Inhibition of prostaglandin synthesis in human endothelial cells treated with metabolic inhibitors.

Endothelial cell injury is often associated with increased synthesis of prostaglandin (PG)I2. We observed, however, that endothelial cells treated with metabolic inhibitors which reduce cellular ATP content develop an injury pattern characterized by reduced PGI2 synthesis. This study examined the relationship between cell injury, arachidonic acid metabolism and ATP content in human umbilical vein endothelial cells treated with 2-deoxyglucose (2DG), a glycolytic inhibitor, and oligomycin (OG), a respiratory chain inhibitor. Either inhibitor alone significantly reduced cellular ATP concentrations, but only OG reduced basal PG synthesis. The combination of 2DG and OG, however, was more effective than either agent alone in reducing cellular ATP content (greater than or equal to 50% of control) and inhibiting basal and agonist-stimulated PGI2 synthesis. This reduced PGI2 synthesis preceded 51chromium release, lactic dehydrogenase release and was not associated with a net release of arachidonic acid from cell membranes. Histamine, A23187 and bradykinin stimulated PGI2 synthesis in untreated but not in 2DG and OG treated cells. Exogenous arachidonic acid increased PGI2 synthesis to a similar extent in both 2DG and OG treated and untreated cells. Therefore, reduced PG synthesis in 2DG and OG treated endothelial cells is not due to inhibition of cyclooxygenase. Furthermore, reduced PG synthesis in these cells occurs prior to cell injury and is not strictly associated with cellular ATP depletion.     

Inhibition of prostaglandin synthesis in human endothelial cells treated with metabolic inhibitors

Endothelial cell injury is often associated with increased synthesis of prostaglandin (PG)I₂. We observed, however, that endothelial cells treated with metabolic inhibitors which reduce cellular ATP content develop an injury pattern characterized by reduced PGI₂ synthesis. This study examined the relationship between cell injury, arachidonic acid metabolism and ATP content in human umbilical vein endothelial cells treated with 2-deoxyglucose (2DG), a glycolytic inhibitor, and oligomycin (OG), a respiratory chain inhibitor. Either inhibitor alone significantly reduced cellular ATP concentrations, but only OG reduced basal PG synthesis. The combination of 2DG and OG, however, was more effective than either agent alone in reducing cellular ATP content (≥ 50% of control) and inhibiting basal and agonist-stimulated PGI₂ synthesis. This reduced PGI₂ synthesis preceded ⁵¹ chromium release, lactic dehydrogenase release and was not associated with a net release of arachidonic acid from cell membranes. Histamine, A23187 and bradykinin stimulated PGI₂ synthesis in untreated but not in 2DG and OG treated cells. Exogenous arachidonic acid increased PGI₂ synthesis to a similar extent in both 2DG and OG treated and untreated cells. Therefore, reduced PG synthesis in 2DG and OG treated endothelial cells is not due to inhibition of cyclooxygenase. Furthermore, reduced PG synthesis in these cells occurs prior to cell injury and is not strictly associated with cellular ATP depletion.     

Prostaglandin synthesis by human glomerular cells in culture

PG synthesis by cultured human glomerular mesangial and epithelial cells incubated with [1- 14C] arachidonic acid was determined by radioimmunoassay (RIA) after high performance liquid chromatography purification. Both dissociated cells and cell monolayers were studied under basal conditions. PG synthesis by epithelial cells was undetectable. Mesangial cells produced low amounts of PGE2, PGF2 alpha and 6 keto-PGF1 alpha and no TXB2. We also examined the effects of several agents on PG synthesis in these two types of cells scraped away from their flasks using direct RIA. Arachidonic acid produced a slight stimulation only with mesangial cells whereas angiotensin II, cyclic AMP and calcium ionophore were inactive with both cell lines. Homogenization of the cells did not enhance the stimulatory effect of arachidonic acid. Alkalinization of the incubation medium produced an increase of PG production by mesangial cells. These results suggest that two types of human glomerular cells, particularly epithelial cells, possess low cyclooxygenase activity. The low capacity of human mesangial and epithelial cells to produce PG may have consequences for the endocrine control of the glomerular microcirculation in man.     

Activity of prostaglandin biosynthetic pathways in rat pancreatic islets

Isolated pancreatic islets of the rat were either prelabeled with [3H]arachidonic acid, or were incubated over the short term with the concomitant addition of radiolabeled arachidonic acid and a stimulatory concentration of glucose (17mM) for prostaglandin (PG) analysis. In prelabeled islets, radiolabel in 6-keto-PGF1 alpha, PGE2, and 15-keto-13,14-dihydro-PGF2 alpha increased in response to a 5 min glucose (17mM) challenge. In islets not prelabeled with arachidonic acid, label incorporation in 6-keto-PGF1 alpha increased, whereas label in PGE2 decreased during a 5 min glucose stimulation; after 30-45 min of glucose stimulation labeled PGE levels increased compared to control (2.8mM glucose) levels. Enhanced labelling of PGF2 alpha was not detected in glucose-stimulated islets prelabeled or not. Isotope dilution with endogenous arachidonic acid probably occurs early in the stimulus response in islets not prelabeled. D-Galactose (17mM) or 2-deoxyglucose (17mM) did not alter PG production. Indomethacin inhibited islet PG turnover and potentiated glucose-stimulated insulin release. Islets also converted the endoperoxide [3H]PGH2 to 6-keto-PGF1 alpha, PGF2 alpha, PGE2 and PGD2, in a time-dependent manner and in proportions similar to arachidonic acid-derived PGs. In dispersed islet cells, the calcium ionophore ionomycin, but not glucose, enhanced the production of labeled PGs from arachidonic acid. Insulin release paralleled PG production in dispersed cells, however, indomethacin did not inhibit ionomycin-stimulated insulin release, suggesting that PG synthesis was not required for secretion. In confirmation of islet PGI2 turnover indicated by 6-keto-PGF1 alpha production, islet cell PGI2-like products inhibited platelet aggregation induced by ADP. These results suggest that biosynthesis of specific PGs early in the glucose secretion response may play a modulatory role in islet hormone secretion, and that different pools of cellular arachidonic acid may contribute to PG biosynthesis in the microenvironment of the islet.     

Selective inhibition of arachidonic acid metabolite release from human lung tissue by antiinflammatory steroids

The effects of antiinflammatory steroids on arachidonic acid metabolite release from human lung fragments were analyzed. Incubation of lung fragments for 24 hr with 10(-6) M dexamethasone inhibited the net release of the prostacyclin metabolite 6-keto-PGF1 alpha, PGE2, and PGF2 alpha from lung fragments stimulated with anti-IgE but failed to inhibit the anti-IgE-induced release of PGD2, TXB2, and iLTC4. The IC50 of dexamethasone for inhibition of both spontaneous and anti-IgE-induced 6-keto-PGF1 alpha release was approximately 2 X 10(-8) M, and a 6-hr preincubation with the drug was required for 50% inhibition of prostaglandin release. Other agents were tested for activity in stimulating arachidonic acid metabolite release from human lung fragments. FMLP (fmet-leu-phe) stimulated the release of all metabolites tested (6-keto-PGF1 alpha, PGD2, PGE2, PGF2 alpha, TXB2, iLTC4); platelet-activating factor (PAF), but not lysoPAF, stimulated the release of PGD2, TXB2, and iLTC4. In contrast to the case with anti-IgE, where dexamethasone failed to inhibit net PGD2 and TXB2 release, the steroid inhibited the release of these metabolites stimulated by both FMLP and PAF. The steroid inhibited iLTC4 release induced by the highest concentration of PAF (10(-6)M) but did not inhibit iLTC4 release stimulated by either 10(-7) M PAF, FMLP, or anti-IgE. Because neither FMLP nor PAF caused the release of PGD2 or TXB2 from purified human lung mast cells, and because they also failed to induce histamine release from lung fragments, it is suggested that these stimuli produce PGD2 and TXB2 release in lung fragments through an action on a cell distinct from the mast cell. This suggestion is supported by the selective inhibition of the release of these arachidonic acid metabolites by dexamethasone. We suggest that the inhibitory action of steroids on arachidonic acid metabolite in human lung fragments contributes to their therapeutic efficacy in pulmonary diseases.     

Decreased prostaglandin synthesis in postmortem cerebral cortex from patients with Alzheimer's disease.

The syntheses of prostaglandin (PG) F2 alpha, E2 and D2, and thromboxane (TX) B2 from [14C]arachidonic acid were studied in frontal cortex of human control and Alzheimer's disease (AD) brains using the microsomal fractions. Under the assay conditions employed, it was found that the major metabolite of [14C]arachidonic acid was PGE2 accounting for 63% of total prostanoid production; PGF2 alpha accounted for 21.5%, TXB2 for 9%, and PGD2 for 6.5%. When AD samples were compared to control samples, microsomal PG synthesis was significantly decreased, with reduced production of PGE2, PGF2 alpha and PGD2. Such decreases in AD brain seem unrelated to age, sex, postmortem delay and, as far as could be determined, antemortem state. In both control and Alzheimer groups, a history of anti-inflammatory therapy seemed to correlate with increased PG synthesis.     

Metabolism of prostaglandin endoperoxide by microsomes from cat lung

It has been reported that the prostaglandin (PG) precursor, arachidonic acid, produces divergent hemodynamic responses in the feline pulmonary vascular bed. However, the pattern of arachidonic acid products formed in the lung of this species is unknown. In order to determine the type and activity of terminal enzymes in the lung, prostaglandin biosynthesis by microsomes from cat lung was studied using the prostaglandin endoperoxide, PGH2, as a substrate. The major products of incubations of PGH2 with microsomes were thromboxane (TX) B2 (the major metabolite of TXA2), 6-keto-PGF1 alpha (the breakdown product of PGI2) and 12L-hydroxy-5,8,10-heptadecatrienoic acid (HHT). Formation of TXB2 was markedly reduced by imidazole. Tranylcypromine decreased the formation of TXB2 and HHT and inhibited the formation of 6-keto-PGF1 alpha. At low PGH2 concentrations, equal production of TXB2 and 6-keto-PGF1 alpha was observed. However, as PGH2 concentration increased, 6-keto-PGF1 alpha production approached early saturation while TXB2 production increased in a linear fashion. These results suggest that enzymatic formation of TXA2 and PGI2 is a function of substrate availability in the lung. These findings provide a possible explanation for the divergent hemodynamic responses to arachidonic acid infusions at high and low concentrations in the feline pulmonary vascular bed.     

Effect of arachidonic acid-rich oil on lipids and arachidonate metabolites in ethanol- treated rats.

The effects of dietary arachidonic acid-rich oil (AAoil) on lipids and arachidonate metabolites in the liver and plasma were evaluated in ethanol-treated rats. Rats were fed a purified diet containing 10% weight of lard or AAoil for 14 days. Ethanol was administered by gavage at a single daily dose of 3 g/kg body weight. Comparing with the lard group, a decrease was observed in liver fatty vacuoles in the AAoil group. Plasma 6-keto-prostaglandin (PG) F1 alpha and thromboxane (TX) B(2)levels and the 6-keto-PGF1 alpha/TXB(2)ratio increased significantly in the AAoil group. Liver 6-keto-PGF1 alpha also increased but not leukotriene B(4)in the AAoil group. In the phospholipid fraction of liver tissue, plasma and red blood cells, arachidonic acid (20:4n-6) and docosatetraenoic acid (22:4n-6) increased and oleic acid (18:1n-9) and linoleic acid (18:2n-6) decreased significantly in the AAoil group compared with the lard group. These observations suggest that AAoil supplementation reduces liver injury of ethanol-treated rats, although longer observation will be necessary for confirmation.     

Attenuation of LPS-induced neutrophil thromboxane b2 release and chemiluminescence.

Polymorphonuclear leukocytes (PMN) may play a key role in acute lung injury and ARDS. The mechanisms of PMN-mediated lung injury include the release of inflammatory mediators, such as oxygen free radicals which cause direct tissue injury, and arachidonic acid metabolites which cause pulmonary vasoconstriction and increased vascular permeability. The goals of this in vitro study were 1) to assess the effects of PMN-activating agents (lipopolysaccharide, LPS; phorbol myristate acetate, PMA; tumor necrosis factor, TNF) on PMN thromboxane B2 (TXB2) release and oxygen free radical production and 2) to determine the effects of agents purported to suppress PMN activity (pentoxifylline, PTX; adenosine; dibutyryl cyclic AMP, DBcAMP; and terbutaline, TBN) on activator-induced PMN TXB2 release and oxygen free radical production. PMN TXB2 release was determined by radioimmunoassay and oxygen free radical production was monitored by chemiluminescence. Our results show that 1) LPS and PMA significantly increase PMN TXB2 release, whereas tumor necrosis factor (TNF) has no effect; 2) LPS and PMA significantly increase PMN chemiluminescence; 3) DBcAMP and TBN significantly reduce LPS-induced PMN TXB2 release whereas PTX and adenosine do not; 4) TBN significantly reduces PMA-induced PMN TXB2 release whereas other agents do not; 5) All agents (PTX, adenosine, DBcAMP, and TBN) significantly reduce LPS-induced PMN chemiluminescence but none attenuate PMA-induced PMN chemiluminescence. We conclude that: LPS and PMA activate PMN manifested by TXB2 release and chemiluminescence. Additionally, all the PMN suppressing agents do attenuate some PMN functions. Of interest, PTX, adenosine, DBcAMP, and TBN have different effects depending upon functional assay and activating agent. It will be important to investigate the mechanisms by which PMN suppressing agents alter signal transduction resulting in differential effects on PMN function.     

Interleukin-1 alpha stimulates prostaglandin biosynthesis in serum-activated mesangial cells by induction of a non-pancreatic (type II) phospholipase A2

Enhanced prostaglandin (PG) biosynthesis is a hallmark of inflammation, and interleukin-1 (IL), a proinflammatory cytokine, is a potent stimulus of PG production. We investigated the mechanisms of IL-1 alpha-enhanced PG synthesis in serum-stimulated mesangial cells. The rIL-1-stimulated increase in PGE2 synthesis was dose- and time-dependent and inhibited by both cycloheximide and actinomycin D. Phospholipase (PL) activity was increased 5- to 10-fold in acid extracts of rIL-1-treated cells as measured by arachidonate release from exogenous [14C]arachidonyl-phosphatidyl-ethanolamine. This induced phospholipase activity was Ca(2+)-dependent and inhibited by the PLA2 inhibitors, aristocholic acid, 7,7-dimethyl-5,8-eicosadienoic acid, and p-bromophenacylbromide, but not by the 1,2-diacylglycerol lipase inhibitor RHC 80267. The rIL-1-stimulated PLA2 had an alkaline pH optimum, and phosphatidylethanolamine was preferred over phosphatidylcholine as substrate. The PLA2 activity increased by rIL-1 was inhibited in cells coincubated with cycloheximide and was measurable after 6 h. A sensitive and specific solution hybridization assay demonstrated a coordinate time-dependent induction of non-pancreatic PLA2 mRNA expression which was increased at least 6-fold by 24 h. In whole cells, IL-1 had no effect on basal [3H]arachidonic acid release but vasopressin (1 microM)-stimulated release was potentiated 2- to 3-fold, suggesting that IL-1 may prime cells for increased PG synthesis via increased PLA2 activity. Thus IL-1 directly stimulates, as well as primes cells for, enhanced PG synthesis, in part, by increasing PLA2 activity through new synthesis of a non-pancreatic (Type II) PLA2.     

Zymosan-activated plasma-mediated thromboxane production by the perfused rabbit liver and isolated hepatocytes: Involvement of calcium

The present study investigates the mechanism of zymosan-activated plasma (ZAP)-mediated eicosanoid production by the isolated, perfused rabbit liver and describes ZAP-induced eicosanoid stimulation in cultured hepatocytes. Perfused livers receiving untreated plasma demonstrated no significant changes in portal venous pressure or the rates of release of lactic dehydrogenase or acid phosphatase activity (indicator of cellular injury). The control group livers demonstrated stable rates of release for 6-keto PGF1α and thromboxane B2 (TXB2). In contrast, the infusion of ZAP alone resulted in a rapid but transient releaes of TXB2 from the livers. No significant changes in perfusion pressure or enzyme release were observed following ZAP administration. Perfusion of livers with a calcium-free buffer decreased the basal rates of both 6-keto PGF1α and TXB2 production and significantly, but not completely, attenuated the ZAP-mediated increase in hepatic TXB2 production. Perfusion of livers with nifedipine (3 μM) had no effect on ZAP-mediated TXB2 production in this model. Isolated hepatocytes responded to ZAP-treatment with significant increases in TXB2 production. These data suggest that activated fluid phase complement components induce thromboxane production by specific cells within the liver and that this stimulation is partially dependent upon the release of intracellular calcium but independent of complement-mediated cellular injuiry.     

Effects of increasing dietary linoleic acid on phospholipid fatty acid composition and eicosanoid production in leucocytes and gill cells of Atlantic salmon (Salmo salar).

Diets containing linoleic acid at 10, 25 and 45% of total dietary fatty acids were fed to three groups of post-smolt Atlantic salmon (Salmo salar) for 18 weeks. Incorporation of linoleic acid into membrane phospholipids of leucocytes and gills increased in response to dietary intake. In general, there was an increase in arachidonic acid and a decrease in eicosapentaenoic acid in the individual phospholipids of both cell types in response to increasing dietary linoleic acid. These changes in eicosanoid precursors were reflected in significantly increased plasma concentrations of 6-keto-PGF1 alpha and TXB2 in salmon given the highest dietary linoleic acid. In whole blood stimulated with the calcium ionophore A23187, LTB4, 12-HETE and TXB2 were significantly increased and 12-HEPE significantly decreased in response to increasing dietary linoleic acid. In isolated gill cells stimulated with A23187, 12-HEPE, 12-HETE, 14-HDHE and TXB2 were all decreased in response to increasing dietary linoleic acid, although the ratio of 12-HEPE/12-HETE was also decreased.     

Prostaglandin production in myotube cultures. Influence on protein turnover.

The production of prostaglandins (PG) E2 and F2 alpha and their possible role in regulation of protein turnover in cultured skeletal-muscle cells were examined. Primary chick myoblasts and myotubes, and L8 myotubes, produced PGE2 and PGF2 alpha from endogenous arachidonic acid. PG production by all three cell types was increased manyfold by the addition of exogenous arachidonic acid. Arachidonate-stimulated PG production was inhibited by the addition of indomethacin (0.1 mM). When L8 and chick myotubes were treated with PGE2, PGF2 alpha, arachidonic acid (0.01 mM) or indomethacin (0.1 mM), no significant alterations in rates of protein synthesis or degradation were observed. Rates of protein synthesis and degradation in these cells were responsive to the addition of 10% fetal-bovine serum under identical experimental conditions. Thus, in contrast with incubated adult skeletal muscle, it appears that the production of prostaglandin metabolites from arachidonic acid is unrelated to regulation of protein turnover in cultured muscle cells.     

Altered myocardial prostaglandin synthesis in spontaneously diabetic rats

Patients with diabetes mellitus have an increased susceptibility to heart disease. The exact mechanism for this phenomenon is unclear. Abnormalities in prostaglandin (PG) production have been suggested as a possible cause. In this connection, we examined the PG synthetic capacity of cardiac microsomes from spontaneously diabetic rats. Cardiac microsomes from diabetic and control rats produced varying amounts of 6-keto-PGF1 alpha (stable degradation product of PGI2), PGE2, PGD2, PGF2 alpha, and TXB2 (stable breakdown product of TXA2). In both instances the production of 6-keto-PGF1 alpha predominated, however, microsomes from diabetic rats showed markedly greater conversion of arachidonic acid to all the PG products, especially 6-keto-PGF1 alpha. When PGF2 alpha metabolism was detected between diabetic and control heart preparations. These results show an enhanced cyclooxygenase activity in diabetic rat hearts without any change in prostaglandin dehydrogenase activity. Such a change may promote some of the cardiac alterations seen in diabetic mellitus.     

Fc gamma 2b receptor-mediated prostaglandin synthesis by a murine macrophage cell line (P388D1)

The nature of signals transmitted by two types of Fc gamma receptors (one specific for IgG2b and the other for IgG2a) present on the surface of a murine macrophage cell line (P388D1) was investigated. Specific binding of IgG2b (presented as EA2b) to cell surface Fc gamma 2br triggered the release of 3H-arachidonic acid and 3H-prostaglandins (PG) from P388D1 cells that were prelabeled with 3H-arachidonate. The release of 3H-arachidonic acid, which increased in a dose-dependent manner, was enhanced by exogenous Ca++ (1.25 mM) and was completely blocked by ethylenediaminetetraacetate (EDTA) (4 mM) or a phospholipase A2 inhibitor, p-bromophenacylbromide (7 microgram/ml). A cyclooxygenase inhibitor, indomethacin (9 microgram/ml), reduced the 3H-arachidonic acid release and completely blocked the conversion of arachidonate into PG. Cytochalasin D (1 microgram/ml), which inhibited the phagocytosis of immune complexes by 90% of P388D1 cells, did not affect the Fc gamma 2bR-triggered release of arachidonic acid. Specific binding of IgG2a (presented as EA2a) to cell surface Fc gamma 2aR did not trigger the release of either 3H-arachidonic acid or 3H-PG from P388D1 cells. Our data demonstrate a signal for the activation of the arachidonic acid metabolic cascade is transmitted by Fc gamma 2bR, but not by Fc gamma 2aR, on the surface of P388D1 cells, probably through the initial activation of the phospholipase A2 activity associated with Fc gamma 2bR.     

Up-regulation of prostaglandin E2 binding and of prostanoid release in rabbits producing antibodies against prostaglandin E2

German Giant rabbits successfully immunized against prostaglandin (PG) E2 as shown by a rise in antibody titers developed gastric mucosal lesions. Enzymatically dispersed gastric mucosal cells of these animals had a significantly enhanced production of PG E2 and PG I2 as measured by specific radioimmunoassays. This may be explained by an increased supply with endogenous arachidonic acid (as indicated by an enhanced phospholipase A2/LAT ratio) and by a higher activity of the subsequent PG forming enzymes (as indicated by a more effective stimulation of PG production by exogenous arachidonic acid). Gastric mucosal plasma membranes of immunized rabbits had significantly higher PG E2 binding capacity (108 +/- 9 fmol/mg protein) than those of nonimmunized rabbits (72 +/- 5 fmol/mg protein). The ligand affinity was not affected by immunization. Neither histamine-stimulated 14C-amino-pyrine uptake of isolated parietal cells as a marker for acid production nor its inhibition by PG E2 were influenced by receptor up-regulation. The increased eicosanoid release can be regarded as an endogenous defense mechanism against increased mucosal vulnerability caused by PG E2 scavenging. The potential role of PG E2 receptor up-regulation in support of this process remains to be established.     

The effect of temporary hypoxia on prostaglandin synthesis in mouse brain cell cultures during development

Cultures of dissociated brain cells of new born mice represent a model for the study of brain development. One and two weeks old, they correspond in regard to oligodendrocyte differentiation to about the developmental stage of a human newborn and a six months old infant respectively. Such cultures were used to establish the developmental prostaglandin pattern and to study early and late recovery of prostaglandin synthesis from temporary hypoxia. Basal and bradykinin stimulated prostaglandin release were examined. Most prominently in stimulated release, the developmental prostaglandin pattern at one week showed a prevalence of PGE2 (33 +/- 4%) over PGD2 (12 +/- 5%), which in two weeks old cultures changes to an opposite distribution (PGE2 10 +/- 4%; PGD2 25 +/- 6%). This change goes parallel with the number and differentiation of oligodendrocytes. During the first day post hypoxia, imposed at the end of one week, the production of 6 oxo PGF1 alpha, PGE2, PGD2 and TXB2 was significantly decreased in two study series and increased compared to control in another. Since the arachidonic acid uptake was the same in all three series, this differential observation indicates differential early recovery. 8 days post hypoxia (late recovery), PG release was not different from control, indicating complete recovery at that time. During early recovery from hypoxia on 14 days old cultures, basal PG release was not significantly different from control, however bradykinin stimulated release was significantly inhibited in all three series. This may indicate that mainly regulatory influences on PG release in older cultures are compromised by hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prostanoid synthesis and vascular responses to exogenous arachidonic acid following cerebral ischemia in piglets

In newborn pigs, cerebral ischemia abolishes both increased cerebral prostanoid production and cerebral vasodilation in response to hypercapnia and hypotension. Attenuation of prostaglandin endoperoxide synthase activity could account for the failure to increase prostanoid synthesis and loss of responses to these stimuli. To test this possibility, arachidonic acid (3, 6, or 30 micrograms/ml) was placed under cranial windows in newborn pigs that had been exposed to 20 min of cerebral ischemia. The conversion to prostanoids and pial arteriolar responses to the arachidonic acid were measured. At all three concentrations, arachidonic acid caused similar increases in pial arteriolar diameter in sham control piglets and piglets 1 hr postischemia. Topical arachidonic acid caused dose-dependent increases of PGE2 in cortical periarachnoid cerebral spinal fluid. 6-keto-PGF1 alpha and TXB2 only increased at the highest concentration of arachidonic acid (30 micrograms/ml). Cerebral ischemia did not decrease the conversion of any concentration of arachidonic acid to PGE2, 6-keto-PGF1 alpha, or TXB2. We conclude that ischemia and subsequent reperfusion do not result in inhibition of prostaglandin endoperoxide synthase in the newborn pig brain. Therefore, the mechanism for the impaired prostanoid production in response to hypercapnia and hypotension following cerebral ischemia appears to involve reduction in release of free arachidonic acid.     

Inhibition of COX activity by NSAIDs or ascorbate increases cAMP levels and enhances differentiation in 1alpha,25-dihydroxyvitamin D3-induced HL-60 cells

Arachidonic acid metabolism is modulated during differentiation induced by 1alpha,25(OH)(2)D(3) in HL-60 cells. Antioxidants that affect arachidonic acid metabolism enhance this differentiation program. Ascorbate also enhances differentiation in 1alpha,25(OH)(2)D(3)-induced cells depending on the induction of cAMP. The aim of this work was to study if this cAMP rise depends on modulation of arachidonic acid metabolism by ascorbate. Cyclooxygenase inhibitors, indomethacin and aspirin, increased cAMP levels and also enhanced 1alpha,25(OH)(2)D(3)-induced differentiation in HL-60 cells. Ascorbate did not affect the release of arachidonic acid-derived metabolites but decreased the levels of TXB(2) and PGE(2), suggesting the inhibition of cyclooxygenase. On the other hand, free arachidonic acid increased both cAMP levels and differentiation in the absence or presence of 1alpha,25(OH)(2)D(3). Neither cyclooxygenase inhibitors nor ascorbate modified AA effect. Then, inhibition of cyclooxygenase activity by ascorbate could accumulate free arachidonic acid or other metabolites that increase cAMP levels and enhance differentiation in 1alpha,25(OH)(2)D(3)-induced HL-60 cells.     

Labelling of lipids and phospholipids with [3H]arachidonic acid and the biosynthesis of eicosanoids in U937 cells differentiated by phorbol ester

Phorbol esters induce morphologic and biochemical differentiation in U937 cells, a monocyte/macrophage-like line derived from a human histiocytic lymphoma. We are interested in the phorbol ester-stimulated release of arachidonic acid from cellular membranes and the subsequent synthesis of eicosanoids, as it may prove to correlate with the induced cellular differentiation. Undifferentiated log-phase U937 cells released little recently incorporated [3H]arachidonic acid, but phorbol 12-myristate 13-acetate increased its apparent rate of release to that of cells differentiated by exposure to phorbol myristate acetate for 3 days. Exposure of washed differentiated cells immediately prelabelled with [3H]arachidonic acid to additional phorbol myristate acetate did not augment the release of [3H]arachidonic acid. The basal release of nonradioactive fatty acids from differentiated cells was 5-10 times that of undifferentiated cells, and phorbol myristate acetate increased their release from both types of cell 2- to 3-fold. Differentiated cells immediately prelabelled with [3H]arachidonic acid exhibited greater incorporation into phosphatidylinositol and phosphatidylcholine, and contained more radioactive free arachidonic acid, compared with undifferentiated cells. Undifferentiated cells contained more radioactivity in phosphatidylserine, phosphatidylethanolamine and neutral lipids. Phorbol myristate acetate caused differentiated cells to release [3H]arachidonic acid from phosphatidylinositol, phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine, but release from neutral lipids was reduced, and the content of [3H]arachidonic acid increased. In undifferentiated cells incubated with phorbol myristate acetate, radioactivity associated with phosphatidylserine, phosphatidylethanolamine and neutral lipid was reduced and [3H]arachidonic acid was unchanged. Synthesis of cyclooxygenase products exceeded that of lipoxygenase products in both differentiated and undifferentiated cells. Phorbol myristate acetate increased the synthesis of both types of product, cyclooxygenase-dependent more than lipoxygenase-dependent, especially in differentiated cells. The biological significance of these changes in lipid metabolism that accompany phorbol myristate acetate-induced differentiation are yet to be established.