David C. Henke

Metabolism of arachidonic acid by human nasal and bronchial epithelial cells

Nasal and bronchial epithelium from normal human nasal turbinates was isolated from surgical specimens and used to study arachidonic acid metabolism. High-performance liquid chromatography analysis of cell incubations in the presence of calcium ionophore, A23187, showed the formation of 15-lipoxygenase products. The major arachidonic acid metabolite with bronchial and nasal tissue was 15-HETE identified by uv spectroscopy, coelution with the authentic standards by HPLC, and GC-mass spectrometry. The second major metabolite, formed from either arachidonic acid or 15-HPETE, was identified as 13-hydroxy-14,15-epoxy-5,8,11-eicosatetraenoic acid (15-alpha-HEPA) by uv spectroscopy, coelution with the authentic standard, and GC-mass spectrometry. In addition, two 8,15-diHETEs and two 8,15-LTs were identified by uv spectroscopy and coelution with the authentic standards by HPLC on both reverse-phase and normal-phase HPLC. Also isolated and identified were 14,15-diHETEs, and 12-HETE. Nasal epithelial cells appear to be more active than nasal bronchial cells in oxidizing arachidonic acid. However, the profile of metabolites from these normal tissue preparations was similar. The addition of 15-lipoxygenase products to nasal epithelium weakly stimulated Cl- ion secretion. These studies indicate that human pulmonary epithelial cells selectively oxidize arachidonic acid to 15-lipoxygenase metabolites.

Diphenylamine-2-carboxylate (DPC) inhibits both Cl- conductance and cyclooxygenase of canine tracheal epithelium

Diphenylamine-2-carboxylate (DPC) decreases Cl− conductance (GCl) in epithelia and cells of several tissues, an effect which has been ascribed to blockade of conductive Cl− channels. However, one DPC derivative, flufenamic acid, is a clinically useful non-steroidal antiinflammatory agent, the mechanism of action of which involves the blockade of arachidonic acid metabolism by cyclooxygenase. BecauseGCl in canine tracheal epithelium is stimulated by exogenous prostaglandins and induction of cyclooxygenase activity, we tested the hypothesi that DPC inhibits dog tracheal epitheliumGCl through inhibition of cyclooxygenase. DPC inhibited the short circuit current of amiloride-pretreated tissues by 50% at 0.138 mmol/l and by more than 95% at 3 mmol/l. Isoproterenol reversed the inhibition seen at 0.1 mmol/l DPC and stimulated current above control (indomethacin-pretreated) levels. Higher concentrations of DPC diminished the stimulation of current by subsequent exposure to isoproterenol, such that there was little effect of isoproterenol in the presence of 3 mmol/l DPC. DPC, 0.1 mmol/l, also blocked stimulation of current by exogenous arachidonic acid, but not of exogenous prostaglandins PGE or PGD. The metabolism of3H-arachidonic acid to3H-PGD2, monitored by HPLC, was completely blocked by 0.1 mmol/l DPC. We conclude that the isoproterenol/prostaglandin reversible blockade ofGCl by DPC can be attributed to inhibition of arachidonic acid metabolism.

High-performance liquid chromatography separation of phospholipid classes and arachidonic acid on cyanopropyl columns

An HPLC method for the separation and analysis of arachidonic acid and eight phospholipid classes is described: phosphatidylglycerol, phosphatidylinositol, cardiolipin, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, and 2-lysophosphatidylcholine. The separation is carried out at 60 degrees C on 2 cyanopropyl columns using a gradient of acetonitrile and 5 mM sodium acetate (pH 5.0). Cyanopropyl columns require a lower proportion of water in the mobile phase to elute the more polar phospholipids than other types of columns and are thus less prone to equilibration problems. The method is highly reproducible (average coefficient of variation for each retention time less than or equal to 3.5%) and permits analysis of peaks by phosphorus content. Data obtained by analyzing lipid extracts from rat alveolar macrophages prelabeled with [G-3H]-arachidonic acid were analyzed by this HPLC method and compared to standard analysis by TLC. There was a significant correlation between the radioactivity profiles obtained with the two chromatographic methods (HPLC versus TLC) by linear regression analysis [HPLC = 0.83 (TLC) + 3.58, n = 25, r = 0.95, P less than 0.001].

Arachidonic acid metabolism in respiratory epithelial cells

1. Arachidonic acid metabolism by respiratory epithelial cells either freshly isolated or maintained in culture is discussed. 2. A comparison is made between rat, rabbit, canine and human cells and illustrates the considerable variation in the metabolism between various species. 3. The relationship between arachidonic acid metabolism and ion movement in these cells is reviewed.

Endothelial cells can synthesize leukotriene B4

Leukotriene B4 (LTB4) from vascular endothelium may play a key role in the genesis of atherosclerotic lesions. However, the ability of this tissue to synthesize LTB4 is controversial. To resolve this issue arachidonic acid metabolism was characterized in cultures of confluent monolayers of a rabbit aortic endothelial cell line by use of both high-pressure liquid chromatography and radioimmunoassay. Cells were grown to confluence in Dulbeccos modified Eagles medium/Hams F12 with 5% fetal bovine serum. Lipoxygenase activity was studied by placing the cells in Hanks balanced salt solution with 2 mumol/L indomethacin. After 30 minutes preincubation with indomethacin cells were exposed to either arachidonic acid (10 mumol/L) or arachidonic acid labeled with radioactive carbon (14C) (1 microCi; SA 58 mCi/mmol) and then stimulated with 9.5 mumol/L calcium ionophore A23187 for 55 minutes. Studies of the cyclooxygenase activity were performed without preincubating with indomethacin. Samples were prepared for high-pressure liquid chromatography by evaporation to dryness under a vacuum and resuspending in 2 ml of 1:1 methanol/water. Tritium-labeled standards were added before loading the 14C-labeled samples on the column. Radiolabeled arachidonic acid metabolites were separated by high-pressure liquid chromatography and detected by means of a dual channel flow-through radiodetector that monitors both 14C and 3H. Based on coelution with authentic standards three lipoxygenase metabolites of arachidonic acid have been identified: LTB4, 12- and 5-hydroxyeicosatetraneoic acid. Leukotriene B4 was further characterized by ultraviolet spectral analysis and inhibition studies with use of nordihydroguaiaretic acid. Quantitation was facilitated by commercially available radioimmunoassay kits. An average of 600 pg LTB4/10(6) cells was measured from separate experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

Macrolide Use Leads to Clinical and Radiological Improvement in Patients with Cryptogenic Organizing Pneumonia

Cryptogenic organizing pneumonia is an idiopathic form of organizing pneumonia (also known as bronchiolitis obliterans organizing pneumonia). Because cryptogenic organizing pneumonia is considered an inflammatory disease, it characteristically responds to the broad-spectrum antiinflammatory corticosteroids, although relapse is common on discontinued use. Additionally, long-term use of corticosteroids has many side effects. In severe cases in which corticosteroids have failed, either cytotoxic therapy or macrolide therapy is used. Because of the toxicity and adverse effects of cytotoxic therapy (e.g., cyclophosphamide), this therapy option cannot be used long term in refractory cases. Macrolide therapy has been shown to be an effective antiinflammatory agent that is relatively safe when used on a long-term basis in patients with cryptogenic organizing pneumonia.