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The major eicosanoid produced within the rat liver, prostaglandin (PG) D2, wa studied for its ability to interact with the various liver cell types. It appeared that PGD2 bound specifically to parenchymal liver cells, whereas the binding of PGD2 to Kupffer and endothelial liver cells was quantitatively unimportant. Maximally 700 pg of PGD2/mg of parenchymal-cell protein could be bound by a high-affinity site (1 x 10(6) PGD2-binding sites/cell). The recognition site for PGD2 is probably a protein because trypsin treatment of the cells virtually abolished the high-affinity binding. High-affinity binding of PGD2 was a prerequisite for the induction of a metabolic effect in isolated parenchymal liver cells, i.e. the induction of glycogenolysis. High-affinity binding of PGD2 by parenchymal cells was coupled to the conversion of PGD2 into three metabolites, whereas no conversion of PGD2 by Kupffer and endothelial liver cells was noticed. The temperature-sensitivity of the conversion of PGD2 was consistent with a conversion of PGD2 on or in the vicinity of the cell membrane. One of the PGD2 metabolites could be identified as 9 alpha, 11 beta-PGF2. It can be calculated that the conversion rate of PGD2 by parenchymal liver cells exceeds the production rate of PGD2 by Kupffer plus endothelial liver cells, indicating that PGD2 is meant to exert its activity within the liver. The present finding that PGD2 formed by the non-parenchymal liver cells is recognized by a specific receptor on parenchymal liver cells and that binding, conversion and metabolic effect of PGD2 are interlinked by this receptor provides further support for the specific role of PGD2 in the intercellular communication in the liver.

In an investigation of the pathogenesis of acute necrotizing pancreatitis (ANP) the plasma levels of TXB2, 6-keto-PGF1 alpha, and PGE2 were measured in rats. After induction of ANP by injection of 5% sodium taurocholate into the pancreatic duct, a marked increase in TXB2 levels and a slight increase in 6-keto-PGF 1 alpha levels were found. PGE2 levels decreased. Mortality was 100% within 30 h. Pretreatment with chloroquine, a phospholipase A2 inhibitor, led to a inhibition of TXB2 production, whereas 6-keto-PGF1 alpha and PGE2 levels showed a surprising slight elevation in the first 6 h. Pretreatment with chloroquine decreased mortality by 30%. Pretreatment with FPL 55712, a leukotriene synthesis blocker, caused an increase in TXB2 and PGE2 levels, whereas the formation of 6-keto-PGF1 alpha remained unaltered. Two out of nine animals survived after pretreatment with FPL 55712. The results of the present study indicate that arachidonate end products are involved in ANP. The significance of the high TXB2 levels, decreased PGE2 levels, and only slightly elevated 6-keto-PGF1 alpha levels during ANP requires further investigation. The thromboxane A2 to prostacyclin ratio may be important.

Eicosanoid synthesis by alveolar macrophages (AM), harvested from tumor bearing animals, was measured after tumor inoculation in rats treated with or without carrageenan (carra), an immunomodulating agent. After incubaticn of the cells with [14]C-arachidonic acid and the Ca-ionophore A23187, samples were measured by high pressure liquid chromatography (HPLC). From the HPLC profiles the lypoxygenase products, 12-hydroxyeicosatetraenoic acid (12-HETE), 15-HETE, and leukotriene-B 4 (LTB 4 ) were determined as well as the cyclooxygenase products, prostaglandin (PG)E 2 , PGF 2α and TXB 2 . After tumor inoculation AM-synthesis of lipoxygenase products tended to increase to values twice those of the base line values, whereas cyclooxygenase products showed subnormal values. In the non treated animals, 10 days after tumor inoculation, statistically significant increases in 12- and 15-HETE, LTB 4 and PGE 2 were observed when compared with carra treated animals. Later measurements did not show these differences in AM metabolism. AM metabolism was (negatively) correlated with the number of macrophages, which was particularly evident in the correlation with 12-HETE synthesis.

The effect of adrenalectomy on the formation of cyclooxygenase and lipoxygenase products by activated peritoneal rat macrophages was determined. After isolation, the cells were incubated with [1-14C]arachidonic acid and the calcium ionophore A23187 and the metabolites isolated by HPLC chromatography. The main components formed in the controls are 6-keto-prostaglandin F1 alpha, thromboxane B2 and 12-HETE. One peak represents 5,12-di-HETE. Smaller amounts of prostaglandin F2 alpha, prostaglandin E2, prostaglandin D2, leukotriene B4 and 15-HETE are also present. After adrenalectomy, a considerable increase occurs in the amounts of leukotriene B4, 15-HETE and 12-HETE. The increase in the prostaglandins is smaller. The compounds formed from endogenous arachidonic acid are also determined. In the cells of the controls, 6-keto-prostaglandin F1 alpha and thromboxane B2 are produced in higher amounts than leukotriene B4. After adrenalectomy, the formation of leukotriene B4 is much more increased than that of 6-keto-prostaglandin F1 alpha. These effects are most probably related to a diminished amount or inactivation of lipocortin, a glucocorticosteroid-induced peptide with phospholipase A2 inhibitory activity in adrenalectomized animals.

Ascites was collected from six patients with liver cirrhosis and the cells isolated. These cells, mainly macrophages, were labelled with 14C-arachidonic acid and stimulated with the calcium ionophore A23187. The metabolites formed were separated by HPLC. The main substances formed by the ascites cells were leukotriene B4, 5-hydroxy-6,8,11,14 eicosatetraenoic acid and leukotriene C4. Smaller amounts of thromboxane B2, 12-hydroxy-5,8,10 heptodecatrienoic acid and 6-keto-prostaglandin F1 alpha were isolated. Human peritoneal macrophages are therefore capable of producing leukotrienes and prostaglandins. Production of these substances might play a role in some of the complications of patients with liver cirrhosis and ascites.

Induction of glycogenolysis in the perfused liver by platelet activating factor (PAF) was blocked by the cyclooxygenase inhibitor indomethacin. 3H-labeled PAF was shown to interact in the perfused liver primarily with Kupffer cells. The addition of PAF to Kupffer cells resulted in a dose-dependent stimulation of prostaglandin D2 (PGD2) production, which was identified as the main eicosanoid formed after PAF stimulation of the Kupffer cells. PGD2 was able to induce a dose-dependent stimulation of glycogenolysis both in the perfused liver and in isolated parenchymal cells. The time-dependency of the PGD2 production and the glucose output by the perfused liver is consistent with a primary interaction of PAF with the Kupffer cells, followed by PGD2 formation, which subsequently stimulates glucose production in parenchymal cells.

In experimental animals endotoxin administration causes increased levels of thromboxane B2 and prostaglandins. Liver cirrhosis is often complicated by endotoxemia. In sixteen patients with alcoholic liver cirrhosis, we measured plasma thromboxane B2 levels. In twelve patients we found on one or more occasions raised plasma thromboxane B2 levels. Raised plasma thromboxane B2 levels were associated with significantly higher serum levels of urea, alkaline phosphatase, gamma glutamyl transpeptidase and lower antiplasmin and antithrombin III levels. It is possible that some of the complications in patients with alcoholic liver cirrhosis are mediated by thromboxanes.

Paw oedema, induced by carrageenan, was potentiated in normal rats by arachidonic acid and bishomo-γ-linoleic acid, but not by 5,8,11-eicosatrienoic acid. The latter is not an endogenous prostaglandin precursor, but replaces the other two in essential fatty acid deficient (EFAD) rats. Carrageenan oedema was partially suppressed in these EFAD rats. Aspirin exhibited equal suppression of carrageenan oedema in both normal and EFAD rats, despite the fact that, in the latter, prostaglandins are of negligible importance. The anti-inflammatory effect of dexamethasone was also identical in both normal and EFAD rats. The view that interference with the prostaglandin-system explains the acute anti-inflammatory effects of the two drugs, is discussed, in relation to the present results.

A metabolite of ticlopidine, PCR 3787, in concentrations from 25-200 micrograms/ml inhibited the aggregation of human platelets, induced by different amounts of collagen. The effect of PCR 3787 on PG formation was tested in rat platelets. After incubation with PCR 3787, platelets were labelled with 1-14C arachidonic acid and aggregation induced with collagen. The sum of the PG formed (PGF2 alpha + PGE2 + PGD2) was higher in the platelets incubated with PCR 3787 than in the controls. The aggregation-inhibiting effects of PGI2 (1 ng/ml) and PGD2 (10 ng/ml) on human platelets were enhanced by the addition of PCR 3787 (100 micrograms/ml). A stable derivative of PGI2, ciloprost was used. These results indicate, that part of the aggregation-inhibiting effect of ticlopidine can be attributed to the formation of this metabolite.

Small doses of endotoxin markedly increase the survival rate of adult rats exposed to 98% oxygen for periods that are normally lethal. The lysine salt of acetyl salicylic acid (L-ASA) partially reverses this protective effect of endotoxin. In this pilot study we investigated the level of eicosanoid production by broncho-alveolar lavage (BAL) cells and found that BAL cells of endotoxin protected rats, present in abundance, have an equal or increased capacity of HHT, 15-HETE, 12-HETE, LTB4 and 5-HETE production. These data suggest that production of the lipoxygenase products by BAL cells does not seem to play an important role in the pathogenesis of pulmonary oxygen toxicity. We did not find any indication for the occurrence of shunting of arachidonic acid metabolism to the lipoxygenase pathway as an explanation for the reversal of endotoxin's protective action by L-ASA.

Growth of BN175, a malignant fibrosarcoma, was correlated with high plasma TXB 2 and PGE 2 levels. This statistically significant increase was first detected 17 days after inoculation of the tumor, at which time the tumors were 20 mms in diameter. A further increase in tumor size was associated with still higher PGE 2 and TXB 2 values. At the same time, progressive alterations in platelet function, as measured by ADP-induced platelet aggregation, were observed.6-keto-PGF 1α levels remained normal throughout the whole experiment. It was concluded that tumor growth was associated with changes in PG synthesis and platelet function, although it remains unclear whether these changes were caused by some host immunological response towards the tumor or were predominantly the result of tumor PG-synthesis.

We studied the effect of pretreatment with two doses of the thromboxane aniagonist BM 13.177 and its solvent on the development of electrically induced coronary artery thrombosis in pigs. Results were compared with those obtained in animals pretreated with intravenously administered acetylsalicylate and its solvent The effects of both compounds on the overall cardiovascular performance (heart rate, mean arterial blood pressure, cardiac output, systemic vascular resistance) were minimal. In the animals receiving solvent or acetylsalicylate the time to occlusive coronary thrombosis was 33 ± 4 and 32 ±6 min, respectively. BM 13.177, in a dose of 5 mg · kg −1 , did not modify the time to thrombotic occlusion (35 ± 7 min), but in six of the eight animals that had received 10 mg · kg −1 BM 13.177, there was no occlusion within 120 min. In the acetylsalicylate-treated animals, collagen-indaced platelet aggregation and plasma thromboxane B 2 declined by 72 and 82%, respectively. The decreases were 46 and 20%, respectively, with the higher dose of BM 13.177. It is concluded that, in this porcine model of coronary artery thrombosis, the thromboxane antagonist BM 13.177 effectively suppressed formation of occlusive thrombi whereas acetylsalicylate was ineffective at a dose that lowered arterial thromboxane levels.

A comparison was made of the contractile effects of exogenously added and endogenously formed TxA2 on guinea pig lung parenchyma. Aggregating platelets were used as a source of exogenous TxA2. After the addition to the lung strip, the contractions were measured. Endogenous formation of TxA 2 was induced by adding either 1 μ g PlA 2 or 50 ng LTD 4 to the strip. It was found that the ratio of potency of action of endogenous/ exogenous TxA 2 was 5.1±0.62 for PlA 2 and 5.0±1.3 for LTD 4 .

The activation of macrophages by several agents results in the formation of arachidonic acid metabolites via the cyclooxygenase and lipoxygenase pathways. It has been shown that in human alveolar and peritoneal macrophages after stimulation by the calcium ionophore A23187, the main substances formed are LTB 4 and 5-HETE and also TxB 2. In rat this profile of eicosanoid formation was identical in alveolar macrophages, however a pronounced difference was observed in peritoneal macrophages, which predominantly generated the cyclooxygenase products 6kPGFI~ and TxB 2 [1]. Several of these eicosanoids have effects on the contractility and vascular permeability of lung tissue. Furthermore it has been reported that they are involved in the pathological effects of asthma and inflammatory diseases. In the experiments described here, the effects of the chemotactic peptide FMLP (formyl-methionylleucyl-phenylalanine) and PAF-acether (platelet activating factor) on the formation of eicosanoids from [1-14C]-arachidonic acid labelled resting and stimulated guinea-pig alveolar macrophages were determined. It was the aim of the study to investigate whether substances released from these macrophages could contribute to the bronchoconstriction induced by PAF-acether [2].

Human and rat peritoneal macrophages and rat Kupffer cells were labelled with [1-14C] arachidonic acid and stimulated with the calcium ionophore A23187. The metabolites formed were separated by high pressure liquid chromatography (HPLC). Human peritoneal macrophages formed especially leukotriene B4, 5-hydroxy-6,8,11,14 eicosatetraenoic acid and small amounts of leukotriene C4 and thromboxane B2, 12-hydroxy-5,8,10 heptadecatrienoic acid and 6-keto-prostaglandin F1 alpha, whereas rat peritoneal macrophages mainly produced cyclooxygenase products and in particular thromboxane B2 and 12-hydroxy-5,8,10 heptadecatrienoic acid. Rat Kupffer cells synthesized mainly cyclooxygenase products such as prostaglandin F2 alpha, prostaglandin D2 and prostaglandin E2. These results indicate that the profile of eicosanoids production by macrophages is dependent both on the species and on the tissue from which the macrophage is derived.

The effect of adrenalectomy on the formation of cyclooxygenase and lipoxygenase products from [1-14C]-arachidonic acid in rat kidneys after incubation with the Calcium-ionophore A23187 has been determined. The metabolites were isolated by HPLC. The main components formed are PGD2, PGE2, PGF2 alpha, LTB4, 5,12 di-HETE and 5-, 12- and 15-HETE. After adrenalectomy, an increase occurs in the formation of PG and LT, which is highest in that of PGD2 and 12-HETE. These effects are most probably related to a diminished formation or inactivation of lipocortin in adrenalectomized animals, a glucocorticosteroid induced peptide with phospholipase A2 inhibitory activity.

The formation of metabolites of arachidonic acid by the cyclo-oxygenase and lipoxygenase pathways were determined in human lung tissue, obtained from surgery. In this measurement the chopped tissue was incubated with the calcium ionophore A23187. Formation of metabolites from [1-14C] arachidonic acid was also determined. The metabolites were extracted, separated by HPLC and identified by measurement of the absorption spectrum at 280 nm, radioactivity, biological activity and by radioimmunoassay. 6-keto-prostaglandin F1 alpha (6-ketoPGF1 alpha), the metabolite of prostacyclin, is the cyclo-oxygenase product present in the highest amount (400 +/- 49 ng g-1), followed by PGD2 (162 +/- 59 ng g-1) thromboxane B2 (102 +/- 32 ng g-1) PGE2 (104 +/- 46 ng g-1) and PGF2 alpha (58 +/- 26 ng g-1). The amounts of the lipoxygenase products are: leukotriene B4 (LTB4), 163 +/- 100 ng g-1; LTC4, 63 +/- 31 ng g-1 and LTE4 121 +/- 34 ng g-1. From [1-14C] arachidonic acid higher amounts of the cyclooxygenase than of the lipoxygenase products were formed, with the exception of PGE2. The effects of several of these substances on the contraction of human small airway smooth muscle were measured. The contractions, induced by equivalent amounts of LTC4 and a synthetic analogue of thromboxane T X A2 were approximately one hundred times those induced by PGD2, PGF2 alpha and histamine. These results suggest that thromboxane A2 and LTC4 are the most important arachidonic acid metabolites that induce bronchoconstriction in the human lung.

The results of these and other experiments mentioned indicate that the actions of glucocorticosteroids on the metabolism of arachidonic acid have different aspects, one of which may be prevailing depending on the tissue studied. The relationship between these different activities is a matter of further investigation in this field.

The possible role of thromboxane A2 (TXA2) in acute necrotizing pancreatitis (ANP) was investigated in rats. After ANP was induced by injecting sodium taurocholate (5% w/v) into the pancreatic duct, the thromboxane B2 (TXB2) levels in plasma increased significantly. The effects of indomethacin, a general blocker of prostaglandin synthesis, on survival time and on plasma TXB2 levels were compared with those of dazoxiben, a more specific blocker of TXA2 synthesis, and Flunarizine, a calcium entry blocker known to inhibit the effects of TXA2. In a test group without any treatment, all animals died within 30 h of ANP induction. Although TXB2 levels were lowered by the administration of indomethacin, dazoxiben, and Flunarizine, survival times were not significantly altered. Indomethacin pretreatment had no beneficial effect, whereas 30% and 40% of the animals survived for 36 h after treatment with Flunarizine and dazoxiben, respectively. The results of the present study indicate that inhibition of TXA2 synthesis alone does not dramatically alter survival time. However, a potential role for other arachidonate metabolites in ANP cannot be ruled out by this study.

Background During the chronic stage of Complex Regional Pain Syndrome (CRPS), impaired microcirculation is related to increased vasoconstriction, tissue hypoxia, and metabolic tissue acidosis in the affected limb. Several mechanisms may be responsible for the ischemia and pain in chronic cold CPRS. Discussion The diminished blood flow may be caused by either sympathetic dysfunction, hypersensitivity to circulating catecholamines, or endothelial dysfunction. The pain may be of neuropathic, inflammatory, nociceptive, or functional nature, or of mixed origin. Summary The origin of the pain should be the basis of the symptomatic therapy. Since the difference in temperature between both hands fluctuates over time in cold CRPS, when in doubt, the clinician should prioritize the patient's report of a persistent cold extremity over clinical tests that show no difference. Future research should focus on developing easily applied methods for clinical use to differentiate between central and peripheral blood flow regulation disorders in individual patients.

Milrinone and salbutamol inhibit antigen induced stimulated arachidonic acid metabolism, probably at a site before the separation in lipoxygenase and cyclooxygenase pathways

These effects of malotilate on eicosanoid formation differ from those of known lipoxygenase inhibitors such as BW 755C (IC50 of 5-lipoxygenase 35 μM, 12-lipoxygenase >100 μM and 15-lipoxygenase 1.2 μM), nordihydroguiaretic acid (IC50 of 5-lipoxygenase 1.4 μM, 12-lipoxygenase 26 μM and 15-lipoxygenase 1 μM) and ketoconazole (5-lipoxygenase 28 μM, 12-lipoxygenase not affected and 15-lipoxygenase increased) [5]. The differential effects of malotilate on the 5-, 12- and 15-lipoxygenases and also on the generation of the compounds of the cyclooxygenase, have not previously been reported. The suppression of leukotriene productionin vitro occurred at concentrations found following normal therapeutic dosesin vivo. Inhibition of the production of the chemotactic substance LTB4 and the vasoconstrictive TxA2 provide a possible explanation for the useful effects of this drug on liver necrosis and liver fibrosis.