Small bowel mucosa from celiac patients generates 15-hydroxyeicosatetraenoic acid (15-HETE) after in vitro challenge with gluten

Celiac disease (gluten-sensitive enteropathy [GSE]) is a disorder characterized by small intestinal mucosal injury caused by dietary exposure to wheat gluten and similar proteins. There is evidence that the mucosal injury is immunologically mediated and there is an inflammatory infiltrate present in the mucosa. It is postulated that release of lipid-derived inflammatory mediators may be involved in the pathogenesis of the mucosal injury. Jejunal mucosal biopsy samples from patients with GSE and from a group of patients who were subsequently shown to have normal jejunal mucosa were incubated with tritiated arachidonate and a peptic/tryptic digest of either gluten or casein. Generation of lipid-derived inflammatory mediators was measured by beta-scintillation counting after separation of metabolites by reverse-phase high performance liquid chromatography with two different buffer systems. The predominant arachidonic acid metabolite generated was 15-hydroxyeicosatetraenoic acid (15-HETE). Mucosa from newly diagnosed GSE patients on a normal diet generated more 15-HETE than either control patients or GSE patients maintained on a gluten-free diet. In addition, gluten acted as a specific stimulus to 15-HETE production by mucosa from the GSE patients on a normal diet. 15-HETE has a number of biologic effects that could contribute to the mucosal changes seen in GSE, and the specific release of 15-HETE by gluten suggests involvement in the pathogenesis of the disorder.     

20-hydroxyeicosatetraenoic acid (20-HETE) metabolism in coronary endothelial cells

We have investigated the role of endothelial cells in the metabolism of 20-hydroxyeicosatetraenoic acid (20-HETE), a vasoactive mediator synthesized from arachidonic acid by cytochrome P450 omega-oxidases. Porcine coronary artery endothelial cells (PCEC) incorporated 20-[(3)H]HETE primarily into the sn-2 position of phospholipids through a coenzyme A-dependent process. The incorporation was reduced by equimolar amounts of arachidonic, eicosapentaenoic or 8,9-epoxyeicosatrienoic acids, but some uptake persisted even when a 10-fold excess of arachidonic acid was available. The retention of 20-[(3)H]HETE increased substantially when methyl arachidonoyl fluorophosphonate, but not bromoenol lactone, was added, suggesting that a Ca(2+)-dependent cytosolic phospholipase A(2) released the 20-HETE contained in PCEC phospholipids. Addition of calcium ionophore A23187 produced a rapid release of 20-[(3)H]HETE from the PCEC, a finding that also is consistent with a Ca(2+)-dependent mobilization process. PCEC also converted 20-[(3)H]HETE to 20-carboxy-arachidonic acid (20-COOH-AA) and 18-, 16-, and 14-carbon beta-oxidation products. 20-COOH-AA produced vasodilation in porcine coronary arterioles, but 20-HETE was inactive. These results suggest that the incorporation of 20-HETE and its subsequent conversion to 20-COOH-AA in the endothelium may be important in modulating coronary vascular function.     

Atrial reflexes during high frequency oscillatory ventilation

High frequency oscillatory ventilation (HFOV) is a new method of artificial ventilation which has been advocated for use in critically ill individuals. It alters the discharge in pulmonary stretch receptors (SAR) from a phasic to a continuous pattern. Since some cardiovascular neurones in the medulla are influenced by the discharge from SAR, experiments were undertaken to determine whether the reflexes from the left atrial (volume) receptors (LAR) were influenced by HFOV. The reflex increases in heart rate and urine flow which result from activation of the (LAR) were examined during both intermittent positive pressure ventilation (IPPV) and HFOV. In five dogs, the increase in heart rate was 23.9 +/- 4.3 and 24.5 +/- 5.4 beats/min during IPPV and HFOV, respectively. In six dogs the response of an increase in urine flow was examined and this response also was not altered by HFOV. It is concluded that the integrity of these reflexes was unaffected by HFOV in the anesthetized dog model.     

15(S)-hydroxyeicosatetraenoic acid (15-HETE), a product of arachidonic acid peroxidation, is an active component of hemozoin toxicity to monocytes

Several studies have shown that human and murine hemozoin-fed phagocytes are functionally impaired. Unpurified hemozoin contains unspecifically attached unsaturated fatty acids such as arachidonic and linolenic acids. The presence in unpurified hemozoin of large quantities of ferric heme with small amounts of free iron makes hemozoin a generator of oxidative radicals capable of forming lipoperoxides or other breakdown products from polyunsaturated fatty acids. Here we show that delipidized hemozoin had reduced toxicity to monocytes. Phorbol myristate acetate (PMA)-elicited burst was poorly affected by delipidized hemozoin (ca. 17% and 21% burst inhibition by delipidized hemozoin vs ca. 75% and 65% burst inhibition by native hemozoin at 20 min or 17 h post-phagocytosis, respectively). Analysis of the lipid fraction isolated from native hemozoin by HPLC and chiral-phase HPLC showed equimolar amounts of 15(R)- and 15(S)-HETE (HETE, 15-hydroxy-6,8,11,13-eicosatetraenoic acid), most likely by-products of non-enzymatic peroxidation of arachidonic acid. The biologically active isomer, 15(S)-HETE, the product of 15-lipoxygenase, is a powerful mediator of inflammation and the effector of a large number of bioactions. 15(R,S)-HETE was found in native hemozoin (0.24 millimole/mole hemozoin heme), in supernatants of hemozoin-fed monocytes (87 nMol) and in hemozoin-fed monocytes (9.6 microMol). Approximately 84% of 15-HETE attached to hemozoin was in the esterified form. A large preponderance of esterified over free 15-HETE was also noted in supernatants of hemozoin-fed monocytes and in hemozoin-fed monocytes. In the latter cells, remarkable levels of the substance were attained. A dose-dependent curve of inhibition of PMA-elicited oxidative burst was observed. Assuming homogenuous distribution of 15-HETE in hemozoin-fed monocytes, 15(S)-HETE concentrations measured in hemozoin-fed monocytes (8 muMol) would bring about ca. 85% inhibition of PMA-elicited burst. In conclusion, derivatives of lipoperoxidation of unsaturated fatty acids such as 4-hydroxynonenal, 15-HETE and others now under study, appear to be relevant causes of hemozoin toxicity.     

20-hydroxyeicosatetraenoic acid (20-HETE): structural determinants for renal vasoconstriction

The effects of natural and synthetic eicosanoids on the diameter of rat interlobular arteries studied in vitro were compared to that of the potent, endogenous vasoconstrictor 20-HETE. Vasoconstrictor activity was optimum for chain lengths of 20-22 carbons with at least one olefin or epoxide between located between C(13)-C(15) and an oxygen substituent at C(20)-C(22). The presence of delta (Zou et al. Am. J. Physiol. 1996, 270, R228; Gebremedhin, D. et al. Am. J. Physiol. 1998, 507, 771)-, delta (Carroll et al. Am. J. Physiol. 1996, 271, R863; Vazquez et al. Life Sci. 1995, 56, 1455)-, or delta (Imig et al. Hypertension 2000, 35, 307; Lopez et al. Amer. J. Physiol. 2001, 281, F420)-olefins had no influence on the vasoconstrictor response whereas the introduction of a C(7)-thiomethylene enhanced potency. A sulfonamide or alcohol, but not a lactone, could replace the C(1)-carboxylate. These data were used to construct a putative binding domain map of the 20-HETE receptor consisting of: (i) a comparatively open, hydrophilic binding site accommodating the C(1)-functionality; (ii) a hydrophobic trough spanning the olefins; (iii) a shallow pocket containing a critical pi-pi binding site in the vicinity of the pi (Ito et al. Am. J. Physiol. 1998, 274, F395; Quigley, R.; Baum, M.; Reddy, K. M.; Griener, J. C.; Falck, J. R. Am. J. Physiol. 2000, 278, F949)-olefin; and (iv) an oxyphilic binding site proximate to the omega-terminus.     

Enhanced esterification process of 5-hydroxyeicosatetraenoic acid (5-HETE) in PMN from asthmatic patients

Polymorphonuclear neutrophils (PMN) generate 5-HETE which can be retained within cells as free metabolites or esterified into cellular lipids. Since this metabolite has been shown to have certain inflammatory porperties, we compared the generation and distribution profile of 5-HETE in A 23187-stimulated PMN from asthmatic patients (AP) and normal subjects (NS). 5-HETE was analyzed using RP-HPLC. After 5 min , total 5 HETE generation was similar in the two populations. However, esterified 5-HETE was significantly enhanced in AP (72 ± 3 % versus 47 ± 2 % of the total synthesis, p < 0.005), whereas intracellular free 5-HETE was decreased (13 ± 3 % versus 37 ± 4 %, p < 0.005) and similar low release was observed. Kinetic studies showed that PMN from AP esterified 5-HETE more rapidly and to a greater extent than PMN from NS. By contrast, more intracellular free 5-HETE was recovered in PMN from NS. Esterification seems to be the major pathway of 5-HETE metabolism in PMN from AP. Moreover, we showed that most of the 5-HETE added exogenously was esterified into cellular lipids. In these experimental conditions, PAF-induced migration of PMN was increased. The enhanced ability of PMN to migrate could be due to the increase of 5-HETE esterification process.     

Regulation of T-lymphocyte mitogenesis by the leukocyte product 15-hydroxy-eicosatetraenoic acid (15-HETE)

Synthesis of lipoxygenase metabolites of [¹⁴C]arachidonic acid by mouse spleen lymphocyte cultures was inhibited by the leukocyte product 15-hydroxy-eicosatetraenoic acid (15-HETE) in a dose-dependent manner. In parallel experiments, the influence of 15-HETE on mitogenesis in spleen lymphocyte cultures was examined. 15-HETE at concentrations similar to those which inhibited cellular lipoxygenases progressively inhibited mitogenesis induced by the T-cell mitogen PHA but had no significant effect on the mitogenic response to the B-cell mitogen LPS. The inhibitory response was maximal when 15-HETE was added within 8 hr of exposure to PHA. Several analogs of 15-HETE having progressively fewer double bonds were tested in the same systems. 15-OH,20:3 had approximately the same potency as 15-HETE in inhibiting both mitogenesis and formation of metabolites from [¹⁴C]arachidonic acid. 15-OH, 20:2 and 15-OH,20:0 were much less active in either assay. Mitogenesis, induced in spleen cell cultures by the tumor promoter phorbol myristate acetate, was also blocked by 15-HETE. These experiments indicate that lipoxygenase metabolites of arachidonic acid may play an important role in T-lymphocyte blastogenesis and suggest that 15-HETE, via its ability to selectively inhibit cellular lipoxygenases, may function as an endogenous regulator of T-lymphocyte responses.     

Enhanced esterification process of 5-hydroxyeicosatetraenoic acid (5-HETE) in PMN from asthmatic patients.

Polymorphonuclear neutrophils (PMN) generate 5-HETE which can be retained within cells as free metabolites or esterified into cellular lipids. Since this metabolite has been shown to have certain inflammatory properties, we compared the generation and distribution profile of 5-HETE in A 23187-stimulated PMN from asthmatic patients (AP) and normal subjects (NS). 5-HETE was analyzed using RP-HPLC. After 5 min, total 5 HETE generation was similar in the two populations. However, esterified 5-HETE was significantly enhanced in AP (72 +/- 3% versus 47 +/- 2% of the total synthesis, p less than 0.005), whereas intracellular free 5-HETE was decreased (13 +/- 3% versus 37 +/- 4%, p less than 0.005) and similar low release was observed. Kinetic studies showed that PMN from AP esterified 5-HETE more rapidly and to a greater extent than PMN from NS. By contrast, more intracellular free 5-HETE was recovered in PMN from NS. Esterification seems to be the major pathway of 5-HETE metabolism in PMN from AP. Moreover, we showed that most of the 5-HETE added exogenously was esterified into cellular lipids. In these experimental conditions, PAF-induced migration of PMN was increased. The enhanced ability of PMN to migrate could be due to the increase of 5-HETE esterification process.     

Regulation of 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) binding sites on human epidermal cells by interferon-gamma

We recently detected specific high-affinity binding sites for 12(S)-HETE, the main arachidonic acid metabolite in skin, on epidermal cells. The putative receptor is involved in keratinocyte chemotaxis toward 12(S)-HETE, which points to its participation in wound healing. In an effort to further characterize the 12(S)-HETE receptor, we investigated its regulation by various cytokines. Of the tested cytokines, only interferon (IFN)-gamma led to a massive induction of the 12(S)-HETE receptors. The effect was dose and time dependent and blocked by cycloheximide. The up-regulation of 12(S)-HETE receptors by IFN-gamma may represent an amplification mechanism of the assumed role of 12(S)-HETE in skin wound repair.     

15-Hydroxy-eicosatetraenoic acid (15-HETE) inhibits carrageenan-induced experimental arthritis and reduces synovial fluid leukotriene B4 (LTB4)

15-Hydroxy-eicosatetraenoic acid (15-HETE), a product of arachidonic acid, has no proinflammatory capacity, but can inhibit the formation and the chemotactic response of neutrophils to leukotriene B4 (LTB4), a potent mediator of inflammation. The purpose of the present study was to determine whether intraarticular administration of 15-HETE in carrageenan-induced acute arthritis might decrease the levels of LTB4 in synovial fluid and modify the arthritis. A bilateral acute knee joint arthritis was established in 7 dogs by intraarticular injections of carrageenan every third day. To the right joints, 15-HETE was administered both concomitantly with the carrageenan injections and continuously via an osmotic pump. In samples of synovial fluid obtained on day 0, 3 and 10 PGE2 and LTB4 were determined using reversed phase high performance liquid chromatography combined with radioimmunoassays and neutrophil chemokinesis. In the presence of 15-HETE the clinical severity of arthritis was significantly reduced and the volume of synovial effusate was decreased on an average by 42%. Furthermore, the relative number of neutrophils in histological sections of synovial tissue was decreased by 58%. Intraarticular caragheenan injections induced LTB4 formation, and maximum levels were obtained on day 3 (279.2 +/- 148.2 pg/joint). PGE2 was also present on day 3, but maximum levels were detected on day 10 (9.5 +/- 4.8 ng/joint). In joints injected with both carragheenan and 15-HETE the levels of LTB4 on days 3 and 10 were inhibited by 90% and 83%, respectively. For PGE2 a small but insignificant decrease was found on both day 3 and on day 10. These results show that LTB4 may be an important mediator of acute arthritis induced by carragheenan in dogs, and that intraarticular administration of 15-HETE can modify this arthritis by inhibiting LTB4 formation.     

Evidence for pulmonary release of 5-hydroxyeicosatetraenoic acid (5-HETE) during endotoxemia in unanesthetized sheep

Leukocyte trapping in the pulmonary circulation may be an important component of the lung vascular injury response to endotoxin, but mediators of the pulmonary leukostasis and increased lung vascular permeability are unknown. The leukocyte 5-lipoxygenation pathway of arachidonic acid metabolism yields highly biologically active products including leukotrienes C₄ and D₄ (formerly slow reacting substance of anaphylaxis) and the potent chemotaxin, leukotriene B₄. A major product of 5-lipoxygenation is 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), for which a sensitive, stable isotope dilution assay employing combined gas chromatography-mass spectrometry is available. This assay was used to test the hypothesis that 5-lipoxygenation products might participate in pulmonary vascular responses to endotoxin. We measured 5-HETE concentrations in lung lymph at three intervals during endotoxemia in unanesthetized sheep. Concentrations of 5-HETE in lung lymph exceeded those in aortic blood plasma. Lymph 5-HETE concentrations increased from 1.7±0.3 (mean ± SEM, N = 7) ng/ml during baseline to peak values of 6.1±1.8 ng/ml (p < 0.05) during the hours after endotoxemia and preceeding the steady state increased lung vascular permeability response. During the increased permeability steady state from 240 to 270 minutes after endotoxin, lymph 5-HETE concentrations (1.4±0.3 ng/ml) and lymph 5-HETE flow (i.e., 5-HETE concentration x lung lynph flow rate) returned to baseline values. Although these observations are consistent with the hypothesis that 5-lipoxygenation products participate in the pulmonary vascular injury response to endotoxin, lymph 5-HETE concentrations did not correlate with any of the other experimental measurements. It may be only coincidence that the increase in lymph 5-HETE concentrations appeared contemporaneous with the onset of lung vascular injury.     

20-hydroxyeicosatetraenoic acid (20-HETE) activates mouse TRPC6 channels expressed in HEK293 cells

In the present study, we show that the eicosanoid compound, 20-hydroxyeicosatetraenoic acid (20-HETE), an important arachidonic acid metabolite, activates mouse TRPC6 in a stable, overexpressing HEK293 cell line, Hek-t6.11. Application of 20-HETE rapidly induced an inward, non-selective current in whole-cell recordings, which was inhibited by N-methyl-d-glucamine, 1.8 mm Ca2+, and 100 microM Gd3+ but remained unaffected by flufenamate and indomethacin. The current-voltage relationship obtained at low concentrations of 20-HETE (1-10 microM) demonstrated slight inward rectification, whereas the highest concentration of 20-HETE tested (30 microM) showed outward rectification, as shown previously for these channels using 100 microM 1-oleoyl-2-acetyl-sn-glycerol. Dose-response curves indicate that 20-HETE activated TRPC6 channels with an EC50 = 0.8 microM. Single channel analysis using inside-out patches revealed that 20-HETE increased open probability of mouse TRPC6 channels approximately 3-fold, and this was in a membrane-delimited fashion. Interestingly, 20-HETE did not provoke changes in intracellular Ca2+ concentrations. Thus, we have identified an arachidonic acid metabolite, 20-HETE, as a novel activator for a TRP family member, TRPC6.     

Omega-oxidation of 20-hydroxyeicosatetraenoic acid (20-HETE) in cerebral microvascular smooth muscle and endothelium by alcohol dehydrogenase 4

20-Carboxyeicosatetraenoic acid (20-COOH-AA) is a bioactive metabolite of 20-hydroxyeicosatetraenoic acid (20-HETE), an eicosanoid that produces vasoconstriction in the cerebral circulation. We found that smooth muscle (MSMC) and endothelial (MEC) cultures obtained from mouse brain microvessels convert [3H]20-HETE to 20-COOH-AA, indicating that the cerebral vasculature can produce this metabolite. The [3H]20-COOH-AA accumulated primarily in the culture medium, together with additional radiolabeled metabolites identified as the chain-shortened dicarboxylic acids 18-COOH-18:4, 18-COOH-18:3, and 16-COOH-16:3. N-Heptylformamide, a potent inhibitor of alcohol dehydrogenase (ADH), decreased the conversion of [3H]20-HETE to 20-COOH-AA by the MSMC and MEC and also by isolated mouse brain microvessels. Purified mouse and human ADH4, human ADH3, and horse liver ADH1 efficiently oxidized 20-HETE, and ADH4 and ADH3 were detected in MSMC and MEC by Western blotting. N-Heptylformamide inhibited the oxidation of 20-HETE by mouse and human ADH4 but not by ADH3. These results demonstrated that cerebral microvessels convert 20-HETE to 20-COOH-AA and that ADH catalyzes the reaction. Although ADH4 and ADH3 are expressed in MSMC and MEC, the inhibition produced by N-heptylformamide suggests that ADH4 is primarily responsible for 20-COOH-AA formation in the cerebral microvasculature.     

Specific enhancement of LTD4-induced contractions of isolated guinea pig trachea by 5-hydroxyeicosatetraenoic acid (5-HETE)

In view of the likely production of monohydroxyeicosatetraenoic acids (HETEs) in bronchial asthma, the role of these lipoxygenase products in the development of a classical clinical element of airway disease, namely airway hyperreactivity, has been investigated. Tracheas removed from guinea-pigs actively sensitized to ovalbumin produced, upon antigenic challenge (0.01 microgram/ml), a 17-fold increase (0.97 +/- 0.34 ng/ml to 16.73 +/- 1.58 ng/ml) in the amount of 5-hydroxyeicosatetraenoic acid (5-HETE) as measured by radioimmunoassay of the tissue-bath fluid, indicating that this tissue is capable of producing 5-HETE. While 5-HETE alone, at concentrations equal to or greater than those found during the above antigenic response (0.001 to 1.0 microM), failed to produce intrinsic contractions of normal, nonsensitized guinea-pig trachea, a 30 min pretreatment with 5-HETE (1.0 microM) enhanced subsequent LTD4-induced contractions. Pretreatment with either 12- or 15-HETE, at similar concentrations and conditions, failed to potentiate LTD4 concentration-response curves. The effect of 5-HETE was time-dependent, since pretreatment for either 15 or 60 min had little or no effect on subsequent LTD4 responses. Also, the 5-HETE-induced enhancement seemed specific for LTD4, since contractions to LTC4 (in the presence of I-serine borate), acetylcholine, histamine, PGD2 or U-46619 were unaffected by 5-HETE. Therefore, 5-HETE may have a role in the development of airway hyperreactivity by interacting with released LTD4 to exacerbate airway smooth muscle contraction in asthma.     

Formation of 11-hydroxyeicosatetraenoic acid and 15-hydroxyeicosatetraenoic acid in human umbilical arteries is catalyzed by cyclooxygenase

Human umbilical arteries convert arachidonic acid into three hydroxy-eicosatetraenoic acids as well as 6-ketoprostaglandin F1 alpha, prostaglandins E2, F2 alpha and D2 and thromboxane B2. Two of these hydroxy derivatives of arachidonic acid were purified by reverse-phase HPLC and identified by GC-MS as 11-hydroxyeicosatetraenoic acid (11-HETE) and 15-hydroxyeicosatetraenoic acid (15-HETE) while a third, presumed dihydroxy derivative has not yet been identified. Both the cyclooxygenase and HETE synthesizing activities were found to be localized mainly in the microsomal fraction (100 000 X g pellet) (51 and 61% of total, respectively), and approx. 25% of both activities was found in the 10 000 X g pellet. The formation of these HETEs was inhibited by the cyclooxygenase inhibitors indomethacin and aspirin but not by the lipoxygenase inhibitor nordihydroguaiaretic acid. Production of immunoreactive 15-HETE as well as 6-ketoprostaglandin F1 alpha were also decreased significantly when arterial segments were incubated in the presence of either indomethacin or aspirin. Indomethacin inhibited the formation of both prostanoids and HETEs by microsomes in a concentration-dependent and time-dependent manner. The ID50 values for indomethacin against HETE synthesizing activity and against cyclooxygenase were 4.5 and 3.8 microM, respectively. The inactivation constants were found to be 0.09 and 0.08 min-1 for HETE synthesizing activity and cyclooxygenase, respectively. These two microsomal activities were solubilized in parallel with Tween-20. Incubation with three distinct monoclonal antibodies against different epitopes on cyclooxygenase precipitated both cyclooxygenase and HETE synthesizing activity. Each of these activities was recovered in the immune pellets. These studies demonstrate that in human umbilical arteries 11-HETE, 15-HETE and a presumed di-HETE are the products of cyclooxygenase.     

Metabolism of 15-hydroxyeicosatetraenoic acid by Caco-2 cells

Monolayers of Caco-2 cells, a human enterocyte cell line, were incubated with [1-14C]15-hydroxyeicosatetraenoic acid (15-HETE), a lipid mediator of inflammation, and [1-14C]arachidonic acid. Both fatty acids were taken up readily and metabolized by Caco-2 cells. [1-14C]Arachidonic acid was directly esterified in cellular phospholipids and, to a lesser extent, in triglycerides. When [1-14C]15-hydroxyeicosatetraenoic acid was incubated with Caco-2 cells, about 10% was directly esterified into cellular lipids but most (55%) was beta-oxidized to ketone bodies, CO2, and acetate, with very little accumulation of shorter carbon chain products of partial beta-oxidation. The radiolabeled acetate generated from beta-oxidation of [1-14C]15-hydroxyeicosatetraenoic acid was incorporated into the synthesis of new fatty acids, primarily [14C]palmitate, which in turn was esterified into cellular phospholipids, with lesser amounts in triglycerides. Caco-2 cells were also incubated with [5,6,8,9,11,12,14,15-3H]15-hydroxyeicosatetraenoic acid; most of the radiolabel was recovered either in ketone bodies or in [3H]palmitate esterified in phospholipids and triglycerides, demonstrating that most of the [3H]15-hydroxyeicosatetraenoic acid underwent several cycles of beta-oxidation. The binding of both 15-hydroxyeicosatetraenoic acid and arachidonic acid to hepatic fatty acid binding protein, the only fatty acid binding protein in Caco-2 cells, was measured. The Kd (6.0 microM) for 15-HETE was three-fold higher than that for arachidonate (2.1 microM).     

Specific enhancement of LTD4-induced contractions of isolated guinea pig trachea by 5-hydroxyeicosatetraenoic acid (5-HETE)

In view of the likely production of monohydroxyeicosatetraenoic acid (HETE's) in bronchial asthma, the role of these lipoxygenase products in the development of a classical clinical element of airway disease, namely airway hyperreactivity, has been investigated. Tracheas removed from guinea-pigs actively sensitized to ovalbumin produced, upon antigenic challenge (0.01 μg/ml), a 17-fold increase (0.97 ± 0.34 ng/ml to 16.73 ± 1.58 ng/ml) in the amount of 5-hydroxyeicosatetraenoic acid (5-HETE) as measured by radioimmunoassay of the tissue-bath fluid, indicating that this tissue is capable of producing 5-HETE. While 5-HETE alone, at concentrations equal to or greater than those found during the above antigenic response (0.001 to 1.0 μM), failed to produce intrinsic contractions of normal, nonsensitized guinea-pig trachea, a 30 min pretreatment with 5-HETE (1.0 μM) enhanced subsequent LTD₄-induced contractions. Pretreatment with either 12- or 15-HETE, at similar concentrations and conditions, failed to potentiate LTD₄ concentration-response curves. The effect of 5-HETE was time-dependent, since pretreatment for either 15 or 60 min had little or no effect on subsequent LTD₄ responses. Also, the 5-HETE-induced enhancement seemed specific fot LTD₄, since contractions to LTC₄ (in the presence of l-serine borate), acetylcholine, histamine, PGD₂ or U-46619 were unaffected by 5-HETE. Therefore, 5-HETE may have a role in the development of airway hyperreactivity by interacting with released LTD₄ to exacerbate airway smooth muscle contraction in asthma.     

Conversion of 15-hydroxyeicosatetraenoic acid to 11-hydroxyhexadecatrienoic acid by endothelial cells

Cultured endothelial cells take up 15-hydroxyeicosatetraenoic acid (15-HETE), a lipoxygenase product formed from arachidonic acid, and incorporate it into cellular phospholipids and glycerides. Uptake can occur from either the apical or basolateral surface. A substantial amount of the 15-HETE incorporated into phospholipids is present in the inositol phosphoglycerides. 15-HETE is converted into several metabolic products that accumulate in teh extracellular fluid; this conversion does not require stimulation by agonists. The main product has been identified as 11-hydroxyhexadecatrienoic acid [16:3(11-OH)], a metabolite of 15-HETE that has not been described previously. Formation of 16:3(11-OH) decreases when 4-pentenoic acid is present, suggesting that it is produced by beta-oxidation. The endothelial cells can take up 16:3(11-OH) only 25% as effectively as 15-HETE, and 16:3(11-OH) is almost entirely excluded from the inositol phosphoglycerides. These results suggest that the endothelial cells can incorporate 15-HETE when it is released into their environment. Through partial oxidation, the endothelium can process 15-HETE to a novel metabolite that is less effectively taken up and, in particular, is excluded from the inositol phosphoglycerides.     

Amniotic fluid 5-hydroxyeicosatetraenoic acid in preterm labor

5-Hydroxyeicosatetraenoic acid (5-HETE) is an arachidonate lipoxygenase product capable of stimulating human uterine contractility in a dose-dependent manner in vitro. The purpose of this study was to determine if preterm labor is associated with changes in the concentration of this metabolite in amniotic acid. Amniotic fluid was obtained by transabdominal amniocentesis from three groups of women with preterm labor: group 1 — women without intraamniotic infection who responded to tocolysis (n = 32); group 2 — women without intraamniotic infection who failed to respond to tocolysis (n = 22); and group 3 — women with intraamniotic infection (n = 14). 5-HETE was determined by radioimmunoassay. The median amniotic fluid concentration of 5-HETE in women who responded to tocolysis (median = 1412 pg/ml; range: 111–3547) was significantly lower than in women who delivered despite tocolysis (median = 2052 pg/ml; range 136–7774) and women with intraamniotic infection (median = 1876 pg/ml; range: 543–7033) [p < 0.05]). No difference in amniotic fluid concentrations' of 5-HETE were found between women in groups 2 and 3 (p > 0.05).     

Amniotic fluid 5-hydroxyeicosatetraenoic acid in preterm labor

5-Hydroxyeicosatetraenoic acid (5-HETE) is an arachidonate lipoxygenase product capable of stimulating human uterine contractility in a dose-dependent manner in vitro. The purpose of this study was to determine if preterm labor is associated with changes in the concentration of this metabolite in amniotic fluid. Amniotic fluid was obtained by transabdominal amniocentesis from three groups of women with preterm labor: group 1 - women without intraamniotic infection who responded to tocolysis (n = 32); group 2 - women without intraamniotic infection who failed to respond to tocolysis (n = 22); and group 3 - women with intraamniotic infection (n = 14). 5-HETE was determined by radioimmunoassay. The median amniotic fluid concentration of 5-HETE in women who responded to tocolysis (median = 1412 pg/ml; range: 111-3547) was significantly lower than in women who delivered despite tocolysis (median = 2052 pg/ml; range: 136-7774) and women with intraamniotic infection (median = 1876 pg/ml; range: 543-7033) [p less than 0.05]). No difference in amniotic fluid concentrations' of 5-HETE were found between women in groups 2 and 3 (p greater than 0.05).