Kinetics of prostanoid synthesis by macrophages is regulated by arachidonic acid sources.

The dependence of prostanoid synthesis on the nature of free arachidonic acid (AA) appearance was investigated in mouse peritoneal macrophages. AA delivery from intracellular sources to the constitutive prostaglandin (PG)H synthase was provided by action of calcium-ionophore A23187; and from extracellular sources by AA addition to the culture medium. It was found that the kinetics of prostanoid synthesis dramatically depends on the sources of AA. Free AA concentration used for prostanoid synthesis is either a constant or a variable value depending upon the sources. The kinetics of cellular prostanoid synthesis can be regulated by the following processes: (a) the irreversible inactivation of PGH-synthase in the course of the reaction (kin), (b) prostanoid metabolism (kr), and (c) incorporation of exogenous AA into cellular membranes (ka). From our experiments and mathematical calculation these parameters were found to be kin = 0.20 +/- 0.02 min-1, kr = 0.17 +/- 0.03 min-1 in the case of stimulation with A23187, and kin = 0.0156 min-1, kr = 0. 0134 min-1, ka = 0.0025 min-1 in the case of exogenous AA addition. The studies of prostanoid biosynthesis by macrophage microsomes led to independent determination of kin = 0.20 +/- 0.02 min-1. This value perfectly fits the kinetics of the prostanoid cell synthesis under endogenous AA supply but shows a 10-fold decrease in the case of exogenous AA supply. Our study on the kinetics of prostanoid synthesis by mouse peritoneal macrophages clearly demonstrate that AA is able to regulate cellular prostanoid synthesis in the presence of constitutive PGH-synthase only. A regulation mechanism based on the co-operation of the constitutive PGH-synthase isoform and the availability of free AA is proposed and could be confirmed by mathematical modelling.     

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.     

Glycerylprostaglandin synthesis by resident peritoneal macrophages in response to a zymosan stimulus

Cyclooxygenase (COX)-2 oxygenates arachidonic acid (AA) and 2-arachidonylglycerol (2-AG) to endoperoxides, which are subsequently transformed to prostaglandins (PGs) and glycerylprostaglandins (PG-Gs). PG-G formation has not been demonstrated in intact cells treated with a physiological agonist. Resident peritoneal macrophages, which express COX-1, were pretreated with lipopolysaccharide to induce COX-2. Addition of zymosan caused release of 2-AG and production of the glyceryl esters of PGE2 and PGI2 over 60 min. The total quantity of PG-Gs (16 +/- 6 pmol/10(7) cells) was much lower than that of the corresponding PGs produced from AA (21,000 +/- 7,000 pmol/10(7) cells). The differences in PG-G and PG production were partially explained by differences in the amounts of 2-AG and AA released in response to zymosan. The selective COX-2 inhibitor, SC236, reduced PG-G and PG production by 49 and 17%, respectively, indicating a significant role for COX-1 in PG-G and especially PG synthesis. Time course studies indicated that COX-2-dependent oxygenation rapidly declined 20 min after zymosan addition. When exogenous 2-AG was added to macrophages, a substantial portion was hydrolyzed to AA and converted to PGs; 1 microm 2-AG yielded 820 +/- 200 pmol of PGs/10(7) cells and 78 +/- 41 pmol of PG-Gs/10(7) cells. SC236 reduced PG-G and PG production from exogenous 2-AG by 88 and 76%, respectively, indicating a more significant role for COX-2 in the utilization of exogenous substrate. In conclusion, lipopolysaccharide-pretreated macrophages produce PG-Gs from endogenous 2-AG during zymosan phagocytosis, but PG-G formation is limited by substrate hydrolysis and inactivation of COX-2.     

PGE(2) is generated by specific COX-2 activity and increases VEGF production in COX-2-expressing human pancreatic cancer cells

In some cancers cyclooxygenase (COX) inhibition appears to be anti-mitogenic and anti-angiogenic, but the actions of COX-derived prostaglandins in pancreatic cancer (PaCa) are unknown. In this study COX-2 was detected in three of six PaCa cell lines while COX-1 was identified in all cell lines. COX-2 expression correlated with basal and arachidonic acid (AA) stimulated PGE(2) production. PGE(2) production was inhibited by the COX-2 inhibitor nimesulide. In COX-2 expressing cells, exogenous AA and PGE(2) increased VEGF synthesis via the EP(2) receptor. Whereas PGE(2) stimulated intracellular cAMP formation in COX-2 positive and negative cells, 8-bromo cAMP stimulated VEGF production only in COX-2 expressing cells. Stimulating COX-2 expressing PaCa cell lines with AA enhanced migration of endothelial cells, an effect which was inhibited by a COX-2 inhibitor and EP(2) receptor antagonist. These data identify a subset of human PaCa cell lines that express functional COX-2 enzyme. PGE(2) generated by specific COX-2 activity increases VEGF secretion in human PaCa cells through an autocrine mechanism.     

Bovine placental prostaglandin synthesis: principal cell synthesis as modulated by the binucleate cell

Bovine placentomes were collected during late gestation, prepartum, and immediately postpartum. Postpartum tissues were collected prior to fetal placental release. A procedure for separating fetal placental principal cells from fetal binucleate cells (BNC) was developed. Dispersed fetal placental cells (mixed types), principal cells, and BNC were each examined for their ability to produce prostaglandins (PGs) from arachidonic acid (AA) and to metabolize prostaglandin E2 (PGE2) and prostaglandin F2 alpha (PGF2 alpha) in vitro. Dispersed fetal placental cells obtained prepartum produced predominantly PGs of the E series (PGEs) from AA (p less than 0.05). PGE synthesis predominated (p less than .05) in cells from postpartum tissue if the fetal placental membranes were subsequently retained, whereas synthesis of PGs of the F series (PGFs) predominated (p less than 0.05) if the fetal membranes were subsequently released. Principal cells were the primary source of fetal placental PG synthesis from AA (p less than 0.05). BNC exhibited a lesser ability to synthesize PGs from AA (p less than 0.05), but were able to convert PGF2 alpha to PGE2. Dispersed fetal placental cells exhibited greater ability to convert PGF2 alpha to PGE2 (p less than 0.05) than did the separated cells. These data suggest the function of a two-cell system within the fetal villi such that the BNC modulate the output of principal cell PG synthesis and/or metabolism.     

Pathways of arachidonic acid metabolism in human amnion cells at term

We have compared the metabolism of (3H) arachidonic acid by monolayers of human amnion, cells obtained prior to or following labor at term. Radiolabel was either added exogenously or previously incorporated into cellular phospholipid pools to compare metabolism of arachidonic acid from different substrate sources. Cells obtained both prior to and following labor synthesized metabolites co-chromatographing on HPLC with di- and mono-HETEs and also a metabolite with polarity corresponding to a epoxyeicosatrienoic acid. Both types of cells were able to synthesize PGE2 when (3H) arachidonic acid was added exogenously. However, only those cells obtained following labor synthesized PGE2 from (3H) arachidonic acid incorporated into intracellular pools. These findings suggest that the cyclooxygenase and PGE2 isomerase enzymes are present in amnion prior to delivery but that exogenous arachidonic acid would be required for PGE2 synthesis at that time as the enzymes do not appear to be linked to a source of endogenous arachidonic acid. At the time of parturition, there may be a switching on of an enzyme system to generate arachidonic acid from intracellular pools specifically for PGE2 synthesis or alternatively coupling of such a system to a cyclooxygenase-PGE2 isomerase system resulting in PGE2 synthesis. These findings raise intriguing new possibilities for the regulation of eicosanoid synthesis in amnion which may include membrane topography, substrate pool-enzyme linking and regulation of specific phospholipase enzymes.     

Prostaglandins in human breast cancer. Identification of a cytosolic prostaglandin-9-keto-reductase activity

Endogenous sources of prostaglandin production in human breast tumors were investigated by radioimmunoassay analysis of PGE2 and PGF2a productions and 3H-PGE2 conversion. PG synthetase located within the microsomal fraction mainly produced PGE2, while little PGF2a synthesis occured. In cytosol preparations. PGE2 is converted into PGF2a. In 15 tumor specimens, no relationship was observed between PGE2 production and the metabolic activity which varied widely from sample to sample. These findings demonstrate the presence of PG-9-keto-reductase in the cytosol from human breast tumors. A way of PGE2 inactivation by this enzyme is suggested since no less polar PGE2 metabolites were detected. It is concluded that PGE2 production by the microsomes will reflect the PG synthetase activity of a given human mammary carcinoma while metabolic conversion of PGE2 within the cytosol reflects the metabolic activity of the same sample. Both activities were otherwise apparently unlinked.     

Concomitant activation of extracellular signal-regulated kinase and induction of COX-2 stimulates maximum prostaglandin E2 synthesis in human airway epithelial cells

The intracellular regulation and kinetics of prostaglandin (PG)E(2) synthesis in human airway epithelial (NCI-H292) cells was investigated. Interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha and lipopolysaccharide (LPS) all induced PGE(2) synthesis (p<0.001) and transient (5-15 min) phosphorylation of extracellular signal-regulated kinase (ERK). Phorbol myristate acetate (PMA) and calcium ionophore, A23187 further enhanced PGE(2) synthesis (p<0.001) and caused phosphorylation of ERK that was sustained for up to 16 h. COX-2 protein expression and PGE(2) synthesis were increased following exposure to combinations of stimuli that increased intracellular Ca(2+), and activated protein kinase C as well as ERK. Inhibition of ERK almost completely abrogated PGE(2) synthesis in response to all stimuli. Sustained, maximum PGE(2) synthesis was observed when cells were stimulated such that ERK phosphorylation was concomitant with increased COX-2 protein expression. These results argue against redundancy in pathways for PGE(2) synthesis, and suggest that at various stages of inflammation different stimuli may influence ERK activation and COX-2 expression, so as to tightly regulate the kinetics and amount of PGE(2) produced by airway epithelial cells in response to lung inflammation.     

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.     

Patterns of prostaglandin synthesis and degradation in isolated superficial and proliferative colonic epithelial cells compared to residual colon

Prostaglandin (PG) synthesis and degradation were examined in different regions (epithelial versus non-epithelial structures) of the rat distal colon by both HPLC analysis of [14C] arachidonate (AA) metabolites and by specific radioimmunoassays. Intact isolated colonic epithelial cells synthesized mainly PGF2 alpha and TXA2, as monitored from the formation of its stable degradation product TXB2 (PGF2 alpha greater than TXB2 greater than 6-keto-PGF1 alpha, the stable degradation product of PGI2 = PGD2 = PGE2 = 13,14-dihydro-15-keto-PGF2 alpha). The profile of PG products of isolated surface epithelial cells was identical to that of proliferative epithelial cells. However, generation of PGs by surface epithelium was 2 to 3-fold higher than by proliferative cells both basally and in the presence of a maximal stimulating concentration (0.1 mM) of AA. The latter implied a greater synthetic capacity of surface epithelium, rather than differences due to endogenous AA availability. The major sites of PG synthesis in colon clearly resided in submucosal structures; the residual colon devoid of epithelial cells accounted for at least 99% of the total PGs produced by intact distal colon. The profile of AA metabolites formed by submucosal structures also differed markedly from that of the epithelium. The dominant submucosal product was PGE2. PGE2 and its degradation product 13,14-dihydro-15-keto-PGE2 accounted for 63% of the PG products formed by submucosal structures (PGE2 much greater than PGD2 greater than 13,14-dihydro-15-keto-PGE2 greater than PGF2 alpha = TXB2 = 6-keto-PGF1 alpha greater than 13,14-dihydro-15-keto-PGF2 alpha). By contrast, epithelial cells, and particularly surface epithelium, contributed disproportionately to the PG degradative capacity of colon, as assessed from the metabolism of either PGE2 or PGF2 alpha. When expressed as a percentage, epithelial cells accounted for 71% of total colonic PGE2 degradative capacity but only 23% of total colonic protein. Approximately 15% of [3H] PGE2 added to the serosal side of everted colonic loops crossed to the mucosal side intact. Thus, at least a portion of the PGE2 formed in the submucosa reaches, and thereby can potentially influence functions of the epithelium.     

Eicosanoid production by the coronary microvascular endothelium

Cultured rabbit coronary microvessel endothelial (RCME) cells have been used as an in vitro model to study the regulation of microvascular endothelial cell prostaglandin (PG) production by hormones, vasoactive drugs, and inflammatory mediators in an environment that can be tightly controlled and that is unaffected by interactions with other cell types, physical stimulation, or alterations in oxygenation. The most potent stimuli for RCME cell PG secretion were substances associated with inflammation, including histamine, interleukin 1, leukotriene D4, fMet-Leu-Phe, interferon-gamma, and exogenous phospholipases. Inhibition of calcium availability by lower [Ca2+]o or by treatment with calcium channel blockers reduced A23187-stimulated PG release but increased PG synthesis from exogenous arachidonic acid (AA). These observations suggest that Ca2+ may regulate several steps in the pathway leading to PG synthesis and release. Elevated intracellular [Ca2+] may, on the one hand, promote PG production by stimulating phospholipase A2 leading to AA release and, on the other hand, limit the magnitude of the response by increasing the rate of AA reacylation. Glucocorticoids reduce PG production by RCME cells via an action that requires new protein and mRNA synthesis and appears to involve the production of an endothelial cell-derived phospholipase inhibitory protein, or "endocortin." Thus, microvascular endothelial cells can both contribute to (by the release of PGs and possibly platelet-activating factor-acether) and limit (by the production of endocortins) the degree of a local inflammatory response in the heart.     

The effect of progesterone and human interferon alpha-2 on the release of PGF2 alpha and PGE from epithelial cells of human proliferative endometrium.

Progesterone and interferon-like trophoblastic proteins modulate prostaglandin (PG) synthesis from endometrium in early ovine and bovine pregnancy. Enriched epithelial cells were prepared from human endometrium removed in the proliferative phase of menstrual cycle (n = 8). Progesterone at a concentration of 1 microM suppressed PGE release from the cells during the first 24 hours in culture. After 48 hours in culture progesterone at a dose of 100 nM and 1 microM suppressed both the release of PGF2 alpha and PGE from the cells and this suppression was maintained for a further two days. Addition of exogenous 30 microM arachidonic acid (AA) abolished this effect of progesterone on both PGF2 alpha and PGE release. Interferon alpha-2 did not suppress the basal release of PGF2 alpha nor PGE. In the presence of progesterone, interferon alpha-2 attenuated the progesterone mediated suppression of PGF2 alpha but not PGE release from endometrial cells. These findings suggest that progesterone suppresses the basal release of PGs from human endometrium, but unlike the sheep, interferon alpha-2 does not exert this action on human endometrium.     

Intracellular measurement of prostaglandin E2: effect of anti-inflammatory drugs on cyclooxygenase activity and prostanoid expression.

Cyclooxygenase (COX) converts arachidonic acid to prostaglandin (PG) H2, which is further metabolized to various prostaglandins, prostacyclin and thromboxane A2. COX exists in at least two different isoforms. COX-1 is constitutively expressed, whereas COX-2 is induced by proinflammatory stimuli. Prostaglandin E2 is a major metabolite of COX activation. In order to compare the activity of target ligands and COX inhibitors on PGE2 synthesis and release, the responsiveness of several cell lines to the calcium ionophore A23187, bacterial lipopolysaccharide (LPS), nonsteroidal anti-inflammatory drugs (NSAIDs), and the glucocorticoid, dexamethasone, were investigated. For intracellular measurements, the culture supernatant was aspirated, and the cells were thoroughly washed and lysed with dodecyltrimethylammonium bromide. Intracellular and secreted PGE2 were measured with an enzyme immunoassay. A23187 and LPS increased intracellular PGE2 in a dose-dependent manner. Kinetic experiments with A23187-stimulated mouse 3T3 fibroblast cells revealed a distinct biphasic response in COX activity. In the presence of NSAIDs or dexamethasone, there was a dose-dependent inhibition in intracellular PGE2 with A23187-stimulated 3T3 cells. Inhibitory studies demonstrated an apparent increased sensitivity of COX activity to the action of inhibitors when measuring intracellular PGE2 compared with using cell culture supernatants. Indeed, intracellular PGE2 levels were comprehensively reduced in the presence of low concentrations of inhibitor. The utilization of cell culture lysates and, in particular, measurement of intracellular PGE2 should prove useful for identifying new COX inhibitors.     

Prostaglandin profile and synthetic capacity of the colon: comparison of tissue sources and subcellular fractions.

Although there has been intense interest in the physiology and pathophysiology of prostaglandins (PGs) synthesized in the colon, little is known about the PG profile and synthetic capacity of different tissue sources and subcellular fractions as enzyme sources. Subcellular fractions prepared from the mucosa and muscle layer of rat colon were incubated with or without exogenous arachidonic acid ([3H]20:4n-6) for 30 min. In experiments with exogenous [3H]20:4n-6, the prostaglandin synthetic capacity of the colonic muscle layer was significantly higher than that of the mucosa. Among the subcellular fractions, microsomes had the highest PG synthetic capacity in both mucosa and muscle. The major PG product was PGI2 and PGD2 in the mucosal microsomes and PGI2 and PGE2 in the muscularis microsomes. However, production of PGI2 in the mucosa and PGE2 in the muscle was significantly reduced in the fractions containing both cytosol and microsome, resulting in an alteration of the PG profile. Substrate availability (exogenous vs endogenous supply) appears to influence the PG profile of the colon. In the colonic mucosa with exogenous [3H]20:4n-6, the production of PGI2 was 5 times higher than that of PGE2, whereas the production of PGE2 was twice higher than that of PGI2 in experiments with endogenous 20:4n-6. These observations indicate: 1) different PG profile and synthetic capacity of tissue sources and subcellular fractions; 2) alteration of PG profile due to the variation of 20:4n-6 availability. Thus, the outcome of experiments on the physiological role of PG in the colon may be determined, in part, by the tissue source and subcellular fraction selected for analysis. The present study also suggests that the variation of substrate availability in physiological and pathophysiological processes may affect the PG profile of the colon.     

Zymosan-induced glycerylprostaglandin and prostaglandin synthesis in resident peritoneal macrophages: roles of cyclo-oxygenase-1 and -2

COX [cyclo-oxygenase; PG (prostaglandin) G/H synthase] oxygenates AA (arachidonic acid) and 2-AG (2-arachidonylglycerol) to endoperoxides that are converted into PGs and PG-Gs (glycerylprostaglandins) respectively. In vitro, 2-AG is a selective substrate for COX-2, but in zymosan-stimulated peritoneal macrophages, PG-G synthesis is not sensitive to selective COX-2 inhibition. This suggests that COX-1 oxygenates 2-AG, so studies were carried out to identify enzymes involved in zymosan-dependent PG-G and PG synthesis. When macrophages from COX-1-/- or COX-2-/- mice were treated with zymosan, 20-25% and 10-15% of the PG and PG-G synthesis observed in wild-type cells respectively was COX-2 dependent. When exogenous AA and 2-AG were supplied to COX-2-/- macrophages, PG and PG-G synthesis was reduced as compared with wild-type cells. In contrast, when exogenous substrates were provided to COX-1-/- macrophages, PG-G but not PG synthesis was reduced. Product synthesis also was evaluated in macrophages from cPLA(2alpha) (cytosolic phospholipase A2alpha)-/- mice, in which zymosan-induced PG synthesis was markedly reduced, and PG-G synthesis was increased approx. 2-fold. These studies confirm that peritoneal macrophages synthesize PG-Gs in response to zymosan, but that this process is primarily COX-1-dependent, as is the synthesis of PGs. They also indicate that the 2-AG and AA used for PG-G and PG synthesis respectively are derived from independent pathways.     

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.     

Role of Ca(2+)-independent phospholipase A(2) and cyclooxygenase/lipoxygenase pathways in the nitric oxide production by murine macrophages stimulated by lipopolysaccharides.

There is evidence of molecular cross talk between inflammatory mediators such as nitric oxide (NO) and prostaglandins (PG), which may regulate tissue homeostasis and contribute to pathophysiological processes. Here we examine the role of endogenous arachidonic acid (AA) and its AA metabolites in the regulation of NO release by lipopolysaccharide (LPS)-stimulated macrophages RAW 264.7. Our results suggest that bromoenol lactone-sensitive phospholipase A(2) is involved in AA release and the subsequent PG and leukotriene (LT) production. The cyclooxygenase inhibitor, indomethacin, and lipoxygenase inhibitors such as baicalein and zileuton blocked the dose-dependent PGE(2) or LTB(4) and nitrite (NO(2)(-)) production induced by LPS. Furthermore, the effects of indomethacin were reverted by exogenous PGE(2) and forskolin, whereas AH23848B, an EP(4) PGE(2) subtype receptor antagonist, decreased NO(2)(-) release. On the other hand, the effect of baicalein on NO(-)(2) production was reverted by exogenous LTB(4) and the fibrate WY 14,643, a natural and a synthetic peroxisome proliferator-activated receptor alpha (PPAR alpha), respectively. Thus, PGE(2) via EP(4) receptor/cAMP and LTB(4) via PPAR alpha may be involved in the control of NO synthesis by LPS in macrophage RAW 264.7 cultures.     

Conversion of arachidonic acid to cyclooxygenase and lypoxygenase products,and incorporation into phospholipids in the mouse neuroblastoma clone,Neuro-2A

Arachidonic acid (AA) incorporation into phospholipids and cyclooxygenase and lipoxygenase mediated metabolism of arachidonic acid were studied in homogenized and intact Neuro-2A cells. When 3H8-AA was added to homogenized cells and incubated 20 minutes, 39% of the label was converted to prostaglandins (PGs), 10% to hydroxy-eicosatetraenoic acid (HETE) and 26% was incorporated into phospholipids. PGE2 and PGF2a were the major PGs produced. Synthesis of PGs was blocked by 10 microM indomethacin and synthesis of PGs and HETE was blocked by 10 microM eicosatetraynoic acid (ETYA). The cell homogenate produced the 13,14-dihydro-15-keto metabolites of PGE2 and PGF2a from 3H8-AA and also converted exogenous 3H7-PGE2 and 3H8-PGF2a to metabolites. When intact cells were labeled for 24 hours with 14C1-AA and the cells and media then analyzed, 75% of the radioactivity was incorporated into cellular phospholipids, 0.8% was converted to PGs and metabolites and 0.7% converted to HETE. Cells prelabeled for 24 hours were washed and incubated for 30 minutes in fatty acid free media. There was a 23% release of AA from phospholipids. One-fifth of the released AA was converted to HETE. PG synthesis in the intact resting cells was low. In summary, the Neuro-2A cell provides a good model system for studying arachidonic acid metabolism and incorporation into phospholipids in cells of neuronal origin.     

Inhibition of phospholipase activity in human monocytes by IFN-gamma blocks endogenous prostaglandin E2-dependent collagenase production

Previous studies from our laboratory have demonstrated that exposure of human monocytes to a stimulant, such as Con A, results in the production of the enzyme collagenase through PGE2-dependent pathway. Inasmuch as rIFN-gamma has been shown to modulate monocyte/macrophage PG synthesis, we examined the effect of rIFN-gamma on the activation sequence leading to collagenase production. The addition of rIFN-gamma (10 to 1000 U/ml) to Con A-stimulated monocytes resulted in a dose-dependent inhibition of PGE2 and collagenase synthesis. The suppression of collagenase production by rIFN-gamma was related to its ability to reduce PGE2 levels as demonstrated by the restoration of collagenase activity by the addition of PGE2. HPLC analysis of the arachidonic acid (AA) metabolites released by monocytes showed that rIFN-gamma caused a reduction in the release of AA and products of the cyclooxygenase and lipoxygenase pathways. These data indicated that rIFN-gamma decreased eicosanoid production by inhibiting the release of AA from phospholipids. This conclusion was supported by the reduction in membrane bound phospholipase activity in rIFN-gamma-treated monocytes. Moreover, the inhibition by rIFN-gamma of PGE2 and collagenase was reversed by the addition of phospholipase A2. Our findings demonstrate that rIFN-gamma inhibits phospholipase activity in activated monocytes and as a result blocks PGE2-dependent collagenase synthesis.