Aspirin inhibits arachidonic acid metabolism via lipoxygenase and cyclo-oxygenase in hamster isolated lungs

The effect of aspirin on the fate of exogenous arachidonic acid (AA) was investigated in isolated perfused lungs of female hamsters. During pulmonary infusion of aspirin (10 μM, 100 μM or 1 mM) 45 nmol of ¹⁴C-AA was infused in two minutes into the pulmonary circulation. The nonrecirculating perfusion effluent was collected for 6 minutes after the beginning of the AA infusion. Arachidonate infusion increased the perfusion pressure. This pressor response was completely abolished by 1 mM aspirin. When aspirin was infused into the pulmonary circulation, the amount of radioactivity was increased in the perfused lungs and decreased dose dependently in the nonrecirculating perfusion effluent. The amount of unmetabolized free arachidonate was not changed significantly by aspirin in the perfused lungs or in the perfusion effluent. In the effluent the amounts of all arachidonate metabolites, which were extracted with ethyl acetate first at pH 7.4 and then at pH 3.5 and analysed by thin layer chromatography, were decreased quite similarly by aspirin. The formation of arachidonate metabolites was completely inhibited by 1 mM aspirin. In the perfused lung tissue the amount of ¹⁴C-AA was increased by aspirin in phospholipids and neutral lipids. The present study indicates that the metabolism of arachidonic acid is inhibited by aspirin in hamster lungs not only via cyclo-oxygenase but also via other lipoxygenases.     

The metabolism of arachidonic acid in isolated perfused fetal and neonatal rabbit lungs

The developmental pattern of fetal and neonatal rabbit lungs to metabolize arachidonic acid (AA) to different cyclo-oxygenase products was studied in isolated rabbit lungs, which were perfused with Krebs bicarbonate buffer. ¹⁴C-AA (66 nmol) was injected into the pulmonary circulation and the nonrecirculating perfusion effluent was collected for four minutes. About ten per cent of the injected radioactivity was found in the 0–4 min perfusion effluent. The metabolites of AA in the effluent were analyzed by thin layer chromatography. The major metabolites of AA were PGE₂ and its 15-keto-derivates, but also PGF_2α and its 15-keto-derivates, TXB₂ and 6-keto-PGF_1α were found in the effluent. The most drastic developmental change was the increase in the amount of 15-keto-metabolites of PGE₂ from late fetal period to the lungs of one day old rabbits (1.8 fold increase between birth and first postnatal day). Smaller changes were detected in the amounts of other cyclo-oxygenase products.     

The metabolism of arachidonic acid in isolated perfused fetal and neonatal rabbit lungs

The developmental pattern of fetal and neonatal rabbit lungs to metabolize arachidonic acid (AA) to different cyclo-oxygenase products was studied in isolated rabbit lungs, which were perfused with Krebs bicarbonate buffer. 14C-AA (66 nmol) was injected into the pulmonary circulation and the nonrecirculating perfusion effluent was collected for four minutes. About ten per cent of the injected radioactivity was found in the 0-4 min perfusion effluent. The metabolites of AA in the effluent were analyzed by thin layer chromatography. The major metabolites of AA were PGE2 and its 15-keto-derivates, but also PGF2 alpha and its 15-keto-derivates, TXB2 and 6-keto-PGF1 alpha were found in the effluent. The most drastic developmental change was the increase in the amount of 15-keto-metabolites of PGE2 from late fetal period to the lungs of one day old rabbits (1.8 fold increase between birth and first postnatal day). Smaller changes were detected in the amounts of other cyclo-oxygenase products.     

The effect of cigarette smoke on the metabolism of arachidonic acid in isolated hamster lungs

The effects of cigarette smoke on the metabolism of exogenous arachidonic acid (AA) were investigated in isolated hamster lungs. Arachidonate was injected into the pulmonary circulation and the metabolites were analysed from the nonrecirculating perfusion effluent by thin layer chromatography. After the pulmonary injection of 66 nmol of ¹⁴C-AA about 20 % of the injected radioactivity appreated in the perfusion effluent mostly as metabolites in six minutes. When isolated lungs were ventilated with cigarette smoke during the perfusion, the amounts of PGF_2α, PGE₂ and two unidentified metabolite groups increased in the lung effluent. In two other experimental series hamsters were exposed to cigarette smoke before the lung perfusion either once for 30 min or during one hour daily for ten consecutive days. Neither pre-exposures caused any changes in the amounts of arachidonate metabolites in the lung effluent.     

The metabolism of arachidonic acid to an antiaggregatory compound in isolated lungs of fetal and neonatal rabbits

The developmental pattern of fetal and neonatal rabbit lungs to generate an antiaggregatory compound from arachidonic acid (AA) was studied in isolated rabbit lungs, which were perfused with Krebs bicarbonate buffer. The antiaggregatory effect of the nonrecirculating perfussion effluent was tested by adding a small portion of the effluent to human platelet rich plasma (PRP) in a Born-type aggregometer before the aggregation was induced by ADP. The production of an antiaggregatory compound was minimal, when exogenous AA was not infused into the pulmonary circulation. When arachidonate (40 nmol/min) was infused into the pulmonary circulation of rabbits which were 1 day or 1 week old, the perfusion effluent significantly inhibited the ADP induced aggregation of PRP. Perfused lungs from fetal rabbits (gestation age 28–31 days) formed also an antiaggregatory compound fro AA, but the antiaggregatory effect was not as great as 1 day after birth. It seems that neonatal rabbit lungs metabolize AA more to an antiaggregatory compound than late fetal lungs. The fact that the AA induced production of an antiaggregatory compound is inhibited by simultaneous infusion of indomethacin favours the hypothesis that this antiaggregatory compound could he PGI₂.     

The metabolism of prostaglandin E2 in perinatal rabbit lungs

The inactivation of prostaglandin E₂ (PGE₂) was studied in isolated perfused lungs of fetal and neonatal rabbits. 200 nmol of ¹⁴C-PGE₂ was infused into the pulmonary circulation and the metabolites of PGE₂ were analysed from the nonrecirculating perfusion effluent. The amount of the main metabolite, 13,14-dihydro-15-keto-PGE₂, increased significantly between the 28th and 30th day of fetal life, remained relatively constant at the time of birth and increased again between 1st and 7th postnatal day. In contrast the amount of 15-keto-PGE₂ remained relatively stable during the studied period. The activity of NAD⁺-dependent 15-hydroxyprostaglandin dehydrogenase (15-OH-PGDH) was determined from the 100.000 g supernatant fraction of fetal, neonatal and maternal rabbit lungs using ¹⁴C-PGE₂ (20 μM) as the substrate. In the lungs of late fetal rabbits the activity of 15-OH-PGDH was significantly higher compared to the early postnatal period. Maternal rabbit lungs possessed, however, very high activities compared to the studied perinatal lungs. The results show, that the activity of the pulmonary 15-OH-PGDH is high already during the late fetal period. The inactivation of PGE₂ in isolated perfused lungs seems, however, to increase during the last prenatal days. Thus it seems possible that the uptake mechanism could be the rate limiting step in the metabolism of PGE₂ in rabbit lungs during the perinatal period.     

Prostacyclic production in rabbit arteries in situ: Inhibition by arachidonic acid-induced endothelial cell damage or by low-dose aspirin

The central artery of the rabbit ear was perfused in situ and effluent fractions from the artery were assayed for 6-keto-prostaglandin F_1α (6-K-PGF_1α) and thromboxane B₂ (TxB₂), the stable metabolites of prostacyclin (PGI₂) and TxA₂, using specific radioimmunoassays. These metabolites of arachidonic acid (AA) were not detected in the effluent during infusion of Tyrode's solution but both metabolites were detected when small amounts of AA were infused into the artery. Examination of the arteries by scanning electron microscopy revealed that high concentrations of AA which caused a short burst of 6-K-PGF_1α and TxB₂ production damaged the endothelial cells while lower concentrations which stimulated continuous production did not cause damage. When a non-damaging concentration of AA was infused into an artery that the previously received a damaging concentration, PG production was greatly reduced. Pretreatment of the rabbits with 4 mg/kg acetyl-salicyclic acid (ASA) inhibited 6-K-PGF_1α production by the rabbit ear artery in response to AA and 70% inhibition was still evident 18 hours after ASA.     

Placental Transfer of Lactate,Glucose and 2-deoxyglucose in Control and Diabetic Wistar Rats

Placental transfer of lactate, glucose and 2-deoxyglucose was examined employing the in situ perfused placenta. Control and streptozotocin induced diabetic Wistar rats were infused with [U¹⁴C]-glucose and [³H]-2-deoxyglucose (2DG). The fetal side of the placenta was perfuseci with a cell free medium and glucose uptake was calculated in the adjacent fetuses. Despite the 5-fold higher maternal plasma glucose concentration in the diabetic dams the calculated fetal glucose metabolic index was not significantly different between the 2 groups. Placental blood flow was reduced in the diabetic animals compared with controls but reduction of transfer of [U¹⁴C]-glucose and [³H]-2-deoxyglucose and endogenously derived [¹⁴C]-Lactate to the fetal compartment, could not be accounted for by reduced placental blood flow alone. There was no significant net production or uptake of lactate into the perfusion medium that had perfused the fetal side of the placenta in either group. The plasma lactate levels in the fetuses adjacent to the perfused placenta were found to be higher than in the maternal plasma and significantly higher in the fetuses of the diabetic group compared with control group. In this model the in situ perfused placenta does not secrete significant quantities of lactate into the fetal compartment in either the control or diabetic group.     

The effect of dietary eicosapentaenoic acid on arachidonic acid incorporation and metabolism in rat leukocytes

Arachidonic Acid (AA) released from membrane phospholipids by phospholipase A₂ during cell activation is the major polyunsaturated fatty acid precursor in mammals for the cyclooxygenase and lipoxygenase pathways. Eicosaspentaenoic acid (EPA), a major polyunsaturated fatty acid in fish oils competes with AA for these enzymes. The resulting products from EPa are generally less potent than the corresponding AA metabolites which may explain the beneficial effects of this oil in reducing thrombotic and inflammatory responses. This study compares the incorporation of ¹⁴C-AA into leukocyte phospholipids and its release and metabolism by the cyclooxygenase and lipoxygenase pathways in rats fed a ‘Max EPA’ fish oil rich diet (EPA group) and a hydrogenated coconut/safflower oil control diet. More than 75% of radiolabel was incorporated into leukocytes with no difference seen between dietary groups. Upon stimulation with calcium ionophore, the EPA group released significantly more radiolabelled AA than the control group. The EPA diet showed a significant increase in the formation of 5-hydroxyeicosatetraenoic acid and 6-keto-prostaglandin F_1α but no difference was seen in leukotriene B₄ formation. The majority of radiolabel released was free AA, this being significantly higher in the EPA grou than in the control. The percentage of radiolabel remaining after stimulation in phosphatidylglycerol, phosphatidylethanolamine and neutral lipids was significantly less in EPA fed rats. As the release and metabolism of endogenous AA may not be the same as ¹⁴C-AA, these results do not necessarily indicate that the mass of AA available for eicosanoid biosynthesis has been altered by the EPA diet.     

Metabolism of C-arachidonate in rat isolated lung

Metabolism of ¹⁴C-arachidonate was investigated in rat isolated lungs perfused via the pulmonary circulation with Krebs solution. Only 10% of the radioactivity derived from an infusion of ¹⁴C-arachidonate through the pulmonary circulation of rat isolated lungs appeared in the effluent by 10 minutes. At 10 min, the major component of effluent radioactivity and 20–40% of that retained in lung was unchanged arachidonate. Between 10 and 20 min of perfusion, a further small amount of radioactivity was lost in lung effluent and at 20 min the retained radioactivity showed a decrease in the proportion present as free arachidonate. Between 20 and 60 min, there was no further loss of radioactivity in effluent and no further change in the distribution in lung. Addition of albumin to the Krebs solution perfusate during the infusion of ¹⁴C-arachidonate increased effluent radioactivity to 80%, but albumin added after 10 min only caused the efflux of a small amount of radioactivity (10%). Treatment of labelled lung at 20 min with the calcium ionophore A23187 released biologically active metabolites of arachidonate but very little radioactivity. Metabolism of arachidonate, either during the infusion or after retention in lung, in rat lung was closer to that in human lung than to that in guinea-pig lung.     

Metabolism of 14C-arachidonate in rat isolated lung

Metabolism of ¹⁴C-arachidonate was investigated in rat isolated lungs perfused via the pulmonary circulation with Krebs solution. Only 10% of the radioactivity derived from an infusion of ¹⁴C-arachidonate through the pulmonary circulation of rat isolated lungs appeared in the effluent by 10 minutes. At 10 min, the major component of effluent radioactivity and 20–40% of that retained in lung was unchanged arachidonate. Between 10 and 20 min of perfusion, a further small amount of radioactivity was lost in lung effluent and at 20 min the retained radioactivity showed a decrease in the proportion present as free arachidonate. Between 20 and 60 min, there was no further loss of radioactivity in effluent and no further change in the distribution in lung. Addition of albumin to the Krebs solution perfusate during the infusion of ¹⁴C-arachidonate increased effluent radioactivity to 80%, but albumin added after 10 min only caused the efflux of a small amount of radioactivity (10%). Treatment of labelled lung at 20 min with the calcium ionophore A23187 released biologically active metabolites of arachidonate but very little radioactivity. Metabolism of arachidonate, either during the infusion or after retention in lung, in rat lung was closer to that in human lung than to that in guinea-pig lung.     

Hydrocortisone selectively inhibits IgE-dependent arachidonic acid release from rat peritoneal mast cells

Purified rat mast cells were used to study the effects of anti-inflammatory steroids on the release of [1-¹⁴C]-arachidonic acid ([1-¹⁴C]AA) and metabolites. Mast cells were incubated overnight with glucocorticoids, [1-¹⁴C]AA incorporated into cellular phospholipids and the release of [1-¹⁴C]AA, and metabolites determined using a variety of secretagogues. Release of [1-¹⁴C]AA and metabolites by concanavalin A, the antigen ovalbumin and anti-immunoglobulin in E antibody was markedly reduced by glucocorticoid treatment. Neither the total incorporation of [1-¹⁴C]AA nor the distribution into phospholipids was altered by hydrocortisone pretreatment. Glucocorticoid pretreatment did not alter [1-¹⁴C]AA release stimulated by somatostatin, compound 48/80, or the calcium ionophore, A23187. These data indicate that antiinflammatory steroids selectively inhibit immunoglobulin dependent release of arachidonic acid from rat mast cells. These findings question the role of lipomodulin and macrocortin as general phospholipase inhibitors and suggest that they may be restricted to immunoglobulin stimuli.     

Atherosclerosis decreased prostacyclin formation in rabbit lungs and kidneys

Metabolism of arachidonic acid (AA) was studied in perfused lungs and kidneys of normal and atherosclerotic rabbits by determination of PGE₂, PGF_2α and the stable metabolites of PGI₂ (6-keto-PGF_1α) and TXA₂ (TXB₂). PGI₂ was the main AA metabolite formed by normal lungs and kidneys. Atherosclerosis reduced the formation of PGI₂ by about 50 % in both organs. TXA₂ formation was similarily decreased in lungs. In kidneys, the decrease in PGI₂ formation was accompanied by an increase in PGE₂ formation.     

Gestational diabetes mellitus decreases placental uptake of long-chain polyunsaturated fatty acids: involvement of long-chain acyl-CoA synthetase

The long-chain polyunsaturated fatty acids (LC-PUFAs) arachidonic (AA) and docosahexaenoic (DHA) acids are essential for fetal development. Gestational diabetes mellitus (GDM) is a pregnancy disorder associated with perinatal and lifelong risk complications for both the mother and the newborn. Our aim was to investigate the influence of GDM, and some of its associated conditions, upon the placental uptake of AA and DHA. Uptake of ¹⁴C-AA and ¹⁴C-DHA by human trophoblasts obtained from normal pregnancies (NTB cells) was mediated by both saturable (for lower substrate concentrations) and non-saturable (for higher substrate concentrations) mechanisms. Uptake of both fatty acids was inhibited by other LC-PUFAs and, markedly, by the long-chain acyl-CoA synthetase (ACSL) inhibitor, triacsin C. Human trophoblasts obtained from GDM pregnancies (DTB cells) showed a significantly lower ¹⁴C-AA and ¹⁴C-DHA accumulation, through a decrease in both the saturable and the non-saturable components of uptake, which was associated with a decrease in ACSL1 mRNA levels. Uptake of LC-PUFAs by NTB cells increased (by 20–25%) after short-term exposure to TNF-α (¹⁴C-AA and ¹⁴C-DHA) and insulin (¹⁴C-DHA). In conclusion, GDM, distinctly from its associated conditions, markedly decreases placental uptake of LC-PUFAs, which probably contributes to the deleterious effects of this disease for the newborn.     

Effect of radiation on the biosynthesis of L-Tryptophan from 14C-Anthranilic acid in kohlrabi (Brassica oleracea L., var.gongylodes L.)

The metabolism of¹⁴C-anthranilic acid (¹⁴C-AA) in kohlrabi (Brassica oleracea L. var.gongylodes L.) and the effect of radiation gamma⁶⁰Co on this metabolism was investigated. In hypocotylar segmnents of seven days old etiolated seedlings¹⁴C-AA was metabolised par, tially to its detoxication product¹⁴C-β-glucoside of AA. Simultaneously L-tryptophan was also formed, which in these plants is a precursor of indolic glucosinolates glucobrassicin and neoglucobrassicin. The metabolism of¹⁴C-AA was followed for 97 h. Radiation, applied both to seeds and to seven days old plants did not affect the metabolism of¹⁴C-AA substantially. The intermediary reaction AA → L-tryptophan in the biosynthesis of L-tryptophan is not a radiosensitive part of the synthesis of this amino acid. A not too high radiation sensitivity (max. 45%) was observed in the metabolic pathway leading from L-tryptophan to glucobrassicin.     

Pulmonary uptake of PGE2 is inhibited by dipyridamole in rat isolated lungs

The metabolism of prostaglandin E₂ (PGE₂) is decreased by dipyridamole (20 μM) in rat isolated perfused lungs. The inhibition of the metabolism is reversible as the decreased metabolism returned to the control level when pulmonary infusion of dipyridamole was abolished. After pulmonary injection of ¹⁴C-PGE₂ (10 nmol) the radioactivity appeared more rapidly in the effluent when dipyridamole was infused into pulmonary circulation. Dipyridamole in vitro did not change the activity of 15-hydroxyprostaglandin dehydrogenase (15-OH-PGDH) in the 100, 000 × g supernatant fraction of homogenized lungs. Thus, the decreased metabolism seems to be due to the inhibition of the uptake of PGE₂ into the lungs. When the rats were pretreated with dipyridamole in drinking water for one week the activity of 15-OH-PGDH in the 100, 000 × g supernatant fraction of the lungs was not changed significantly.     

Mobilization of arachidonic acid in collagen-stimulated human platelets.

Stimulation of platelets with collagen results in the mobilization of arachidonic acid (AA) from phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI). In this study the effect of aspirin, indomethacin, BW755C and prostaglandin H2 (PGH2) on labelled AA release in response to varied concentrations of collagen was investigated. Our results indicate that aspirin (0.56 mM) and indomethacin (5.6 microM) not only inhibited the collagen-mediated formation of cyclo-oxygenase metabolites, but also caused a significant reduction in the accumulation of free labelled AA and 12-hydroxyeicosatetraenoic acid (12-HETE) (21-64%). Aspirin and indomethacin also inhibited the release of [3H]AA from PC (37-75%) and PI (33-63%). The inhibition of AA release caused by aspirin was reversed partially by PGH2 (1 microM). In contrast, a smaller/no inhibition of collagen-stimulated labelled AA and 12-HETE accumulation (0-11%) and of collagen-stimulated AA loss from PC and PI was observed in the presence of BW755C. The results obtained in the presence of aspirin, indomethacin and BW755C at lower concentrations of collagen further demonstrate that AA release from PI (45-61% inhibition at 10 micrograms of collagen), but not from PC, was affected by the inhibition of cyclo-oxygenase. The results obtained on the effect of PGH2 further support that deacylation of phospholipids occurs independently of cyclo-oxygenase metabolites, particularly at higher concentrations of collagen. These results also demonstrate that aspirin and indomethacin, but not BW755C, cause a direct inhibition of collagen-induced [3H]AA liberation from PC as well as from PI. We also conclude that the diacylglycerol lipase pathway is a minor, but important, route for AA release from PI in collagen-stimulated human platelets. The mechanisms underlying the regulation of AA release by collagen in the absence of cyclo-oxygenase metabolites are not clear.     

Arachidonic acid metabolites induced β-adrenoceptor densitization in rat lung in vitro

The possible involvement of arachidonic acid (AA) or its metabolites in β-adrenoceptor desensitization has been studied in rat lung parenchyma both from a functional and a biochemical point of view. In vitro perfusion of rat lungs with AA (3×10^?5M for 20 min) reduced the relaxant effect of isoproterenol (ISO) on lung parenchymal strips, shown by a shift to the right of ISO dose-response curve, similar to that obtained using desensitizing concentration of specific β-agonist. Moreover, AA treatment reduced the capacity of ISO to stimulate adenylate-cyclase activity, whereas the number of β-receptor binding sites was not significantly modified. Inhibition of cyclo-oxygenase pathway by indomethacin (INDO) (1.5 × 10^?5M) prevented both the loss of ISO-relaxing capacity and the decrease of adenylate-cyclase activity induced by AA treatment. In order to support the role of eicosanoids in β-adrenoceptor desensitization, changes of endogenous free AA levels have also been studied in lung homogenates. Perfusion of rat lung with ISO (10^?6M for 20 min) decreased by about 50% the levels of free AA and the pretreatment with BW755C (9×10^?5M), a lipo- and cyclo-oxygenase inhibitor, prevented this phenomenon. On the basis of these results, we suggest that the activation of AA cascade is actually involved in β-adrenoceptor desensitization in lung tissues with a possible interference at the site beyond the drug-receptor interaction.     

Atherosclerosis decreased prostacyclin formation in rabbit lungs and kidneys.

Metabolism of arachidonic acid (AA) was studied in perfused lungs and kidneys of normal and atherosclerotic rabbits by determination of PGE2, PGF2 alpha and the stable metabolites of PGI2 (6-keto-PGF1 alpha) and TXA2 (TXB2). PGI2 was the main AA metabolite formed by normal lungs and kidneys. Atherosclerosis reduced the formation of PGI2 by about 50 % in both organs. TXA2 formation was similarily decreased in lungs. In kidneys, the decrease in PGI2 formation was accompanied by an increase in PGE2 formation.     

Effects of arachidonic acid and prostaglandin F2 alpha in isolated rat lungs

Isolated rat lungs were ventilated and perfused by saline-Ficoll perfusate at a constant flow. The baseline perfusion pressure (PAP) correlated with the concentration of 6-keto-PGF1 alpha the stable metabolite of PGI2 (r = 0.83) and with the 6-keto-PGF1 alpha/TXB2 ratio (r = 0.82). A bolus of 10 micrograms exogenous arachidonic acid (AA) injected into the arterial cannula of the isolated lungs caused significant decrease in pulmonary vascular resistance (PVR) which was followed by a progressive increase of PVR and edema formation. Changes in perfusion pressure induced by AA injection also correlated with concentrations of the stable metabolites (6-keto-PGF1 alpha: r = -0.77, TxB2: -0.76), and their ratio: (6-keto-PGF1 alpha/TXB2: r = -0.73). Injection of 10 and 100 micrograms of PGF2 alpha into the pulmonary artery stimulated the dose-dependent production of TXB2 and 6-keto-PGF1 alpha. No significant correlations were found between the perfusion pressure (PAP) which was increased by the PGF2 alpha and the concentrations of the former stable metabolites. The results show that AA has a biphasic effect on the isolated lung vasculature even in low dose. The most potent vasoactive metabolites of cyclooxygenase, prostacyclin and thromboxane A2 influence substantially not only the basal but also the increased tone of the pulmonary vessels.