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.     

Release of eicosanoids from cultured rat aortic endothelial cells; studies with arachidonic acid and calcium ionophore A23187

The release of prostanoids from the three different vascular cell types derived from rat aortic explants has been studied in vitro. Under resting conditions and when incubated with exogenous arachidonic acid (AA, 10 microM), the endothelial cells (EC) produced the highest concentration of prostacyclin (PGI2 PGE2 PGF2 alpha TxA2). In contrast, PGE2 was the major prostanoid produced by the smooth muscle cells and fibroblasts. Pretreatment of EC with aspirin (10 microM) or indomethacin (10 microM) effectively inhibited the production of prostanoids by these cells. Incubation with the calcium ionophore A23187 (10 microM) did not stimulate production of PGI2 or leukotriene B4 (LTB4) by EC. However, treatment of EC with a combination of A23187 and AA led to production of amounts of both PGI2 and LTB4 which were greater than the summed values for the different drug treatments. These findings indicate that the concentration of substrate, AA, is a limiting factor in prostanoid formation by these cultured vascular cells but that rat EC are relatively poor in the enzymes required for leukotriene formation.     

Production of prostaglandin E2 and prostacyclin by thymic nurse cells in culture

To better define the thymic microenvironment, we have examined a specific population of thymic stromal cells, thymic nurse cells (TNC) for production of eicosanoids. TNC were prepared from BALB/c mice, cultured in complete medium, and culture supernatants were analyzed for the presence of various metabolites of arachidonic acid. Freshly isolated TNC produced large quantities of PGE2 and 6-keto PGF1 alpha (a stable metabolite of prostacyclin, PGI2). Both of these eicosanoids were produced continuously in culture, after an initial lag period of approximately 2 h. No significant production of the eicosanoids PGD2, thromboxane B2, or leukotrienes B4, C4/D4/E4 was seen in these cultures. Production of PGE2 and PGI2 by TNC was not stimulated by treatment with the calcium ionophore A23187, or by cell-cell interactions resulting from coculture of the TNC with free thymocytes. Eicosanoid production in these cultures was not due to production of these substances by cells likely to be present as contaminants, such as T rosettes or free thymocytes. These findings raise the possibility that PGE2 and/or PGI2 may provide signals to thymocytes at a specific developmental stage.     

Epidermal growth factor (EGF), tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) and calcium ionophore A23187 increase cytoplasmic free calcium and stimulate arachidonic acid release and PGE2/6-keto PGF1 alpha production in cultured porcine thyroid cells

Epidermal growth factor (EGF), 12-O-tetradecanoylphorbol 13-acetate (TPA) and calcium ionophore A23187 increase cytoplasmic free calcium ([Ca2+]i) and stimulate arachidonic acid release and production of PGE2 and 6-keto PGF1 alpha, an end metabolite of PGI2, in cultured porcine thyroid cells. Addition of EGF, TPA or A23187 to the cells loaded with fura-2, a fluorescent Ca2+ indicator, causes an immediate increase in [Ca2+]i, which is the earliest event after mitogen stimulation. This [Ca2+]i response occurs immediately, reaching a maximum within several seconds. EGF, TPA and A23187 stimulate arachidonic acid release and PGE2 and 6-keto PGF1 alpha production; the maximum effects are obtained after 2-4 h incubation. EGF, TPA and A23187 increase [Ca2+]i and then stimulate arachidonic acid release and PG production.     

Effect of human plasma lipoproteins on prostacyclin production by cultured endothelial cells

Prostacyclin (PGI2) production by bovine aortic or human umbilical vein endothelial cells increased when either human high density lipoproteins3 (HDL3) or low density lipoproteins (LDL) were added to a serum-free culture medium. At low concentrations and short incubation times, HDL3 produced more PGI2 than LDL, but LDL was just as effective as HDL3 in 18-hr incubations with high concentrations of lipoproteins. Neither lipoprotein was toxic to the cultures as assessed by [3H]leucine incorporation into cell protein. The stimulatory effect of HDL3 and LDL on PGI2 production decreased as growing cultures became confluent. Incubation with lipoproteins neither enhanced arachidonic acid release nor increased PGI2 formation when the cells were stimulated subsequently with ionophore A23187, indicating that the lipoproteins do not affect the intracellular processes involved in PGI2 production. The addition of albumin reduced the amount of PGI2 formation elicited by HDL3 or LDL. As compared with albumin-bound arachidonic acid, from 6- to 13-fold less PGI2 was produced during incubation with the lipoproteins. Furthermore, the amount of PGI2 formation elicited by the lipoproteins in 18 hr was 4-fold less than that produced during incubation with a fatty acid mixture containing only 5% arachidonic acid, and 3-fold less than when the cells were stimulated with the ionophore A23187 for 20 min. Taken together, our results indicate that human HDL and LDL contribute to endothelial PGI2 production only in a modest way and suggest that this process is not specific for either of these two plasma lipoproteins. In view of the greater participation of albumin-bound arachidonic acid in PGI2 production, plasma lipoproteins may not play as important a role in endothelial prostaglandin formation as has been suggested.     

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.     

Profile of eicosanoids produced by human saphenous vein endothelial cells and the effect of dietary fatty acids

Human saphenous vein endothelial cells (HSVECs) derived from primary cultures of adult human veins constitute an excellent in vitro model for studying human endothelial metabolism. In this study we report the (14)C-labelled prostanoid profile of HSVECs under resting and stimulated conditions and the effect of the n-3 polyunsaturated fatty acids eicosapentaenoic acid and docosahexaenoic acid on them. Results indicate that HSVECs while under resting conditions produce mainly prostaglandin F(2alpha)(PGF(2alpha)). After stimulation with calcium ionophore A23187, the cells were found to synthesise PGI(2), PGE(2)and PGF(2alpha) as major products and thromboxane B(2)and PGD(2)as minor products. Production of (14)C-labelled hydroxyeicosatetraenoic acids was not detected. Eicosapentaenoic acid was found to inhibit basal and stimulated prostanoid production whereas docosahexaenoic acid inhibited basal but strongly increased stimulated prostanoid production. These results may offer the basis for further studies aiming to investigate targets for pharmacological intervention in inflammatory conditions.     

Regulation of prostaglandin production in cultured gastric mucosal cells

The aims of this study were to investigate whether exogenous prostaglandin modulates prostaglandin biosynthesis by cultured gastric mucosal cells, and to clarify the role of cyclic nucleotides in the possible modulation of prostaglandin production. After pretreatment for 30 min with buffer alone (control) or 1 to 100ng/ml PGE2, cells were incubated with 4 uM arachidonic acid for 30 min. Pretreatments with greater than 5ng/ml PGE2 inhibited arachidonate-induced PGE2 and PGI2 production in a dose-dependent fashion, as compared with control, with inhibition by 64 +/- 8% and 75 +/- 4% respectively, at 100ng/ml PGE2. PGE2, at 100ng/ml, significantly increased intracellular cAMP accumulation, but pretreatment with dibutyryl cAMP (0.01-mM) did not alter the amounts of arachidonate-induced PGE2 production. Furthermore, while greater than 10ng/ml PGE2 increased cGMP production dose-dependently, preincubation with dibutyryl cGMP (0.001-0.1mM) also failed to affect PGE2 synthesis significantly. In addition, pretreatment with isobutyl-methyl-xanthine, while increasing accumulation of cellular cyclic nucleotides, did not significantly change PGE2 production. Calcium ionophore A23187-induced PGE2 production was also inhibited by pretreatment with PGE2. These results indicate that exogenous PG inhibits subsequent arachidonate or A23187-induced PG biosynthesis in rat gastric mucosal cells, and suggest the possibility that PG regulates its own biosynthesis via feedback inhibition independent of cyclic nucleotides in these cells.     

Calcium ionophore A23187 elevates angiotensin-converting enzyme in cultured bovine endothelial cells

Calcium ionophore A23187 (0.3-0.4 microM) elevated cellular angiotensin-converting enzyme activity (ACE) 2-7-fold after 48 h incubation with bovine pulmonary artery endothelial cells in culture. Cycloheximide (0.1 micrograms/ml) blocked the elevation in ACE produced by A23187. The increase in ACE was inhibited by 0.2 mM EGTA, 50 microM verapamil and 50 microM nifedipine, and was not associated with changes in cellular cAMP. Melittin, a phospholipase A2 activator, or addition of exogenous arachidonic acid failed to reproduce the elevation, and indomethacin only partially blocked the A23187 effect. The elevation of ACE was also inhibited by the calcium-calmodulin inhibitor, calmidazolium. Thus, we postulate that the ionophore A23187 elevates ACE in endothelial cells through a calcium-dependent mechanism other than phospholipase A2 activation. The elevation depends on new protein synthesis and involves calcium-calmodulin-dependent cellular mechanisms.     

Production of prostaglandins by fetal rat lung type II pneumonocytes and fibroblasts

The output of prostaglandins I2, E2, F2 alpha and 13,14-dihydro-15-keto-PGF2 alpha (PGFM) from third passage day 20 rat fetal fibroblasts and type II alveolar pneumonocytes was studied. In 2 h incubations, the output levels for each cell type were: PGI2 greater than PGE2 much greater than PGF2 alpha = PGFM when cells were incubated with Ca2+ ionophore A23187 (10 microM) or arachidonic acid (1 microgram/ml).     

Prostaglandin synthesis by cells comprising the calf pulmonary artery

Prostaglandin (PG) production was evaluated in the three cell types (endothelial, smooth muscle, and fibroblast) comprising the bovine pulmonary artery. Prostacyclin (PGI2) was the predominant prostaglandin (PG) produced by endothelial, smooth muscle, and fibroblast cells as they exist in culture or in freshly excised tissue fragments. In addition to PGI2, measurable amounts of PGE2, PGF2a, and thromboxane A2 (TXA2) were also produced by these cells. Endothelial cells were the most active producers of PGs. However, the type of PG produced was characteristic of the particular cell type, while the level of production was dependent on external factors. Prostaglandin production by cultured cells, both under basal conditions and in response to stimulatory agents, was quite similar to that of the respective freshly excised tissue fragments containing a given cell type. These cells in culture could be stimulated to produce PGI2 by both angiotensin and bradykinin at very low (physiological) concentrations, a further indication of the retention of the physiological responsiveness of these cells in culture. Endothelial cells and fibroblasts were activated by bradykinin at concentrations as low as 10(-12) M but did not respond to angiotensin. Smooth muscle cells in primary and first passage cultures were activated by both bradykinin and angiotensin at 10(-12) M concentrations. Serial subcultivations of smooth muscle cells resulted in a progressive loss in their responsiveness to bradykinin stimulation. The state of cell growth proved to be an important determinant of PG production. Actively growing cells in culture synthesized less PG when compared to cells which had entered into a "quiescent" nongrowth state.     

Ca2+ requirement of prostanoid but not of superoxide production by rat Kupffer cells

The release of the prostanoids prostaglandin D2 (PGD2), prostaglandin E2 (PGE2) and thromboxane induced by zymosan and phorbol ester in cultured rat Kupffer cells was found to depend on the extracellular concentration of Ca2+ to some extent. Prostanoid formation following the addition of the calcium ionophore A 23187 was totally inhibited when calcium ions were withdrawn from the medium whereas the prostanoid synthesis from added arachidonic acid was independent of Ca2+. A half-maximal rate of PGE2 release by cells treated with zymosan, phorbol ester or A23187 was obtained at 0.6-0.7 microM free extracellular Ca2+ and greater than or equal to 100 microM free Ca2+ was required to stimulate PGE2 formation maximally. The calmodulin antagonist R24571 partially inhibited the release of PGE2 elicited by zymosan and A23187 but not by phorbol ester or arachidonic acid. Verapamil and nifedipine, two calcium channel blockers, had no effect on the formation of PGE2 irrespective of the stimulus. TMB 8 [3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester] an intracellular calcium antagonist, inhibited the synthesis of PGE2 induced by zymosan and phorbol ester. The superoxide formation following the addition of zymosan and phorbol ester was not influenced by removal of calcium ions from the medium or by addition of the various calcium antagonists. The data presented here suggest that Ca2+-dependent reactions are involved in the synthesis of prostanoids induced by zymosan and phorbol ester and that both extracellular Ca2+ and mobilization of Ca2+ from intracellular stores are needed to induce maximally the production of prostanoids in cultured rat Kupffer cells.     

Effect of fatty acid modification on prostaglandin production by cultured 3T3 cells

We have investigated the extent to which modifications in the essential fatty acid content of mammalian cells can affect prostaglandin production. Swiss mouse 3T3 cells stimulated with the calcium ionophore A23187 produced 1.7 to 7 times more prostaglandin E(2) (PGE(2)) when the cultures were supplemented with linoleic acid. Increases in PGE(2) production as a result of linoleic acid supplementation occurred under all culture conditions except during the first 24 hr after attachment, when prostaglandin production was very high. Arachidonic acid supplementation produced a similar enhancement in the capacity of the cells to produce PGE(2), but no appreciable increase occurred when the cultures were supplemented with oleic acid. The phospholipids of the cells exposed to the linoleate-enriched medium contained 4 times more arachidonic acid and twice as much linoleic acid as compared with the corresponding controls. The choline phosphoglycerides were most highly enriched in arachidonic acid, but 2- to 3-fold increases also occurred in the inositol and ethanolamine phosphoglycerides. When cultures initially enriched with linoleic acid were transferred to an unsupplemented medium, the fatty acid composition as well as the capacity of the cells to produce PGE(2) reverted almost to control values. The amount of exogenous arachidonic acid converted to PGE(2) as measured by radioimmunoassay also was greater when the cells were enriched with linoleic acid. Studies with radioactive arachidonic acid indicated that the distribution of prostaglandin metabolites was not affected appreciably by linoleic acid enrichment. These findings suggest that at least two factors contribute to the increased capacity of the cultures supplemented with linoleate to produce PGE(2). One is enrichment of the phospholipid substrate pools with arachidonic acid. The other is an increased ability of the cells to synthesize PGE(2) from unesterified arachidonic acid, perhaps because the prostaglandin-forming enzymes are more active.-Denning, G. M., P. H. Figard, and A. A. Spector. Effect of fatty acid modification on prostaglandin production by cultured 3T3 cells.     

Bradykinin and not cholecystokinin stimulates exaggerated prostanoid release from the inflamed rabbit gallbladder.

The relationship of bradykinin and cholecystokinin (CCK) to inflamed gallbladder prostanoid synthesis and release was examined in rabbits treated with common bile duct ligation (BDL) for 24 or 72 h. Gallbladders removed from control and BDL groups were incubated in oxygenated Krebs buffer at 37 degrees C (pH 7.4) for 60 min. The slices were then placed every 20 min in vials containing increasing doses of bradykinin (30-3000 ng) or CCK (30-1000 ng). Incubation fluid was analyzed by RIA for 6-keto-prostaglandin (PG)F1 alpha (PGI2 metabolite), PGE2 and thromboxane (TX) B2. Bradykinin stimulated control gallbladder 6-keto-PGF1 alpha and PGE2 release was modest. Gallbladders from 24- and 72-h BDL groups released 3- to 10-fold higher levels of 6-keto-PGF1 alpha and PGE2 (not TXB2) following bradykinin stimulation when compared to controls, which was abolished with indomethacin pretreatment. CCK did not stimulate gallbladder prostanoid release in the control or BDL groups. These data show that bradykinin and not CCK stimulated PGI2 and PGE2 release from inflamed rabbit gallbladder. Increased BDL gallbladder PGI2 release may be prolonged or augmented by bradykinin as gallbladder distention and progressive acute inflammation stimulate local bradykinin formation.     

Human keratinocyte sensitivity towards inflammatory cytokines varies with culture time

Proliferating keratinocyte cultures have been reported to synthesize higher concentrations of prostaglandin (PG) E than confluent ones. As interleukin-1 (IL-1) stimulates keratinocyte PGE synthesis we investigated whether the degree of confluency of the keratinocyte culture modified the response of the cells to IL-1. It was found that IL-1alpha (100 U/ml) stimulated PGE(2) synthesis by proliferating (7 days in culture) but not differentiating (14 days in culture) keratinocytes. Similar effects were observed using tumour necrosis factor-alpha. Both arachidonic acid (AA) and the calcium ionophore A23187 stimulated PGE(2) synthesis by 7 and 14 day cultures although the increase was greatest when 7 day cultures were used. Our data indicate that there is a specific down-regulation of the mechanism(s) by which some inflammatory cytokines stimulate keratinocyte eicosanoid synthesis as cultured keratinocytes begin to differentiate.     

Synthesis of prostaglandins and cyclic AMP by cultured embryonic palate mesenchyme at various population densities

During embryonic development, facial and palate mesenchymal cells exhibit differential growth rates. Normal palatal growth is regulated in part by hormones and growth factors. Because hormonal responsiveness of some cells correlates with their cell density, we have investigated the relationship between embryonic palate mesenchymal cell population density and their ability to synthesize prostaglandins (PGs) and cyclic AMP. Primary cultures of palate mesenchymal cells exhibited typical lag, log, and stationary phases of growth with a doubling time of 32-34 hrs. The ability of cells to produce PGE2 in response to a calcium ionophore (A23187), an activator of phospholipase A2 (melittin), arachidonic acid, or serum was maximal during the period of early exponential growth. Prostaglandin F2 alpha synthesis in response to A23187 or arachidonic acid showed a similar transient increase also corresponding temporally to the period of early exponential growth. The ability to synthesize PGF2 alpha in response to melittin, however, failed to diminish after early exponential growth. The pattern of cAMP synthesis in response to isoproterenol and PGE1 was different from that seen for induced prostaglandin synthesis. A transient increase in sensitivity to isoproterenol and PGE1 was seen that corresponded temporally to the period of late exponential growth just prior to attainment of confluency. Decreased sensitivity to stimulation of either prostaglandin or cAMP production as the cells became confluent was shown to be a density-dependent phenomenon; confluent cultures that were subcultured to reestablish logarithmic growth exhibited density-dependent hormonal responses identical to those seen in primary cultures. The ability of palate mesenchymal cells to synthesize both prostaglandins and cAMP, thought to be critical for proper palatal development, might thus be related to local differential craniofacial growth rates.     

Regulation of prostaglandin production by osteoblast-rich calvarial cells

The effect of various factors upon prostaglandin (PG) production by the osteoblast was examined using osteoblast-rich populations of cells prepared from newborn rat calvaria. Bradykinin and serum, and to a lesser extent, thrombin, were all shown to stimulate PGE2 and 6-keto-PGF1 alpha (the hydration product of PGI2) secretion by the osteoblastic cells. Several inhibitors of prostanoid synthesis, dexamethasone, indomethacin, dazoxiben and nafazatrom, were tested for their effects on the calvarial cells. All inhibited PGE2 and PGI2 (the major arachidonic acid metabolites of these cells) production with half-maximal inhibition by all four substances occurring at approximately 10(-7) M. For dazoxiben and nafazatrom, this was in contrast to published results from experiments in vivo which have indicated that the compounds stimulated PGI2 production. Finally, since the osteoblast is responsive to bone-resorbing hormones, these were tested. Only epidermal growth factor (EGF) was shown to modify PG production. At early times EGF stimulated PGE2 release, however, the predominant effect of the growth factor was an inhibition of both PGE2 and PGI2 production by the osteoblastic cells. The present results suggest that the bone-resorbing hormones do not act to cause an increase in PG by the osteoblast and that any increase in PG production by these cells may be in response to vascular agents.     

Influence of morphology and malignancy of three new human melanoma cell lines on the cyclooxygenase pathway

Three newly established human melanoma cell lines (WU-BI, PN-JC, MJ-ZJ) of different morphology and different stage of malignancy were incubated with ionophore A23187 (2.5 to 40 microM) or arachidonic acid (AA, 6.25 to 100 microM). PGF2 alpha, 6-keto-PGF1 alpha, PGE2, TXB2 and 2,3-dinor-TXB2 from isolated cells and supernatants were measured by negative ion chemical ionization gas chromatography/mass spectrometry (GC/MS). PGE2 decreased in the fibroblastoid MJ-ZJ cells from 36.7 ng/mg cell protein about 70% (A23187) and about 20% (AA), respectively. However, in the cell supernatant PGE2 increased up to 295.4 +/- 66.5 ng/mg cell protein. Production of PGF2 alpha and PGE2 increased up to 5.7 +/- 1.2 ng/mg cell protein for polydendritic WU-BI cells and spindle shaped PN-JC cells. Up to 9.3 +/- 4.3 ng PGF2 alpha and 13.4 +/- 4.7 ng PGE2 was measured for WU-BI and PN-JC in the cell supernatants. All three melanoma cell lines completely lacked formation of 6-keto-PGF1 alpha, TXB2, and 2,3-dinor-TXB2.     

Purification of natural killer-like Kurloff cells and arachidonic acid metabolism.

Pulmonary and splenic Kurloff cells have been purified from estrogen-treated guinea pig. Enzymatic digestion of lung tissue and mechanical dispersion of cells yielded about 650 x 10(6) viable cells. After centrifugal elutriation and centrifugation on continuous Percoll gradient, a population of high-density (1,100 g/ml) pulmonary Kurloff cells were obtained with high viability (approximately 99%) and purity (approximately 99%). Splenic Kurloff cells have been isolated by disruption of spleen tissue and centrifugation on continuous Percoll gradient. High-density splenic Kurloff cells (150 x 10(6) cells per spleen) were also obtained with high purity (approximately 99%) and viability (approximately 99%). Pulmonary and splenic Kurloff cells were incubated with various concentrations of arachidonic acid (10, 30 and 100 microM) in the absence or presence of 2 microM ionophore A23187. With 10 microM arachidonic acid the relative production of cyclooxygenase products was the following: TxB2 greater than PGE2 approximately PGI2. For an arachidonic acid concentration superior to 10 microM, the profile of release was PGE2 much greater than TxB2 greater than PGI2. Arachidonic acid metabolism through the 5-lipoxygenase pathway was also studied by incubating pulmonary or splenic Kurloff cells with 10 microM arachidonic acid in the absence or presence of 2 microM ionophore A23187, or in some experiments, with 2.5 microM leukotriene A4. Reverse phase HPLC profiles clearly indicated that high-density Kurloff cells did not express 5-lipoxygenase activity. However, these cells showed the ability to convert exogenous leukotriene A4 into leukotriene B4 suggesting the presence of LTA4 hydrolase activity. These data have been confirmed by a sensitive RIA method. This study constitutes the first report on the purification of pulmonary Kurloff cells and on arachidonic acid metabolism by these cells. The possible implications of Kurloff cells in various biological events are discussed.     

Prostanoid synthesis by vascular slices and cultured vascular cells of piglet aorta

Biosynthesis of prostanoids was studies in vascular slices of human umbilical arteries, piglet aorta and vena cava as well as in cultured vascular cells of piglet aorta. After preincubation with radioactive labeled arachidonic acid, prostanoids in the incubation media of slices or cultured cells were measured by radioimmunoassay or by radioactivity determination of labeled compounds following separation on reserved-phase high performance liquid chromatography. In all vascular slices 6-keto-PGF1α was the main prostanoid found, followed by PGE2. Thromboxane B2 and PGF2α were also formed, but only in trace amounts. In cultured cells taken from the three layers of the vascular wall, the prostanoid profiles differed markedly from those obtained from vascular slices. Each cell strain showed a specific prostanoid pattern. Endothelial cells synthesized predominantly 6-keto-PGF1α and PGF2α. In smooth muscle cells no 6-keto-PGF1α could be detected; here the predominant prostanoid was PGE2. PGF2α was formed in smaller quantities. Fibroblasts synthesized all prostanoids (PGE2, PGF2α, TXB2, 6-keto-PGF1α), PGE2 and PGF2α being the major products. In vascular slices and in cultured endothelial cells, the predominant prostacyclin derivative detected was 6-keto-PGF1α; the enzymatic PGI2-metabolite, 6,15-diketo-PGF1α, could be detected only in piglet vena cava slices in small amounts.