calcimycin and 12-hydroxy-5-8-10-heptadecatrienoic-acid

As a Chinese traditional herbal medicine, leaves of Platycladus orientalis (Linnaeus) Franco (LPO) are used to treat coughs, excessive mucus secretion, chronic bronchitis, bronchiectasis, and asthma, etc. The experiments were carried out to investigate their anti-inflammatory properties and mechanisms, which could support the Chinese traditional uses of treating inflammatory airway diseases.. The anti-inflammatory activities of the chloroform fraction (CHL) and pure compounds of LPO were evaluated for their abilities to inhibit pro-inflammatory enzymes in vitro, and production of tumor necrosis factor-α (TNF-α) and nitric oxide in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Furthermore, the arachidonic acid metabolites, stimulated by calcium ionophore A23187, were also determined by HPLC.. For the first time, the assays of eicosanoids in intact cells showed that the CHL, hinokiol, and acacetin had significant inhibitory effects on 5-hydroxy-eicosa-tetra-enoic acid (5-HETE) and leukotriene B(4) (LTB4) formations. And cell-free enzyme assays (5-lipoxygenase, leukotriene A(4)-hydrolase, cyclooxgenase-2) demonstrated the potent inhibitory effects of the CHL, hinokiol and acacetin on 5-lipoxygenase (5-LOX). Then, the inhibitions of the CHL, hinokiol on NO biosynthesis and the inhibitions of the CHL, 8(14),15-pimaradien-3β,18-diol, and hinokiol on TNF-α release were also confirmed in the RAW264.7 murine macrophages.. The data indicate that the inhibitory effects of the CHL and its components (hinokiol and acacetin) on 5-LOX contribute to the anti-inflammatory activity of LPO. Moreover, the CHL and its components also show beneficial effects on NO and TNF-α production. Consequently, these results provide a rationale for LPO's traditional applications in the treatment of inflammatory airway diseases.

Topics: Animals; Anti-Inflammatory Agents; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Calcimycin; Calcium Ionophores; Cell Line; Chloroform; Chromatography, High Pressure Liquid; Cupressaceae; Cyclooxygenase 2; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Epoxide Hydrolases; Fatty Acids, Unsaturated; Flavones; Hydroxyeicosatetraenoic Acids; Inflammation Mediators; Leukotriene B4; Lipoxygenase Inhibitors; Macrophages; Medicine, Chinese Traditional; Mice; Nitric Oxide; Plant Leaves; Plants, Medicinal; Rats; Rats, Sprague-Dawley; Solvents; Tumor Necrosis Factor-alpha

Platelets of patients with uremia develop a defective platelet function and have a decreased production of thromboxane B2 (TxB2). Activated platelets generate thromboxane from free arachidonate that is previously released from the membrane phospholipids (PLs) by phospholipases. Phospholipase A2 (PLA2) release up to 70% of the arachidonate in normal platelets, and to date, the activity of this enzyme in uremia is unknown. This work studied the PLA2 activity in the platelets of nine uremic patients and nine healthy volunteers. Washed platelets were labelled with [(14)C]arachidonic acid and activated with calcium ionophore A-23187 (4 microgr/ml). Lipids were resolved by TLC and identified by autoradiography. The distribution of [(14)C]arachidonic acid in the five major platelet phospholipids was found to be normal. Uremic platelets released more radioactivity than normal platelets (19.0 +/- 5.2% versus 11.3 +/- 1.6%, P = 0.001). The production of both, radioactive thromboxane B2 and hydroxyheptadecatrienoic acid was normal (2.6 +/- 1.2% and 3.5 +/- 1.6% of total radioactivity respectively), but the formation of the lipoxygenase metabolite hydroxyeicosatetraenoic acid was increased with respect to the controls (12.9 +/- 4.6% vs 7.0 +/- 1.3% of total radioactivity, P = 0002). In conclusion, platelets of patients with uremia have an increased activity of phospholipase A2 and produce increased amounts of hydroxyeicosatetraenoic acid, an inhibitor of the platelet function.

Topics: Arachidonic Acid; Blood Platelets; Calcimycin; Case-Control Studies; Fatty Acids, Unsaturated; Humans; Kidney Failure, Chronic; Phospholipases A; Phospholipases A2; Platelet Function Tests; Thromboxane B2; Uremia

We analyzed the effect of the PAF receptor antagonist (+)-cis-3,5-dimethyl-2-(3-pyridyl)thiazolidin-4-one hydrochloride (SM-12502) on human platelet aggregation as well as mediator release. After incubation of human platelet with different concentrations of SM-12502 the cells were subsequently stimulated with either the Ca ionophore A23187, with human thrombin, or with an activator of heterotrimeric G-proteins, sodium fluoride (NaF, in the presence of Al3+). Preincubation of platelets with the PAF receptor antagonist led to an inhibition of 12-lipoxygenase derived 12(S)-HETE and cyclooxygenase derived 12(S)-HHT. Pretreatment of platelets with the PAF receptor antagonist SM-12502 prior to activation with the Ca ionophore A23187 or PAF also inhibited platelet aggregation. Our data clearly indicate an inhibitory effect of the new PAF receptor antagonist SM-12502 on the formation of platelet derived inflammatory mediators of the lipoxygenase pathway as well as of the cyclooxygenase pathway, and furtherone, treatment with the PAF receptor antagonist diminished platelet aggregation after subsequent specific and unspecific activation.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Aluminum; Arachidonate 12-Lipoxygenase; Calcimycin; Calcium; Fatty Acids, Unsaturated; Gene Expression Regulation; GTP-Binding Proteins; Humans; Ionophores; Platelet Activating Factor; Platelet Activation; Platelet Aggregation; Platelet Aggregation Inhibitors; Platelet Membrane Glycoproteins; Prostaglandin-Endoperoxide Synthases; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Signal Transduction; Sodium Fluoride; Thiazoles; Thiazolidines; Thrombin

Studies were made on the effects of stilbene derivatives isolated from medicinal plants on arachidonate metabolism and degranulation in human polymorphonuclear leukocytes (PMN-L). Resveratrol (3,4',5-trihydroxystilbene) isolated from the roots of Reynoutria japonica was found to inhibit the 5-lipoxygenase products 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), 5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid (5,12-diHETE) and leukotriene C4(LTC4); its concentrations for 50% inhibition (IC50) were 8.90 x 10(-6) M, 6.70 x 10(-6) M and 1.37 x 10(-6) M, respectively. The IC50 of 5-HETE, 5,12-diHETE and LTC4 formations of synthetic 3,3',4-trihydroxystilbene were 5.90 x 10(-6) M, 6.30 x 10(-7) M and 8.80 x 10(-7) M, respectively. Moreover, they inhibited the release of lysosomal enzyme such as lysozyme and beta-glucuronidase induced by calcium ionophore A 23187 from human PMN-L at 10(-3)-10(-4) M.

Topics: Arachidonic Acid; Autoradiography; Calcimycin; Cell Degranulation; Cyclic AMP; Fatty Acids, Unsaturated; Glucuronidase; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Leukotriene C4; Lysosomes; Muramidase; Neutrophils; Plant Roots; Plants, Medicinal; Resveratrol; Stilbenes; Structure-Activity Relationship

Cells were incubated in the presence of the Ca2+ ionophore A23187 (10 microM) and arachidonic acid (AA, 80 microM). The release of eicosanoids from subcultivated cardiac endothelial and fibroblast-like cells amounted to 23.3 +/- 4.5 and 2.0 +/- 0.4 nmol/mg cellular protein per 30 min, respectively. The release from isolated cardiomyocytes remained below the detection limit of the high-performance liquid chromatography assay (< 0.00015 nmol/assay). When a very sensitive radioimmunoassay was applied, cardiomyocytes released 0.002 +/- 0.0001 nmol prostacyclin per milligram cellular protein per 30 min. Prostaglandin (PG) E2 and PGF2 alpha, 12-hydroxyheptadecatrienoic acid, 11- and 15-hydroxyeicosatetraenoic acid, and thromboxane B2 were the main eicosanoids released by endothelial cells. The stable product of prostacyclin, 6-keto-PGF1 alpha, contributed relatively little to the total amount of eicosanoids formed by endothelial cells. Fibroblast-like cells released predominantly PGE2 and 6-keto-PGF1 alpha and, to a lesser extent, 12-hydroxyheptadecatrienoic and 15-hydroxyeicosatetraenoic acids. Neither endothelial cells nor fibroblast-like cells released leukotrienes. A23187 stimulated eicosanoid release from endothelial cells when exogenous AA was below 40 microM. Addition of albumin reduced the amount of eicosanoids produced. Histamine and bradykinin did not influence 6-keto-PGF1 alpha and PGE2 production in cardiomyocytes. Histamine only gave rise to a slight but significantly higher release of 6-keto-PGF1 alpha in endothelial cells.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Arachidonic Acid; Calcimycin; Cells, Cultured; Dinoprost; Dinoprostone; Endothelium; Fatty Acids, Unsaturated; Fibroblasts; Heart; Hydroxyeicosatetraenoic Acids; Indomethacin; Male; Microscopy, Electron; Myocardium; Rats; Rats, Inbred Lew; Rats, Wistar

The synthesis of 14C labelled arachidonic acid metabolites was measured in colonic tissues obtained from mice, rats, guinea pigs, rabbits, piglets and in colonic biopsies from humans during colonoscopy. The main eicosanoids formed after stimulation with calcium ionophore A23187 were: in humans, 15-hydroxy-eicosatetraenoic acid (15-HETE); in mice, 12-HETE; in rats, 12-HETE, 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 6-keto-prostaglandin F1 alpha (6kPGF1 alpha); in guinea pigs, PGD2; in rabbits, 6kPGF1 alpha, PGE2 and 15-HETE; and in pigs PGE2 and 12-HETE. In inflamed 15-HETE production was increased in man, HHT and 12-HETE production in rats and overall eicosanoid production in mice.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 6-Ketoprostaglandin F1 alpha; Animals; Calcimycin; Colitis, Ulcerative; Colon; Dinoprostone; Eicosanoids; Fatty Acids, Unsaturated; Guinea Pigs; Humans; Hydroxyeicosatetraenoic Acids; Intestinal Mucosa; Mice; Mice, Inbred BALB C; Prostaglandin D2; Rabbits; Rats; Rats, Wistar; Species Specificity

Stimulation of platelets induces a rapid release of arachidonate from specific phospholipids and subsequent remodeling of arachidonate-containing phospholipids. This process is accompanied by transformation of released arachidonate by cyclooxygenase and lipoxygenase enzymes. We addressed the question of whether the cyclooxygenase and the lipoxygenase products originated from the same arachidonate-containing phospholipids. [14C]Arachidonate prelabeled platelets were stimulated by thrombin or by ionophore A 23187. We monitored the cyclooxygenase pathway by following 12-hydroxy-5,8,10-heptadecatrienoic acid [12(S)-HHT] formation and the lipoxygenase pathway by following 12-hydroxy-5,8,10,14-eicosatetraenoic acid [12(S)-HETE] formation and compared specific activities. The data showed that the same pool of released arachidonate can be utilized by either cyclooxygenase or by lipoxygenase. Indeed, the specific activity of both products was identical when both enzymes were acting. Since cyclooxygenase was rapidly deactivated while lipoxygenase continued to be active, the specific activity of 12(S)-HETE became lower than the specific activity of 12(S)-HHT when large amounts of 12(S)-HETE were synthesized. Based on comparison of specific activity between phospholipids and oxygenated products, the pools of arachidonate-containing phospholipids involved in the synthesis of oxygenated products are dependent on the amount of arachidonate released.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acids; Blood Platelets; Calcimycin; Chromatography, High Pressure Liquid; Fatty Acids, Unsaturated; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Kinetics; Lipoxygenase; Prostaglandin-Endoperoxide Synthases; Rats; Thrombin

The in vitro release of arachidonic acid (AA) metabolites from caprine alveolar macrophages (CAM) stimulated with the calcium ionophore A23187 or opsonized zymosan was examined. Leukotriene B4 [5(S),12(R)-6,14-cis-8,10-trans-dihydroxyeicosatetraenoic acid] was the major AA metabolite elicited with either agonist; smaller amounts of 12- and 5-mono-hydroxyeicosatetraenoic acid (HETE), and 12-hydroxyheptadecatrienoic acid (HHT) were also detected. Zymosan stimulation also caused the release of very small quantities of prostaglandins E2 and F2 alpha, and thromboxane B2. Our report is the first to describe arachidonic acid metabolism in CAM.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Bronchoalveolar Lavage Fluid; Calcimycin; Fatty Acids, Unsaturated; Goats; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lipid Metabolism; Macrophages; Zymosan

In previous studies on arachidonic acid (AA) metabolism by pulmonary macrophages in vitro, we observed that the presence of serum in the culture medium influenced the profile of AA metabolites released. To further characterize this phenomenon, rat alveolar macrophages were placed in plastic tissue culture dishes and allowed to adhere in the presence or absence of 7.5% fetal bovine serum (FBS) for 1 h. Adherent cells were then maintained in medium (equilibration) with or without FBS for 3.5 h before stimulation with the calcium ionophore A23187. The release of thromboxane B2 (TXB2) (the stable metabolite of TXA2) and leukotriene B4 (LTB4) during culture was measured by radioimmunoassay and reverse-phase high pressure liquid chromatography, respectively, at the end of each culture step. Cell adhesion to the plastic substrate in FBS-free medium induced an intense stimulation of AA metabolism, with the release of both TXB2 and LTB4. Adhesion and the accompanying TXB2 release appear to be mediated by trypsin-sensitive components since trypsin-pretreated macrophages showed a dramatic reduction in both adherence and TXB2 synthesis. The presence of FBS during the attachment phase of culture reduced both adhesion and release of TXB2 and LTB4 by more than 50%. On the other hand, addition of FBS to cells that had completed adhesion in serum-free medium stimulated release of both metabolites. When challenged with calcium ionophore after 4.5 h of culture, macrophages that had adhered in FBS-free medium released a much smaller amount of TXB2 than did macrophages that had been cultured in the presence of FBS.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Blood; Calcimycin; Cattle; Cell Adhesion; Cells, Cultured; Fatty Acids, Unsaturated; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Macrophages; Plastics; Pulmonary Alveoli; Rats; Thromboxane B2

12-Hydroxyheptadecatrienoic acid (HHT), a UV chromophore, has been used to assess cyclooxygenase (CO) pathway metabolism of arachidonic acid (AA) by rat peritoneal cells. Simultaneous monitoring at 235 and 280 nm after HPLC permits the measurement of both CO and 5-lipoxygenase (5-LO) pathway fluxes in a single system.

Topics: Animals; Arachidonate 5-Lipoxygenase; Arachidonic Acids; Ascitic Fluid; Calcimycin; Chromatography, High Pressure Liquid; Cyclooxygenase Inhibitors; Eosinophils; Fatty Acids, Unsaturated; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lipoxygenase Inhibitors; Macrophages; Male; Mast Cells; Prostaglandin Antagonists; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Inbred Strains; Reference Standards; Spectrophotometry, Ultraviolet

Washed human platelets prelabeled with [14C]arachidonic acid and then exposed to the Ca2+ ionophore A23187 mobilized [14C]arachidonic acid from phospholipids and formed 14C-labeled thromboxane B2, 12-hydroxy-5-8,10-heptadecatrienoic acid, and 12-hydroxy-5,8,10,14-eicosatetraenoic acid. Addition of phorbol myristate acetate (PMA) by itself at concentrations from 10 to 1000 ng/ml did not release arachidonic acid or cause the formation of any of its metabolites, nor did it affect the metabolism of exogenously added arachidonic acid. When 1 microM A23187 was added to platelets pretreated with 100 ng of PMA/ml for 10 min, the release of arachidonic acid, and the amount of all arachidonic acid metabolites formed, were greatly increased (average 4.1 +/- 0.5-fold in eight experiments). This effect of PMA was mimicked by other stimulators of protein kinase C, such as phorbol dibutyrate and oleoyl acetoyl glycerol, but not by 4-alpha-phorbol 12,13-didecanoate, which does not stimulate protein kinase C. However, phosphorylation of the cytosolic 47-kDa protein, the major substrate for protein kinase C in platelets, was produced at lower concentrations of PMA and at a much higher rate than enhancement of arachidonic acid release by PMA, suggesting that 47-kDa protein phosphorylation is not directly involved in mobilization of the fatty acid. PMA also potentiated arachidonic acid release when stimulation of phospholipase C by the ionophore (which is due to thromboxane A2 and/or secreted ADP) was blocked by aspirin plus ADP scavengers, i.e. apyrase or creatine phosphate/creatine phosphokinase. Increased release of arachidonic acid was attributable to loss of [14C]arachidonic acid primarily from phosphatidylcholine (79%) with lesser amounts derived from phosphatidylinositol (12%) and phosphatidylethanolamine (8%). Phosphatidic acid, whose production is a sensitive indicator of phospholipase C activation, was not formed. Thus, the potentiation of arachidonic acid release by PMA appeared to be due to phospholipase A2 activity. These results suggest that diacylglycerol formed in response to stimulation of platelet receptors by agonists may cooperatively promote release of arachidonic acid via a Ca2+/phospholipase A2-dependent pathway.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acid; Arachidonic Acids; Blood Platelets; Calcimycin; Diglycerides; Enzyme Activation; Fatty Acids, Unsaturated; Glycerides; Humans; Hydroxyeicosatetraenoic Acids; Kinetics; Phorbols; Phospholipases A; Phospholipases A2; Phospholipids; Phosphoproteins; Protein Kinase C; Tetradecanoylphorbol Acetate; Thromboxane B2

Rat neutrophils isolated from three-hour carrageenan pleural exudates actively metabolize arachidonic acid into three major metabolites, HHT, 11-HETE and 15-HETE. However, in the presence of the calcium ionophore, A23187, or the non-ionic detergent, BRIJ 56, these cells also produce 5-HETE and LTB. The production of these lipoxygenase products is calcium dependent. While non-steroidal anti-inflammatory drugs do not affect 5-HETE or LTB production, BW 755C and ETYA inhibit formation of these metabolites from exogenously added arachidonic acid.

Topics: Animals; Arachidonic Acids; Calcimycin; Carrageenan; Cetomacrogol; Exudates and Transudates; Fatty Acids, Unsaturated; Hydroxy Acids; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lipoxygenase; Male; Neutrophils; Pleura; Rats