Metabolism Of Exogenous Arachidonic Acid Research Articles

Restricted diet modifies carbohydrate metabolism in immature rats

The aim of the present work was to study the effect of a restricted diet on carbohydrate metabolism in submandibular glands of female prepuber rats and the influence of arachidonic acid derivatives. Rats of 21 days of age were divided into three groups. Group I: normally fed rats. Group II: restricted diet (50% of the normal food intake). Group III: normally restricted diet with re-feeding. The baseline concentration of tissue glycogen was significantly higher in Group II than in I or III and after 60 min of incubation in a glucose free medium all groups showed a glycogen drop. In addition, the glucose metabolism was increased in Group II. Cycloxigenase inhibitors failed to alter 14CO 2 levels in Groups I and III. In Group II, indomethacin and acetylsalicylic acid inhibited glucose metabolism, which was reverted by PGE 2 addition. The exogenous arachidonic acid metabolism and different eicosanoids showed that restricted diet significantly increased the production of PGE 2 but diminished PGF 2 α production. Our results suggest that a restricted diet would lead to a new dynamic equilibrium of glucose homeostasis. Prostaglandins E 2 and F 2 α would participate by adapting the source of energy to tissue demands while maintaining the metabolic features that characterize puberty.

Upregulation of 8-Lipoxygenase in the Dermatitis of IκB-α-Deficient Mice

Neonatal mice deficient in IkappaB-alpha, an inhibitor of the ubiquitous transcription factor NF-kappaB, develop severe and widespread dermatitis shortly after birth. In humans, inflammatory skin disorders such as psoriasis are associated with accumulation in the skin of the unusual arachidonic acid metabolite 12R-hydroxyeicosatetraenoic acid (12R-HETE), a product of the enzyme 12R-lipoxygenase. To examine the etiology of the murine IkappaB-alpha-deficient skin phenotype, we investigated the expression of lipoxygenases and the metabolism of exogenous arachidonic acid in the skin. In the IkappaB-alpha-deficient animals, the major lipoxygenase metabolite was 8S-HETE, formed together with a minor amount of 12S-HETE; 12R-HETE synthesis was undetectable. Skin from the wild-type littermates formed 12S-HETE as the almost exclusive lipoxygenase metabolite. Upregulation of 8S-lipoxygenase (8-LOX) in IkappaB-alpha-deficient mice was confirmed at the transcriptional and translational level using ribonuclease protection assay and western analysis. In immunohistochemical studies, increased expression of 8-LOX was detected in the stratum granulosum of the epidermis. In the stratum granulosum, 8-LOX may be involved in the terminal differentiation of keratinocytes. Although mouse 8S-lipoxygenase and human 12R-lipoxygenase are not ortholog genes, we speculate that in mouse and humans the two different enzymes may fulfill equivalent functions in the progression of inflammatory dermatoses.

Effect of fasting on the contractile activity and metabolism of labelled glucose and arachidonic acid in uteri isolated from intact and ovariectomized rat

The effects of fasting for 4 days on the isometric developed tension (IDT) and on the metabolism of labelled glucose and arachidonic acid in uteri from intact and spayed (25 days) rats, were explored. Starvation produces a fall in the contractile activity of intact rats, while in ovariectomized ones, no differences can be see with respect to their controls. Fasting produces a fall in the glucose metabolism of both intact and ovariectomized rats, being more noticeable in the former group. Indomethacin (5 × 10 −6 M) increases the metabolism of labelled glucose in all experimental groups, significantly. The metabolism of exogenous arachidonic acid into different eicosanoids, PGE 2, PGF 2α, 6-keto-F 1α and TXB 2, shows that total food deprivation diminishes significantly the production of PGE 2 in intact rats. In contrast, in ovariectomized starved rats, PGE 2 increases markedly. The rest of the metabolites studied are not influenced by fasting. These results show that the effects of fasting on the contractile activity and on the release of some metabolites from arachidonic acid by the uteri isolated from intact rats are not seen in ovariectomized animals.

Increases in 5-lipoxygenase activating protein expression account for enhanced capacity for 5-lipoxygenase metabolism that accompanies differentiation of peripheral blood monocytes into alveolar macrophages.

The capacity for 5-lipoxygenase (5-LO) metabolism of endogenous arachidonic acid (AA) is greater in alveolar macrophages (AM) than in their circulating precursors, peripheral blood monocytes (PBM); however, the ability of PBM to metabolize exogenous AA to 5-LO products is comparable to that of AM. In the present study, we examined the enzymatic mechanisms by which 5-LO metabolism of AA is altered during differentiation of PBM in the lung. Resting human AM exhibited greater steady-state levels of 5-LO (7-fold) and LTA4 hydrolase (2-fold) proteins than autologous PBM; moreover, they differed from PBM in that they contained a significant amount of 5-LO associated with the particulate fraction. Importantly, AM contained 40-fold more 5-lipoxygenase activating protein (FLAP) than did PBM, which correlated well with the relative abilities of intact AM and PBM to metabolize endogenous AA to leukotrienes. The FLAP inhibitor MK-886 was unable to block leukotriene synthesis from exogenous AA in the two cell types, despite its ability to completely inhibit 5-LO metabolism of endogenous AA. These observations indicate that, although FLAP is essential for the synthesis of leukotrienes from endogenous AA, perhaps by presenting AA to 5-LO, it is not required for 5-LO metabolism of exogenous AA. The differing roles of FLAP in 5-LO metabolism of endogenous versus exogenous AA are consistent with the conclusion that it is the markedly greater expression of FLAP, rather than of 5-LO, that is primarily responsible for the increased leukotriene synthesis from endogenous AA that accompanies PBM differentiation into AM.

Influence of fatty acid anilides present in toxic oils on the metabolism of exogenous arachidonic acid in cultured human endothelial cells

The effect of fatty acid anilides (FAA) on the exogenous arachidonic acid (AA) metabolism and toxicity of isolated human endothelial cells was studied to clarify their possible role in the etiology of toxic oil syndrome. Confluent cells were incubated with and without linoleic acid anilide (LAA), oleic acid anilide (OAA) and two unrelated samples for 2–24 h prior to the addition of [1- 14C]AA alone or with calcium ionophore A-23178. The eicosanoids produced were analyzed by RP-HPLC. A dual stimulatory and inhibitory effect on the conversion of exogenous AA as a function of preincubation time with anilides (100 and 1000 μM) was observed. Treated cells significantly increased (1–3-fold) the production of the main cyclooxygenase-derived prostanoids (6-keto-PGF 1α and PGF 2α) formed by these cells, with a maximum stimulatory effect after 2—3 h, only when AA was used alone. However, afterwards a time- and dose-dependent decrease in prostanoid formation was observed with LAA ( P < 0.05 at 24 h), either in the absence or presence of ionophore A-23187 in the incubation mixture. This inhibitory effect on cyclooxygenase was not observed with OAA, which still stimulate after 24 h of treatment. The changes in prostanoid synthesis were not followed with a parallel release in the lactate dehydrogenase activity in the medium (except with unrelated samples). Moreover, anilide treatment increased the appearance of cytosolic lipid droplets or vacuoles after 2 and 5 h of contact with LAA and OAA, respectively. From these results, it was suggested that anilides impair prostanoid synthesis in endothelial cells; their stimulatory effect could be explained by an unspecific effect on cell membrane, not related to cell toxicity and the inhibitory effect by an inhibition of the cyclooxygenase activity. These observations further contribute to our understanding of the possible role of anilides in the etiology of the toxic oil syndrome.

Effects of cigarette smoking on the function of metabolizing arachidonic acid and angiotensin I in the isolated perfused rat lungs.

The effects of acute and chronic cigarette smoking on the metabolism of exogenous arachidonic acid (AA) and angiotensin I (AI) in perfused isolated rat lungs were studied. The results showed that acute cigarette smoking did not alter the contents of 6-keto-PGF1 alpha (the stable metabolite of PGI2) and TXB2 (the stable metabolite of TXA2) in the effluent and the increment of pulmonary artery pressure (delta Ppa) caused by AA. The conversion of A I into A II was significantly increased (P < 0.01), while the delta Ppa induced by A I injection was obviously decreased as compared with controls (P < 0.05). After cigarette smoke exposure for 30 days, the delta Ppa caused by AA or A I did not differ from that of controls, but the contents of 6-keto-PGF1 alpha and A II increased more markedly than those in non-smoking rats (P < 0.05). It is suggested that acute and chronic cigarette smoking in rats can promote the lung function of converting A I into A II, chronic smoking can increase the lung function of metabolizing AA into PGI2.

Influence of N-phenyllinoleamide from toxic oil samples on the lipoxygenase metabolism of exogenous arachidonic acid in mouse peritoneal macrophages

N-phenyllinoleamide (NPLA) is a useful marker for adulterated oil samples associated with cases of toxic oil syndrome (TOS). To date, NPLA has not reproduced the human poisoning episode in experimental animal models and, thus, its pathological role in the syndrome remains controversial. The present report describes the effect of NPLA on the lipoxygenase metabolism of exogenous arachidonic acid (AA) in mouse peritoneal macrophages (MPM). Results show that MPM cells exposed to 1mM NPLA for 2 h, when subsequently incubated with exogenous 3H-AA, undergo a significant increase in the biosynthesis of 3H-12-hydroxyeicosatetraenoic acid ( 3H-12-HETE) whereas levels of 3H-15-HETE are relatively stable. These data indicate that NPLA selectively potentiates the lipoxygenase metabolism of exogenous AA, supporting the possible implication of lipid peroxidative processes in the ethiopathology of TOS, although the relatively high NPLA concentration required ‘in vitro’ makes it unlikely that this xenobiotic could be directly related to human toxicity.

Epidermal Cell—Polymorphonuclear Leukocyte Cooperation in the Formation of Leukotriene B4 by Transcellular Biosynthesis

The cellular origin of Leukotriene B4, a potent pro-inflammatory agent that is present in psoriatic lesions, has not been completely ascertained. The present study was performed in order to assess the possible contribution of epidermal cells to leukotriene B4 synthesis through 5-lipoxygenase or by means of transcellular metabolism of the epoxide intermediate leukotriene A4 from activated polymorphonuclear leukocytes. The metabolism of exogenous arachidonic acid in fresh human epidermal cell, polymorphonuclear leukocyte or mixed suspensions was determined by means of high-performance liquid chromatography. Epidermal cells transformed arachidonic acid mainly into 12-hydroxy-eicosatetraenoic acid and prostaglandin E2. Formation of prostaglandins F2 alpha and D2, 12-hydroxy-eptadecatrienoic acid, and 15- and 11-hydroxy-eicosatetraenoic acids was also detected. We did not detect any eicosanoid derived from 5-lipoxygenase pathway. Mixed suspensions of polymorphonuclear leukocytes and epidermal cells (ratio 1:4) produced 1.72 times more leukotriene B4 than leukocytes alone under the same experimental conditions. Epidermal cells incubated with 5 microM authentic leukotriene A4 for 3 min yielded 2.954 +/- 0.27 pmoles/10(6) cells of leukotriene B4, which was characterized by co-elution with authentic standard and its ultraviolet absorption spectrum. These data demonstrate the existence of a leukotriene A4 epoxide hydrolase activity in human epidermal cells. Our results suggest that epidermal cells could cooperate in leukotriene B4 biosynthesis by transcellular metabolism of leukotriene A4 in lesions of psoriasis, and possibly other inflammatory dermatoses characterized by increased leukotriene B4 levels and prominent polymorphonuclear leukocyte infiltrates.

Metabolism of exogenous arachidonic acid by polymorphonuclear leukocytes from psoriatic patients

We evaluated the metabolism of exogenous arachidonic acid in polymorphonuclear leukocytes from psoriatic patients in comparison with those from normal subjects. The leukocytes were incubated with 10 microM labelled arachidonic acid and 5 microM A23187 for varying periods of time. We evaluated all the compounds derived from the 5-lipoxygenase activity, including the products of non-enzymatic transformation of leukotriene A4 and w-oxidized leukotriene B4. In the experimental conditions used, there was no significant difference in the formation of any compound at any time of incubation. These results indicate that there is no intrinsic alteration either in 5-lipoxygenase activity or in w-oxidation ability of circulating polymorphonuclear leukocytes in psoriatic patients.

Exogenous arachidonic acid metabolism in platelets from psoriatic patients.

Several reports have been published on platelet hyperaggregation in psoriatic patients, which might be related to alterations in arachidonic acid (AA) metabolism by platelets. We have studied the AA metabolism pattern, total eicosanoid formation and biosynthesis rate in platelet from psoriatic patients and from normal subjects after incubation with exogenous AA. We have found no difference in the metabolic pattern of platelets. Total formation of 12-HETE was higher in the control group (p less than 0.01). The rate of synthesis of cyclooxygenase products was higher in the psoriatic group, but only that of thromboxane B2 was statistically significant (p less than 0.02). There was a close correlation between thromboxane B2 formation rate and the lag time of platelet aggregation in response to 1 mM AA (p less than 0.01). The percentage of aggregation of platelets from psoriatics was significantly higher than that from normal subjects (p less than 0.05) and the lag time was lower in psoriatic group, but the difference was not statistically significant.

Inhibitory effect of stearidonic acid (18∶4 n−3) on platelet aggregation and arachidonate oxygenation

The effect of stearidonic acid (18:4 n-3) present in fish and some plant oils, such as black currant seed oil, was studied on human platelets. When added to platelets simultaneously with collagen, arachidonic acid or endoperoxide mimetic U46619, 18:4 n-3 appeared as a weak inhibitor of platelet aggregation. In addition, 18:4 n-3 did not alter the metabolism of exogenous arachidonic acid. In contrast, when preincubated with platelets after precoating onto albumin, 18:4 n-3 inhibited platelet aggregation induced by thrombin, collagen, arachidonic acid or U46619, and was as potent as eicosapentaenoic acid (20:5 n-3) tested under similar conditions. Stearidonic acid also altered the endogenous arachidonate oxygenation stimulated by low doses of thrombin, but to a significantly lesser extent than did 20:5 n-3. It seems therefore that, in addition to competing with endogenous arachidonate metabolism, 18:4n-3 may affect platelet aggregation by another mechanism.

Effect of Lonapalene on Metabolism of Exogenous Arachidonic Acid in Human Platelets

Lonapalene was studied as a topical antipsoriatic agent on the basis of its inhibitory effect on 5-lipoxygenase. We studied the effect of lonapalene on the metabolism of exogenous arachidonic acid in washed platelet suspensions by RP-HPLC. Lonapalene was shown to inhibit platelet cyclooxygenase, with substrate diversion towards 12-HETE production. The pattern of inhibition was similar to that of indometacin, although lonapalene was about 1,000 times weaker. The dual inhibition of lonapalene and concomitant deviation of arachidonic acid metabolism may have potential implications, with regard to therapeutic indications and side effects, which deserve further study.

The interaction of ethanol and exogenous arachidonic acid in the generation of extracellular messengers by mouse peritoneal macrophages

It is increasingly recognized that macrophages play a crucial role in the development of chronic inflammatory states such as alcoholic liver disease. These cells can metabolize free arachidonic acid in the absence of a discernible trigger. The present study was undertaken to examine the short-term effects of ethanol on the generation of these exogenous arachidonate-derived extracellular mediators. Ethanol caused a dose-dependent decrease in the production of both cyclooxygenase and lipoxygenase metabolites. Similar effects were observed on the esterification of exogenous arachidonate into cellular lipids. To characterize further the effects of ethanol on exogenous arachidonic acid metabolism, we studied the short-term responses displayed by macrophages challenged with another soluble stimulus; the tumor-promoting agent phorbol myristate acetate. We observed an inhibition by ethanol of the superoxide anion response triggered by phorbol myristate acetate similar to that observed for exogenous arachidonate oxygenation. Our results show that ethanol can inhibit these soluble stimuli-elicited responses, possibly through its disorganizing effect on plasma membrane.

Metabolism of exogenous arachidonic acid by mouse peritoneal macrophages

On incubation of resident mouse peritoneal macrophages with arachidonic acid several hydroxyacyl derivatives detectable in cellular supernatants are formed. As main products monohydroxyarachidonic acids (monoHETE's) were identified. In addition, smaller amounts of dihydroxyarachidonic acids (diHETE's) supernatants by reversed phase HPLC, normal phase HPLC in combination with UV-spectroscopy and combined gas-chromatography / masspectrometry revealed the presence of 5-, 8-, 12- and 15 - mono-HETE's, two distinct 5, 12-diHETE's, several 8, 15-diHETE's and 14, 15-diHETE. Among the 5, 12-diHETE's, only small amounts of a compound with the characteristics of LTB, were detected. Under the conditions employed, the cycloxygenase products PGE 2 and PGI 2 (as 6-keto-PGF 1g) were only minor metabolites. In contrast, when macrophage cultures were stimulated with the phagocytic stimulus zymosan, PGI 2, PGE 2 and LTC 4 were found as the major conversion products of arachidonic acid, whereas mono- and diHETE's were not formed in detectable amounts.

Hemorheological Changes in Elderly Subjects-Effect of Pentosan Polysulfate and Possible Role of Leucocyte Arachidonic Acid Metabolism

The effects of pentosan polysulfate (PPS) on various hemorheological parameters were studied in a group of very elderly subjects in good general health. Alterations in blood viscosity and filterability were detected in these patients, without any concomitant changes in factors which are known to affect these parameters: notably hematocrit, fibrinogen and plasma lipid levels. The hemorheological abnormalities were considerably improved by twice daily treatment with 50 mg of PPS (i.m.). Apart from its anticoagulant activity, PPS has been shown to have an anti-inflammatory action. We were interested to investigate its effects on metabolism of exogenous arachidonic acid (AA) by both platelets and leucocytes. It is becoming increasingly recognized that metabolites of AA via the 5 LO pathway appear to play a role in inflammatory processes. In this study, PPS was found to inhibit leucocyte 5 LO activity. Reduction in the levels of these metabolites may therefore have an effect on whole blood rheology.

Lipoxygenase metabolism of endogenous and exogenous arachidonate in leukocytes: GC-MS analyses of incubations in H 218O buffers

Fatty acids are labeled with 18O in the carboxyl group during ester hydrolysis in H 2 18O. We utilized this principle to develop a novel mass spectrometric method for study of the turnover of arachidonic acid in intact cell systems. Analysis of 18O incorporations was by negative ion chemical ionization GC-MS. The use of deuterium-labeled exogenous substrates allowed the metabolic fate of exogenous and intrinsic compounds to be distinguished. Thus, it was shown that the preferred route of 5-lipoxygenase metabolism of exogenous arachidonic acid in ionophorestimulated human neutrophils is via direct reaction with the enzyme and not via incorporation into cellular lipids. The re-uptake of 5-HETE was also studied. For investigation of intracellular reactions which involve ester hydrolysis, the 18O labeling method is unique in providing direct evidence of the intermediate metabolic pathway of endogenous and exogenous products associated with the arachidonic acid cascade.

Effect of serum on the metabolism of exogenous arachidonic acid by phagocytic cells of the mouse thymic reticulum

Phagocytic cells derived from the mouse thymic reticulum (PTR) were used to study the effect of ( 14C on the metabolism of arachidonic acid (AA). After labeling with ( C)-AA in the presence of 10 % serum, we failed to detect in the culture medium the presence of significant amounts of radiolabeled prostaglandins. In contrast, when the labeling period was carried out in serum-free medium, we observed the secretion of both cyclooxygenase and lipooxygenase derivatives. In addition, the pattern of arachidonate incorporation into cell lipids was different in the two culture conditions. In the presence of serum, the great majority of the radioactivity was found associated with phospholipids, whereas in serum-free medium, almost 50 % of the incorporated fatty acid was associated with triglycerides. Since serum albumin is known to play a major role in the control of fatty acid uptake, we have studied the effect of the addition of 2 % BSA to cells prelabeled in the absence of serum. This treatment switches the patterns of metabolite release and lipid labeling towards those of serum-treated cells. In addition, we showed that the effects of glucocorticoids on AA release differ markedly according to the composition of the culture medium.

Effects of dipyridamole, nifedipine, verapamil, hydralazine and propranolol on the formation of prostacyclin and thromboxane in a coupled system of platelets and aorta

The effects of some vasodilatory (dipyridamole, nifedipine, verapamil) and antihypertensive (hydralazine, propranolol) drugs on the metabolism of exogenous arachidonic acid (AA), and on the production of thromboxane from endogenous AA stores were examined. In a coupled system of platelets and rat aorta, dipyridamole (100 μM), nifedipine (300 μM) and hydralazine (250 μM) potentiated the formation of 6-keto-F 1α from exogenous AA. Hydralazine in this system reduced the formation of TxB2. At higher concentrations the drugs (dipyridamole 500 μM; hydralazine 1 mM, verapamil 2mM; propranolol 2mM) were effective in reducing the formation of TxB2. Moreover, the drugs at lower concentrations produced reduced amounts of TxB2 in clotting blood. A dose-dependent inhibition of platelet aggregation induced by ADP, epinephrine, collagen and arachidonate was observed with these drugs. The results demonstrate that some of the beneficial effects of the drugs in the cardiovascular system can be explained on the basis of their effects on platelet aggregation and on AA metabolism.

Altered aortic and cremaster muscle prostaglandin synthesis in diabetic rats.

Alterations in the synthesis and release of prostaglandins have been reported in humans and animal models of diabetes mellitus. In the present study synthesis and release of prostaglandins by thoracic aorta and cremaster muscle of rats with streptozotocin-induced diabetes of 8 wk duration was compared with age-matched controls. Prostaglandin synthesis was assessed by the measurement of immunoreactive prostaglandin E2 (PGE2) and 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha) release and by quantifying metabolism of exogenous [1-14C]arachidonic acid by thoracic aortic rings and minced cremaster muscle. The cremaster muscles from diabetic rats released significantly greater quantities of PGE2 and 6-keto-PGF1 alpha and PGE2. In contrast, the aortas from diabetic rats released smaller quantities of 6-keto-PGF1 alpha and PGE2 and exhibited reduced 6-[1-14C]keto-PGF1 alpha. These studies indicate that diminished prostacyclin (PGI2) and/or PGE2 production is not a general feature of all diabetic vascular tissues, suggesting that large and small blood vessels may not be similarly affected by diabetes in regard to the metabolism of exogenous arachidonic acid and the synthesis and release of prostaglandins. Furthermore, the vascular changes often observed in conjunction with diabetes, i.e., alterations in vascular reactivity and microangiopathy in small blood vessels and atherosclerosis of large blood vessels may be related in some way to the segmental differences observed in prostaglandin synthesis.

Transformations of exogenous arachidonic acid in human platelets in the presence of oleic- and eicosapentaenoic acids

Effects of various concentrations (12.5–50 μM) of eicosapentaenoic acid (EPA) on the metabolism of exogenous arachidonic acid (AA) in washed human platelets were examined under two different incubation conditions. First platelets were pretreated with EPA before exposing them to labelled AA. In the second incubation platelets were exposed to a mixture of EPA (12.5 and 25 μM) and labelled AA. At all concentrations EPA reduced thromboxane B2 (TxB2) formation, and at 25 and 50 μM this fatty acid reduced also prostaglandins (PGE 2, PGF 2α). A reduced formation of PGs and TxB2 was confirmed by a decreased formation of PG-endoperoxides. While in EPA treated platelets less CO-(TxB2, HHT, PGs) and lipoxygenase (HETE) products were formed at 25 and 50 μM, more HETE seemed to be generated at lower EPA concentration (12.5 μM). Oleic acid reduced TxB 2 formation at high concentration (100 μM). EPA showed a dose-dependent inhibition of platelet aggregation induced by arachidonate, epinephrine and collagen; it was most effective against AA-induced aggregation.