin release by 90%. These findings indicate that ET-1 can directly stimulate prostacyclin release from endothelial cells probably through a receptor mediated mechanism.     

Endothelin stimulates the release of prostacyclin from rat mesenteric arteries

The effect of endothelin on the release of prostacyclin was examined in perfused rat mesenteric arteries with or without their pretreatment with indomethacin. Porcine endothelin at 10 pmol (a subpressor dose) and 40 pmol stimulated the release of 6-keto-PGF1 alpha, a stable metabolite of prostacyclin. Rat endothelin also stimulated its release, but less than porcine endothelin. Pretreatment with indomethacin completely inhibited this 6-keto-PGF1 alpha release. These results indicate that endothelin stimulates the release of prostacyclin from mesenteric arteries. This release may modulate the action of endothelin locally.     

Phosphodiesterase 5 inhibitors augment UT-15C-stimulated ATP release from erythrocytes of humans with pulmonary arterial hypertension

Both prostacyclin analogs and phosphodiesterase 5 (PDE5) inhibitors are effective treatments for pulmonary arterial hypertension (PAH). In addition to direct effects on vascular smooth muscle, prostacyclin analogs increase cAMP levels and ATP release from healthy human erythrocytes. We hypothesized that UT-15C, an orally available form of the prostacyclin analog, treprostinil, would stimulate ATP release from erythrocytes of humans with PAH and that this release would be augmented by PDE5 inhibitors. Erythrocytes were isolated and the effect of UT-15C on cAMP levels and ATP release were measured in the presence and absence of the PDE5 inhibitors, zaprinast or tadalafil. In addition, the ability of a soluble guanylyl cyclase inhibitor to prevent the effects of tadalafil was determined. Erythrocytes of healthy humans and humans with PAH respond to UT-15C with increases in cAMP levels and ATP release. In both groups, UT-15C-induced ATP release was potentiated by zaprinast and tadalafil. The effect of tadalafil was prevented by pre-treatment with an inhibitor of soluble guanylyl cyclase in healthy human erythrocytes. Importantly, UT-15C-induced ATP release was greater in PAH erythrocytes than in healthy human erythrocytes in both the presence and the absence of PDE5 inhibitors. The finding that prostacyclin analogs and PDE5 inhibitors work synergistically to enhance release of the potent vasodilator ATP from PAH erythrocytes provides a new rationale for the co-administration of these drugs in this disease. Moreover, these results suggest that the erythrocyte is a novel target for future drug development for the treatment of PAH.     

Effect of long-term hypoxia on cultured aortic and pulmonary arterial endothelial cells

In a previous study, we found a marked difference in the release of a cytokine, neutrophil chemoattractant activity (NCA), from cultured endothelial cells exposed to acute decreases in ambient oxygen, depending on the vascular bed of origin. In the current study, we used this cytokine to evaluate the effect of long-term exposure to decreased oxygen on endothelial cell function. We found that, in aortic and pulmonary arterial endothelial cells maintained for months in decreased ambient oxygen (10 or 3% oxygen), exposure to acute decreases in ambient oxygen caused a change in the pattern of NCA release; however, the differential response between the two cell types persisted. Aortic endothelial cells release NCA when exposed acutely to a level of oxygen below that in which they have been chronically maintained. In contrast, pulmonary arterial endothelial cells release NCA only when exposed to 0% oxygen acutely, but only if grown chronically in 10% oxygen; otherwise there was no release of NCA. As another indicator of endothelial cell function, we evaluated the effects of acute hypoxic exposure on prostacyclin production by endothelial cells maintained in 21 or 3% oxygen. If grown in 21% oxygen, both cell types decreased prostacyclin production upon exposure to 0% oxygen. However, when grown in 3% oxygen, only aortic endothelial cells decreased prostacyclin production when exposed acutely to 0% oxygen; pulmonary arterial endothelial cell prostacyclin production did not change. This study demonstrating the persistence of a differential pattern of NCA release and the appearance of a differential pattern of prostacyclin production after a long-term decrease in environmental oxygen suggests that the capacity of certain vascular endothelial cells to respond to decreases in oxygen concentration is carried by the cell throughout its existence. Thus, in certain situations, vascular endothelial cells may be important in sensing acute decreases in ambient oxygen.     

Effects of uterine stimulant drugs on prostacyclin production by the pregnant rat myometrium. I. Oxytocin, bradykinin and PGF2α

The release of prostacyclin from chopped myometrial fractions of 18–20 day pregnant rats was assayed by inhibition of ADP-induced aggregation of citrated rabbit platelet-rich plasma. Preincubation of myometrial tissue with oxytocin 10 mU/ml increased prostacyclin generation from 2.25 ± 0.48 (control) to 3.75 ± 0.73 ng/mg over 15 minutes. Bradykinin 20 μg/ml also caused a significant increase in myometrial prostacyclin output from 2.26 ± 0.19 to 4.26 ± 0.64 ng/mg. PGF_2α 1 μg/ml did not increase prostacyclin release significantly. Pretreatment of myometrial tissue with the phospholipase inhibitor mepacrine significantly reduced the peptide-stimulated release of prostacyclin. The results suggest that prostacyclin production may play an important role in modulating the actions of oxytocin and bradykinin in the pregnant rat myometrium.     

Synthesis and metabolism of leukotrienes by human endothelial cells: influence on prostacyclin release

The synthesis and metabolism of leukotrienes (LTs) by endothelial cells was investigated using reverse-phase high-performance liquid chromatography. Cells were incubated with [14C]arachidonic acid. LTA4 or [3H]LTA4 and stimulated with ionophore A23187. The cells did not synthesize leukotrienes from [14C]arachidonic acid. LTA4 and [3H]LTA4 were converted to LTC4, LTD4, LTE4 and 5,12-diHETE. Endothelial cells metabolized [3H]LTC4 to [3H]LTD4 and [3H]LTE4. The metabolism of [3H]LTC4 was inhibited by L-serine-borate complex, phenobarbital and acivicin in a concentration-related manner, with maximal inhibition occurring at a concentration of 0.1 M, 0.01 M and 0.01 M, respectively. LTC4, LTB4 and LTD4 stimulated the synthesis of prostacyclin, measured by radioimmunoassays as 6-keto-PGF1 alpha. The stimulation by LTC4 was greater than that by LTD4 or LTB4. LTE4, 14,15-LTC4 and 14,15-LTD4 failed to stimulate the synthesis of prostacyclin. LTD4 and LTB4 also stimulated the release of PGE2, whereas LTC4 did not. Serine-borate and phenobarbital inhibited LTC4-stimulated synthesis of prostacyclin in a concentration-related manner. They also inhibited the release of prostacyclin by histamine, A23187 and arachidonic acid. Acivicin had no effect on the release of prostacyclin by LTC4, histamine or A23187. Furthermore, FPL-55712, an LT receptor antagonist, inhibited LTC4-stimulated prostacyclin synthesis but had no effect on histamine-stimulated release of prostacyclin or PGE2. Indomethacin inhibited both LTC4- and histamine-stimulated release. The results show that (a) endothelial cells metabolize LTA4, LTC4 and LTD4 but do not synthesize LTs from arachidonic acid; (b) LTC4 act directly at the leukotriene receptor to stimulation prostacyclin synthesis; (c) the presence of the glutathione moiety at the C-6 position of the eicosatetraenoic acid skeleton is necessary for leukotriene stimulation of prostacyclin release; and (d) the metabolism of LTC4 to LTD4 and LTE4 does not appear to alter the ability of LTC4 to stimulate the synthesis of PGI2.     

Effect of elastase on prostacyclin synthesis in aortic smooth muscle cells

The effects of elastase on prostacyclin biosynthesis in cultured rat aortic smooth muscle cells were investigated. Prostacyclin is the major product formed from arachidonic acid by aortic smooth muscle cells. When intact cells were incubated with elastase, a significant stimulatory effect on prostacyclin biosynthetic activity in cells was evident. However, the addition of elastase directly to the cell-free homogenates did not show any effects on prostacyclin biosynthesis. The maximal effect of elastase on the stimulation of prostacyclin biosynthesis without any cellular damage was observed at a concentration of 50 unit/ml elastase. Elastase also caused a marked release of arachidonic acid. At higher concentrations of elastase (75-100 units/ml), the release of arachidonic acid and prostacyclin synthesis was observed, but, at these concentrations of elastase, cells were slightly damaged. On the other hand, the releases of prostacyclin and arachidonic acid were markedly enhanced, when cells were preincubated with elastase (1 unit/ml) for 3 days. These results indicate that elastase, even at low concentrations, causes the releases of arachidonic acid and prostacyclin, especially when aortic smooth muscle cells are pre-treated with elastase.     

In SHR aorta, calcium ionophore A-23187 releases prostacyclin and thromboxane A2 as endothelium-derived contracting factors

In mature spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY), acetylcholine and the calcium ionophore A-23187 release endothelium-derived contracting factors (EDCFs), cyclooxygenase derivatives that activate thromboxane-endoperoxide (TP) receptors on vascular smooth muscle. The EDCFs released by acetylcholine are most likely prostacyclin and prostaglandin (PG)H(2), whereas those released by A-23187 remain to be identified. Isometric tension and the release of PGs were measured in rings of isolated aortas of WKY and SHR. A-23187 evoked the endothelium-dependent release of prostacyclin, thromboxane A(2), PGF(2alpha), PGE(2), and possibly PGH(2) (PGI(2) > thromboxane A(2) = PGF(2alpha) = PGE(2)). In SHR aortas, the release of prostacyclin and thromboxane A(2) was significantly larger in response to A-23187 than to acetylcholine. In response to the calcium ionophore, the release of thromboxane A(2) was significantly larger in aortas of SHR than in those of WKY. In both strains of rat, the inhibition of cyclooxygenase-1 prevented the release of PGs and the occurrence of endothelium-dependent contractions. Dazoxiben, the thromboxane synthase inhibitor, abolished the A-23187-dependent production of thromboxane A(2) and inhibited by approximately one-half the endothelium-dependent contractions. U-51605, an inhibitor of PGI synthase, reduced the release of prostacyclin elicited by A-23187 but induced a parallel increase in the production of PGE(2) and PGF(2alpha), suggestive of a PGH(2) spillover, which was associated with the enhancement of the endothelium-dependent contractions. These results indicate that in the aorta of SHR and WKY, the endothelium-dependent contractions elicited by A-23187 involve the release of thromboxane A(2) and prostacyclin with a most likely concomitant contribution of PGH(2).     

Effects of prostacyclin and its stable analog, iloprost, upon insulin secretion in isolated pancreatic islets

We investigated the effects of prostacylin in three concentrations of 2.7 nM, 53.8 nM and 267 nM on insulin release by isolated rat islets. The lowest concentration produced no significant effects. The middle concentration led to stimulation followed by inhibition of the reaction studied, while the highest concentration strongly depressed insulin release. These effects of prostacyclin appeared to be specified, because they were mimicked by its stable analog, iloprost, but not by its metabolite, 6-keto-PGF_1α. The results suggest that prostacyclin generated in islets might exert locally an influencing upon insulin release.     

Respiratory movements alter the generation of prostacyclin and thromboxane A2 in isolated rat lungs: The influence of arachidonic acid-pathway inhibitors on the ratio between pulmonary prostacyclin and thromboxane A2

The influence of hyperventilation on the spontaneous generation of prostacyclin and thromboxane A₂ by isolated rat lungs was studied. Both prostacyclin and thromboxane A₂, as measured by RIA of their stable end-products, 6-oxo-PGF_1α and TXB₂ respectively, were continuously released into the perfusate. However, the concentration of prostacyclin in the perfusate was higher than thromboxane A₂. Under normal ventilation at a rate 40–50 breaths/min, the ratio between these two compounds was 5:1. Increasing the rate of respiration to 100 breaths/min preferentially stimulated the release of prostacyclin. During hyperventilation, the ratio between 6-oxo-PGF_1α and TXB₂ was 12:1. Aspirin and indomethacin suppressed both basal and hyperventilation-stimulated release of prostacyclin and thromboxane A₂. Hydroperoxy-fatty acids and tranylcypromine inhibited only the release of prostacyclin but did not affect the generation of thromboxane A₂. Our findings confirm that the lung generates prostacyclin predominantly, and provide direct evidence that respiratory movements are involved in generation of pulmonary prostacyclin and thromboxane A₂.     

The role of thrombin in the regulation of the endothelial prostaglandin production

Prostaglandin synthesis in endothelial cells may be initiated by the addition of exogenous substrate (arachidonic acid) or by addition of thrombin or the CA2+-ionophore A23187, which leads to prostacyclin formation from endogenous substrates. We noticed that endothelial cells produce more than twice the amount of prostacyclin when incubated with thrombin and arachidonic acid together than with arachidonic acid alone. In addition, it was found that the thrombin-induced conversion of endogenous substrates was inhibited by exogenous arachidonic acid. This means that the conversion of exogenous added arachidonic acid to prostacyclin was stimulated by thrombin. This activation of the enzymes involved in prostacyclin synthesis lasted about 5 min and could be inhibited by phospholipase inhibitors such as mepacrine and p-bromophenyl-acylbromide but not by the cAMP analogue dibutyryl cAMP, an inhibitor of arachidonic acid release from cellular phospholipids. These data demonstrate that, in addition to causing release of endogenous substrate, thrombin and the Ca2+-ionophore also activate the enzyme system involved in the further transformation of arachidonic acid.     

The effect of immune mediators (cytokines) on the release of endothelium-derived relaxing factor (EDRF) and of prostacyclin by freshly harvested endothelial cells.

The effect of recombinant tumor necrosis factor and other cytokines stimulated by LPS (lipopolysaccharide), on the release of endothelial-derived relaxing factor and of prostacyclin was investigated using freshly harvested endothelial cells attached to plastic microcarrier beads. The results show that the cytokines failed to interfere with the release of EDRF and prostacyclin under the conditions of these experiments.