Increased pulmonary vascular resistance and permeability due to arachidonate metabolism in isolated rabbit lungs

Liberation and metabolism of arachidonic acid may be the common final pathway of different stimuli on the pulmonary vascular bed. In a model of isolated, ventilated rabbit lungs, perfused with Krebs Henseleit albumin buffer in a recirculating system, changes of pulmonary vascular resistance and of vascular permeability are monitored continuously. The addition of free arachidonic acid or of the Ca-ionophore A 23187 to the perfusion fluid consistently evokes a biphasic increase in vascular resistance as well as an initially reversible increase in vascular permeability, followed by pulmonary edema. Both phases of increased vascular resistance are completely suppressed by inhibition of the cyclooxygenase, decreased to a large degree by inhibitors of thromboxane synthetase, and markedly augmented by short preincubation of arachidonic acid with ram seminal vesicular microsomes and by sulfhydryl reagents. The increased pulmonary vascular permeability is augmented by inhibition of cyclooxygenase and reduced by simultaneous lipoxygenase inhibition. Antagonists of histamine, serotonin and sympathic or parasympathic activity do not have any influence. PG F2alpha., TxB2, PG E2 and PG I2 alter the pulmonary vascular resistance, but do not increase vascular permeability. In conclusion, increased availability of free arachidonic acid evokes a rise in pulmonary vascular resistance, which can be ascribed to cyclooxygenase products, especially to thromboxane, and causes a rise in vascular permeability which can be ascribed to lipoxygenase products. The findings may be related to acute pulmonary lesions with increase in vascular resistance and with vascular leakage.     

Influence of tocopherol, its chromane compound, phytyl chains and superoxide dismutase on increased vascular resistance and permeability due to arachidonate metabolism in isolated rabbit lung

In the model of isolated, ventilated rabbit lungs, perfused with isoionic and isooncotic fluid, the addition of arachidonic acid to the perfusion fluid or the liberation of arachidonic acid by the Ca-ionophore A 23187 result in an increase in pulmonary vascular resistance and permeability. The former can be ascribed to cyclooxygenase products, the latter to lipoxygenase products of arachidonic acid. The effect of alpha-tocopherol, its chromane compound, alpha-tocopherolquinone, phytol, 2-methyl-1,4-naphthoquinone, 2-methyl-3-phytyl-1,4-naphthoquinone and of superoxide dismutase (SOD) on the increase in pulmonary vascular resistance and permeability was investigated. A membrane effect of the phytyl side chain and an antioxidative effect of the chromane compound can be distinguished: phytol increase the arachidonate-induced rise of pulmonary vascular resistance and permeability, whereas the chromane compound decreases both to a large degree. Methyl-phytyl-naphthoquinone and methyl-naphthoquinone gave equivalent results. SOD decreases the enhanced vascular resistance and the vascular leakage. The possibility of antioxidative therapy in acute pulmonary lesions with vascular leakage and increased vascular resistance is discussed.     

Influence of tocopherol,its chromane compound,phytyl chains and superoxide dismu8tase on increased vascular resistance and permeability due to arachidonate metabolism in isolated rabbit lung

In the model of isolated, ventilated rabbit lungs, perfused with isoionic and isooncotic fluid, the addition of arachindonic acid to the perfusion fluid or the liberation of arachidonic acid by the Ca-ionophore A 23187 results in an increase in pulmonary vascular resistance and permeability. The former can be ascribed to cyclooxygenase products, the later to lipoxygenase products of arachidonic acid. The effects of α-tocopherol, its chromane compound, α-tocopherolquinone, phytol, 2-methyl-1,4-napthoquinone, 2-methyl-3-phytyl-1, 4-naphthoquinone and of superoxide dismutase (SOD) on the increase in pulmonary vascular resistance and permeability was investigated. A membrane effect of the phytyl side chain and an antioxidative effect of the chromane compound can be distinguished: phytol increases the arachidonate-induced rise of pulmunary vascular resistance and permeability, whereas the chromane compound decreases both to a large degree. Methyl-phytyl-naphthoquinone and methyl-naphthoquinone gave equivalent results. SOD decreases the enhanced vascular resistance and the vascular leakage. The possibility of antioxidative therapy in acute pulmonary lesions with vascular leakage and increased vascular resistance is discussed.     

Edema from cyclooxygenase products of endogenous arachidonic acid in isolated lung

We infused A23187, a calcium ionophore, into the pulmonary circulation of dextran-salt-perfused isolated rabbit lungs to release endogenous arachidonic acid. This led to elevations in pulmonary arterial pressure and to pulmonary edema as measured by extravascular wet-to-dry weight ratios. The increase in pressure and edema was prevented by indomethacin, a cyclooxygenase enzyme inhibitor, and by 1-benzylimidazole, a selective inhibitor of thromboxane (Tx) A2 synthesis. Transvascular flux of 125I-albumin from vascular to extravascular spaces of the lung was not elevated by A23187 but was elevated by infusion of oleic acid, an agent known to produce permeability pulmonary edema. We confirmed that A23187 leads to elevations in cyclooxygenase products and that indomethacin and 1-benzylimidazole inhibit synthesis of all cyclooxygenase products and TxA2, respectively, by measuring perfusate levels of prostaglandin (PG) I2 as 6-ketoprostaglandin F1 alpha, PGE2, and PGF2 alpha and TxA2 as TxB2. We conclude that release of endogenous pulmonary arachidonic acid can lead to pulmonary edema from conversion of such arachidonic acid to cyclooxygenase products, most notably TxA2. This edema was most likely from a net hydrostatic accumulation of extravascular lung water with an unchanged permeability of the vascular space, since an index of permeability-surface area product (i.e., transvascular albumin flux) was not increased.     

Reactive oxygen production associated with arachidonic acid metabolism by peritoneal macrophages

The nature of the calcium-dependent chemiluminescence observed in peritoneal macrophages after exposure to the calcium ionophore A23187 or during the phagocytosis of zymosan has been investigated. Eicosatetraynoic acid, an inhibitor of the lipoxygenase and cyclooxygenase pathways of arachidonic acid metabolism, inhibited the calcium-dependent chemiluminescence whereas indomethacin, a selective inhibitor of the cyclooxygenase pathway, did not. Arachidonic acid induced chemiluminescence only in phagocytosing cells, whilst 15-HPETE, an intermediate of the lipoxygenase pathway, generated a similar, transient chemiluminescent response in either unstimulated or phagocytosing cells. The results suggest that the lipoxygenase pathway may be a significant source of the reactive species of oxygen that give rise to chemiluminescence. Prostaglandin E₁ inhibited the chemiluminescence induced by zymosan and A23187, but did not affect that generated in response to 15-HPETE or arachidonic acid, suggesting that the inhibition is directed at a step either connected with or occurring prior to the release of free arachidonic acid by the cells.     

Mechanism of phosgene-induced lung toxicity: role of arachidonate mediators

We have previously shown that phosgene markedly increases lung weight gain and pulmonary vascular permeability in rabbits. The current experiments were designed to determine whether cyclooxygenase- and lipoxygenase-derived mediators contribute to the phosgene induced lung injury. We exposed rabbits to phosgene (1,500 ppm/min), killed the animals 30 min later, and then perfused the lungs with a saline buffer for 90 min. Phosgene markedly increased lung weight gain, did not appear to increase the synthesis of cyclooxygenase metabolites, but increased 10-fold the synthesis of lipoxygenase products. Pre- or posttreatment with indomethacin decreased thromboxane and prostacyclin levels without affecting leukotriene synthesis and partially reduced the lung weight gain caused by phosgene. Methylprednisolone pretreatment completely blocked the increase in leukotriene synthesis and lung weight gain. Posttreatment with 5,8,11,14-eicosatetraynoic acid (ETYA), a nonmetabolized competitive inhibitor of arachidonic acid metabolism, or the leukotriene receptor blockers, FPL 55712 and LY 171883, also dramatically reduced the lung weight gain caused by phosgene. These results suggest that lipoxygenase products contribute to the phosgene-induced lung damage. Because phosgene exposure did not increase cyclooxygenase synthesis or pulmonary arterial pressure, we tested whether phosgene affects the lung's ability to generate or to react to thromboxane. Infusing arachidonic acid increased thromboxane synthesis to the same extent in phosgene-exposed lungs as in control lungs; however, phosgene exposure significantly reduced pulmonary vascular reactivity to thromboxane but not to angiotension II and KCl.     

Hydroperoxide-induced chemiluminescence in rabbit lungs: role of arachidonic acid enzymes

Low-level chemiluminescence (C) is thought to be an index of oxidant stress. We measured the relationship between low-level C, pulmonary arterial pressure, and perfusate concentration of thromboxane B2 (TxB2) in isolated perfused rabbit lungs during challenge with tert-butyl hydroperoxide (t-bu-OOH). We also measured glutathione release as another index of oxidant stress. We found that C was correlated with each variable, suggesting that oxidant stress measured by C and by glutathione release stimulated TxB2 production and pulmonary vasoconstriction. We also investigated the contribution of active O2 metabolites produced by prostaglandin (PG) peroxidase to oxidant stress by studying the effects of t-bu-OOH before and after the use of cyclooxygenase and lipoxygenase inhibitors. We found that C was augmented after inhibition, perhaps due to metabolism of t-bu-OOH by peroxidases of both arachidonic acid (AA) metabolic pathways in the absence of their normal substrates. We studied phenylbutazone, thought to inhibit peroxidases, and AA. C during t-bu-OOH administration was not augmented after phenylbutazone and was markedly inhibited after AA administration perhaps because AA competes with t-bu-OOH. To further study the role of peroxidases we pretreated the lungs with the antioxidant dithiothreitol, which inhibits peroxidases involved in both the cyclooxygenase and lipoxygenase pathways. Dithiothreitol nearly abolished C produced by t-bu-OOH and also prevented the increased light caused by eicosatetrynoic acid. We directly tested the hypothesis that C occurred as a result of the interaction of t-bu-OOH and the cyclooxygenase and lipoxygenase enzymes; we measured C when t-bu-OOH was added to purified PGH2 synthase or soybean lipoxygenase. The combination of t-bu-OOH with PGH2 synthase or lipoxygenase led to C that was inhibited by dithiothreitol and by the antioxidant phenol. These results suggest that enzymes involved in AA metabolism can interact with t-bu-OOH and that the action of these enzymes on t-bu-OOH leads to C. The results may mean that lipid peroxides can indirectly contribute to tissue oxidant stress due to production of active O2 metabolites as by-products of their metabolism by AA peroxidases.     

Cyclooxygenase inhibition prevents PMA-induced increases in lung vascular permeability

The effect of cyclooxygenase inhibition in phorbol myristate acetate (PMA)-induced acute lung injury was studied in isolated constant-flow blood-perfused rabbit lungs. PMA caused a 51% increase in pulmonary arterial pressure (localized in the arterial and middle segments as measured by vascular occlusion pressures), a 71% increase in microvascular permeability (measured by the microvascular fluid filtration coefficient, Kf), and a nearly threefold increase in perfusate thromboxane (Tx) B2 levels. Cyclooxygenase inhibition with three chemically dissimilar inhibitors, indomethacin (10(-7) and 10(-6) M), meclofenamate (10(-6) M), and ibuprofen (10(-5) M), prevented the Kf increase without affecting the pulmonary arterial pressure increase or resistance distribution changes after PMA administration. The specific role of TxA2 was investigated by pretreatment with OKY-046, a specific Tx synthase inhibitor, or infusion of SQ 29548, a TxA2 receptor antagonist; both compounds failed to protect against either the PMA-induced permeability or the vascular resistance increase. These results indicate that cyclooxygenase-mediated products of arachidonic acid other than TxA2 mediate the PMA-induced permeability increase but not the hypertension.     

Cyclooxygenase inhibition prevents PMA-induced increase in pulmonary vascular permeability to albumin.

In a previous study, we demonstrated that phorbol myristate acetate-(PMA) induced injury in isolated blood-perfused rabbit lungs was characterized by increased pulmonary vascular resistance (PVR) and permeability to water as measured by fluid filtration coefficient (Kf). The Kf increase was prevented by pretreatment with three cyclooxygenase inhibitors, indomethacin, ibuprofen, and meclofenamate. Other studies have shown that PMA causes a decrease in pulmonary vascular surface area, probably due to the increase in arterial resistance. Measurement of Kf requires increased microvascular pressure, and therefore Kf estimates the permeability of the entire vascular bed. Thus the permeability of the flowing vessels may be overestimated by Kf. In this study, we chose to investigate the effect of PMA on vascular permeability to protein by measuring albumin leak. Because this measurement does not require a hydraulic stress, it is more likely to reflect the permeability of flowing vessels. PMA administration (5 x 10(-8) M) caused significant increases in both PVR and 125I-labeled bovine serum albumin leak. Cyclooxygenase inhibition with indomethacin, ibuprofen, or meclofenamate prevented the PMA-induced increase in albumin leak without affecting the PVR increase. These results suggest that cyclooxygenase-mediated products of arachidonic acid mediate the PMA-induced increase in vascular permeability to both water and protein.     

Thromboxane-induced pulmonary vasoconstriction: involvement of calcium

Infusion of tert-butyl hydroperoxide (t-bu-OOH) or arachidonic acid into rabbit pulmonary arteries stimulated thromboxane B2 (TxB2) production and caused pulmonary vasoconstriction. Both phenomena were blocked by cyclooxygenase inhibitors or a thromboxane synthase inhibitor. The increase in pulmonary arterial pressure caused by either t-bu-OOH or arachidonic acid infusion correlated with the concentration of TxB2 in the effluent perfusate. The concentration of TxB2 in the effluent perfusate, however, was always 10-fold greater after arachidonic acid infusion. In the rabbit pulmonary vascular bed lipoxygenase products did not appear involved in the vasoactive response to t-bu-OOH or exogenous arachidonic acid infusion. Calcium entry blockers or a calcium-free perfusate prevented the thromboxane-induced pulmonary vasoconstriction. Calmodulin inhibitors also blocked the pulmonary vasoconstriction induced by t-bu-OOH without affecting the production of TxB2 or prostacyclin. These results suggest that thromboxane causes pulmonary vasoconstriction by increasing cytosol calcium concentration.     

Effect of Bicuculline-Induced Status Epilepticus on Prostaglandins and Hydroxyeicosatetraenoic Acids in Rat Brain Subcellular Fractions

Abstract: Rat cerebrum, prelabeled in vivo by intraventric-ular injection of [1-¹⁴C]arachidonic acid, was used to assess cyclooxygenase and lipoxygenase reaction products in total homogenates, cytosol, synaptosomes, and microsomes. Effects of bicuculline-induced status epilepticus on arachi-donic acid metabolism in synaptosomes and microsomes were also measured. Lipoxygenase activity, resulting in the synthesis of hydroxyeicosatetraenoic acids (HETEs), and cyclooxygenase activity, resulting in the synthesis of prostaglandins (PGs), were measured by reverse-phase and normal-phase HPLC with flow scintillation detection. Endogenous lipoxygenase products in synaptosomes were identified by capillary gas chromatography-mass spectrometry. PGs and HETEs were detected in all subcellular fractions. The synaptosomal fraction showed the highest lipoxygenase activity, with 5-HETE, 12-HETE, and leukotriene B₄ as the major products. Following bicuculline-induced status epilepticus, endogenous free arachidonic acid and other fatty acids accumulated in synaptosomes, but not in microsomes. Incorporation of [1-^l4C]arachidonic acid into synaptosomal and microsomal phospholipids was decreased after bicuculline treatment. Bicuculline-induced status epilepticus resulted in increased synthesis of HETEs in synaptosomes. PG synthesis increased in the microsomal fraction. When [1-¹⁴C]arachidonic acid-labeled synaptosomes and microsomes were incubated for 1 h at 37°C the synthesis of eicosa-noids, particularly PGD₂, was increased significantly in bi-cuculline-treated rats, as compared with untreated rats. Depolarization (45 mM K⁺) of synaptosomes induced a loss of [1-¹⁴C]arachidonic acid from phosphatidylinositol, and increased the synthesis of PGD₂ and HETEs, an effect that was enhanced in bicuculline-treated rats. This study localizes changes in arachidonic acid metabolism and lipoxygenase activity resulting from bicuculline-induced status epilepticus in the brain subcellular fraction enriched in nerve endings.     

Lipoxygenase and cyclooxygenase blockade by BW755C does not prevent the secondary phase of septic pulmonary hypertension

The infusion of Group B beta hemolytic streptococci (GBS) in newborn animals generates a dual phase pulmonary hypertensive response. The initial, acute phase responds to cyclooxygenase or thromboxane inhibition, and appears to be thromboxane mediated. The second phase is characterized by a more moderate rise in pulmonary vascular resistance, accompanied by an increase in microvascular permeability. It has been speculated that this phase may be leukotriene mediated. In an attempt to clarify this, we have studied and compared the effects of the thromboxane synthetase inhibitor, Dazmegrel (DAZ), and the combined cyclooxygenase/lipoxygenase inhibitor, BW755C, on the cardiopulmonary hemodynamics of the secondary phase of GBS induced pulmonary hypertension in newborn piglets. Ten piglets were infused with GBS, and all animals developed a significant increase in pulmonary artery pressure (to 39 +/- 5 and 36 +/- 5 mmHg for DAZ and BW755C animals respectively). After one hour of GBS, either DAZ or BW755C was administered. Data were collected for another two hours following drug administration. GBS infusion was continued throughout. Both DAZ and BW755C were associated with transient, acute reductions in pulmonary artery pressure (to 22 +/- 5 and 22 +/- 8 mmHg, respectively). However, after 60 minutes, PAP again began to rise in both groups (PAP 30 +/- 5 and 30 +/- 11 mmHg respectively by 240 minutes). There were no differences between the groups at any time. These data do not support a significant role for lipoxygenase products in mediating the secondary phase of septic pulmonary hypertension.     

Action of arachidonic acid in ureteral-ligated kidney of the anesthetized canine: possible lipoxygenase activity

In anesthetized dogs 48 h after unilateral ureteral ligation, intra-arterial injection of arachidonic acid produced a transient increase followed by a prolonged decrease of resistance in the ureteral-ligated kidney; whereas, in the control kidney, only the prolonged decrease in resistance was observed in response to arachidonate. Indomethacin blocked not only the arachidonate-induced renal efflux of both immunoreactive 6-keto-prostaglandin F1 alpha and thromboxane B2 but also vasodilation in both kidneys. In contrast, the initial vasoconstriction in the obstructed kidney was not affected by pretreatment with the cyclo-oxygenase inhibitor. Infusion of 5,8,11,14-eicosatetraynoic acid, an inhibitor of lipoxygenase activity, into the ureteral-ligated kidney after indomethacin markedly reduced the initial vasoconstrictor response to arachidonate. These data demonstrate that vascular reactivity to arachidonic acid is altered in the ureteral-obstructed kidney and are consistent with the hypothesis that formation of lipoxygenase as well as cyclooxygenase derivatives may participate in the hemodynamic responses to arachidonic acid in this pathophysiologic model.     

Partial characterization of the PAF-induced soluble factors which mimic the activity of 'early pregnancy factor'

Platelet-activating factor (PAF) stimulated mouse spleen cells to release soluble factors (termed S2 factors) which were capable of inducing increased rosette inhibition titres when applied to fresh mouse spleen cells in the rosette inhibition assay. In this ability the S2 factors mimic that of pregnancy serum, an action previously ascribed to 'early pregnancy factor'. The PAF-stimulated production of these S2 factors was not influenced by inhibitors of cyclooxygenase metabolism, but was completely inhibited by the lipoxygenase inhibitors, diethyl carbamazine and nordihydroguaiaretic acid. The S2 factors had a lipid-like character in that they were extractable in organic solvents. The calcium ionophore A23187 also stimulated the production of these factors which may well be products of the lipoxygenase pathway of arachidonic acid metabolism.     

The effects of neutrophils and phospholipase A2 on transvascular albumin flux in isolated rabbit lungs

In this study, addition of phospholipase A2 (PLA2) to salt-perfused isolated rabbit lungs containing rabbit polymorphonuclear leukocytes leads to an increase in pulmonary capillary permeability. We add 1.5 X 10(8) polymorphonuclear leukocytes to the perfusate. Next, indomethacin is added to the perfusate and 40 units of PLA2 are infused into the pulmonary arterial inflow of the lungs. At the end of the study, a lung sample is removed for measurement of transvascular albumin flux using I125-albumin as a measure of the permeability-surface area product. Control studies demonstrate no increase in transvascular albumin flux. Addition of a dual cyclooxygenase and lipoxygenase inhibitor, BW755C, to the perfusate prevents the increase in transvascular albumin flux. We conclude that PLA2 interacts with polymorphonuclear leukocytes to increase protein permeability. Since PLA2 can release endogenous arachidonic acid and platelet-activating factor from cells, this suggests that release of such products may contribute to an increase in pulmonary capillary permeability from polymorphonuclear leukocytes. The ability of BW755C to prevent the increase suggests the possibility that lipoxygenase products contribute.     

Arachidonic Acid Metabolites in bFGF-, PDGF-, and Serum-Stimulated Vascular Cell Growth

The signal transduction pathways through which growth factors regulate vascular cell growth are not fully understood. Recent studies suggest that metabolites of the lipoxygenase pathway may be involved in vascular cell growth. We have measured the effect of the lipoxygenase pathway inhibitors nordihydroguiaretic acid (NDGA), 5,6-dehydroarachidonic acid, and baicalein on bovine capillary endothelial cell (EC) and aortic smooth muscle cell (SMC) growth in the presence or the absence of growth factors. NDGA totally suppressed serum-stimulated EC and SMC growth as well as growth factor-stimulated proliferation over a 9-day time course. Removal of the inhibitor revealed that the inhibitory effect of NDGA was reversible and not due to cytotoxicity. The morphology of NDGA-treated EC was changed in a reversible manner from the characteristic polygonal to spindle shape. The 5-lipoxygenase inhibitor 5,6-dehydroarachidonic acid had no effect on vascular cell proliferation, but inhibition of 12-lipoxygenase with baicalein blocked both EC and SMC cell growth in a dose-dependent manner, in the presence and the absence of growth factors. Indomethacin, an inhibitor of the cyclooxygenase pathway, had no effect on EC and SMC proliferation. Quinacrine and oleyloxyethylphosphorycholine inhibition of the phospholipase A₂-catalyzed release of arachidonic acid from membrane phospholipids blocked growth factor- and serum-stimulated proliferation of EC and SMC. These results suggested that arachidonic acid metabolites are critical intermediaries in the regulation of vascular cell growth.     

Comparative evaluation of the action of prostanoid inhibitors on uterine contractile function

Indomethacin and substance BW-755C in experiments on isolated myometrium striae of pregnant white rats exert an inhibiting effect on the contractile uterus function due to inhibition of cyclooxygenase or lipoxygenase ways of the arachidonic acid transformation. Prostaglandin F2 alpha is sensitive to functioning of the cyclooxygenase and lipoxygenase ways of the arachidonic acid transformation, while oxytocin--only lipoxygenase one. Conclusions rest on results from multiparametric analysis of the contractile uterus function suggested by authors and confirmed by the pattern recognition method--the Karunen-Loev orthogonal decomposition.     

Platelet lipoxygenase-dependent oxygen burst. Evidence for differential activation of lipoxygenase in intact and disrupted human platelets

The metabolism of arachidonic acid in platelets by both cyclooxygenase and lipoxygenase involves the rapid consumption of molecular oxygen. However, selective inhibition of cyclooxygenase completely abolishes the arachidonate-induced oxygen burst in intact platelets. This is in contrast to platelet lysates, in which approximately 50% of the arachidonate-induced oxygen burst remains detectable following inhibition of cyclooxygenase with acetylsalicylic acid. This lipoxygenase oxygen burst is blocked by preincubation of the platelets with ETYA, which inhibits both cyclooxygenase and lipoxygenase. In cell-free 100000 x g supernatants of platelet lysates, which contain only lipoxygenase activity, arachidonate induces an oxygen burst which is not blunted by preincubation with aspirin but is completely abolished by preincubation with ETYA. The finding of a lipoxygenase-dependent oxygen burst in platelet lysates but not in intact platelet suspensions suggests differential activation or differential availability of platelet lipoxygenase in intact and disrupted platelets. This was confirmed by a 5 min lag in the generation of [14C]HETE (the major lipoxygenase product) from [14C]arachidonic acid in intact platelets, but an almost immediate initiation of [14C]HETE production in platelet lysates. In contrast, the synthesis of [14C]thromboxane B2 (the major cyclooxygenase product) from [14C]arachidonic acid began immediately in both intact and disrupted platelet preparations and peaked within 5 min. These observations provide new insight into factors controlling platelet hydroxy acid production and help to explain the nature of the platelet oxygen burst.     

On the role of arachidonic acid metabolites in mast-cell mediated mitogenesis in the rat

We investigated whether the mitogenic response induced by local mast-cell secretion in the rat mesentery was affected by suppression of phospholipase A2, lipoxygenase, or cyclooxygenase in arachidonic acid metabolism. Enzyme inhibitor was given in a single intravenous dose 5 min before intraperitoneal injection of the mast-cell secretagogue 48/80. Mepacrine, a phospholipase A2 inhibitor, suppressed the generation of both leukotrienes (SRS) and prostaglandins (PG), whereas the lipoxygenase inhibitor BW 755C reduced the generation of SRS, and the cyclooxygenase inhibitor indomethacin significantly suppressed the generation of PG. None of the enzyme inhibitors affected the basal mesenteric histamine content or histamine release in the mesentery after exposure to 48/80, and none of them affected mast-cell-mediated mitogenesis in the mesentery as judged by specific DNA activity and mitosis counting. The stimulation of DNA synthesis and mitosis initiated by secreting mast cells is apparently not mediated or modulated by synthesis of leukotrienes, prostaglandins, or other known arachidonic acid metabolites.     

Relation between arachidonic acid metabolism and development of thymocytes in fetal thymic organ cultures

Because products of arachidonic acid metabolism, particularly the PG, have been implicated as modulators of growth and differentiation of adult thymocytes, we investigated relations between metabolism of arachidonic acid and growth, as well as differentiation, of thymocytes during fetal thymic organ culture. Fetal thymic cells synthesized immunoreactive PGE2 during organ culture and were found to be capable of metabolizing exogenous arachidonic acid to products that cochromatographed with authentic 6-keto-PGF1 alpha, PGE2, PGF2 alpha. Synthesis of these products and growth and expression of Thy-1 and Lyt-1 Ag were inhibited by culture of fetal thymic lobes with indomethacin, a cyclooxygenase inhibitor, as well as meclofenamate and eicosatetraynoic acid, inhibitors of cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism. Only indomethacin inhibited expression of Lyt-2. Culture with eicosatetraynoic acid also inhibited the capacity of thymic lobes to synthesize 15-hydroxyeicosatetraenoic acid-like products. The inhibitory effects of indomethacin on growth and expression of Thy-1 were partially reversed by simultaneous addition of arachidonic acid. Thus, fetal thymic cells appear to require an intact cyclooxygenase, and possibly lipoxygenase, pathway of arachidonic acid metabolism for growth and differentiation. These data also provide evidence that Lyt-1 and Lyt-2 may be regulated by different requirements with respect to arachidonic acid metabolism.