Products Of Arachidonic Metabolism Research Articles

Thromboxane A 2 Receptors Contribute to the Exaggerated Exercise Pressor Reflex in Rats with Heart Failure

Background Mechanical and metabolic signals associated with skeletal muscle contraction stimulate the sensory endings of thin fiber skeletal muscle afferents. During exercise, stimulation of these afferents initiates a reflex, termed the exercise pressor reflex (EPR), which contributes importantly to reflex increases in sympathetic nerve activity (SNA) and mean arterial blood pressure (MAP). In patients and animals with heart failure with reduced ejection fraction (HF-rEF), activation of the EPR contributes to exaggerated increases in SNA and is linked to decreased exercise tolerance and increased mortality. The exaggerated EPR in HF-rEF is attributed, at least in part, to an increased responsiveness of thin fiber skeletal muscle afferents produced by cyclooxygenase (COX) products of arachidonic metabolism. However, the COX metabolite receptors on the sensory endings that mediate the COX-induced afferent sensitization in HF-rEF are unknown. Purpose & Hypothesi The purpose of this investigation was to determine the role played by one of the major receptors for products of COX metabolism present on sensory neurons, namely thromboxane A2 receptors (TxA2-Rs), in the exaggerated EPR in HF-rEF rats. We tested the hypothesis that injection of the TxA2-R antagonist daltroban (80μg) into the arterial supply of the hindlimb would reduce the reflex increase in renal SNA (RSNA) and MAP evoked in response to 30 seconds of 1 Hz dynamic hindlimb muscle contraction in HF-rEF rats, but not control rats. Methods Experiments were performed on male Sprague-Dawley rats subjected to either a coronary artery ligation surgery to produce myocardial infarction and HF-rEF or a sham ligation. At least six weeks after the initial surgery, in decerebrate, unanesthetized rats we compared the EPR evoked by electrically induced hindlimb skeletal muscle contraction before and after injection of daltroban into the arterial supply of the hindlimb. Results Ejection fraction was significantly reduced in HF-rEF (47±3%) compared to sham (84±1%, p<0.01). We found that daltroban reduced the EPR response in HF-rEF (n=8; peak ΔMAP pre: 17±4; post: 12±2mmHg; P=0.04; peak ΔRSNA pre: 135±55; post: 46±13%; P=0.07), but not in SHAM rats (n=10; peak ΔMAP pre: 13±3; post: 11±2mmHg; P=0.48; peak ΔRSNA pre: 64±17; post: 63±15%; P=0.96;) rats. In a separate group of HF-rEF rats (n=4), we determined whether systemic circulation of daltroban accounted for the attenuating effects on EPR activation seen when daltroban was injected into the hindlimb arterial supply. We found that intravenous injection of daltroban had no effect on the EPR (peak ΔMAP pre: 26±7; post: 25±7mmHg; P=0.50). In these same four HF-rEF rats we also found that hindlimb arterial injection of the vehicle for daltroban (0.4mL saline containing 1% DMSO) had no effect on the EPR (peak ΔMAP pre: 23±8; post: 20±6mmHg; P=0.40). Conclusions These data suggest that that TxA2-R receptors on the sensory endings of muscle afferents contribute to the exaggerated EPR in HF-rEF. Our data enhance the understanding of the sympathetic and cardiovascular adjustments to exercise in the ~6 million Americans with HF-rEF.

Involvement of 5-lipoxygenase in survival of Epstein–Barr virus (EBV)-converted B lymphoma cells

Epstein–Barr Virus (EBV) is involved in the progression of lymphomas through still unknown mechanism involving increased resistance to induced apoptosis. We show here that in a set of apoptosis-resistant EBV-converted Burkitt’s lymphoma clones, 5- and 12-lipoxygenases (LOXs) are over-expressed. Further investigations on 5-LOX showed that resistance to apoptosis increases parallely with the expression of 5-lipoxygenase (5-LOX). Inhibitors of 5-LOX: (a) decrease peroxides level, indicating that this enzyme promotes the generation of oxidative stress in EBV+ cells, and (b) potently induce apoptosis in the EBV resistant cell line E2R. 5- and 15-HETE, the products of the 5 and 15-LOXs, respectively, counteract 5-LOX inhibitor induced apoptosis, indicating that products of arachidonate metabolism, rather than peroxides, trigger a signal transduction that is required for survival of the EBV-converted cells. These findings suggest that 5- and, to a lesser extent, other LOXs, that are involved in tumor progression of several cell types, may also participate in lymphomagenesis, especially that EBV-mediated.

Multiple roles of COX-2 in tumor angiogenesis: a target for antiangiogenic therapy

Angiogenesis is required for multistage carcinogenesis. The inducible enzyme cyclooxygenase-2 (COX-2) is an important mediator of angiogenesis and tumor growth. COX-2 expression occurs in a wide range of preneoplastic and malignant conditions; and the enzyme has been localized to the neoplastic cells, endothelial cells, immune cells, and stromal fibroblasts within tumors. The proangiogenic effects of COX-2 are mediated primarily by three products of arachidonic metabolism: thromboxane A 2 (TXA 2), prostaglandin E 2 (PGE 2), and prostaglandin I 2 (PGI 2). Downstream proangiogenic actions of these eicosanoid products include: (1) production of vascular endothelial growth factor; (2) promotion of vascular sprouting, migration, and tube formation; (3) enhanced endothelial cell survival via Bcl-2 expression and Akt signaling; (4) induction of matrix metalloproteinases; (5) activation of epidermal growth factor receptor-mediated angiogenesis; and (6) suppression of interleukin-12 production. Selective inhibition of COX-2 activity has been shown to suppress angiogenesis in vitro and in vivo. Because these agents are safe and well tolerated, selective COX-2 inhibitors could have clinical utility as antiangiogenic agents for cancer prevention, as well as for intervention in established disease alone or in combination with chemotherapy, radiation, and biological therapies.

Ribozyme-mediated inhibition of rat leukocyte-type 12-lipoxygenase prevents intimal hyperplasia in balloon-injured rat carotid arteries.

12-Lipoxygenase (12-LO) products of arachidonate metabolism have growth and chemotactic effects in vascular smooth muscle cells. We have also recently demonstrated increased 12-LO mRNA and protein expression in the neointima of balloon-injured rat carotid arteries. In this study, we evaluated whether 12-LO activation plays a role in neointimal thickening in this rat model by using a specific ribozyme (Rz) directed to rat 12-LO. We designed a chimeric DNA-RNA hammerhead Rz to cleave rat leukocyte-type 12-LO mRNA. This Rz dose-dependently cleaved a 166-nucleotide target 12-LO mRNA substrate in vitro and reduced 12-LO mRNA and protein expression in rat vascular smooth muscle cells. A control mutant Rz (MRz) with a point mutation in the catalytic site was inactive. To test the in vivo efficacy of the 12-LO Rz, the left common carotid arteries of rats were injured with a balloon catheter. The distal half of the injured arteries was treated with Rz or MRz mixed with lipofectin. The proximal half received only lipofectin. Twelve days after injury, intima-to-media ratios were significantly lower in the Rz-treated sections than in untreated sections from the same rat (0.742+/-0.16 versus 1.749+/-0.12, P:<0.001). In contrast, the MRz had no significant effect. These results indicate the important role of the leukocyte-type 12-LO pathway in restenosis in response to injury.

Intestinal lymphoepithelial communication.

The close anatomic juxtaposition of epithelial cells with lymphocytes lining the intestinal tract facilitates communication between the two cell types. This intercellular dialogue is important for mucosal development and has a conditioning effect on mucosal structure, function, and response to tissue injury. Lymphoepithelial communication is bi-directional, and mediated in large part, by shared ligands and receptors. The chemical messengers involved include cytokines, growth factors, local hormones, and products of arachidonate metabolism. The interdependency between the epithelium and adjacent lymphoid cells is such that the epithelium is considered to have a central role in the mucosal immune system and is an active participant in both the afferent and efferent limbs of the mucosal immune response. The molecular crosstalk between the epithelium and adjacent lymphocytes is just one aspect of a more complex network of intercellular signalling within the intestinal mucosa and upon which the integrity of the mucosa is dependent. Thus, there are extensive interactions between nerve and immune cells and between the enteric flora and the epithelium and amongst intestinal mesenchymal cells including fibroblasts and vascular endothelial cells. Disruption of any aspect of the mucosal microenvironment, as has been achieved with selective genetically engineered murine models, is associated with impaired mucosal defence and inflammation.

Release of calcitonin gene-related peptide in cardiac anaphylaxis.

We have investigated the antigen-stimulated release of calcitonin gene-related peptide (CGRP) from ovalbumin-sensitized guinea-pig isolated hearts and the interaction with other mediators of anaphylaxis released concomitantly. It was found that antigen challenge caused a significant increase of CGRP release (from basal 31.2 +/- 2.9 to 51.6 +/- 4.9 fmol/5 min). Anaphylactic CGRP release was significantly attenuated in the presence of the cyclooxygenase inhibitor indomethacin while the 5-lipoxygenase inhibitor Bay-X1005 ((R)-2-[4-quinolin-2-yl-methoxy)phenyl]-2-cyclopentyl acetic acid) had no significant effect. Combined treatment with the histamine receptor (H1,H2) antagonists mepyramine and cimetidine also significantly attenuated anaphylactic release of CGRP. Under control conditions antigen injection increased release of cysteinyl-leukotrienes (LT), thromboxane (TXB2) and 6-keto-prostaglandin (PG)F1 alpha from basal values of 0.96 +/- 0.09, 2.7 +/- 0.7 and 3.4 +/- 0.28 ng/5 min respectively, to 5.9 +/- 0.9, 48.4 +/- 3.4 and 6.9 +/- 1.4 ng/5 min. Indomethacin abolished the release of cyclooxygenase products of arachidonate metabolism and simultaneously increased cysteinyl-LT release significantly (8.8 +/- 1.4 ng/5 min). Conversely Bay-X1005 completely abolished cysteinyl-LT release and had no significant effect on anaphylactic release of TXB2 and 6-keto-PGF1 alpha. Simultaneous blockade of H1 and H2 receptors abolished release of 6-keto-PGF1 alpha, while release of TXB2 and cysteinyl-LT was not significantly affected. The results indicate that CGRP is not a primary mediator of the immediate hypersensitivity reaction of the heart, but is in turn released by arachidonic acid metabolites of the cyclooxygenase pathway and histamine. In contrast, LT obviously do not contribute to anaphylactic CGRP release. CGRP is a potent coronary vasodilator and could act as endogenous functional antagonist of vasoconstrictor mediators also released during cardiac anaphylaxis such as cysteinyl-LT, platelet activating factor and TXA2.

Eicosanoid production by human aortic endothelial cells in response to endothelin

Endothelial cells actively regulate their environment by secreting humoral substances, including endothelin-1 and a variety of eicosanoids, that have local actions. To elucidate interactions among these local mediators, we measured release of cyclooxygenase and lipoxygenase pathway products of arachidonate metabolism by human aortic endothelial cells after incubation with endothelin-1. Supernatants were collected, extracted, and fractionated using high-performance liquid chromatography. Radioimmunoassays for eicosanoids were performed on the appropriate fractions. After endothelin stimulation, production of the prostacyclin metabolite 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha), the thromboxane (Tx) metabolite TxB2, and prostaglandin E2 (PGE2) were increased (307 +/- 48, 320 +/- 91, and 315 +/- 74% of control, P < 0.05). Leukotriene B4 (LTB4) release was modestly increased (195 +/- 19% of control, P < 0.05). The release of 5-hydroxyeicosatetraenoic acid (5-HETE) was increased (300 +/- 57% of control, P < 0.05); production of 12-HETE and 15-HETE was unchanged. Production of eicosanoids peaked between 30 and 120 min. Preincubation with pertussis toxin prevented increased production of PGE2, LTB4, and 5-HETE after endothelin-1 stimulation; pretreatment with sphingosine had no effect. Interactions between endothelin and eicosanoids may be important components of the complex network that regulates vascular tone, coagulation, and inflammation at the local level.

Effect of platelet-activating factor on porcine pulmonary blood vessels in vitro

Platelet-activating factor (PAF) induced contractions of porcine pulmonary vein strips in a concentration-dependent manner, while porcine pulmonary artery strips were unresponsive. Exposure to the specific PAF-antagonists WEB 2086 or BN 52021 antagonized the contractile responses of pulmonary vein strips. Cysteinyl-leukotrienes (LT) and thromboxane (TX) B2 were not detected in the bath fluid after stimulation with PAF suggesting that these eicosanoids as well as their precursors are not mediators of PAF-induced contractions of porcine pulmonary vein strips. Furthermore, PAF had no significant effect on 6-keto-prostaglandin (PG) F1a release and flurbiprofen did not affect the PAF response, while it inhibited the release of 6-keto-PGF1a. This indicates that PGI2 or any other cyclooxygenase product is unlikely to modulate or mediate the PAF response. Incubation experiments with fragments of pulmonary vascular tissues demonstrated spontaneous release of small amounts of cysteinyl-LT, TXB2 and 6-keto-PGF1a, which was significantly increased during incubation in the presence of ionophore A23187. While these results demonstrate the synthesizing capacity of the porcine pulmonary vascular tissues for various eicosanoids, PAF failed to stimulate eicosanoid release under these experimental conditions. We conclude that PAF causes contractions of porcine pulmonary vein strips, which are not mediated by cysteinyl-LT or cyclooxygenase products of arachidonate metabolism. The specific contractile effect of PAF on pulmonary veins, but not arteries, could contribute to the disturbances of the pulmonary circulation observed after injection of PAF or release of endogenous PAF, e.g. after administration of endotoxin.

Mechanisms of Gastroprotection

Gastric mucosa is constantly exposed to various irritants, but it usually maintains its integrity owing to several lines of defense, including mucus-alkaline secretion, mucosal hydrophobicity, rich mucosal blood flow, stabilization of tissue lysosomes, maintenance of mucosal sulfhydryls, and rapid proliferation and renewal of mucosal cells. Prostaglandins (PG) inhibit experimental gastric mucosal damage and ulcerations induced by a wide variety of agents, hence PG have been proposed to contribute to the overall protective process by activation of various mucosal defence lines--particularly by prevention of vasocongestion, ischemia, and deep hemorrhagic necrosis. The relation between tissue PG generation and mucosal protection does not appear to be closely related, and probably only minute amounts of PG are required to maintain mucosal integrity. In contrast to PG, other products of arachidonate metabolism, such as TxA2, LTC4 or LTD4, and the related lipid, platelet-activating factor, appear to mediate mucosal damage mainly by the disturbance in mucosal microcirculation and tissue ischemia. Gastroprotection can be achieved by stimulation of mucosal biosynthesis of protective PG or by the inhibition of the release or action of the proulcerogenic arachidonate metabolites. Certain natural substances, such as sulfhydryls, epidermal growth factor, or polyamines, protect the mucosa via a PG-independent mechanism, probably by enhancing the tissue repair processes.(ABSTRACT TRUNCATED AT 250 WORDS)

Pulmonary Oxygen Toxicity in the Guinea Pig - Effect of Indomethacin

Deleterious pulmonary effects of hyperoxia are thought, in part, to be mediated by high-energy radicals and inflammatory reactions. It has been suggested that arachidonate metabolites, such as leukotrienes, may be responsible for the latter mechanism. We sought to demonstrate that concurrent exposure to hyperoxia and indomethacin, a potent inhibitor of cyclooxygenase, would increase pulmonary inflammation. A total of 50 female guinea pigs were treated as pairs with 95.6% oxygen or air and separately with 0.3 mg/kg of indomethacin or normal saline every 12 h for 3 days. A significant increase in leukocytes in bronchopulmonary lavage subsequent to combined oxygen and indomethacin treatment was observed when compared with either oxygen, indomethacin, or air treatments alone (p less than 0.05). Although oxygen treatment did alter pulmonary volume-pressure relations (p less than 0.05), hyperoxia alone or with concurrent indomethacin treatment did not alter elastance or histologic structures. These findings suggest that pulmonary inflammation in response to hyperoxia may be augmented by indomethacin. We speculate that increased lipoxygenase products of arachidonate metabolism may be partially responsible for these changes.

The glomerular physiology of lipoxin-A.

A number of renal diseases, such as acute and chronic glomerulonephritis, acute interstitial nephritis, allograft rejection, and others are characterized by the presence inflammatory cellular infiltrates consisting, at various stages, of neutrophils, macrophages, monocytes, eosinophils, and lymphocytes. In addition, these disease processes are often accompanied by impairment of glomerular perfusion, filtration, and permselectivity functions. Despite much controversy as to the mediator systems involved in the initiation and perpetuation of these glomerular functional abnormalities, consensus has emerged as to the central role of leukocyte-derived biologically active cytokines1. In this regard, lipid-derived mediators, including platelet activating factor and the cyclooxygenase and lipoxygenase (LO) products of arachidonate metabolism have received particular attention. An understanding of the role played by these locally released compounds in the pathophysiology of glomerular injury can only result from a close integration of the best available bioanalytic and physiologic technologies, and the application of both approaches to experimental disease models, in conjunction with selective antagonism of specific mediators through receptor antagonists and enzyme inhibitors. Using glomerular micropuncture techniques, we have identified the responses of the glomerular microcirculation to peptidyl leukotrienes2,3 and, utilizing selective antagonists, proposed a role for these compounds in mediating the reductions in renal plasma flow and glomerular filtration, as well as the induction of proteinuria, in endotoxin- and anti-glomerular basement membrane antibody-induced glomerular injury4,5. In more recent studies6, we have examined the effects of exogenously administered lipoxin A (LX-A) on the glomerular microcirculation in the rat. Our results indicate that, in sharp contrast to sulfidopeptide LTs, the predominant action of LX-A is to increase renal perfusion. This response appears to be evoked by selective reduction in preglomerular resistance with a consequent augmentation of single nephron and whole kidney glomerular filtration rate.KeywordsLeft Renal ArteryAcute Interstitial NephritisColloid Osmotic PressureArteriolar ResistanceSingle Nephron Glomerular Filtration RateThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Mediators of C5a-induced bronchoconstriction.

Previous studies had suggested that histamine and products of arachidonate metabolism were mediators of the bronchoconstriction induced in guinea pigs by the complement cleavage product C5a. The present study was conducted to further define the arachidonate metabolite(s) involved. Tracheal airflow and transpulmonary pressure were measured in anesthetized and artificially ventilated guinea pigs and pulmonary resistance and dynamic lung compliance were calculated as a measure of bronchoconstriction. The effect of the peptido-leukotriene antagonist L-649,923 and the thromboxane synthetase inhibitor U-63557A on the C5a-induced bronchoconstriction was determined. Also, the response to C5a was evaluated in animals made tachyphylactic to the bronchoconstrictor actions of LTB4. C5a-induced bronchoconstriction was not altered in animals treated with L-649,923 or made tachyphylactic to LTB4 suggesting that LTB4 and peptido-leukotrienes are not major mediators of the response. C5a challenge caused a significant increase in plasma thromboxane B2 levels which was prevented in part by the thromboxane synthetase inhibitor U-63557A. In addition, C5a-induced bronchoconstriction was significantly inhibited by U-63557A. Thus, these studies suggest that thromboxane is the arachidonate metabolite at least in part responsible for C5a-induced bronchoconstriction.

Leuktriene synthesis by human gastrointestinal tissues

The prostaglandin and leukotriene synthesizing capacity of human gastrointestinal tissues obtained at surgery was investigated using radioimmunoassays for prostaglandin E 2, leukotriene B 4 and sulfidopeptide leukotrienes. The leukotriene inimunoassay data were validated by high-pressure liquid chromatography (HPLC). During incubation at 37°C, fragments of human gastric, jejuno-ileal and colonie mucosa released considerably larger amounts of prostaglandin E 2 than of leukotriene B 4 and sulfidopeptide leukotrienes. Gastrointestinal smooth muscle tissues released even larger amounts of prostaglandin E 2, but smaller amounts of leukotrienes than the corresponding mucosal tissues. Adenocarcinoma tissue released larger amounts of leukotriene B 4, sulfidopeptide leukotrienes and prostaglandin E 2 than normal colonie mucosa. Ionophore A23187 (5 μg/ml) did not stimulate release of prostaglandin E 2 from any of the tissues investigated, but enhanced release of leukotriene B 4 and sulfidopeptide leukotrienes. HPLC analysis demonstrated that immunoreactive leukotriene B 4 co-chromatographed almost exclusively with standard leukotriene B 4, while immunoreactive sulfidopeptide leukotrienes consisted of a mixture of leukotrienes C 4, D 4 and E 4. Leukotriene synthesis by human gastrointestinal tissues was inhibited by the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) and the dual enzyme inhibitor BW755C (3-amino-1-(trifluoromethylphenyl)-2-pyrazoline hydrochloride). Synthesis of prostaglandin E 2 was inhibited by the cyclooxygenase inhibitor indomethacin as well as by BW755C. Incubation of gastrointestinal tissues in the presence of glutathione decreased the amounts of leukotrienes D 4 and E 4, while release of leukotriene C 4 was simultaneously increased. On the other hand, incubation of tritiated leukotriene C 4 with incubation media from human gastric or colonic mucosa resulted in conversion of the substrate to [ 3H]leukotriene D 4 and [ 3H]Ieukotriene E 4. The results indicate the capacity of human gastrointestinal tissues to synthesize the 5-lipoxygenase-derived products of arachidonate metabolism, leukotriene B 4 and sulfidopeptide leukotrienes, in addition to larger amounts of prostaglandin E 2. Furthermore, considerable activities of the sulfidopeptide leukotriene-metabolizing enzymes γ-glutamyl transpeptidase and dipeptidase were detected in human gastrointestinal tissues. These enzymes might play an important role in biological inactivation and/or change of biological profile of sulfidopeptide leukotrienes generated in the human gastrointestinal tract.

Prostanoid mediated effects of centchroman, a non-steroidal oral contraceptive.

The effect of centchroman, a non-steroidal oral contraceptive, has been studied on platelet aggregation and on the products of arachidonate metabolism. Centchroman inhibited platelet aggregation in vitro and ex vivo after acute as well as chronic treatment for one year in laboratory animals. It did not affect aggregation of platelets in women taking centchroman for one year. It is an inhibitor of platelet cyclooxygenase as indicated by the inhibition of malonaldehyde and thromboxane B2 but has no effect on vascular cyclooxygenase activity ex vivo or at low concentration in vitro. Thus, centchroman is a safe antifertility agent without risk of thrombotic episodes associated with hormonal oral contraceptives.

Clinical study of a new antimetastatic compound, Nafazatrom (Bay g 6575). Effects on platelet consumption and monocyte prostaglandin production in patients with advanced cancer.

Nafazatrom (Miles Laboratories, West Haven, CT) is a potent inhibitor of metastasis in several animal model systems. The compound is active whether cancer cells are injected intravenously or are allowed to spontaneously metastasize from established tumors. Before studying the antimetastatic effects of Nafazatrom in a long-term randomized clinical trial, the authors conducted a preliminary study to evaluate its toxicity as well as its effects on certain parameters hat have been associated with the biology of metastasis. Since the interaction of platelets, monocytes, and tumor cells may be important for metastasis formation, the authors serially evaluated plasma levels of factors secreted from activated platelets (beta-thrombo-globulin and platelet factor 4) that are inversely correlated with platelet survival. Products of arachidonate metabolism from monocytes were also measured since Nafazatrom is known to modulate prostaglandin production. Thirty-one patients received doses of Nafazatrom ranging from 75 to 1500 mg/m2 orally three times a day. The dose was escalated every 2 weeks. There was no toxicity at any dose level. No major antitumor responses were observed. There was no consistent change in levels of platelet factors either within individual patients or for the population as a whole. Although Nafazatrom (20 microM) decreased tritiated arachidonate incorporation into peripheral blood monocytes from normal subjects in vitro, this effect was not seen in patients treated with the drug. Release of 3PGE2 and 3HETE from cultured monocytes was also not altered by treatment with Nafazatrom. It was concluded that Nafazatrom is safe and well-tolerated up to total doses of 4500 mg/day. The data suggest that the antimetastatic activity of Nafazatrom in vivo is mediated by mechanisms other than by a decrease in platelet consumption or by modulation of arachidonate metabolism by monocytes.

Ethanol stimulates formation of leukotriene C 4 in rat gastric mucosa

Ethanol-induced gastric mucosal damage is characterized by microcirculatory changes such as statis and plasma leakage. Sluggish blood flow and statis have also been observed after administration of exogenous leukotriene (LT) C 4. The effect of ethanol on the release of LTC 4 from rat gastric mucosa was therefore investigated. It was found that intragastric instillation of ethanol increases gastric mucosal release of LTC 4 in a dose- and time-dependent manner parallel to the production of gastric lesions. The lipoxugenase inhibitor nordihydroguaiaretic acid (NDGA) and the anti-ulcer drug carbenoxolone (CX) inhibited mucosal release of LTC 4 and simultaneously protected against gastric damage caused by ethanol. It is concluded that increased formation of LTC 4 and /or other 5-lipoxygenase-derived products of arachidonate metabolism may be involved in ethanol-induced gastric damage. Furthermore, inhibition of the 5-lipoxygenase pathway may be an important mechanism of action of gastric protective drugs.

Role of circulating granulocytes in sheep lung injury produced by phorbol myristate acetate.

Phorbol myristate acetate (PMA) and endotoxin cause pulmonary granulocyte sequestration and alteration in lung fluid and solute exchange in awake sheep that are felt to be analogous to the adult respiratory distress syndrome in humans. The basic hypothesis that PMA causes lung injury by activating circulating granulocytes has never been tested. The effects of infused PMA on lung mechanics and the cellular constituents of lung lymph have also not been reported. We therefore characterized the effects of intravenous PMA, 5 micrograms/kg, on lung mechanics, pulmonary hemodynamics, lung fluid and solute exchange, pulmonary gas exchange, blood and lymph leukocyte counts, and plasma and lymph cyclooxygenase products of arachidonate metabolism in 10 awake sheep with normal granulocyte counts and after granulocyte depletion with hydroxyurea. PMA significantly altered lung mechanics from base line in both nongranulocyte depleted and granulocyte-depleted sheep. Dynamic compliance decreased by over 50% and resistance to airflow across the lungs increased over threefold acutely following PMA infusion in both sets of experiments. Changes in lung mechanics, pulmonary hemodynamics, lung fluid and solute exchange, pulmonary gas exchange, and plasma and lymph arachidonate metabolites were not significantly affected by greater than 99% depletion of circulating granulocytes. We conclude that the lung injury caused by PMA in chronically instrumented awake sheep probably is not a result of activation of circulating granulocytes.

Role of arachidonic acid metabolism in human platelet activation and irreversible aggregation.

Previous studies from our laboratory have demonstrated that the aggregation response of platelets inhibited by agents blocking cyclooxygenase activity could be restored to a normal state of sensitivity by prior stimulation of alpha-adrenergic receptors. Since cyclooxygenase activity and thromboxane synthesis are not absolutely required for irreversible platelet aggregation, it is important to define precisely what role this pathway serves in platelet physiology. The present study has evaluated the influence of agents that selectively block arachidonic acid conversion at different steps of synthesis. Inhibition of peroxidase, cyclooxygenase, lipoxygenase, and thromboxane synthetase blocked the second wave response of platelets to several agonists, but did not cause dissociation of aggregates preformed by prior exposure to arachidonate (AA) or adenosine diphosphate. Phospholipase (A2/C) inhibitors, similar to prostaglandin inhibitors, blocked the second wave response of platelets to the action of agonists and, in addition, caused dissociation of aggregates induced by aggregating agents. Results of our study demonstrate that when single agonists are tested at threshold concentrations, products of arachidonate metabolism may play a role in the activation process. However, continued generation of these metabolites does not appear to be essential for the maintenance of irreversible aggregation. When a combination of agents or high concentration of physiological agonists are used, both activation and irreversible aggregation can be secured independent of prostaglandin synthesis or the release reaction.

Release of eosinophil chemotactic leukotriene from neutrophils of patients with pseudo-allergic reactions.

Possible alterations in the release of eosinophil chemotactic leukotrienes (ECL) were studied with leukocytes from 14 patients with chronic urticaria due to pseudo-allergic reactions (PAR) and from 14 healthy volunteers. Patients tended to release less ECL, and inhibition of ECL release was more marked in the presence of cyclooxygenase and lipoxygenase inhibitors. The latter compounds did not cause ECL secretion by themselves. Increased release of lipoxygenase products of arachidonate metabolism from blood leukocytes is therefore an unlikely cause for PAR.

Platelet-mediated cardiac ischemia

Although platelets have been associated with angina pectoris, myocardial infarction, and sudden death, the platelet's capacity for induction and propagation of cardiac ischemia remains incompletely defined. We therefore evaluated the effects of platelet activation occurring within the coronary circulation and tested the hypothesis that inhibition of platelet function would prevent platelet-induced cardiac ischemia. Human platelets were isolated from blood obtained from normal donors by Sepharose 2B column chromatography, resuspended in Hepes buffer, and added to the perfusate of a Langendorff rabbit heart (platelet counts > 10,000/μl). Without, and with low dose (10 μM) prostaglandin E 1 (PGE 1), a reversible inhibitor of platelet function, immediate and irreversible global cardiac ischemia, as monitored by NADH fluorescent photography, ensued ( N = 4) following platelet activation with thrombin (0.1 to 1 U/ml). Higher concentrations of PGE 1 (0.1 to 1 m M, N = 2) or aspirin ingestion (1000 mg taken approximately 12, 4, and 1 hr prior to experiment, N = 2) completely prevented this platelet-induced myocardial ischemia. Aspirin, unlike PGE 1, was effective despite its inability to block thrombin-induced platelet aggregation in our in vitro gel-filtered system. We conclude that activation of platelets within the coronary circulation is sufficient for induction of irreversible cardiac ischemia. The efficacy of aspirin, a cyclooxygenase inhibitor, further suggests that the products of arachidonate metabolism (e.g., thromboxanes) have a fundamental role in the genesis of platelet-mediated myocardial ischemia.