The effects of complete ischemia on cerebral arachidonic acid (AA) metabolism were investigated in the isolated perfused rat brain. During 12.5 min of ischemia, AA, 5-hydroxy-6,8,11,14-eicosatetraenoic acid, and 15-hydroxy-5,8,11,13-eicosatetraenoic acid increased 129-, 4-, and 10-fold, respectively, while subsequent reperfusion for 30 min resulted in normalized levels independently of the duration of preceding ischemia. Prostaglandin (PG) F2 alpha, PGE2, PGD2, 6-keto-PGF1 alpha, and thromboxane (Tx) B2 remained at preischemic levels during 12.5 min of complete ischemia. However, at the end of subsequent reperfusion for 30 min, the levels of the prostanoids PGF2 alpha, PGE2, PGD2, 6-keto-PGF1 alpha, and TxB2 increased according to the preceding ischemic time. The levels reached a maximum after 7.5 min of ischemia and were elevated by 7-, 14-, 48-, 3-, and 30-fold, respectively. A prolongation of ischemia of up to 12.5 min was not associated with further increases of prostanoids at the end of reperfusion. The mechanisms underlying the metabolism of eicosanoids are discussed in relation to the changes of cortical direct current potential.

OBJECTIVE

In liver cirrhosis, inflammation triggers portal hypertension. Kupffer cells (KC) produce vasoconstrictors upon activation by bacterial constituents. Here, we hypothesize that the anti-inflammatory action of the cannabinoid receptor 2 (CB2) agonists JWH-133 and GP 1a attenuate portal hypertension.

METHODS

In vivo measurements of portal pressures and non-recirculating liver perfusions were performed in rats 4weeks after bile duct ligation (BDL). Zymosan (150μg/ml, isolated liver perfusion) or LPS (4mg/kgb.w., in vivo) was infused to activate the KC in the absence or presence of JWH-133 (10mg/kgb.w.), GP 1a (2.5mg/kgb.w.) or ZnPP IX (1μM). Isolated KC were treated with Zymosan (0.5mg/ml) in addition to JWH-133 (5μM). The thromboxane (TX) B2 levels in the perfusate and KC media were determined by ELISA. Heme oxygenase-1 (HO-1) and CB2 were analyzed by Western blot or confocal microscopy.

RESULTS

JWH-133 or GP 1a pre-treatment attenuated portal pressures following KC activation in all experimental settings. In parallel, HO-1 expression increased with JWH-133 pre-treatment. However, the inhibition of HO-1 enhanced portal hypertension, indicating the functional role of this novel pathway. In isolated KC, the expression of CB2 and HO-1 increased with Zymosan, LPS and JWH-133 treatment while TXB2 production following KC activation was attenuated by JWH-133 pre-treatment.

CONCLUSIONS

JWH-133 or GP 1a treatment attenuates portal hypertension. HO-1 induction by JWH-133 plays a functional role. Therefore, the administration of JWH-133 or GP 1a represents a promising new treatment option for portal hypertension triggered by microbiological products.

BACKGROUND

Liver grafts from non-heart-beating donors inevitably suffer from warm ischemic injury. In these grafts, large quantities of inflammatory cytokines and arachidonic acid metabolites are induced, further aggravating injury. Cyclooxygenase (COX) is an intracellular enzyme that converts arachidonic acid into prostaglandin (PG)G2 and PGH2. COX has two isoforms: constitutive COX-1 and inducible COX-2. The aim of this study was to evaluate the effects of COX-2 inhibition by FK3311 (FK) on warm ischemic injury in a canine total hepatic vascular exclusion (THVE) model.

METHODS

Sixteen mongrel adult dogs were studied. The portal triad of the hilum and the inferior vena cava above and below the liver was clamped for 1 hour. Splanchnic decompression was achieved by active splenofemorojugular bypass. The animals were divided into two groups. FK (1 mg/kg) was administered in the FK group (n = 8), and saline was administered in the control group (n = 8). Hepatic venous blood was collected to measure serum alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase (LDH), and hyaluronic acid levels. Serum thromboxane (Tx)B2 and 6-keto-PGF1alpha levels were also measured. Hepatic tissue blood flow was estimated simultaneously. Liver specimens were harvested for histologic study and polymorphonuclear neutrophils were counted.

RESULTS

Alanine aminotransferase, aspartate aminotransferase, and hyaluronic acid 2 and 6 hours after reperfusion and LDH 30 minutes and 2 and 6 hours after reperfusion were significantly (p < 0.05) lower in the FK group than in the control group. Hepatic tissue blood flow remained significantly (p < 0.05) higher in the FK group than in the control group 1, 2, and 6 hours after reperfusion. Histologic tissue damage was mild and polymorphonuclear neutrophil infiltration was significantly lower (p < 0.05) in the FK group than in the control group 1 and 6 hours after reperfusion. Thirty minutes after reperfusion, TxB2 was significantly reduced (p < 0.05) in the FK group, and 6-keto-PGF1alpha was not significantly lower.

CONCLUSIONS

FK protected against hepatic warm ischemia-reperfusion injury by marked inhibition of TxA2.

Patients with inflammatory bowel disease (IBD) are at higher risk of venous thromboembolism and coronary artery disease despite having a lower burden of traditional risk factors. Platelets from IBD patients release more soluble CD40 ligand (CD40L), and this has been implicated in IBD platelet hyper-activation. We here measured the urinary F2-isoprostane 8-iso-prostaglandin (PG)2α (8-iso-PGF2α), urinary 11-dehydro-thromboxane (TX) B2 (11-dehydro-TXB2) and plasma CD40L in IBD patients, and explored the in vitro action of anti-tumour necrosis factor (TNF)-α antibody infliximab on IBD differentiating megakaryocytes. Urinary and blood samples were collected from 124 IBD patients and 37 healthy subjects. Thirteen IBD patients were also evaluated before and after 6-week infliximab treatment. The in vitro effect of infliximab on patient-derived megakaryocytes was evaluated by immunoflorescence microscopy and by flow cytometry. IBD patients had significantly (p<0.0001) higher urinary 8-iso-PGF2α and 11-dehydro-TXB2 as well as plasma CD40L levels than controls, with active IBD patients displaying higher urinary and plasma values when compared to inactive patients in remission. A 6-week treatment with infliximab was associated with a significant reduction of the urinary excretion of 8-iso-PGF2α and 11-dehydro-TXB2 (p=0.008) and plasma CD40L (p=0.001). Infliximab induced significantly rescued pro-platelet formation by megakaryocytes derived from IBD patients but not from healthy controls. Our findings provide evidence for enhanced in vivo TX-dependent platelet activation and lipid peroxidation in IBD patients. Anti-TNF-α therapy with infliximab down-regulates in vivo isoprostane generation and TX biosynthesis in responder IBD patients. Further studies are needed to clarify the implication of infliximab induced-proplatelet formation from IBD megakaryocytes.

Pharmacokinetic and pharmacodynamic properties in goats of the non-steroidal anti-inflammatory drug tolfenamic acid (TA), administered both alone and in combination with the fluoroquinolone marbofloxacin (MB), were established in a tissue cage model of acute inflammation. Both drugs were injected intramuscularly at a dose rate of 2 mg kg(-1). After administration of TA alone and TA+MB pharmacokinetic parameters of TA (mean values) were Cmax=1.635 and 1.125 microg ml(-1), AUC=6.451 and 3.967 microgh ml(-1), t1/2K10=2.618 and 2.291 h, Vdarea/F=1.390 and 1.725Lkg(-1), and ClB/F=0.386 and 0.552 L kg(-1) h(-1), respectively. These differences were not statistically significant. Tolfenamic acid inhibited prostaglandin (PG)E2 synthesis in vivo in inflammatory exudate by 53-86% for up to 48 h after both TA treatments. Inhibition of synthesis of serum thromboxane (Tx)B2 ex vivo ranged from 16% to 66% up to 12h after both TA and TA+MB, with no significant differences between the two treatments. From the pharmacokinetic and eicosanoid inhibition data for TA, pharmacodynamic parameters after dosing with TA alone for serum TxB2 and exudate PGE2 expressing efficacy (Emax=69.4 and 89.7%), potency (IC50=0.717 and 0.073 microg ml(-1)), sensitivity (N=3.413 and 1.180) and equilibration time (t1/2Ke0=0.702 and 16.52 h), respectively, were determined by PK-PD modeling using an effect compartment model. In this model TA was a preferential inhibitor of COX-2 (COX-1:COX-2 IC50 ratio=12:1). Tolfenamic acid, both alone and co-administered with MB, did not affect leucocyte numbers in exudate, transudate or blood. Compared to placebo significant attenuation of skin temperature rise over inflamed tissue cages was obtained after administration of TA and TA+MB with no significant differences between the two treatments. Marbofloxacin alone did not significantly affect serum TxB2 and exudate PGE2 concentrations or rise in skin temperature over exudate tissue cages. These data provide a basis for the rational use of TA in combination with MB in goat medicine.

BACKGROUND

Even low doses of oral aspirin inhibit prostacyclin (prostaglandin [PG] I2) formation and cause gastrointestinal toxicity. We examined the skin as a novel route for continuous low-dose aspirin administration and selective inhibition of platelet cyclooxygenase in humans.

RESULTS

Aspirin 250 or 750 mg/d for 10 days induced a dose-dependent inhibition of serum thromboxane (TX) B2. At the highest dose, five of six subjects responded, with a mean reduction in serum TXB2 of 95 +/- 3% (P = .003). Urinary 2,3-dinor TXB2, an index of in vivo TXA2 formation, decreased by 68 +/- 7% and recovered slowly, consistent with inhibition of platelet cyclooxygenase in vivo. In contrast, PGI2 biosynthesis, determined as excretion of 2,3-dinor-6-keto PGF1 alpha, was 81 +/- 5% of baseline at 10 days. Intravenous bradykinin increased PGI2 biosynthesis 5.1 +/- 1.6-fold (n = 4) before aspirin treatment. Oral aspirin 75 mg/d for 14 days abolished bradykinin-induced PGI2 formation, whereas dermal aspirin 750 mg/d had no effect despite similar inhibition of TXA2 biosynthesis. In five subjects, plasma aspirin and salicylate were determined after a single application of 750 mg. Aspirin was absorbed slowly, with peak levels of 0.24 +/- 0.11 micrograms/mL at 3 hours. Salicylate levels peaked at 6 to 12 hours, with plasma levels of 0.79 +/- 0.14 micrograms/mL.

CONCLUSIONS

Thus, it is possible to achieve selective inhibition of platelet cyclooxygenase by aspirin applied to the skin. This approach may be applicable to other antiplatelet agents and be useful in patients at risk for gastrointestinal bleeding or toxicity.

Levels of the stable urinary metabolites of thromboxane A2 and prostacyclin, 11-dehydro-thromboxane B2 (11-dehydro-TXB2) and 2,3-dinor-6-keto-prostaglandin F1alpha (2,3-dinor-6-keto-PGF1alpha) were measured in diabetics to elucidate the relation between the thromboxane A2/prostacyclin (TX/PGI) balance and pathological states of diabetes mellitus. 11-Dehydro-TXB2 and 2,3-dinor-6-keto-PGF1alpha were derivatized to methyl ester-propylamide-dimethylisopropylsilyl ether and methyl ester-methoxime-dimethylisopropylsilyl ether derivatives, respectively, and applied to a gas chromatography/selected ion monitoring. The TX/PGI ratios of diabetics were higher than those of healthy volunteers, suggesting the hypercoagulative states of this disease. The ratios showed positive correlations with the levels of blood glucose. The levels of hemoglobin A1c and triglyceride were correlated weakly with the ratio. Some of the patients who had relatively low levels of blood glucose also showed high TX/PGI ratios. Furthermore, the ratio increased in the order of the groups 1, 2, and 3; group 1 contained patients who did not take medicine for diabetes, group 2 contained those who took oral hypoglycemic agents, and group 3 contained those who received insulin therapy. These observations indicate that the TX/PGI ratio reflects the pathological conditions of diabetes and is a useful marker, having few different features from other markers that are presently used.

Alcohol is known to sometimes cause coronary spasm, the mechanism of which is still unknown. The authors monitored changes in plasma levels of prostanoids (thromboxane [TX B2], 6-keto prostaglandin F1 alpha [PGF1 alpha]), catecholamines (CA), serotonin (5-HT), cyclic nucleotides (cyclic adenosine monophosphate--cAMP, cyclic guanosine monophosphate--cGMP), and platelet aggregation after alcohol ingestion (Japanese rice wine 400 mL) in 8 patients with alcohol-induced variant angina and 8 healthy men as controls. Coronary spasm was confirmed to have been induced in 4 patients nine hours after alcohol challenge (VA[+]), when their plasma ethanol levels had already returned to a null level. Neither CA nor 5-HT levels showed any change after alcohol ingestion either in patients or controls, though controls showed high levels of CA during alcohol ingestion. TX B2 in VA(+) patients increased gradually after alcohol ingestion to reach up to a statistically significantly high level just before attack, as compared with those of controls and VA(-) patients, who, on the contrary, did not show such changes. The levels of 6-keto PGF1 alpha, however, which were significantly lower in patients than in controls before the test, exhibited a gradual increase in VA(+) patients in parallel with the increase in TX B2. No significant changes in cAMP levels between either controls or patients were present. On the contrary, cGMP levels had a gradual decrease in patients after alcohol ingestion. Especially six hours after alcohol ingestion, cGMP levels in VA(+) patients decreased so much as to make a statistically significant difference, as compared with the level in controls. Platelet aggregability in controls showed a decrease after alcohol ingestion, in spite of no change or even increase in patients. These data suggest that low levels of PGF1 alpha and the decrease of cGMP levels from alcohol ingestion play important roles in the mechanism of coronary spasm induced by alcohol ingestion.

OBJECTIVE

Platelet activation plays a major role in cardiovascular events (CVEs). Mediterranean diet (Med-Diet) reduces the incidence of stroke and myocardial infarction but it is still unclear if it affects platelet activation. Aim of the study was to evaluate the effect of Med-Diet on the urinary excretion of 11-dehydro-thromboxane (Tx) B2, a marker of in vivo platelet activation, in patients with atrial fibrillation (AF).

METHODS

Prospective observational cohort study including 801 non-valvular AF patients on chronic treatment with warfarin/acenocumarol referring to I Medical Clinic - Atherothrombosis Center of Sapienza University of Rome, Italy, from February 2008 to December 2013. Adherence to Med-Diet was evaluated by a short nine-items dietary questionnaire. Urinary excretion of 11-dehydro-TxB2 was measured in all patients.

RESULTS

Mean follow-up was 33.9 (±19.8) months, yielding 2223 patient/year of observation. Mean age of patients was 73.3 (±8.9) years, 43.7% were female. Median value of urinary TxB2 was 105.5 [60.0-190.0] ng/mg creatinine. We found a significant inverse correlation between total Med-Diet score and 11-dehydro-TxB2 values (Rs: -0.356, p < 0.001). In a multivariable stepwise linear regression analysis, history of stroke/TIA (β = 0.146, p = 0.003), olive oil (β = -0.130, p = 0.007), wine (β = -0.102, p = 0.036) and antiplatelet drugs (β = -0.098, p = 0.045) were independently associated to 11-dehydro-TxB2. We found no differences in the rate of ischemic or bleeding events across tertiles of Med-Diet score during follow-up.

CONCLUSIONS

Med-Diet adherence is inversely associated to urinary excretion of 11-dehydro-TxB2, suggesting that Med-Diet may favorably affect platelet function in AF patients. Clinical Trial Registration: ClinicalTrials.gov NCT01882114.

The present paper compares the effects of two monounsaturated oils, extra virgin olive oil (EVOO) and high-oleic acid sunflower oil (HOSO), on serum and LDL peroxides, eicosanoid production and the thrombogenic ratio (thromboxane (TX) B2:6-keto-prostaglandin F1alpha) in fourteen non-obese post-menopausal women. The subjects, mean age 63 (SD 11) years, were assigned to two consecutive oleic acid-rich 28 d dietary periods. EVOO and HOSO represented 62 % of the total lipid intake and were used as the only culinary fat during the first and second dietary periods respectively. Serum peroxides, plasma alpha-tocopherol and TXB2 levels in stimulated platelet-rich plasma (PRP-TXB2) were significantly higher (P < 0.01, P < 0.001, and P < 0.05, respectively) after the HOSO diet than after the EVOO diet. The relationship between the serum cholesterol level (< 6.21 mmol/l or>> or = 6.21 mmol/l) and the type of dietary oil on eicosanoids, peroxides and alpha-tocopherol were evaluated by two-way ANOVA. Dietary oil significantly affected (P < 0.05) the PRP-TXB2 level, whereas serum and LDL peroxides were significantly affected (P < 0.001 and P < 0.01, respectively) by the serum cholesterol level. The plasma alpha-tocopherol level was significantly affected by the serum cholesterol level and the type of dietary oil (both P < 0.001). No significant relationships were found between serum cholesterol levels, serum peroxide or LDL peroxide levels, plasma alpha-tocopherol concentrations or alpha-tocopherol intakes with eicosanoid production or the thrombogenic ratio due to dietary changes. However, in spite of their higher alpha-tocopherol levels, hypercholesterolaemic subjects showed increased peroxidation in serum and LDL in comparison with normocholesterolaemic subjects on the HOSO diet in comparison with the EVOO diet. These findings suggest that differences in the type of minor compounds, as well as in the concentration of linoleic acid, in both these monounsaturated oils may play an important role in modulating eicosanoid production and lipoprotein peroxidation when they constitute a large proportion of the diet of post-menopausal women.

We have recently shown that dipyrone (metamizole), a non-opioid analgesic, can nullify aspirin (acetylsalicylic acid; ASA) antiplatelet effects in patients with coronary artery disease (CAD). In this study, we analysed the aspirin and dipyrone drug-drug interaction in order to identify strategies to prevent the dipyrone induced inhibition of asprin antiplatelet effects. Platelet function was measured by arachidonic acid-induced light-transmission aggregometry, thromboxane (TX) B2- formation by immunoassay. Dipyrone metabolite plasma levels were determined by high-performance-liquid-chromatography (HPLC). In seven healthy individuals, in vitro ASA (30 µM/ 100 µM/ 300 µM/ 1,000 µM) and dipyrone (10 µM) coincubation revealed, that the aspirin and dipyrone interaction can be overcome by increasing doses of aspirin. In 36 aspirin and dipyrone comedicated CAD patients, addition of ASA (30 µM/ 100 µM) in vitro inhibited, but did not completely overcome the dipyrone induced reduction of aspirin antiplatelet effects. Notably, the inhibition of thromboxane formation in aspirin and dipyrone comedicated CAD patients coincided with dipyrone plasma levels. In a cross-over designed study in four healthy individuals, we were able to prove that inhibition of aspirin (100 mg/ day) effects by dipyrone (750 mg/ day) was reversible. Furthermore, aspirin (100 mg/ day) medication prior to dipyrone (750 mg/ day) intake prevented the inhibition of antiplatelet effects by dipyrone in 12 healthy individuals. In conclusion, aspirin medication prior to dipyrone intake preserves antiplatelet effects, circumventing the pharmacodynamic drug-drug interaction at the level of cyclooxygenase-1.

BACKGROUND

Increased systemic levels of thromboxane (Tx) during cardiopulmonary bypass (CPB) in humans have been reported. It is not known whether this reflects a general systemic response to the surgical procedure or an increased pulmonary production of Tx in response to ischemia and reperfusion.

METHODS

Thromboxane B2 levels were measured in the right atrium and left atrium of 14 patients undergoing coronary artery bypass grafting for angina. Eight patients (group 1) were without aspirin for at least 15 days before operation, and 6 patients (group 2) were treated with aspirin (100 mg/day) for at least 1 month before operation. Levels of TxB2 were determined by enzyme immunoassay after lipid extraction and separation.

RESULTS

Thromboxane B2 levels were elevated throughout CPB. In group 1, left atrial TxB2 levels were significantly higher (p < 0.05) than right atrial levels at all study points during CPB. After pulmonary reperfusion, TxB2 levels in both atria increased significantly (p < 0.02) compared with the levels before cross-clamping of the aorta, and there was an increasing gradient between the two atria (p < 0.05). Mean plasma TxB2 levels during CPB in group 2 were significantly reduced (p < 0.0001) in the right atrium (by 73%) and in the left atrium (by 69%) compared with levels in group 1.

CONCLUSIONS

The rise in TxB2 levels in the left atrium after CPB in humans reflects production of Tx mainly in the lungs, most probably by ischemic pulmonary tissue and intravascular hematologic components. Aspirin markedly reduces Tx production during CPB, and it might play a major role in preventing pulmonary injury after operations with CPB in humans.

Inhalation of smoke containing acrolein, the most common toxin in urban fires after carbon monoxide, causes vascular injury with non-cardiogenic pulmonary edema containing potentially edematogenic eicosanoids such as thromboxane (Tx) B2, leukotriene (LT) B4, and the sulfidopeptide LTs (LTC4, LTD4, and LTE4). To determine which eicosanoids are important in the acute lung injury, we pretreated sheep with BW-755C (a combined cyclooxygenase and lipoxygenase inhibitor), U-63557A (a specific Tx synthetase inhibitor), or indomethacin (a cyclooxygenase inhibitor) before a 10-min exposure to a synthetic smoke containing carbon particles (4 microns) with acrolein and compared the results with those from control sheep that received only carbon smoke. Acrolein smoke induced a fall in arterial PO2 and rises in peak inspiratory pressure, main pulmonary arterial pressure, pulmonary vascular resistance, lung lymph flow, and the blood-free wet-to-dry weight ratio. BW-755C delayed the rise in peak inspiratory pressure and prevented the fall in arterial PO2, the rise in lymph flow, and the rise in wet-to-dry weight ratio. Neither indomethacin nor U-63557A prevented the increase in lymph flow or wet-to-dry weight ratio, although they did blunt and delay the rise in airway pressure and did prevent the rises in pulmonary arterial pressure and pulmonary vascular resistance. Thus, cyclooxygenase products, probably Tx, are responsible for the pulmonary hypertension after acrolein smoke and to some extent for the increased airway resistance but not the pulmonary edema. Prevention of high-permeability pulmonary edema after smoke with BW-755C suggests that LTB4, may be etiologic, as previous work has eliminated LTC4, LTD4, and LTE4.

OBJECTIVE

Selective cyclooxygenase (COX)-2 inhibitors do not adversely affect renal function in experimental cirrhosis. In the current study, we investigated the molecular mechanisms underlying the effects of the selective COX-2 inhibitor, celecoxib, and assessed the influence of albumin on its actions.

METHODS

Rat mesangial cells (RMC) were incubated with celecoxib in the absence or presence of albumin, and levels of selected vasoconstrictor eicosanoids, renin release and alpha-smooth muscle actin (alpha-SMA) expression were determined. The effects of celecoxib on PPARgamma were assessed in RMC co-transfected with PPARgamma and luciferase reporter constructs.

RESULTS

Under resting conditions, RMC expressed COX-1, COX-2 and 12/15-lipoxygenase and mainly generated prostaglandin (PG)E2, thromboxane (TX)B2, 12-hydroxyeicosatetraenoic acid (12-HETE) and 8-epi-PGF2alpha. Celecoxib, in addition to reducing PGE2, significantly decreased 8-epi-PGF2alpha formation. In the presence of albumin, celecoxib also reduced TXB2 and 12-HETE. Albumin per se inhibited PGE2 as well as renin release. In trans-activation assays, celecoxib acted as a PPARgamma agonist whereas albumin inhibited PPARgamma as well as 15d-PGJ2-induced PPARgamma activation. Finally, celecoxib and albumin potentiated the inhibitory effect of 15d-PGJ2 on alpha-SMA expression.

CONCLUSIONS

These data provide novel molecular mechanisms of celecoxib and their modulation by albumin, that may be relevant to prevent renal dysfunction in conditions of unbalanced effective blood volume.

Interleukin-6 (IL-6) is a cytokine involved in the differentiation of B-cells to antibody secreting plasma cells, the activation of T-cells, and the stimulation of hepatocyte production of acute phase proteins. Because of the pro-inflammatory effects of this cytokine, we investigated the ability of the fatty acid arachidonic acid (AA) to regulate the release of IL-6 from rat resident peritoneal macrophages (M phi) in vitro. AA (0.5-16 microM) stimulated IL-6 release during a 4 h incubation period in a biphasic manner, with 4 microM AA generating a peak of IL-6 release (3-5-fold). AA (0.5-16 microM) also induced an increasing release of the AA metabolite thromboxane B2 (TXB2). The AA-induced release of IL-6 occurred within 1-2 h of incubation, whereas TXB2 concentrations were elevated within 5 min of AA treatment. The TX synthetase inhibitor CGS 12970 (4.0 microM and 40.0 microM) effectively blocked the generation of TXB2, but increased prostacyclin (PGI2) generation and potentiated the release of IL-6. In addition, PGI2, as well as the PGI2 agonists iloprost and cicaprost, stimulated IL-6 release from M phi by greater than 5-fold over vehicle-treated basal levels. These data suggest that PGI2 (but not TXA2) is involved in AA-induced IL-6 release from peritoneal M phi.

The effects of two anti-thrombotic and anti-lipidemic oils, evening primrose oil and fish oil, on glucose and lipid metabolism, prostaglandin (PG) levels and body composition were studied in patients with non-insulin-dependent diabetes. Seven patients were administered 4 g evening primrose oil, 2.4 g sardine oil and 200 mg vitamin E for 4 weeks. Fasting plasma glucose, hemoglobin A1c, total cholesterol, body weight and % body fat mass were significantly decreased after the treatment, and levels of changes in these parameters were not different from 11 patients who did not receive the oils. In the treatment group, concentrations of (e) icosapentaenoic acid (EPA) increased significantly in all the lipoprotein fractions, but dihomo-gamma-linolenic acid (DGLA) increased only in the high-density lipoprotein (HDL) fraction. The treatment decreased urinary 11-dehydro-thromboxane B2 excretion (32.7% decrease, P < 0.05), but did not alter significantly plasma PGE1 or 6-keto-PGF1 alpha levels. The ratio of 6-keto-PGF1 alpha and PGE1 to 11-dehydro-thromboxane B2 increased significantly after the treatment. These results suggest that these oil treatments are useful in improving abnormal lipid and thromboxane (TX)A2 metabolism in diabetic patients.

Eicosanoids have been shown to be major mediators of airway inflammation. Platelet-activating factor (PAF), a potent bronchoconstrictor and stimulator of respiratory mucous secretion, may mediate some of its effects via eicosanoid production. To explore eicosanoid generation by cultured feline tracheal explants, eicosanoids were measured following PAF stimulation. After labeling the explants with [3H]arachidonic acid, supernatant from control and PAF treated explants was fractionated by reverse phase high-performance liquid chromatography (HPLC). The resulting elution pattern suggested the release of arachidonic acid (AA), 15-hydroxyeicosatetraenoic acid (HETE), leukotriene (LT)B4, C4, prostaglandin (PG) D2/E2/F2 alpha, and 6-keto-PGF1 alpha. Radioimmunoassay (RIA) following HPLC resolution confirmed that PAF induced a significantly increased release of peptido-leukotrienes, PGD2, PGE2, PGF2 alpha, LTB4, and 5-HETE, as well as thromboxane (TX) B2. The most remarkable increase was LTC4/D4/E4 (15 x control) and PGD2 (4 x control). The PAF antagonist Ro 19-3704 had an inhibitory effect on the PAF-stimulated release of peptido-leukotrienes. We conclude that PAF stimulates the production of a variety of lipoxygenase and cyclooxygenase pathway metabolites in feline airways.

Arachidonic acid (AA) metabolism in nuclei of human pro-myelocytic leukemia (HL-60) cells was investigated during retinoic acid (RA)-induced granulocytic differentiation and 1 alpha, 25 dihydroxy-vitamin D3-induced monocytic differentiation. The whole control HL-60 cells and their nuclei hardly converted [1-14C]-AA to any metabolites comigrating with authentic prostaglandins (PGs). On the other hand, RA-treated HL-60 cells acquired the ability to convert [1-14C]-AA to PGE2 predominantly and thromboxane B2 (TXB2) to a small degree, whereas the nuclei of the differentiated cells acquired the ability to convert predominantly to TXB2. In contrast, 1 alpha, 25-dihydroxy-vitamin D3-treated HL-60 cells acquired the ability to convert [1-14C]-AA to PGE2, PGF2 alpha, TXB2 and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT), whereas the nuclei of the differentiated cells acquired the ability to convert to PGF2 alpha, TXB2 and HHT. The significance of the acquisition of cyclooxygenase and TX synthetase by the nucleus is unclear, but there may be a specific relationship between the specific PGs formed by the nuclear membrane and nuclear events during HL-60 cell differentiation.

In this study Mongolian gerbils were submitted to a normothermic bilateral carotid ligation lasting 5 min. A noncompetitive antagonist of NMDA receptors, MK-801, 0.8 mg/kg, was injected i.p. 30 min before ischemia, or the ganglioside GM1, 30 mg/kg, was given i.p. for 3 days, twice a day. The morphology of the hippocampal CA1 neurones and the brain content of cyclooxygenase metabolites of arachidonic acid: prostaglandin 6-keto PGF1 alpha and thromboxane Tx B2 were studied. Untreated ischemia induced the accumulation in brain of the 6-keto PGF1 alpha and Tx B2 immunoreactive materials, and resulted in a lesion of 70% of CA1 neurones. In the MK-801- and GM1-pretreated groups the postischemic levels of Tx B2 were significantly decreased. However MK-801 and GM1 did not prevent damage to the CA1 neurones in gerbils normothermic after ischemia, whereas a partial neuroprotection was observed in hypothermic, MK-801 treated gerbils. The results of this study indicate that NMDA receptors may participate in the mechanism of postischemic release of eicosanoids in brain. They also confirm a potential modulatory role of gangliosides. These results are discussed in terms of the involvement of cyclooxygenase metabolites of arachidonic acid in the mechanism of a selective delayed neuronal damage to the hippocampus CA1 after ischemia.

This study investigates whether increased levels of cytokines and lipid mediators may be associated with complications after coronary artery bypass grafting (CABG) with extracorporeal circulation (ECC). Hemodynamic measurements and blood samples were obtained in 32 patients before and after the end of ECC and at the 6th and the 24th postoperative hours. Coagulation and pulmonary and cardiovascular functions were specifically assessed postoperatively at the same time. Patients with cardiovascular dysfunction had higher interleukin 8 (IL-8) levels. Higher platelet-activating factor (PAF) and decreased PAF acetylhydrolase activity (AHA, the enzyme that inactivates PAF) levels were found in patients with moderate cardiovascular dysfunction. Interleukin 6 (IL-6), IL-8, and AHA levels correlated with most hemodynamic parameters and creatine phosphokinase myocardial band levels obtained after surgery. Patients with severe lung injury had lower PAF, 6-keto prostaglandin (Pg)F1alpha, and PgE2 levels and higher thromboxane (Tx) B2 concentrations compared with patients without lung injury. Increased IL6 levels were only associated with moderate lung injury. Impaired hemostasis was associated with higher IL6 levels. AHA, IL-6, and IL-8 seem to be associated with cardiovascular dysfunction. The IL-6 blood levels and the ratio of TxB2/6 keto-PgF1alpha blood levels are increased during post-CABG lung injury. These results identify an association between specific post-CABG complications and the systemic inflammatory response. The clinical significance of this association remains to be evaluated.

CONCLUSIONS

Patients with pulmonary, cardiovascular, or hemostasis dysfunction after cardiopulmonary bypass demonstrate aberrancies in a variety of cytokines and lipid mediators in arterial blood or plasma. The relationship between these findings and inflammatory response-induced complications remains to be determined.

In two groups of medical students (group A n=13, group B n=9, aged 23 to 28 years) we estimated the levels of PGE, PGF2 alpha, 6-keto-PGF1 alpha and thromboxane(TX)B2 by means of radioimmunoassay in the peripheral venous blood immediately before (BES) and 15-30 min after (AES) examination stress and compared these to control levels. Furthermore we determined the circulating platelet aggregates, the heart rate and blood pressure changes. Heart rate and systolic blood pressure increased in the BES period expressing a stressful situation. In BES the plasma level of PGE increased from 84 to 249 pg/ml (p less than 0.001), whereas PGF2 alpha was unchanged. The increase of PGE level seems to be an expression of the increased sympathetic nervous activity existing in the stressful situation. The level of 6-keto-PGF1 alpha, the stable metabolite of prostacyclin, behaved differently in the two groups. In group A there was a statistically significant increase (p less than 0.05), whereas in group B the 6-keto-PGF1 alpha level was unchanged. In AES the TXB2 level increased from 113 to 167 pg/ml (p less than 0.01), whereas under BES conditions the TXB2 level was not changed. Simultaneously with the increased TXB2 level the circulating platelet aggregates were increased. The increase in TXB2 level occurring only after the end of the examination may offer a possible explanation of the frequent appearance of heart attacks after stressful situations.

The cardiovascular and metabolic effects of an endotoxin derived from Serratia marcescens were examined in anaesthetized, spontaneously-breathing cats. There was a marked initial elevation of right atrial pressure (the result of pulmonary vasoconstriction) and decreases in systemic arterial pressure and in arterial PO2. The 'delayed' effects of endotoxin shock in this species (1-8 h) consisted of a reduced cardiac output and decreased urinary excretion. Blood pressure and myocardial contractility (assessed from measurement of left ventricular (LV) dP/dt and LV end-diastolic pressure) were maintained throughout this phase. There was evidence of a metabolic (lactic) acidosis largely compensated by hyperventilation. Plasma levels (both arterial and mixed venous blood samples) of prostaglandin (PG)E2, PGF2 alpha, 6-keto PGF1 alpha and thromboxane (TX)B2 were measured by radioimmunoassay techniques. Endotoxin administration caused substantial increases in the plasma levels of all four derivatives of arachidonic acid, especially between 1 and 6 h. Separation of the endotoxin-treated cats into survivors and non-survivors showed that the non-survivors had significantly higher circulating levels of PGE2, TXB2 and PGF2 alpha. It is suggested that TXB2 and PGF2 alpha might contribute to some of the detrimental effects of endotoxin (e.g. pulmonary, mesenteric, renal vasoconstriction; platelet aggregation with resulting organ failure) and that prostacyclin may be beneficial in endotoxin shock in this species.

Hypertension is the main side effect developing in patients suffering from renal anemia who are treated with recombinant human erythropoietin (rhEPO). We investigated the effect of rhEPO on the vascular tone of rabbit aorta. rhEPO had no direct vasoconstrictor effect, but it enhanced norepinephrine (NE)-induced contractions of rabbit aortic rings. Relaxations to acetylcholine (ACh, 1 microM) were unaltered in the presence or absence of rhEPO, indicating that the endothelium-dependent NO pathway was not affected by rhEPO. In rings of human renal artery and rabbit aorta, rhEPO (200 U/ml) increased the synthesis of constrictor prostanoids. The cyclooxygenase inhibitors indomethacin and aspirin abolished the increase in prostanoid production. However, they did not completely suppress the rhEPO-induced enhancement of NE contractions in rabbit aorta. We further investigated the effect of rhEPO on prostanoid and endothelin-1 synthesis in cultured human endothelial cells. Endothelial cells from human umbilical veins (HUVEC) were isolated and cultured. After incubation with rhEPO, the formation of prostaglandin (PG) I2 (analyzed as its stable metabolite 6-keto-PGF1 alpha), PGF2 alpha, PGE2, thromboxane (Tx) B2, and of endothelin-1 (ET-1) was measured by radioimmunoassay (RIA). rhEPO (200 U/ml) increased the formation of PGF2 alpha and TxB2 and decreased the formation of PGI2 in HUVEC. The release of ET-1 was increased by nearly 90% in the presence of rhEPO (200 U/ml). We conclude that a shift in the balance of constrictor and relaxing prostanoids as well as an increased synthesis of ET-1 may contribute to the hypertensive side effect of rhEPO therapy. ET-1 may at least in part be responsible for the unexpectedly low inhibitory effect of indomethacin on rhEPO-enhanced contractions of rabbit aorta.

We have already demonstrated that arachidonic acid (AA) inhibits carrageenin-induced granuloma growth in vivo and that this effect is related to an increase in prostaglandin E2 formation. As prostaglandin E1 has been shown to be more effective in inhibiting granuloma growth than prostaglandin E2 we investigated the effect of linoleic acid (LA) (18:2 omega 6) and dihomo-gamma-linolenic acid (DHGLA) (20:3 omega 6), potential precursors of prostaglandin E1, in this model. LA and DHGLA inhibited the development of carrageenin-induced granulomas in the rat when injected locally. Both fatty acids (FA) stimulated the release of prostaglandin (PG) E1 from granuloma macrophages (M phi) in vitro, DHGLA being most effective. LA had little effect on the release of PGE2, 6 keto PGF1 alpha, the stable product of prostacyclin (PGI2) or thromboxane (Tx) B2, the stable metabolite of TxA2. DHGLA had no effect on the release of 6 keto PGF1 alpha, but inhibited PGE2 and, to a lesser extent, TxB2 synthesis.