Alpha 1-adrenergic stimulation of arachidonic acid release and metabolism in a rat thyroid cell line. Mediation of cell replication by prostaglandin E2

The rat thyroid cell line, FRTL-5, expresses an alpha 1-adrenergic receptor when exposed to thyrotropin. We have found that occupation of this alpha 1-adrenergic receptor by norepinephrine stimulated the release of [3H]arachidonic acid from prelabeled cells. Arachidonic acid was metabolized primarily to prostaglandin E2 and to much smaller amounts of 11-hydroxy-5,8,11,13-eicosatetraenoic acid, 15-hydroxy-5,8,11,13-eicosatetraenoic acid, prostaglandin D2, and thromboxane B2. Synthesis of all these metabolites was inhibited by the cyclooxygenase inhibitor indomethacin. When FRTL-5 cells were starved of thyrotropin for 24 h, norepinephrine nearly doubled [3H]thymidine uptake into DNA. Cyclooxygenase inhibitors inhibited norepinephrine-stimulated thymidine uptake by 60-70%. Of several arachidonic acid metabolites tested, none was able to stimulate thymidine uptake directly in the presence of indomethacin. Prostaglandin E2, however, was able to restore [3H]thymidine uptake when added together with norepinephrine in the presence of indomethacin. Thus, occupation of an alpha 1-adrenergic receptor in a functional rat thyroid cell line leads to arachidonic acid release. Subsequent metabolism of the arachidonic acid by the cyclooxygenase pathway leads to synthesis of prostaglandin E2, which mediates a norepinephrine-stimulated activity related to cell replication.     

Disturbance of pulmonary prostaglandin metabolism in fetuses of alloxan-diabetic rabbits

[14C]Arachidonic acid conversion in lung homogenates of 28-day fetuses from control and alloxan-diabetic rabbits was studied. The major metabolites were 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid and prostaglandin E2. Small amounts of 6-ketoprostaglandin F1 alpha, prostaglandin F2 alpha, and thromboxane B2 were also observed. Lung homogenates from fetuses of alloxan-diabetic rabbits convert significantly less [14C]arachidonic acid to prostaglandin E2, whereas all other metabolites were present in similar quantities compared to fetuses of non-diabetic rabbits. These studies suggest that the decreased arachidonic acid conversion to prostaglandin E2 could be partially responsible for the functional delay of lung maturation in offspring of alloxan-diabetic rabbits.     

Comparison of arachidonic acid metabolism between myofibroblasts and fibroblasts isolated from rat palatal mucoperiosteum

Myofibroblasts were cultured successfully from experimental wound tissue in rat palatal mucoperiosteum. Arachidonic acid metabolizing activity in cultured myofibroblasts was compared with that in fibroblasts cultured from normal mucoperiosteum. Prostaglandins biosynthesized from [14C]arachidonic acid in cell-free homogenates of both myofibroblasts and fibroblasts were prostaglandins D2, E2 and F2 alpha, and the activity producing each prostaglandin was not significantly different between the myofibroblasts and the fibroblasts, whereas smooth muscle cells, which are histologically similar to myofibroblasts, produced mainly 6-ketoprostaglandin F1 alpha, and relatively small amounts of prostaglandin E2. The release of arachidonic acid from cells prelabeled with [14C]arachidonic acid was compared among three types of cell. The calcium ionophore A23187 strongly enhanced arachidonic acid release in all three cell types. Bradykinin, 5-hydroxytryptamine and prostaglandin F2 alpha affected the stimulation of arachidonic acid release in the fibroblasts but were less or not effective in the myofibroblasts and smooth muscle cells. In addition, prostaglandin E2 biosynthesized in response to several stimuli was measured by radioimmunoassay. The content of prostaglandin E2 correlated closely with arachidonic acid release. In this study, we showed homogeneity between the myofibroblasts and fibroblasts in prostaglandin synthesizing activity and similarity in response to various stimuli between the myofibroblasts and smooth muscle cells, from the standpoint of arachidonic acid metabolism.     

Arachidonic acid metabolic pathways in the rabbit pericardium

Minced rabbit pericardium actively converts [1-14C]arachidonic acid into the known prostaglandins (6-[1-14C]ketoprostaglandin F1 alpha, [1-14C]prostaglandin E2 and [1-14C]prostaglandin F2 alpha) and into several unidentified metabolites. The major metabolite was separated by C18 reverse-phase high-pressure liquid chromatography (HPLC) and identified by gas chromatography-mass spectrometry (GC-MS) to be 6,15-[1-14C]diketo-13,14-dihydroprostaglandin F1 alpha. The other nonpolar metabolites were 15-[1-14C]hydroxy-5,8,11,13-eicosa-tetraenoic acid (15-HETE), 11-[1-14C]hydroxy-5,8,12,14-eicosatetraenoic acid (11-HETE) and 12-[1-14C]hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE). Arachidonic acid metabolites actively produced by the pericardium could influence the tone of surface blood vessels on the myocardium.     

Conversion of ethanolamine, monomethylethanolamine and dimethylethanolamine to choline-containing compounds by neurons in culture and by the rat brain.

The incubation of neurons from chick embryos in primary culture with [3H]ethanolamine revealed the conversion of this base into monomethyl, dimethyl and choline derivatives, including the corresponding free bases. Labelling with [methyl-3H]monomethylethanolamine and [methyl-3H]dimethylethanolamine supported the conclusion that in chick neuron cultures, phosphoethanolamine appears to be the preferential substrate for methylation, rather than ethanolamine or phosphatidylethanolamine. The methylation of the latter two compounds, in particular that of phosphatidylethanolamine, was seemingly stopped at the level of their monomethyl derivatives. Fetal rat neurons in primary culture incubated with [3H]ethanolamine showed similar results to those observed with chick neurones. However, phosphoethanolamine and phosphatidylethanolamine and, to a lesser extent, free ethanolamine, appeared to be possible substrates for methylation reactions. The methylation of water-soluble ethanolamine compounds de novo was further confirmed by experiments performed in vivo by intraventricular injection of [3H]ethanolamine. Phosphocholine and the monomethyl and dimethyl derivatives of ethanolamine were detected in the brain 15 min after injection.     

Prostaglandin I2 synthesis and elevation of cyclic AMP levels in 3T3 fibroblasts

Arachidonic acid and prostaglandin H2 elevate the levels of adenosine 3':5'-monophosphate (cyclic AMP) in Balb/c 3T3 fibroblasts. This effect was inhibited by 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid, an inhibitor of prostaglandin I2 synthase (Claesson, H.-E., Lindgren, J.A. and Hammarstr!om, S. (1977) FEBS Lett. 81, 415-418). After addition of arachidonic acid to 3T3 cultures, cellular cyclic AMP levels and growth medium concentrations of 6-ketoprostaglandin F1 alpha (degradation product of prostaglandin I2) were quantitatively determined. The stimulatory effect of exogenously-added prostaglandin I2 on cellular cyclic AMP levels was also determined. The results indicate that the endogenous production of prostaglandin I2 is sufficient to explain the stimulatory action of arachidonic acid on cyclic AMP formation in 3T3 fibroblasts.     

Metabolism of arachidonic acid and prostanoids in human endometrial stromal cells in monolayer culture

In the present investigation, we compared the metabolism of arachidonic acid in human endometrial stromal cells maintained in monolayer culture with that in human decidual tissues. By gas-chromatographic analysis, the distribution of arachidonic acid in glycerophospholipids and in the neutral lipids of decidual tissues and stromal cells in culture was similar. After the addition of [14C]arachidonic acid to the culture medium, steady-state conditions with respect to radioactive labeling of the lipids of the cells were attained after 24 h, except for phosphatidylethanolamine and neutral lipids. The percentage distribution of [14C]arachidonic acid in the lipids of the cells in culture was as follows: phosphatidylcholine, 41%; phosphatidylserine, 5%; phosphatidylinositol, 19%; phosphatidylethanolamine, 22%; neutral lipids, 11%. This distribution of arachidonic acid among the lipids is similar to that in decidual tissue, except for that in phosphatidylethanolamine. The amount of radioactivity in phosphatidylethanolamine continued to increase up to 72 h whereas that in neutral lipids declined after a maximum amount was present at 4 h. In the cells in monolayer culture, [14C]prostaglandin E2 and [14C]prostaglandin F2 alpha were produced from [14C]arachidonic acid, as is true in superfused decidual tissue. The similarities in arachidonic acid metabolism in these cells to that in decidual tissue are supportive of the proposition that endometrial stromal cells in monolayer culture are an appropriate model for the study of the regulation of arachidonic acid release and prostaglandin formation by endometrium and decidua vera.     

Appearance of the arachidonic acid metabolic pathway in human promyelocytic leukemia (HL-60) cells during monocytic differentiation: enhancement of thromboxane synthesis by 1 alpha,25-dihydroxyvitamin D-3

The appearance of the arachidonic acid metabolic pathway in human promyelocytic leukemia (HL-60) cells was investigated during 1 alpha,25-dihydroxyvitamin D-3-induced monocytic differentiation. 1 alpha,25-Dihydroxyvitamin D-3-treated HL-60 cells acquired the ability to convert [1-14C]arachidonic acid to thromboxane B2 and prostaglandin E2 during monocytic differentiation. The major cyclooxygenase product synthesized by the HL-60 cells after 3-4 days exposure to 1 alpha,25- dihydroxyvitamin D-3 (48 nM) was thromboxane B2 and its production was about 19-25-times higher than that of untreated HL-60 cells. The percent conversion of thromboxane B2 from [1-14C]arachidonic acid in the 1 alpha,25-dihydroxyvitamin D-3 (48 nM, 3 day exposure)-treated HL-60 cells was about 4.4% as compared to that (about 0.3%) of the untreated cells, whereas the percent conversion of thromboxane B2 from [1-14C]prostaglandin H2 in the 1 alpha,25-dihydroxyvitamin D-3-treated cell homogenate was about 22.4% as compared to that (about 13.6%) of the untreated cell homogenate. The stimulatory effect of 1 alpha,25-dihydroxyvitamin D-3 on thromboxane B2 production from [1-14C]arachidonic acid and from [1-14C]prostaglandin H2 in HL-60 cells was inhibited by the addition of cycloheximide (1 microgram/ml). However, 1 alpha,25-dihydroxyvitamin D-3 (48 nM) did not significantly stimulate the arachidonic acid release either in HL-60 cells or in 1 alpha,25-dihydroxyvitamin D-3-induced cells. These results suggest that the stimulatory effect of 1 alpha,25-dihydroxyvitamin D-3 on the thromboxane production in HL-60 cells was not due to the activation of phospholipase A2 but due to the induction of fatty acid cyclooxygenase and thromboxane synthetase activities. Thromboxane A2 actively produced during the monocytic differentiation of HL-60 cells could influence the cell adhesiveness of the monocyte-macrophage-differentiated cells.     

Ca2(+)-dependent synthesis of prostaglandin I2 and mobilization of arachidonic acid from phospholipids in cultured endothelial cells permeabilized with saponin

The feasibility of using saponin as a permeabilization agent to study the effect of free Ca2+ concentration ([Ca2+]f) on prostaglandin I2 (PGI2) synthesis and mobilization of arachidonic acid from membrane phospholipids was investigated in cultured bovine pulmonary artery endothelial cells (BPAEC). Treatment of BPAEC with 20 micrograms/ml saponin caused selective permeabilization of the plasma membrane as determined by measurements of the release of lactate dehydrogenase and beta-hexosaminidase. In cells prelabeled with [3H]arachidonic acid for 22 h, permeabilization with 20 micrograms/ml saponin induced PGI2 synthesis and release of [3H]arachidonic acid from membrane phospholipids. These effects were dependent upon [Ca2+]f in the range 72 nM to 5 microM. Release of [3H]arachidonic acid from phospholipid classes was determined in suspensions of BPAEC prelabeled with [3H]arachidonic acid and permeabilized with 20 micrograms/ml saponin. At [Ca2+]f optimal for PGI2 synthesis, 16.2% of the total incorporated [3H]arachidonic acid was released from phosphatidylinositol (3.4%), phosphatidylethanolamine (3.5%) and phosphatidylcholine (9.3%). The time course and dependence upon [Ca2+]f of [3H]arachidonic acid release from phospholipids correlated with PGI2 synthesis. The amount of PGI2 synthesized in permeabilized BPAEC was similar to that in cell cultures treated with the calcium ionophore A23187. In comparison, however, PGI2 synthesis induced by A23187 was associated with less release of [3H]arachidonic acid from membrane phospholipids, e.g., 2.3% versus 16.2%. The greater loss of [3H]arachidonic acid from phospholipids in saponin-permeabilized BPAEC was most likely due to the loss of cell integrity and/or nonspecific effects of the detergent on phospholipases. Despite these limitations, the Ca2+ dependence observed for PGI2 synthesis and [3H]arachidonic acid mobilization suggest that saponin-permeabilization may provide a useful system for studies of the intracellular events triggered by the rise in intracellular Ca2+ which culminate in PGI2 synthesis.     

Zero extracellular K+ and prostaglandin E release in the guinea-pig taenia coli

Prostaglandin E release rates from isolated strips of guinea-pig taenia coli increased during exposure to zero K+ bathing fluid, from control values of 0.78 +/- 0.11 ng/g per min to levels as high as 29.2 ng/per min. Release rates increased for 40-50 min and then remained constant or fell despite progressive increases in intracellular sodium [Nai+] or fall in intracellular potassium [Ki+]. Readmittance of K+ to the bathing solution resulted in rapid reversal of elevated prostaglandin E release rates. [Nai+] and [Ki+] were markedly more abnormal in strips exposed to zero K+ for 70-201 min compared to 30-min exposures. Upon the readdition of K+ after long zero K+ exposure, the rate of prostaglandin E release fell long before [Nai+] and [Ki+] returned to control levels. After K+ was readded to the bathing solution, the ion concentration of tissues exposed to zero K+ for 30 min returned to normal much more quickly than did those of tissues exposed for the longer time periods, yet the exponential rate constants for fall of prostaglandin E release rate after K+ was added were not significantly different after short or long zero K+ exposure. Thus there was a dissociation between the return of [Nai+] and [Ki+] and the fall of prostaglandin E release rate to control levels. Ouabain augmented prostaglandin E release under conditions where [Ki+] could not fall. Addition of known neurotransmitters present in this tissue to the bathing fluid did not augment prostaglandin E release. Guinea-pig taenia coli strips that had been incubated with [3H]arachidonic acid, constantly released [3H]arachidonic acid and [3H]prostaglandin E and a prostaglandin which cochromatographed with prostaglandin E but could not be converted to prostaglandin B by alkali and was shown to be 6-ketoprostaglandin F1 alpha. Release of [3H]arachidonic acid and [3H]prostaglandin E plus 6-[3H]ketoprostaglandin F1 alpha was increased when strips were exposed to zero K+. Data obtained in this study suggest the augmented prostaglandin E release seen during zero K+ or ouabain is related to increased availability of unbound arachidonic acid at the site of cyclooxygenase in the cell. Augmented prostaglandin E release is apparently not related to alterations in intracellular electrolyte concentrations or release of known neurotransmitters.     

High density lipoprotein-induced cardiac prostacyclin synthesis in vitro: relationship to cardiac arachidonate mobilization

The objectives of this study were to characterize the effects of plasma lipoproteins on prostacyclin (PGI2) production by the Langendorff-perfused rabbit heart, and to determine the mechanism of lipoprotein-induced cardiac PGI2 production. PGI2 production by perfused rabbit hearts was stimulated by injections of rabbit very low density lipoproteins (VLDL), low density lipoproteins (LDL), and high density lipoproteins (HDL). HDL was much more effective than equivalent doses of VLDL or LDL. Infusion of HDL at a physiological concentration stimulated cardiac PGI2 output by 417%, but infusion of VLDL or LDL was ineffective. Cardiac PGI2 production increased from 47% to 340% with increasing doses of HDL. The release of cardiac PGI2 in response to injections or infusions of HDL occurred rapidly; maximal release of PGI2 was reached within 2 min after exposure to HDL. Injections of HDL stimulated the production of [3H]arachidonic acid, [3H]prostaglandin E2, [3H]prostaglandin F2 alpha, and [3H]6-keto-prostaglandin F1 alpha from hearts after prelabeling of cardiac lipids with [3H]arachidonic acid. These results indicate that plasma lipoproteins, specifically HDL, stimulate PGI2 production by the isolated rabbit heart. The mechanism by which HDL increases cardiac PGI2 production may involve the mobilization of cardiac arachidonic acid for PGI2 synthesis.     

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.     

Phosphatidylethanolamine is the donor of the terminal phosphoethanolamine group in trypanosome glycosylphosphatidylinositols.

A variety of eukaryotic cell surface proteins, including the variant surface glycoproteins of African trypanosomes, rely on a covalently attached lipid, glycosylphosphatidylinositol (GPI), for membrane attachment. GPI anchors are synthesized in the endoplasmic reticulum by stepwise glycosylation of phosphatidylinositol (via UDP-GlcNAc and dolichol-P-mannose) followed by the addition of phosphoethanolamine. The experiments described in this paper are aimed at identifying the biosynthetic origin of the terminal phosphoethanolamine group. We show that trypanosome GPIs can be labelled via CDP-[3H]ethanolamine or [beta-32P]CDP-ethanolamine in a cell-free system, indicating that phosphoethanolamine is acquired en bloc. In pulse-chase experiments with CDP-[3H]ethanolamine we show that the GPI phosphoethanolamine is not derived directly from CDP-ethanolamine, but instead from a relatively stable metabolite, such as phosphatidylethanolamine (PE), generated from CDP-ethanolamine in the cell-free system. To test the possibility that PE is the immediate donor of the GPI phosphoethanolamine moiety, we describe metabolic labelling experiments with [3H]serine and show that GPIs can be labelled in the absence of detectable radiolabelled CDP-ethanolamine, presumably via [3H]PE generated from [3H]phosphatidylserine (PS). The data support the proposal that the terminal phosphoethanolamine group in trypanosome GPIs is derived from PE.     

Effect of sphingosine and other amphiphilic amines on the biosynthesis of phosphatidylethanolamine and other glycerolipids in isolated rat hepatocytes.

The importance of ethanolamine and sphingosine as precursors of phosphoethanolamine was investigated by incubating them with [3H]glycerol and isolated rat hepatocytes. Sphingosine (0.1--0.5 mM) stimulated the synthesis of phosphatidylethanolamine from [3H]glycerol, but the stimulation by ethanolamine was more pronounced. Furthermore, more phosphoethanolamine accumulated in the heptatocytes after incubation with ethanolamine than after incubation with sphingosine. It is concluded that ethanolamine is the most important phosphoethanolamine precursor in rat liver. Higher concentrations of sphingosine caused accumulation of [3H]phosphatidate and inhibition of total glycerolipid synthesis in isolated hepatocytes, when incubated in the presence of [3H]glycerol. These effects were very similar to those of fenfluramine and norfenfluramine described previously. Simpler cationic amphiphilic amines, like oleoylamine and octadecyltrimethylammonium bromide, also caused these effects. Variation of alkyl chain length and amphiphile charge showed that both a positive charge and a certain alkyl chain length were necessary for interference with phosphatidate metabolism. A much wider range of compounds inhibited total glycerolipid synthesis from [3H]glycerol.     

Effect of platelet-activating factor on arachidonic acid metabolism in renal epithelial cells

We studied the effects of platelet-activating factor (PAF-acether) on phospholipase activity in renal epithelial cells. When platelet-activating factor was added to renal cells prelabeled with [3H]arachidonic acid, it induced the rapid hydrolysis of phospholipids. Up to 26% of incorporated [3H]arachidonic acid was released into the medium from renal cells. After the addition of PAF-acether, the degradation of phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine were observed. The amount of [3H]arachidonic acid released were comparable to the losses of phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine. In renal cells biosynthetically labeled by incorporation of [3H]choline into cellular phosphatidylcholine, lysophosphatidylcholine and sphingomyelin, the range of concentrations of PAF-acether-induced hydrolysis of labeled phosphatidylcholine were approximately equal to the amounts of lysophosphatidylcholine produced. We also observed a transient rise of diacylglycerol after the addition of platelet-activating factor to these cells. To test for action of phospholipase C, the accumulations of [3H]choline, [3H]inositol and [3H]ethanolamine were determined. The radioactivities in choline and ethanolamine showed little or no change. An increase in inositol was detectable within 1 min and it peaked at 3 min. These results indicate that platelet-activating factor stimulates phospholipase A2 and phosphatidylinositol-specific phospholipase C activity in renal epithelial cells. These phospholipase activities were Ca2+ dependent. Moreover, PAF-acether enhanced changes in cell-associated Ca2+. These results suggest that the increased Ca2+ permeability of cell membrane stimulates phospholipases A2 and C in renal epithelial cells. Prostaglandin biosynthesis was also enhanced in these cells by platelet-activating factor.     

Mechanism of prostaglandin biosynthesis in rabbit kidney medulla. A rate-limiting step and the differential stimulatory actions of L-adrenaline and glutathione.

Microsomal prostaglandin synthase (EC 1.14.99.1) from rabbit kidney medulla was assayed with [5,6,8,9,11,12,14,15-3H]-and [1-14C]-arachidonic acid as the substrate. The ratios of prostaglandin F2 alpha to prostaglandin E2 and to prostaglandin D2 were determined by both 3H and 14C labelling. When 3H was used as a label the ratios were much higher than with 14C labelling indicating that the removal of hydrogen at C-9 or C-11 was the rate-limiting step in the biosynthesis of prostaglandin E2 or prostaglandin D2. This finding shows that the octatritiated arachidonic acid is not the appropriate substrate marker for studying the regulation of the synthesis of different prostaglandins by various agents. When the enzyme assay was carried out in the presence of SnCL2, which was capable of accumulating exclusively prostaglandin F2alpha at the expenses of prostaglandin E2 and prostaglandin D2, the addition of L-adrenaline to the microsomal fraction either alone or with reduced glutathione equally stimulated the formation of prostaglandin F2alpha, whereas the addition of reduced glutathione to the microsomal fraction either alone or with L-adrenaline produced no additional effect. These results suggest that endoperoxide is formed as the common intermediate for the biosynthesis of three different prostaglandins in rabbit kidney medulla, and that L-adrenaline stimulates the synthesis of endoperoxide, whereas reduced glutathione facilitates the formation of prostaglandins from endoperoxide.     

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.     

6-Ketoprostaglandin F1 alpha, prostaglandins E2, F2 alpha and thromboxane B2 production by endothelial cells, smooth muscle cells and fibroblasts cultured from piglet aorta

After [3H]arachidonic acid labeling, cyclooxygenase products were qualitatively analysed in the media of each cultured vascular cell type by reverse-phase high-performance liquid chromatography (rp-HPLC). The prostaglandin E2, prostaglandin F2 alpha, 6-ketoprostaglandin F1 alpha and thromboxane B2 detected in the rp-HPLC radioactive profile were then quantified by radioimmunoassay (RIA) in separate sets of experiments. In preconfluent endothelial cells prostaglandin F2 alpha and 6-ketoprostaglandin F1 alpha were detected in equal amounts (49%), whereas after confluence 6-ketoprostaglandin F1 alpha represented 57% of total secretion (P less than 0.05). Smooth muscle cells secreted mainly prostaglandin F2 alpha (48%) and fibroblasts prostaglandin E2 (44%). Using the bioassay method, antiaggregatory activity was detected only in endothelial cells, though a small percentage of immunoreactive 6-ketoprostaglandin F1 alpha was encountered in smooth muscle cells and fibroblasts (13 and 10%, respectively). Radioimmunological analysis after rp-HPLC separation of the medium of endothelial cells showed that the anti-6-ketoprostaglandin F1 alpha antibody recognized, among other substances, an unidentified compound. Its retention time was similar to that of prostaglandin F2 alpha. This unidentified compound was not detected in the media from smooth muscle cells and fibroblasts.     

The effect of vitreous humour on prostaglandin production by cultured rabbit chorioretinal fibroblasts

Factors in vitreous humour which regulate prostaglandin production were investigated using cultured rabbit chorioretinal fibroblasts. These cells produced predominantly prostaglandin E2, 6-ketoprostaglandin F1 alpha, a compound likely to be a metabolite of prostaglandin E2 and 5-hydroxyeicosatetraenoic acid. The synthesis of 6-ketoprostaglandin F1 alpha was nearly completely inhibited by the cyclooxygenase inhibitor aspirin and partially inhibited by 10(-6) M dexamethasone (49%) and 10(-5) M forskolin (68%). Addition of 10% rabbit vitreous humour to subconfluent cells maintained in Dulbecco's modified Eagle's medium plus 1% fetal bovine serum resulted in stimulation of 6-ketoprostaglandin F1 alpha production by as much as 246% as measured by radioimmunoassay. Chorioretinal fibroblasts labelled by [3H]arachidonic acid incorporation into cellular phospholipids synthesised greater amounts of all labelled arachidonic acid metabolites in response to vitreous humour. It was concluded, therefore, that there are factors present in vitreous humour of molecular weight above 10 kDa which are capable of stimulating cellular cyclooxygenase activity. Confluent cells also responded to a factor(s) present in vitreous humour. The fraction of less than 10 kDa inhibited 6-ketoprostaglandin F1 alpha production by 50% when used at a concentration of 10%. Furthermore, 6-ketoprostaglandin F1 alpha production in confluent cells (but not subconfluent cells) was inhibited to 40% of control levels by vitamin C at a concentration of 1 mg/100 ml. The latter result points to an inhibitory role for vitamin C in vitreous humour. We conclude, therefore, that vitreous humour contains factors important for the regulation of prostaglandin metabolism in the eye.     

Specific incorporation of 5-hydroxy-6,8,11,14-eicosatetraenoic acid into phosphatidylcholine in human endothelial cells

The incorporation of hydroxyeicosatetraenoic acids (HETEs) into cellular lipids was studied in cultures of human umbilical vein endothelial cells. 5-[3H]HETE was incorporated into the phospholipids (8%) and neutral lipids (15.5%). The uptake was at half maximum after 15 min and reached a plateau after 1 h. The incorporation occurred mainly into phosphatidylcholine (6.3%) with minimal uptake into phosphatidylserine and phosphatidylinositol (0.6%) or phosphatidylethanolamine (1.2%). There was no uptake of 12-[3H]HETE, 15-[3H]HETE or [3H]leukotriene B4 into phospholipids. Treatment of the phosphatidylcholine fraction with phospholipase A2 released 64% of the 5-[3H]HETE with 26% remaining in the lysophosphatidylcholine fraction. This indicates that the majority of the 5-HETE was in the sn-2 position. Unlabeled 5-HETE and arachidonic acid inhibited the uptake of 5-[3H]HETE into phosphatidylcholine with an ID50 of 2.5 and 1.25 microM, respectively. Stearic acid and 15-HETE were not effective inhibitors. Histamine, which activates phospholipases, increased the uptake of 5-[3H]HETE into phosphatidylcholine by 3-fold. Both 5-[3H]HETE and 12-[3H]HETE were incorporated into the neutral lipids of the cells. Analysis of the neutral lipid fraction revealed that 5-[3H]HETE was incorporated into mono-, di- and triacylglycerols but not cholesterol esters. Incorporation of 5-HETE into cellular lipids reduced histamine- and arachidonic acid-stimulated synthesis of 6-ketoprostaglandin F1 alpha and prostaglandin E2 in a concentration-related manner. Angiotensin I converting enzyme activity was not changed. Thus, 5-HETE is incorporated specifically into phosphatidylcholine and glycerol esters of human endothelial cells and this incorporation inhibits prostaglandin synthesis in these cells.