Prostaglandin Synthesis In Endothelial Cells Research Articles

Modulation of prostaglandin synthesis in endothelial cells and fibroblasts by corticotropin releasing factor and antagonists: S. Fleisher-Berkovich, G. Rimon and A. Danon. Department of Clinical Pharmacology, Ben-Gurion University, Beer-Sheva 84105, Israel

Modulation of prostaglandin synthesis in endothelial cells and fibroblasts by corticotropin releasing factor and antagonists: S. Fleisher-Berkovich, G. Rimon and A. Danon. Department of Clinical Pharmacology, Ben-Gurion University, Beer-Sheva 84105, Israel

Effect of corticotropin-releasing factor on prostaglandin synthesis in endothelial cells and fibroblasts

Recent evidence suggests that not only the end product of the hypothalamic-pituitary-adrenal axis, but also other hormones in the axis may be involved in regulation of the inflammatory response. We investigated the role of CRF in the regulation of prostaglandin (PG) synthesis in fibroblasts and endothelial cells. Recombinant human interleukin-1 alpha (IL-1 alpha) increased prostacyclin synthesis in endothelial cells by 66% and prostaglandin E2 (PGE2) synthesis in fibroblasts by 91%. The PG response to IL-1 alpha was suppressed to about 50% by simultaneous addition of CRF in endothelial cells (75.6 +/- 6.2 vs. 159.7 +/- 14.9 ng 6-keto-PGF1 alpha/mg protein) and fibroblasts (115.5 +/- 23 vs. 233.6 +/- 42 ng PGE2/mg protein). IL-1 alpha enhanced phospholipase A2 activity by 30% and prostaglandin H synthase activity by 60%, and the two effects were completely blocked by CRF. It is concluded that CRF suppresses IL-1 alpha-induced PG synthesis through actions on both phospholipase A2 and cyclooxygenase. In view of the essential role of central PGE2 in IL-1 alpha-induced CRF/ACTH release, these findings suggest a novel regulatory cascade in immune-neuroendocrine interactions.

Effect of corticotropin-releasing factor on prostaglandin synthesis in endothelial cells and fibroblasts.

Recent evidence suggests that not only the end product of the hypothalamic-pituitary-adrenal axis, but also other hormones in the axis may be involved in regulation of the inflammatory response. We investigated the role of CRF in the regulation of prostaglandin (PG) synthesis in fibroblasts and endothelial cells. Recombinant human interleukin-1 alpha (IL-1 alpha) increased prostacyclin synthesis in endothelial cells by 66% and prostaglandin E2 (PGE2) synthesis in fibroblasts by 91%. The PG response to IL-1 alpha was suppressed to about 50% by simultaneous addition of CRF in endothelial cells (75.6 +/- 6.2 vs. 159.7 +/- 14.9 ng 6-keto-PGF1 alpha/mg protein) and fibroblasts (115.5 +/- 23 vs. 233.6 +/- 42 ng PGE2/mg protein). IL-1 alpha enhanced phospholipase A2 activity by 30% and prostaglandin H synthase activity by 60%, and the two effects were completely blocked by CRF. It is concluded that CRF suppresses IL-1 alpha-induced PG synthesis through actions on both phospholipase A2 and cyclooxygenase. In view of the essential role of central PGE2 in IL-1 alpha-induced CRF/ACTH release, these findings suggest a novel regulatory cascade in immune-neuroendocrine interactions.

Mechanisms by which Candida albicans induces endothelial cell prostaglandin synthesis

One strategy for improving resistance to opportunistic pathogens is to determine host cellular responses during the invasion process and upregulate those responses that are relevant to host defense mechanisms. Within this context, we have shown previously that invasion of endothelial cells by Candida albicans in vitro causes increased production of prostaglandins. As a prerequisite for modulating endothelial cell prostaglandin production, we now characterize the mechanisms through which this process occurs. Endothelial cell invasion by C. albicans appeared to stimulate the conversion of arachidonic acid into prostaglandins by upregulating the synthesis of endothelial cell cyclooxygenase and increasing the activity of the endothelial cell phospholipase. The enhanced activities of these two enzymes were independent of calphostin C-sensitive protein kinase C and resulted in the increased production and extracellular secretion of prostaglandin I2 (PGI2), PGF2 alpha, and PGE2. The secretion of these prostaglandins had no effect on the amount of endothelial cell injury induced by C. albicans. The role of the increased prostaglandin secretion by endothelial cells is likely related to modulation of the leukocyte response at the candida-leukocyte-endothelial cell interface.

Role of protein kinase C in the regulation of prostaglandin synthesis in human endothelium.

The present study specifically addresses the role of protein kinase C (PKC) activation in human endothelial cell Ca2+ mobilization, a response that is functionally coupled to the production of the potent arachidonate (AA) metabolite, prostacyclin (PGI2). Phorbol 12-myristate 13-acetate (PMA), alpha-thrombin, and sodium fluoride (NaF), a direct G-protein activator, produced a rapid and time-dependent translocation of PKC from the cytosol to the membrane. Activation of PKC by brief pretreatment of human umbilical vein endothelial cell (HUVEC) monolayers with PMA resulted in the inhibition of NaF-induced inositol phosphate increases and attenuation of both alpha-thrombin- and NaF-activated increases in intracellular Ca2+ (Ca2+i). Ca2+ mobilization induced by ionophore A23187 was not affected by PKC preactivation, suggesting PKC-dependent negative feedback inhibition of phosphatidylinositol (PI)-specific phospholipase C (PLC). Agonist-stimulated AA release and PGI2 synthesis in PMA-pretreated cultured human endothelial cells, however, was potentiated, and the enhanced PGI2 synthesis produced by A23187, NaF, and alpha-thrombin was dependent upon the dose of PMA. Treatment of HUVEC monolayers with an intracellular Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid-acetoxymethylester (BAPTA-AM), dramatically reduced alpha-thrombin-, NaF-, and A23187-induced PGI2 synthesis, demonstrating the importance of Ca2+i availability in PGI2 synthesis. BAPTA pretreatment did not inhibit PMA-induced PKC activation, and BAPTA-mediated inhibition of agonist-stimulated PGI2 synthesis was partially attenuated by prior PMA pretreatment. Staurosporine, a potent PKC inhibitor, at concentrations that inhibited PKC-induced phosphorylation of histone-1, augmented both alpha-thrombin- and NaF-induced production of inositol phosphates but markedly inhibited alpha-thrombin-, NaF-, and A23187-induced PGI2 synthesis. The downregulation of PKC activity by prolonged PMA treatment (18 h) produced similar inhibition of PGI2 synthesis by these agonists (approximately 50% inhibition). These studies indicate that the integrated phospholipase A2 and PLC activities are under complex regulation by factors that include both PKC activation and [Ca2+i]. PKC exerts dual effects on prostaglandin synthesis via negative regulation of Gp-coupled PI-specific PLC and positive feedback regulation of AA release and PGI2 synthesis. PKC is thus a critical determinant in the regulation of human endothelial cell prostaglandin synthesis by both receptor-mediated and G-protein-dependent cellular activation.

Regulation of endothelial cell prostaglandin synthesis by glutathione.

Prostaglandin synthesis in in vitro systems is dependent on glutathione and peroxide concentrations. We tested the effects of glutathione depletion and H2O2 exposure on prostaglandin synthesis in cultured porcine aortic endothelial cells. Depletion of glutathione using buthionine sulfoximine (BSO), diethylmaleate, and 2,4-chlorodinitrobenzene increased prostaglandin synthetic capacity. Production of prostacyclin, but not prostaglandin E2, from exogenous arachidonic acid was significantly greater than in controls. Glutathione depletion also resulted in enhanced production of prostacyclin from exogenous prostaglandin H2. These responses were not due to direct effects of glutathione-depleting agents on prostaglandin synthetic enzymes. Exposure to H2O2 also altered prostaglandin synthetic capacity in endothelial cells. While 5 microM H2O2 stimulated prostaglandin production from exogenous arachidonate, 25 and 50 microM were found to be inhibitory. Prostaglandin synthetic capacity was greater in BSO-treated cells which were exposed to 5 and 10 microM H2O2 than in cells exposed to H2O2 alone. However, prostaglandin synthetic capacity was greatly reduced in BSO-treated cells exposed to 50 microM H2O2. Thus, normal levels of cellular glutathione exert an inhibitory influence on prostaglandin synthesis. However, glutathione depletion increases the sensitivity of prostaglandin synthesis to inhibition by 50 microM H2O2.

Evidence for the Acceleration of Atherogenesis in Dialysis Patients by Serum Factors Affecting Prostaglandin Synthesis in Endothelial Cells

Evidence for the Acceleration of Atherogenesis in Dialysis Patients by Serum Factors Affecting Prostaglandin Synthesis in Endothelial Cells

Heparin and acidic fibroblast growth factor interact to decrease prostacyclin synthesis in human endothelial cells by affecting both prostaglandin H synthase and prostacyclin synthase

Prostaglandin production by cultured human endothelial cells varies with growth conditions. We observed a marked diminution in both spontaneous and inducible production of prostacyclin (PGI2) by human umbilical vein and saphenous vein endothelial cells when they were cultured in the presence of the heparin-binding growth factor, acidic fibroblast growth factor (aFGF) and heparin, compared with PGI2 production during culture in medium lacking these factors. Decreased PGI2 production was related to duration of exposure of the cells to aFGF and heparin and depended on the concentration of both substances. Heparin (1-100 micrograms/ml) strongly potentiated the effects of aFGF but had a limited and variable effect alone. The decrease in PGI2 production correlated with a reduction in the cellular content of immunoreactive prostaglandin H synthase and prostacyclin synthase. Arachidonate deacylation was not decreased. In addition, the eicosanoid profile of endothelial cells was changed by exposure to aFGF and heparin. These studies indicate that heparin acts as a modulator of prostaglandin synthesis in endothelial cells through its interaction with aFGF, mediated by alterations in two key enzymes in the arachidonate metabolic pathway.

Inhibition of prostaglandin synthesis after metabolism of menadione by cultured porcine endothelial cells.

We have examined the effects of menadione on porcine aortic endothelial cell prostaglandin synthesis. Addition of 1-20 microM menadione caused a dose- and time-dependent inhibition of stimulated prostaglandin synthesis with an IC50 of 5 microM at 15 min. Concentrations greater than 100 microM menadione were necessary to increase 51Cr release from prelabeled cells. Recovery of enzyme inactivated by menadione required a 6-h incubation in 1% serum. In a microsomal preparation, menadione was shown to have no direct effect on conversion of arachidonic acid to prostaglandins. In intact cells menadione caused only a 40% inhibition of the conversion of PGH2 to prostacyclin. Enzymes involved in the incorporation and the release of arachidonic acid were not affected by menadione (20 microM, 15 min). Menadione undergoes oxidation/reduction reactions in intact cells leading to partial reduction of oxygen-forming, reactive oxygen species. In our cells menadione was found to increase KCN-resistant oxygen consumption. Further, an increased accumulation of H2O2 was observed with a time course consistent with menadione-induced inhibition of prostaglandin synthesis. We conclude that menadione at sublethal doses caused inhibition of prostaglandin synthesis. The mechanism involves inactivation of PGH2 synthase by a reactive species resulting from metabolism of menadione by endothelial cells.

Regulation of prostaglandin synthesis in human vascular cells.

Prostacyclin (PGI2) synthesis in human vascular tissue is mainly regulated by the activity of enzymes that metabolize arachidonic acid, and especially by the activity of cyclooxygenase. The activity of this enzyme depends upon the balance between its synthesis and its inactivation by peroxidated metabolites or drugs. Cyclooxygenase in vascular endothelial cells is strongly inhibited by aspirin, but is rapidly resynthesized. The resynthesis proceeds more rapidly in endothelium than in smooth muscle cells. Growth factors for vascular cells strongly influence cyclooxygenase production and activity. PDGF both releases arachidonate and stimulates cyclooxygenase synthesis in fibroblasts and smooth muscle cells, and ECGF enhances new enzyme synthesis. By contrast, we find that the endothelial growth factor depresses PGI2 synthesis in human endothelial cells in a time and concentration-dependent manner. This inhibitory effect is potentiated by heparin. The inhibition correlates with a decrease in the content of immunoreactive cyclooxygenase within the endothelial cells when they are grown in the presence of ECGF and heparin. These findings suggest that proliferation selectively suppresses the function of prostaglandin synthesis in endothelial cells.

Recovery of porcine aortic endothelial cell prostaglandin synthesis following inhibition by sublethal concentrations of hydrogen peroxide

Confluent monolayers of porcine aortic endothelial cells exposed for 10 min to 100 μM H 2O 2 lose their capacity to produce prostaglandins in response to addition of saturating exogenous arachidonic acid. Significant recovery of prostaglandin I 2 and E 2 synthesis occurred within 3 h and full enzymatic capacity returned by 6 h. Reducing the injury by exposure to half the amount of H 2O 2 allowed prostaglandin I 2 production to recover to a greater extent in 3 h, while cells exposed for 60 min to either 0.5 or 1.0 mM H 2O 2 demonstrated no recovery. Pre-treatment with either actinomycin D or cycloheximide also prevented recovery following exposure to 100 μM peroxide. Injured cells did not recover when incubated with balanced salts after removal of peroxide, while incubation with medium 199 allowed for the complete return of synthetic capacity. Addition of 1% fetal calf serum in medium 199 did not facilitate recovery. Production of prostaglandins from endogenous arachidonic acid, released by either bradykinin or the ionophore A23187, was also inhibited by H 2O 2 exposure, however, full recovery of this stimulated synthesis occurred within 3 h. Cycloheximide pre-treatment completely inhibited recovery of bradykinin-induced prostaglandin I 2 synthesis. These data demonstrate that sublethal concentrations of H 2O 2 irreversibly inactivate fatty acid cyclooxygenase and that synthesis of new enzyme is required for recovery. This return of activity occurs more rapidly for production of prostaglandins from endogenous arachidonic acid compared with production following addition of exogenous substrate.

Docosatetraenoic acid in endothelial cells: Formation, retroconversion to arachidonic acid, and effect on prostacyclin production

Cultured bovine aortic endothelial cells convert arachidonic acid to docosatetraenoic acid and also take up docosatetraenoic acid from the extracellular fluid. After a 24-h incubation with biosynthetically prepared [ 3H]docosatetraenoic acid, about 20% of the cellular fatty acid radioactivity was converted to arachidonic acid. Furthermore, in pulse-chase experiments, the decrease in phospholipid docosatetraenoic acid content was accompanied by an increase in arachidonic acid, providing additional evidence for retroconversion. These findings suggest that one possible function of docosatetraenoic acid in endothelial cells is to serve as a source of arachidonic acid. The endothelial cells can release docosatetraenoic acid when they are stimulated with ionophore A23187, but they do not form appreciable amounts of eicosanoids from docosatetraenoic acid. Enrichment of the endothelial cells with docosatetraenoic acid reduced their capacity to produce prostacyclin (PGI 2) in response to ionophore A23187. This may be related to the fact that docosatetraenoic acid enrichment caused a 40% reduction in the arachidonic acid content of the inositol phosphoglycerides. In addition, less prostacyclin was formed when the enriched cells were incubated with arachidonic acid, suggesting that docosatetraenoic acid also may act as an inhibitor of prostaglandin synthesis in endothelial cells.

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 CA 2+-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 Ca 2+-ionophore also activate the enzyme system involved in the further transformation of arachidonic acid.