Arachidonic acid strongly stimulates prostaglandin I3 (PGI3) production from eicosapentaenoic acid in human endothelial cells

Eicosapentaenoic acid (EPA) is a prominent polyunsaturated fatty acid in fish oil which inhibits blood platelet aggregation and thromboxane A2 formation but not prostacyclin-like material generation from vascular endothelium. In this study we investigated interaction between EPA and arachidonic acid (AA) during their oxygenation by cultured endothelial cells. As measured by gas chromatography-mass spectrometry (GC-MS), AA increased markedly prostaglandin I3 (PGI3) production from EPA while that of PGI2 from AA was decreased by EPA. However, increasing the ratio AA/EPA over one almost suppressed the inhibition of PGI2 formation by EPA, and the stimulation of PGI3 production by AA was even higher. The effect of AA on EPA conversion to minor prostaglandins like PGE3 and PGF3 alpha was similar then confirming the stimulating effect and suggesting it is occurring at the cyclooxygenase instead of the prostacyclin synthase level. Altogether these data indicate that, in certain nutritional states where the liberation of EPA from endothelial cells will be accompanied with that of endogenous AA, substantial amounts of PGI3 could contribute to the prostacyclin-like activity of the vessel wall in addition to PGI2.     

Evidence for a bidirectional prostaglandin endoperoxide shunt between platelets and the bovine coronary artery

While platelets have been shown to be capable of supplying prostaglandin (PG) H2 to endothelial cells in culture for PGI2 synthesis, endothelial cells have been shown unable to supply PGH2 to platelets for thromboxane (TX) A2 synthesis. We incubated rings of the bovine coronary artery (BCAR) with human platelets treated with aspirin (to inhibit cyclooxygenase) or CGS 13080 (to inhibit TXA2 synthase) in the presence of 20 microM arachidonic acid. BCAR, with damaged endothelium, produced significantly less PGI2 than that with intact endothelium. However, co-incubation with CGS 13080-treated platelets resulted in an increase in PGI2 independent of endothelium, demonstrating a shunt of PGH2 from platelets to BCAR. Co-incubation of BCAR with aspirin-treated platelets resulted in a net increase in TXA2 demonstrating a shunt of PGH2 from BCAR to platelets. Employing [14C]PGH2 as substrate, BCAR with and without intact endothelium produced similar amounts of 6-keto-[14C]PGF1 alpha. Likewise, homogenates (50 micrograms protein) of intimal and subintimal regions of BCAR and BCAR converted similar amounts of PGH2 to 6-keto-PGF1 alpha. These data suggest that vascular production of PGH2 is more dependent on an intact endothelium than is the conversion of PGH2 to PGI2. These data also suggest a potential for a bidirectional exchange of PGH2 between platelets and vascular wall during platelet-vascular wall interactions.     

The effects of prostacyclin (PGI2) and 6-keto-PGF1 alpha on bovine plasma progesterone and LH concentrations

Injections of 1 mg PGI2 directly into the bovine corpus luteum significantly increased peripheral plasma progesterone concentrations within 5 min. Concentrations were higher in the PGI2-treated heifers than in saline-injected controls between 5 and 150 min and at 3.5, 4, 5, and 7 h post-treatment. Levels tended to remain elevated through 14 h. Saline and 6-keto-PGF1 alpha were without effect on plasma progesterone levels. The luteotrophic effect of PGI2 was not due to alterations in circulating LH concentrations. An in vitro experiment assessed the effects of either PGI2 alone or in combination with LH on progesterone production by dispersed luteal cells. Progesterone accumulation over 2 h for control, 5 ng LH, 1 microgram PGI2, 10 micrograms PGI2, and 10 micrograms PGI2 plus 5 ng LH averaged 99 +/- 42, 353 +/- 70, 152 +/- 35, 252 +/- 45, and 287 +/- 66 ng/ml (n = 4), respectively. Thus PGI2 has luteotrophic effects on the bovine CL both in vivo and in vitro.     

Selenium deficiency inhibits prostacyclin release and enhances production of platelet activating factor by human endothelial cells

Selenium is an essential component of glutathione peroxidase, an enzyme which protects cells against peroxidation and controls concentrations of intracellular peroxides. Since selenium deficiency is clinically associated with an increased degree of atherosclerosis, the effects of selenium deficiency on prostacyclin (PGI2) and platelet activating factor (PAF) production by cultured human umbilical vein endothelial cells (HUVEC) were investigated. In selenium-deficient HUVEC, histamine-induced PGI2 synthesis was significantly decreased when compared to selenium-supplemented HUVEC; in contrast, histamine-induced PAF production was increased by selenium deficiency. Histamine-induced inositol trisphosphate and [Ca2+]i responses and the conversion of PGG2 and PGH2 to PGI2 were not altered by selenium deficiency. However, selenium deficiency decreased the conversion of exogenous arachidonate to PGI2 and markedly suppressed glutathione peroxidase activity. These results suggest that selenium deficiency, by decreasing glutathione peroxidase activity, makes HUVEC susceptible to peroxide-induced inhibition of the cyclooxygenase activity of PGH2 synthase, resulting in decreased PGI2 production. These changes may alter platelet function in vivo and thus play a role in the increased incidence of atherosclerosis reported in selenium-deficient individuals.     

Prostaglandin I2-IP signaling blocks allergic pulmonary inflammation by preventing recruitment of CD4+ Th2 cells into the airways in a mouse model of asthma

PGI(2) plays a key role in limiting Th2-mediated airway inflammation. In studies to investigate the mechanism underlying such regulation, we found that the PGI(2) receptor, IP, is preferentially expressed by effector CD4(+) Th2 cells, when compared with Th1 cells. Adoptive transfer of DO11.10 Th2 cells pretreated with PGI(2) resulted in considerably attenuated pulmonary inflammation and airway hyperreactivity in BALB/c recipient mice in response to OVA inhalation. This suppression was independent of increased cAMP levels, because pretreatment of Th2 cells with dibutyryl cAMP before transfer had no effect on airway inflammation. Moreover, PGI(2) pretreatment of Th2 cells suppressed the ability of the cells to infiltrate the lungs but not the spleen. In vitro studies showed that PGI(2) did not affect IL-4 and IL-5 production or the level of IFN-gamma by the T cells. However, the prostanoid strongly inhibited CCL17-induced chemotaxis of CD4(+) Th2 but not Th1 cells. The IP was implicated in this process since migration of wild-type Th2 cells in response to CCL17 was markedly reduced following treatment with PGI(2), whereas IP-deficient Th2 cells were unaffected and migrated effectively. Collectively, these experiments suggest that PGI(2), which is generated by endothelial cells during lung inflammatory response, serves to limit the influx of Th2 cells to the airways. Our results identify PGI(2)-IP as an important pathway for inhibiting allergic pulmonary inflammation by controlling recruitment of CD4(+) Th2 cells into the inflammatory site.     

Trans fatty acids in partially hydrogenated soybean oil inhibit prostacyclin release by endothelial cells in presence of high level of linoleic acid

Partially hydrogenated soybean oil (PHSBO) and natural soybean oil (SBO) were obtained from a commercial source and their fatty acids were fractionated into saturates, monoenes and diene fractions. The present study compared the effect of the total, monoene and diene fatty acid fractions of PHSBO with those of the SBO on the fatty acid composition of the cell phospholipids (PL) and the prostacyclin (PGI(2)) release by endothelial cells (EC) in culture. Results showed that arachidonic acid (AA) level decreased significantly and linoleic acid (LA) significantly increased in the cells incubated with the diene fraction or the monoene fraction of PHSBO plus 18:2 at 3:1 ratio compared to the cell incubated with those fractions of SBO. These changes were attributed to the inhibition of LA conversion to AA by trans 18:1 and 18:2 isomers present in the monoene or diene fractions of PHSBO leading to a significant decrease of PGI(2) released by the cells incubated with monoene or diene fractions of PHSBO. The cells incubated with the monoene of PHSBO or SBO plus 18:2 at a 1:1 ratio showed no inhibition of LA conversion to AA and the level of AA was almost equal in their PL, but the PGI(2) released by the cells incubated with the monoene of PHSBO was significantly less than the cells incubated with the monoene of SBO. This decrease was not related to the inhibition of PGI(2) synthesizing enzymes or phospholipase (PLA(2)) activities. Our data show that trans acids in PHSBO inhibited the PGI(2) release by the cells through controlling the level of AA as substrate, either by (a) inhibiting the conversion of LA to AA or (b) by shunting the free AA released by the PLA(2) action to metabolism by another pathway leaving less AA available for PGI(2) synthesis.     

Up-regulation of endothelial cyclooxygenase-2 and prostanoid synthesis by platelets. Role of thromboxane A2

Platelet-vascular endothelial cell interactions are central to the maintenance of vascular homeostasis. Thromboxane A2 (TXA2) and prostacyclin (prostaglandin (PG)I2) are the major products of cyclooxygenase (COX) metabolism by platelets and the vascular endothelium, respectively. Here we report the effects of platelet-endothelial interactions on human umbilical vein endothelial cells (HUVECs) COX-2 expression and prostanoid synthesis. Co-incubation of platelets with HUVECs resulted in a dose-dependent induction in COX-2 expression. This was accompanied by a relatively small increase in thromboxane B2 synthesis (2 ng) by comparison to the production of 6-keto-PGF1alpha and PGE2, which increased by approximately 14 and 12 ng, respectively. Abrogation of platelet-HUVEC interactions excluded direct cell-cell contact as a required event. Preincubation of HUVECs with SQ29548, a TXA2 receptor antagonist, dose-dependently inhibited platelet-induced COX-2 expression and prostanoid synthesis. Similarly, if platelet TXA2 synthesis was inhibited no induction of COX-2 was observed. Furthermore, a TXA2 analog, carbocyclic TXA2, induced HUVEC COX-2 expression and the synthesis of 6-keto-PGF1alpha and PGE2. This was also associated with an increase in the expression and activity of PGI synthase and PGE synthase but not TX synthase. Platelet co-incubation (or TXA2) also selectively activated the p44/42 mitogen-activated protein kinase pathway to regulate HUVEC COX-2 expression. Thus it seems that platelet-derived TXA2 can act in a paracrine manner to up-regulate endothelial COX-2 expression and PGI2 synthesis. These observations are of particular importance given the recent observations regarding selective COX-2 inhibitors and the suppression of PGI2 synthesis.     

Epigallocatechin gallate increase the prostacyclin production of bovine aortic endothelial cells

We describe the effect of (-) epigallocatechin gallate (EGCg), one of catechins known in tea, on the prostacyclin (PGI) production by bovine aortic endothelial cells. The amounts of 6-keto-PGF(1alpha) and Delta(17)-6-keto-PGF(1alpha), stable metabolites of PGI(2) and PGI(3), released in culture medium were measured using gas chromatography/selected ion monitoring (GC/SIM). The prostacyclin production of endothelial cells was increased by EGCg in a dose- and time-dependent manner. The effect by EGCg was stronger than any other catechins (catechin, epicatechin, epigallocatechin, and epicatechin gallate). When endothelial cells incubated with EGCg and arachidonic acid (AA) or eicosapentaenoic acid (EPA), PGI(2), and PGI(3) production were increased greater than those incubated with AA or EPA alone. Furthermore, gallic acid, that also has a pyrogallol structure, increased PGI(2) production. These observations indicate that catechins increase the prostacyclin production and that the pyrogallol structure is significant to this function.     

Role of human heme oxygenase-1 in attenuating TNF-alpha-mediated inflammation injury in endothelial cells

Heme oxygenase (HO) is the rate-limiting enzyme in the formation of bilirubin, an antioxidant, and carbon monoxide (CO), a cell cycle modulator and a vasodilator. Cyclooxygenase (COX) is a hemeprotein that catalyzes the conversion of arachidonic acid (AA) to various prostanoids, which play an important role in the regulation of vascular endothelial function in normal and disease states. The influence of suppression or overexpression of HO isoforms on COX expression and synthesis of prostanoids is of considerable physiological importance. Consequently, the goal of the present study was to determine whether the heme-HO system regulates COX enzyme expression and activity in vascular endothelial cells in the absence and presence of TNF-alpha (100 ng/ml). Endothelial cells stably transfected with the retrovirus containing the human HO-1 gene exhibited a several-fold increase in HO-1 protein levels, which was accompanied by an increase in HO activity and a marked decrease in PGE(2) and 6-keto PGF(1alpha) levels. We also assessed the effect of retrovirus-mediated HO-1 gene transfer in the sense and antisense orientation on HO-1 expression and cell cycle progression in human endothelial cells. The levels of CO and HO activity were increased in cells transduced with the HO-1 sense and were greatly suppressed in cells transduced with HO-1 antisense as compared to control sham-transduced cells (P < 0.05). The percentage of the G(1)-phase in cells transduced with HO-1 significantly increased (41.4% +/- 9.1) compared with control endothelial cells (34.8% +/- 4.9). We measured COX activity by determining the levels of PGI(2) and PGE(2). The levels of PGI(2) decreased in cells transduced with HO-1 sense and increased in cells transduced with HO-1 in antisense orientation. The expression of p27 was also studied and showed a marked decrease in cells transduced with HO-1 sense and a marked increase in the HO-1 antisense transduced cells. Cell cycle analysis of endothelial cell DNA distributions indicated that the TNF-alpha-induced decrease in the proportion of G(1)-phase cells and increase in apoptotic cells in control cultures could be abrogated by transfection with HO-1 in the sense orientation. Tin mesoporphyrin (SnMP) reversed the protective effect of HO-1. These results demonstrate that overexpressing HO-1 mitigated the TNF-alpha-mediated changes in cell cycle progression and apoptosis, perhaps by a decrease in the levels of COX activity.     

Arachidonic acid metabolism in thrombocytes and vascular tissues of turkeys

Turkeys are hypertensive compared to mammals of similar size. In vitro synthesis of thrombocyte thromboxane B2 (TxB2), 12L-hydroxy-5,8,10 heptadecatrienoic acid (HHT), 12L-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE) and aortic prostaglandin (PG) production was studied in one to ten month old domestic white turkeys. Compared to normal human platelets, TxB2 production was increased (55.4 vs. 31.4%) and HETE production was markedly reduced (6.5 vs. 34.6%) in control thrombocytes. Similar to human platelets in which cyclooxygenase inhibition with aspirin results in an increase in HETE production, block of the thrombocyte enzyme with aspirin doubled the production of HETE. In vitro conversion of radiolabeled arachidonic acid (AA) showed that the primary PG produced by turkey aorta was PGE2. A 6-keto immunoreactive PG was present which comigrated with authentic 6-keto PGF1 alpha, but failure of the aortic supernatant to inhibit adenosine diphosphate or AA induced platelet aggregation suggested that PGI2 was not produced. The vasodepressor potency of PGE1, PGE2 and PGI2 was altered in awake turkeys with PGE1 and PGE2 having five times the hypotensive effect as PGI2. In addition, conversion of AA to PGE2 by aorta in one month turkeys was greater (17.3 vs. 9.2%) than in ten month old turkeys. Systemic arterial pressure was increased in the ten month old turkeys (188 mmHg) compared to one month old turkeys (143 mmHg). Thus, both vascular AA metabolism and the vasodepressor potencies of PGE2 and PGI2 are altered and the activity of the lipoxygenase pathway in thrombocytes is limited in the turkey.     

Human platelet secreted proteins and prostacyclin production by bovine aortic endothelial cells

The effects of specific human platelet-secreted proteins on prostacyclin (PGI2) production by primary cultures of bovine aortic endothelial cells have been studied. Cells were incubated with various concentrations of highly purified preparations of platelet factor 4 (PF4), low-affinity platelet factor 4 (LA-PF4), beta-thromboglobulin (beta TG), platelet basic protein (PBP), and partially purified platelet-derived growth factor (PDGF) in the presence or absence of arachidonic acid (AA). The amount of 6-Keto-PGF1 alpha, the stable degradation product of PGI2, was determined in the cell incubation medium by means of a specific radioimmunoassay. Short-term (15 min) incubation of cell monolayers with either LA-PF4 or beta TG slightly reduced 6-keto-PGF1 alpha production. The effect was not dose-related and could not be observed after prolonged (24 hr) incubation of the cells with the same proteins. It was not seen in the cell suspensions. Moreover, 6-keto-PGF1 alpha production stimulated by AA was not affected by incubation with either of the proteins. PF4 and PBP had no significant effect on 6-keto-PGF1 alpha production by endothelial cells. Human PDGF showed a slight tendency to stimulate 6-keto-PGF1 alpha release when cells were incubated for 24 hr with the protein; however, PDGF did not potentiate the stimulatory effect of AA on 6-keto-PGF1 alpha release by the cells. We suggest that platelet-derived proteins exert only a moderate and possibly nonspecific effect on PGI2 production by endothelial cells.     

Cytoprotective effect of reduced glutathione in hydrogen peroxide-induced endothelial cell injury

The kinetic effects of hydrogen peroxide (H2O2) on cultured endothelial cells isolated from bovine carotid artery were studied. The cytoprotective effects of glutathione (GSH) on H2O2-induced cell injury were also investigated. H2O2-induced a dose- and time-dependent cell injury in cultured endothelial cells. H2O2-induced cell injury was blocked by simultaneous treatment by catalase, but not by superoxide dismutase. H2O2 also induced endogenous PGI2 biosynthesis, and the maximum PGI2 production was reached after 1 h treatment. Stimulation of PGI2 production was parallel with arachidonate release from H2O2-treated cells. However the prostaglandin biosynthesis enzyme activity in cells was inhibited by H2O2 treatment. When the cells were treated with GSH, the intracellular GSH reached a plateau after 3 h treatment. Both H2O2-induced cell injury and PGI2 production were significantly inhibited by the 3 h pretreatment with GSH. The cytoprotective effect of GSH was completely inhibited by buthionine sulfoximine which is a specific inhibitor of gamma-glutamylcysteine synthetase. The results indicate that the cytoprotective effect of GSH on H2O2-induced cell injury in cultured bovine carotid artery endothelial cells depends on the increase in intracellular GSH content.     

Activation of group VI phospholipase A2 isoforms in cardiac endothelial cells

The endothelium comprises a cellular barrier between the circulation and tissues. We have previously shown that activation of protease-activated receptor 1 (PAR-1) and PAR-2 on the surface of human coronary artery endothelial cells by tryptase or thrombin increases group VIA phospholipase A(2) (iPLA(2)β) activity and results in production of multiple phospholipid-derived inflammatory metabolites. We isolated cardiac endothelial cells from hearts of iPLA(2)β-knockout (iPLA(2)β-KO) and wild-type (WT) mice and measured arachidonic acid (AA), prostaglandin I(2) (PGI(2)), and platelet-activating factor (PAF) production in response to PAR stimulation. Thrombin (0.1 IU/ml) or tryptase (20 ng/ml) stimulation of WT endothelial cells rapidly increased AA and PGI(2) release and increased PAF production. Selective inhibition of iPLA(2)β with (S)-bromoenol lactone (5 μM, 10 min) completely inhibited thrombin- and tryptase-stimulated responses. Thrombin or tryptase stimulation of iPLA(2)β-KO endothelial cells did not result in significant PAF production and inhibited AA and PGI(2) release. Stimulation of cardiac endothelial cells from group VIB (iPLA(2)γ)-KO mice increased PAF production to levels similar to those of WT cells but significantly attenuated PGI(2) release. These results indicate that cardiac endothelial cell PAF production is dependent on iPLA(2)β activation and that both iPLA(2)β and iPLA(2)γ may be involved in PGI(2) release.     

Prostaglandin I2 reduces activation of human arterial smooth muscle cells in-vivo

Patients suffering from peripheral vascular disease have been "ultima ratio"-treated with PGI2 at a rate of 5 ng/kg/min for 6 hours a day and 5 consecutive days i.v. 20 of them underwent surgery thereafter as therapy was not sufficient. A histological examination and quantification of vascular tissue revealed that the number of activated smooth muscle cells was significantly lower in treated patients vascular segments than in untreated ones in all the different age groups. A comparable suppression was found in the intima and the media as well. It is thus concluded, that PGI2 inhibits smooth muscle cell proliferation most probably by inhibiting PDGF-release from the platelets and stimulation of smooth muscle cell cAMP. To achieve a more beneficial PGI2-effect at the vascular level, a prolonged PGI2-therapy looks rather promising.     

Prostaglandin 12 reduces activation of human arterial smooth muscle cells in-vivo

Patients suffering from peripheral vascular disease have been “ultima ratio”-treated with PGI2 at a rate of 5 ng/kg/min for 6 hours a day and 5 consecutive days i.v. 20 of them underwent surgery thereafter as therapy was not sufficient. A histological examination and quantification of vascular tissue revealed that the number of activated smooth muscle cells was significantly lower in treated patients vascular segments than in untreated ones in all the different age groups. A comparable suppression was found in the intima and the media as well. It is thus concluded, that PGI2 inhibits smooth muscle cell proliferation most probably by inhibiting PDGF-release from the platelets and stimulation of smooth muscle cell cAMP. To achieve a more beneficial PGI2-effect at the vascular level, a prolonged PGI2-therapy looks rather promising.     

Expression of prostaglandin G/H synthase (cyclooxygenase) during murine fetal thymic development.

Fetal thymic lobes in organ culture have been shown to have the capacity to metabolize [14C]arachidonic acid (AA) to prostaglandins (PGs), including 6-ketoPGF1 alpha, PGF2 alpha, PGE2, and PGA2. Inhibition of AA metabolism results in inhibition of growth and Thy 1 expression during thymic organ culture. We report herein that freshly-isolated fetal thymic lobes also have the capacity to metabolize [14C]AA to PGs and HETEs at Days 14 and 16 of prenatal murine development. RNA encoding phospholipase A2, which liberates arachidonic acid from membrane phospholipids, and cyclooxygenase (prostaglandin G/H synthase), the first enzyme involved in the conversion of AA to PGs, are expressed during thymic development. We have localized the cyclooxygenase protein to stromal cells in the fetal and adult thymus. Exogenous AA or an analogue of PGI2 (iloprost) stimulated growth of fetal thymocytes in organ culture. These findings, together with our studies of the morphology of thymic lobes cultured with inhibitors of arachidonate metabolism, support the hypothesis that PGs are required for thymocyte proliferation during thymic development.     

Prostaglandin production in cultured gastric mucosal cells: role of cAMP on its modulation

The effect of cAMP on prostaglandin production may depend on cell types. To clarify the relationship between PG and cAMP, we examined arachidonate's effects on PG synthesis and intracellular cAMP accumulation in monolayers of rat gastric mucosal cells. These cells produced PGE2, PGI2 and thromboxaneA2 (TXA2) in amounts of 316 +/- 18, 100 +/- 7 and 30 +/- 5 pg per 10(5) cells in 10 min, respectively, in response to 10 microM arachidonic acid (AA). The production of these PG, however, leveled off subsequently. Cells initially exposed to AA responded poorly to a subsequent stimulation by AA. AA simultaneously stimulated intracellular cAMP accumulation; this stimulatory effect on cAMP production was abolished by the pretreatment with indomethacin. Nevertheless, the pretreatments with dibutyryl cAMP (0.1-5 mM) did not alter the amount of subsequent AA-induced PGE2 production. Furthermore, the preincubation with 1mM isobutyl methyl xanthine also failed to affect PGE2 synthesis, while it increased intracellular cAMP accumulation. Our studies suggest AA stimulates intracellular cAMP formation in cultured gastric mucosal cells, linked with conversion of AA to cyclooxygenase metabolites, AA-induced PG production is limited in these cells, and it seems, however, unlikely that intracellular cAMP modulates AA metabolism to PG.     

IL-1 and related cytokines enhance thrombin-stimulated PGI2 production in cultured endothelial cells without affecting thrombin-stimulated von Willebrand factor secretion or platelet-activating factor biosynthesis

We examined the effects of various cytokines on alpha-thrombin-stimulated prostaglandin (PG) I2 production, von Willebrand factor (vWF) secretion, and platelet-activating factor (PAF) synthesis in cultured human umbilical vein endothelial cells (HUVEC). A 24-h pretreatment with IL-1 beta doubled the low level of constitutive PGI2 production. In contrast, alpha-thrombin increased PGI2 production fivefold in untreated HUVEC. The most striking increase in PGI2 production was observed in IL-1 beta-treated HUVEC that were subsequently stimulated with thrombin. PGI2 production was two to three times greater than in untreated, thrombin-stimulated HUVEC and nearly eightfold greater than in IL-1 beta-treated but unstimulated HUVEC. Enhanced thrombin-stimulated PGI2 production was also observed in HUVEC pretreated with the related cytokines IL-1 alpha, TNF, or lymphotoxin. This cytokine effect was selective for PGI2 production because none of these cytokines altered either constitutive or thrombin-stimulated vWF secretion or PAF biosynthesis. IL-1 beta enhancement of thrombin-stimulated PGI2 production was concentration and time dependent and required protein synthesis. IL-1 beta pretreatment also enhanced PGI2 production in response to another agonist, histamine, and to exogenously added substrates, arachidonic acid or PGH2. Our results indicate that activation by IL-1 and related cytokines selectively primes endothelial cells for enhanced PGI2 production, but not vWF secretion or PAF synthesis, in response to thrombin and histamine. The evidence suggests that this effect is mediated through specific induction of biosynthetic enzymes for PGI2.     

Prostaglandin E2 production is synergistically increased in cultured human glomerular mesangial cells by combinations of IL-1 and tumor necrosis factor-alpha 1

Both IL-1 alpha and IL-1 beta and TNF-alpha induced a time- and dose-dependent release of authentic PGE2 from cultured human glomerular mesangial cells (HMC). This release became significant only after a 4- to 6-h lag phase, and was abolished by inhibition of protein synthesis, and was not related to cell proliferation. Combinations of IL-1 and TNF-alpha when added simultaneously to HMC resulted in a dose-dependent synergistic increase in PGE2 production. These stimulatory effects were specifically inhibited by anticytokine antibodies and the synergistic effect required the simultaneous presence of both IL-1 and TNF-alpha. Arachidonic acid (AA) release experiments and measurement of cyclooxygenase activity, revealed that while both were increased by IL-1 beta and TNF-alpha alone (IL-1 beta greater than TNF-alpha), combinations of IL-1 beta and TNF-alpha resulted in only additive increases in AA release and cyclooxygenase activity. Taken together, these data suggest that stimulation of PGE2 in HMC, by combinations of these cytokines, is not rate limited by AA release or cyclooxygenase activation, but may be related to the induction of the distal enzymes controlling specific PG synthesis.     

The effects of ionizing radiation on the pulmonary endothelial cell uptake of alpha-aminoisobutyric acid and synthesis of prostacyclin

The effects of gamma irradiation (150-3000 rad) on prostacyclin synthesis (PGI2) and Na+-dependent amino acid uptake (alpha-aminoisobutyric acid, AIB) were assessed in vitro in bovine pulmonary artery endothelial cells grown in plastic culture dishes. A dose-dependent increase in both PGI2 synthesis and AIB was found 24 h after irradiation at exposure levels greater than 600 rad. The increase in PGI2 synthesis [297% of sham-irradiated values at 3000 rad, P less than 0.01] was due to an increase in release of arachidonic acid from plasma membrane stores as well as stimulation of cyclooxygenase and/or prostacyclin synthetase enzymes. The increase in AIB uptake (75% increase at 3000 rad compared to sham-exposure values) correlated with the increased synthesis of PGI2 (r = 0.94). There was also a dose-dependent increase in the number of cells that became detached from the culture dishes during the 24-h period after irradiation. The changes in PGI2 synthesis and AIB uptake induced by gamma irradiation differed if the endothelial cells were grown on cover slips, indicating that the endothelial response to irradiation may be dependent on the interaction between the endothelial cell and its extracellular basement membrane matrix.