Eicosapentaenoic acid and prostacyclin production by cultured human endothelial cells

Human umbilical vein endothelial cells incorporate eicosapentaenoic acid (EPA) when this fatty acid is present in the culture medium. From 30 to 70% of the uptake remains as EPA, and much of the remainder is elongated to docosapentaenoic acid. All of the cellular glycerophospholipids become enriched with EPA and docosapentaenoic acid, with the largest increase in EPA occurring in the choline glycerophospholipids. When this fraction is enriched with EPA, it exhibits a large decrease in arachidonic acid content. Cultures exposed to tracer amounts of [1-14C]linolenic acid in 5% fetal bovine serum convert as much as 17% of the radioactivity to EPA. The conversion is reduced, however, in the presence of either 20% fetal bovine serum or 50 microM linolenic acid. Like arachidonic acid, some newly incorporated EPA was released from the endothelial cells when the cultures were exposed to thrombin. However, as compared with arachidonic acid, only very small amounts of EPA were converted to prostaglandins. Cultures enriched with EPA exhibited a 50 to 90% reduction in capacity to release prostacyclin (PGI2) when subsequently stimulated with thrombin, calcium ionophore A23187, or arachidonic acid. The degree of inhibition was dependent on the time of exposure to EPA and the EPA concentration, and it was not prevented by adding a reversible cyclooxygenase inhibitor, ibuprofen, during EPA supplementation. EPA appears to decrease the capacity of the endothelial cells to produce PGI2 in two ways: by reducing the arachidonic acid content of the cell phospholipid precursor pools and by acting as an inhibitor of prostaglandin production. These findings suggest that regimens designed to reduce platelet aggregation and thrombosis by EPA enrichment may also reduce the capacity of the endothelium to produce PGI2.     

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.     

Calcium ionophore A 23187 induces arachidonic acid release from phosphatidylcholine in cultured human endothelial cells

Cultured endothelial cells from human umbilical vein were incubated with (³H)arachidonic acid for 24 hours. The label was incorporated into phospholipids (79.3 %), neutral lipids (15.6 %) and non-esterified fatty acids (4.7 %). Upon challenge with the calcium ionophore A 23187, 5.3 % of the total radioactivity were found in supernatant and corresponded to 6-keto-prostaglandin F_1α (1.6 %) and free arachidonic acid (3.7 %). This release was accompanied by a concomitant and selective decrease of phosphatidylcholine. It is concluded that the entry of calcium promoted by A 23187 activates a phospholipase A₂ regulating the availability of arachidonic acid to the prostacyclin synthetase.     

Prostaglandin thromboxane, and 12-hydroxy-5,8,10,14-eicosatetraenoic acid production by ionophore-stimulated rat serosal mast cells.

Production of several metabolites of arachidonic acid by purified rat serosal mast cells in response to stimulation with the ionophore A23187 was assessed by stable isotope dilution assay using gas chromatography-mass spectrometry. Compounds quantified were prostaglandins D2, E2, F2 alpha, 6-keto-F1 alpha, thromboxane B2, and 12-hydroxy-5,8,10,14-eicosatetraenoic acid. Mast cells incubated at 37 degrees C for 30 min without ionophore produced measurable quantities of all metabolites assayed. 4 microM A23187 resulted in substantial increased synthesis of all metabolites compared to control cells. Of the metabolites quantified, prostaglandin D2 and prostacyclin were the major products derived from arachidonic acid in ionophore-stimulated rat mast cells.     

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 μM 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.     

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.     

Cultured endothelial cells increase their capacity to synthesize prostacyclin following the formation of a contact inhibited cell monolayer

The synthesis of the prostaglandins (PG), prostacyclin (PGI2), PGE2, and thromboxane A2 (TXA2), has been investigated in actively growing and contact-inhibited bovine aortic endothelial cell cultures. Cells were stimulated to synthesize prostaglandins by exposure to exogenous arachidonic acid or to the endoperoxide PGH2 and by the liberation of endogenous arachidonic acid from cellular lipids with melittin or ionophore A23187. Increased capacity of the cells to synthesize PGI2 and PGE2 was observed as a function of time in culture, regardless of the type of stimulation. TXA2 production increased with time only upon stimulation of the cells with ionophore A23187. This increased PG synthetic capacity was independent of cell density since it was mainly observed in confluent, nondividing endothelial cell cultures. The fact that increased PGI2 production in confluent cells was also observed with PGH2, a direct stimulator of PGI2 synthetase, implies that this process is independent of the arachidonate concentration within the cells or in the culture medium. This increased capacity is likely to reflect an increased activity of the PG synthetase system associated with the formation of a contact inhibited endothelial cell monolayer. A similar time-dependent increase in the PGI2 production capacity was also observed during growth of cultured bovine corneal endothelial cells.     

Oligomers of prostaglandin B1 inhibit arachidonic acid mobilization in human neutrophils and endothelial cells

The previous paper (Biochim. Biophys. Acta 1006 (1989) 272-277) has demonstrated that oligomers of prostaglandin B1 are effective in vitro inhibitors of a wide range of both cell-derived and extracellular phospholipases A2. The present study has investigated the effects of prostaglandin oligomers on agonist-stimulated phospholipase activity on intact human cells. PGBx, an oligomer (n = 6) or PGB1, and PGB-trimer inhibit as much as 95% of the A23187-stimulated release of arachidonic acid from human neutrophils. The effect is dose-dependent, with an IC50 of 4-5 microM; near maximal inhibition is obtained with as little as 1 min of preincubation with PGB-trimer. Consistent with its role as a phospholipase A2 inhibitor, PGB-trimer also inhibits the A23187-stimulated incorporation of [3H]acetate into platelet-activating factor. PGBx and PGB-trimer also inhibit the release of arachidonic acid from human umbilical vein endothelial cells stimulated with histamine, thrombin, or ionophore A23187; inhibition of the basal or unstimulated turnover of both arachidonic acid and oleic acid is also observed. Inhibition by PGB-trimer can be blocked by simultaneous addition of 50 microM albumin; cells preincubated with PGB-trimer are not affected by albumin. Furthermore, removal of exogenous PGB-trimer prior to challenge with A23187 does not reverse the inhibition of either endothelial cells and neutrophils. Thus, prostaglandin B1 oligomers are taken up by human neutrophils and vascular endothelial cells and serve as potent inhibitors of arachidonic acid mobilization. One mechanism for the pharmacological effects of PGBx may be inhibition of cell-associated and extracellular phospholipase A2.     

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.     

Docosahexaenoic acid metabolism and effect on prostacyclin production in endothelial cells

Bovine aortic endothelial cultures readily take up docosahexaenoic acid (DHA). Most of the DHA was incorporated into phospholipids, primarily in ethanolamine and choline phosphoglycerides, and plasmalogens accounted for 34% of the DHA contained in the ethanolamine fraction after a 24-h incubation. The retention of DHA in endothelial phospholipids was not greater than other polyunsaturated fatty acids and unlike arachidonic and eicosapentaenoic acids, DHA did not continue to accumulate in the ethanolamine phosphoglycerides after the initial incorporation. About 15% of the [14C(U)]DHA uptake was retroconverted to docosapentaenoic and eicosapentaenoic acids in 24 h. Some of the newly incorporated [14C(U)]DHA was released when the cells were incubated subsequently in a medium containing serum and albumin. The released radioactivity was in the form of free fatty acid and phospholipids and after 24 h, 11% was retroconverted to docosapentaenoic and eicosapentaenoic acids. Total DHA uptake was decreased only 10% by the presence of a 100 microM mixture of physiologic fatty acids, but as little as 10 microM docosatetraenoic acid reduced DHA incorporation into phospholipids by 25%. DHA was not converted to prostaglandins or lipoxygenase products by the endothelial cultures. When DHA was available, however, less arachidonic acid was incorporated into endothelial phospholipids, and less was converted to prostacyclin (PGI2). Enrichment of the endothelial cells with DHA also reduced their capacity to subsequently produce PGI2. These findings indicate that endothelial cells can play a role in DHA metabolism and like eicosapentaenoic acid, DHA can inhibit endothelial PGI2 production when it is available in elevated amounts.     

Stimulation-dependent recruitment of cytosolic phospholipase A2-alpha to EA.hy.926 endothelial cell membranes leads to calcium-independent association.

Cytosolic phospholipase A2-alpha (cPLA2-alpha) is a calcium-activated enzyme involved in agonist-induced arachidonic acid release. In endothelial cells, free arachidonic acid is predominantly converted into prostacyclin, a potent vasodilator and inhibitor of platelet activation. As the rate-limiting step in prostacyclin production is the generation of free arachidonic acid by cPLA2-alpha, this enzyme has become an attractive pharmacological target and the focus of many studies. Following stimulation with calcium-mobilizing agonists, cPLA2-alpha translocates to intracellular phospholipid membranes via its C2 domain. In this study, the calcium-induced association of cPLA2-alpha with EA.hy.926 endothelial cell membranes was investigated. Subcellular fractionation and immunofluorescence studies showed that following stimulation with histamine, thrombin or the calcium ionophore A23187, cPLA2-alpha relocated to intracellular membranes. Treatment of A23187-stimulated cells with EGTA or BAPTA-AM demonstrated that a substantial pool of cPLA2-alpha remained associated with membrane fractions in a calcium-independent manner. Furthermore, immunofluorescence microscopy studies revealed that cells stimulated for periods of greater than 10 min showed a high proportion of calcium-independent membrane-associated cPLA2-alpha. Calcium-independent membrane association of cPLA2-alpha was not due to hydrophobic or cytoskeletal interactions. Finally, the recombinant C2 domain of cPLA2-alpha exhibited calcium-independent membrane binding to membranes isolated from A23187-stimulated cells but not those isolated from nonstimulated cells. These findings suggest that novel mechanisms involving accessory proteins at the target membrane play a role in the regulation of cPLA2-alpha. Such regulatory associations could enable the cell to discriminate between the varying levels of cytosolic calcium induced by different stimuli.     

Eicosapentaenoic acid utilization by bovine aortic endothelial cells: effects on prostacyclin production

We have investigated whether the presence of other fatty acids in physiologic amounts will influence the effects of eicosapentaenoic acid on cellular lipid metabolism and prostaglandin production. Eicosapentaenoic acid uptake by cultured bovine aortic endothelial cells was time and concentration dependent. At concentrations between 1 and 25 microM, most of the eicosapentaenoic acid was incorporated into phospholipids and of this, 60-90% was present in choline phosphoglycerides. Eicosapentaenoic acid inhibited arachidonic acid uptake and conversion to prostacyclin (prostaglandin I2) but was not itself converted to eicosanoids. Only small effects on the uptake of 10 microM eicosapentaenoic acid occurred when palmitic, stearic or oleic acids were added to the medium in concentrations up to 75 microM. In contrast, eicosapentaenoic acid uptake was reduced considerably by the presence of linoleic, n-6 eicosatrienoic, arachidonic or docosahexaenoic acids. Although a 100 microM mixture of palmitic, stearic, oleic and linoleic acid (25:10:50:15) had little effect on the uptake of 10 or 20 microM eicosapentaenoic acid, less of this acid was channeled into endothelial phospholipids. However, the fatty acid mixture did not prevent the inhibitory effect of eicosapentaenoic acid on prostaglandin I2 formation in response to either arachidonic acid or ionophore A23187. An 8 h exposure to eicosapentaenoic acid was required for the inhibition to become appreciable and, after 16 h, prostaglandin I2 production was reduced by as much as 60%. These findings indicate that the capacity of aortic endothelial cells to produce prostaglandin I2 is decreased by continuous exposure to eicosapentaenoic acid. Even if the eicosapentaenoic acid is present as a small percentage of a physiologic fatty acid mixture, it is still readily incorporated into endothelial phospholipids and retains its inhibitory effect against endothelial prostaglandin I2 formation. Therefore, these actions may be representative of the in vivo effects of eicosapentaenoic acid on the endothelium.     

Pathways of arachidonic acid liberation in thrombin and calcium ionophore A23187-stimulated human endothelial cells: respective roles of phospholipids and triacylglycerol and evidence for diacylglycerol generation from phosphatidylcholine

Cultured endothelial cells from human umbilical vein were incubated for 20 h at 37 degrees C in the presence of [U-14C]arachidonic acid. Around 60-70% of the radioactive fatty acid was incorporated into cell lipids and was predominantly found in phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and triacylglycerol (39%, 33%, 13% and 6.5% of total incorporated radioactivity, respectively). Stimulation of the cells with human thrombin (2 U/ml) or calcium ionophore A23187 (5 microM) promoted the release into supernatants of arachidonic acid, 6-ketoprostaglandin F1 alpha, prostaglandins E2 and F2 alpha, in decreasing order of importance. The amount of secreted material was 4-fold higher with A23187, compared to thrombin. Parallel to the liberation process, phosphatidylcholine underwent a rapid decrease of radioactivity with both agonists, suggesting the involvement of a Ca2+-dependent phospholipase A2. Phosphatidylethanolamine displayed a minor decrease with A23187, whereas some reacylation was observed at 10 min with thrombin. Phosphatidylinositol was non-significantly affected in thrombin-stimulated cells, whereas A23187 promoted an early but minor decrease, followed by resynthesis. In contrast to A23187, thrombin was also able to promote a significant hydrolysis of triacylglycerol, which might thus be implicated in the process of arachidonate liberation. Finally, radioactive phosphatidic acid and diacylglycerol appeared in endothelial cells, in response to the two agonists. However, diacylglycerol formation did not parallel that of phosphatidic acid, especially with A23187. Determination of the 14C/3H ratio of the different lipids upon cell labelling with both [14C]arachidonic acid and [3H]palmitic acid revealed that diacylglycerol and phosphatidic acid are hardly derived from inositol-phospholipid breakdown by phospholipase C. Other possible pathways involving for instance phospholipase C splitting of phosphatidylcholine are discussed.     

Induction of prostacyclin formation by sodium n-butyrate in a cloned epithelial liver cell line

The effect of sodium n-butyrate on prostaglandin synthesis in cultured cells was examined. Exposure of BC-90 cells, a clone of an epithelial rat liver cell line, to 1 mM sodium n-butyrate for 40 h induced prostacyclin production. Prostacyclin synthesis was proved by demonstrating: (1) production of labeled 6-ketoprostaglandin F1 alpha by treating [14C]arachidonic acid pre-labeled cells with calcium ionophore A23187, (2) production of unstable substance that inhibited adenosine diphosphate-induced platelet aggregation, and (3) conversion of [14C]arachidonic acid to 6-ketoprostaglandin F1 alpha in homogenates of n-butyrate-treated cells. Untreated control cells showed negligible prostaglandin synthesis. Untreated cell homogenates did not convert [14C]arachidonic acid to any prostaglandins, but they converted [14C]prostaglandin H2 to prostacyclin. Induction of prostacyclin production by n-butyrate was also demonstrated with cells that had been treated with acetylsalicylic acid before n-butyrate treatment in acetylsalicylic acid-free medium. Incorporation of [3H]acetylsalicylic acid by sodium n-butyrate-treated cells increased in accordance with treatment time, while that of untreated cells did not change during culture. There was no difference in the phospholipase A2 activities of n-butyrate-treated and -untreated cells. From these findings, the possibility that n-butyrate induced prostacyclin in BC-90 cells through induction of fatty acid cyclooxygenase activity is discussed.     

Production of arachidonic acid metabolites by endothelial cells in hyperoxia

This study investigated the response of bovine pulmonary artery endothelial cells to incubation in hyperoxia (95% O2-5% CO2). Changes in cell number and morphology, release of lactate dehydrogenase, and production of arachidonic acid metabolites were assessed during continuous exposure of confluent endothelial monolayers to air (air-5% CO2, "controls") or O2 (95% O2-5% CO2, "O2-exposed") for periods of 12-72 h. Control monolayer cell numbers remained constant (approximately 2,000,000 cells/flask), whereas the number of cells in O2-exposed monolayers decreased progressively to 30% of controls (P less than 0.01) by 72 h. As assessed by radioimmunoassay, both control and O2-exposed cells produced the prostacyclin metabolite, 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha), and prostaglandin F2 alpha (PGF2 alpha), but no thromboxane metabolite (TxB2) was detected. The O2-exposed cells released significantly more 6-keto-PGF1 alpha and PGF2 alpha than control cells when apparent net production rates over the entire 72-h period were compared. In addition, both control and O2-exposed (48 h) endothelial monolayers released immunoreactive leukotriene B4 (LTB4) on stimulation with calcium ionophore (10 microM A23187). As with the cyclooxygenase products, O2-exposed cells released more immunoreactive LTB4 than did controls. Both cyclooxygenase and lipoxygenase metabolites of arachidonic acid are released by cultured endothelial cells during the development of O2 toxicity.     

Arachidonic acid release in BW755C-pretreated rat peritoneal mast cells stimulated with A23187, concanavalin A and compound 48/80

Rat peritoneal mast cells respond to various secretagogues, such as ionophore A23187, concanavalin A (Ig E receptor cross-bridging) and compound 48/80 (membrane perturbing), to secrete histamine and to liberate arachidonic acid. Arachidonic acid release was made identifiable by pretreatment with BW755C, an inhibitor of both lipoxygenase and cyclo-oxygenase. The extent of arachidonic acid release varied among these three secretagogues. A23187 appeared to be most potent, whereas compound 48/80 was weakest. The sources of released arachidonic acids may be different depending on the types of stimulants. The stimulation with A23187 released arachidonic acid mainly from phosphatidylcholine and triacylglycerol. After treatment with concanavalin A and compound 48/80, in addition to phosphatidylcholine, phosphatidylinositol also appeared to serve as a donor of arachidonic acid.     

The platelet cyclooxygenase metabolite 12-L-hydroxy-5, 8, 10-hepta-decatrienoic acid (HHT) may modulate primary hemostasis by stimulating prostacyclin production

Although HHT accounts for approximately one third of the arachidonic acid (AA) metabolites produced by stimulated platelets, no well defined function has been attributed to this product. We report that HHT stimulates prostacyclin production by endothelial cells, and have identified the mechanism for this effect. In human umbilical venous endothelial cells, HHT (0.5 and 1 microM) stimulated prostacyclin (RIA for 6KPGF1 alpha) by 32 +/- 22% (1SD) and 42 +/- 38% (P less than 0.05 and less than 0.01). Similar changes were observed when the effect of HHT on exogenous [1-14C] AA metabolism in fetal bovine aortic endothelial cells (FBAECs) was studied. Kinetic analyses revealed that HHT affected vascular cyclooxygenase. HHT (1 microM) increased Vmax in test microsomes (706 +/- 21 pmol/mg/min, mean +/- 1SE) when compared to controls (529 +/- 20; P less than 0.02). No concomitant effect on Km was observed. A further effect of HHT on AA release from endothelial cell membrane phospholipids was noted. Prelabeling experiments revealed that HHT (1 microM) increased the ionophore stimulated release of AA from FBAECs (20952 +/- 555 cpm/well control mean +/- 1SE vs 25848 +/- 557 for paired HHT treated cells; P less than 0.05). The effect of HHT on platelet AA metabolism was next studied. Preincubation of washed platelets with HHT (1 microM) did not enhance thrombin or arachidonic acid induced platelet TXB2 formation. In platelets prelabelled with [1-14C]AA, HHT (1 microM) had no effect on AA release post thrombin stimulation. Conversion to cyclooxygenase metabolites was also not enhanced. HHT stimulates vascular prostacyclin without a concomitant effect on platelet AA metabolism. HHT may thus be an important local modulator of platelet plug formation.     

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.     

Release of arachidonic acid from vascular endothelial cells: fatty acyl specificity is observed with receptor-mediated agonists and with the calcium ionophore A23187 but not with melittin

Vascular endothelial cells respond to a variety of physiological and pharmacological stimuli by releasing free arachidonic acid from membrane phospholipids, thus initiating synthesis of prostacyclin. Previous work in our laboratory has demonstrated that the thrombin-stimulated deacylation is specific for arachidonate and structurally similar polyunsaturated fatty acids that contain a delta-5 double bond. We now report that histamine, bradykinin, and the calcium ionophore A23187 exhibit the same fatty acid specificity as does thrombin. Experiments with both human umbilical vein and calf pulmonary artery endothelial cells indicate that these agonists stimulate the release of previously incorporated [14C]arachidonate but not 8,11,14-[14C]eicosatrienoate or [14C]docosatetraenoate. By contrast, melittin stimulates the release of 8,11,14-eicosatrienoate, docosatetraenoate, and oleate as well as arachidonate. These results suggest that histamine, bradykinin, and A23187 activate a common calcium-dependent phospholipase A2. Melittin appears either to alter the substrate specificity of the receptor-linked phospholipase A2 activity or to activate additional enzymes as well.     

Release of arachidonic acid from human endometrial cells in culture mediated by calcium ionophore (A23187) or fluoride.

Primary cultures of endometrial glands and stromal cells were labelled with [14C]-arachidonic acid for 4 h before exposure to either the calcium ionophore, A23187 (which activates phospholipase A2 (PLA2) by increasing intracellular calcium concentrations) or sodium fluoride (which activates a G-protein). Calcium ionophore (0.5-50 mumol/l) stimulated a dose- and time-dependent release of arachidonic acid from endometrial glands. Incubation with ionophore (10 mumol/l) for 1 h released 22% of the incorporated arachidonic acid. There was a corresponding decrease in phospholipids and no loss from triglycerides. Stromal cells were unresponsive to ionophore. Fluoride (10 mmol/l) stimulated a release of arachidonic acid from stromal cells and endometrial glands (6.5% of the total arachidonic acid incorporated). In stromal cells, arachidonic acid was released from triglycerides in Day-1 cultures and from phospholipids in Day-2 cultures. In both Day-1 and Day-2 cultures of endometrial glands, arachidonic acid was released from phospholipids, but not from triglycerides. Among the phospholipids, phosphatidylcholine was always the major source of arachidonic acid. Arachidonic acid release from endometrial glands and stromal cells may be mediated by activation of PLA2 (or phospholipase C) via a G-protein, but in glands calcium ionophore may have a direct effect on PLA2. The response to calcium ionophore may reflect the differences in calcium requirements of the two endometrial PLA2 isoenzymes.