High Concentrations Of Arachidonic Acid Research Articles

Arachidonic acid and isolated human umbilical vein: Concentration-limited prostacyclin generation and absence of direct toxic effects on endothelial integrity

Previous studies show that administration of high doses of arachidonic acid to rabbits causes disruption of vascular integrity. To examine if similar changes would occur in isolated human vascular tissue, we treated human umbilical veins with arachidonic acid (0, 0.01, 0.1, 1.0 mM). Prostacyclin biosynthesis was quantitated in the supernatants and the endothelial integrity was evaluated by scanning electron microscopy. Prostacyclin synthesis was maximum at low concentrations (0.01 – 0.1 mM) with no additional increase with higher concentrations of arachidonic acid (1mM) confirming previous observations. There was no evidence for endothelial disruption with any concentration of arachidonic acid. This study shows that a) arachidonic acid has no direct effects on the vascular lining of isolated human umbilical vein, b) arachidonic acid-induced sudden death in experimental animals is not due to limitation of prostacyclin synthesis.

The inhibition of arachidonic acid incorporation into human platelet phospholipids by eicosapentaenoic acid

The consumption of a diet enriched in fish or fish oil containing eicosapentaenoic acid results in a decreased content of arachidonic acid in platelet phospholipids which may contribute to the reduced platelet aggregability observed in human subjects on these diets. In order to examine a possible biochemical mechanism for this decreased arachidonate, washed human platelets were incubated with [1− 14C]arachidonic acid in the presence and absence of eicosapentaenoic acid to test its potential as an inhibitor. The incorporation of [1− 14C]arachidonic acid into the total and individual phospholipids was studied using equi-molar concentrations of eicosapentaenoic acid. Reduced incorporation in the presence of eicosapentaenoic acid occurred in total phospholipid as well as phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and phosphatidylcholine at all concentrations. The entry, of [1− 14C]-arachidonic acid was also studied using a fixed concentration of eicosapentaenoic acid and increasing concentrations of [1− 14C]arachidonate. Arachidonate incorporation was significantly inhibited by eicosapentaenoate in total phospholipid, phosphatidylinositol, phosphatidylserine, and phosphatidylcholine. For phosphatidylethanolamine, significant inhibition occurred only at the higher concentrations of arachidonic acid. These results provide evidence that eicosapentaenoic acid has the potential to inhibit the entry of arachidonic acid into the phospholipids which are considered to be active in platelet responses to agonists. This inhibition may be a factor contributing to the lowered content of arachidonate in platelet phospholipids of human subjects consuming fish or fish oils and to the diminished platelet reactivity. Furthermore, the present results indicate that the inhibitory effect of eicosapentaenoate on arachidonate incorporation can differ between individual types of phospholipid.

Platelet Suruival and Function in Rats with Enhanced Thrombotic Tendency

We have investigated the relevance of some laboratory tests of platelet function in predicting conditions of thrombotic tendency. For this purpose, we studied platelet survival, platelet aggregation in response to different stimuli, TxB2 and 6-keto-PGF1 alpha production in serum of rats bearing a nephrotic syndrome induced by adriamycin. These animals show a heavy predisposition to the development of both arterial and venous thrombosis. The mean survival time was normal in nephrotic rats in comparison to controls. As to aggregation tests, a lower aggregating response was found in ADR-treated rats using ADP or collagen as stimulating agents. With arachidonic acid (AA) we observed similar aggregating responses at lower AA concentrations, whereas at higher AA concentrations a significantly lower response was found in nephrotic rats, despite their higher TxB2 production. Also TxB2 and 6-keto-PGF1 alpha levels in serum of nephrotic rats were significantly higher than in controls. No consistent differences were found in PGI2-activity generated by vessels of control or nephrotic rats. These data show that platelet function may appear normal or even impaired in rats with a markedly increased thrombotic tendency. On the other hand, the significance of high TxB2 levels in connection with mechanisms leading to thrombus formation remains a controversial issue.

Arachidonic acid and leukotriene B4 induce aggregation of human peripheral blood mononuclear leucocytes in vitro.

Peripheral human blood mononuclear leucocytes (MNL) aggregated in response to arachidonic acid (AA) in vitro. This phenomenon was similar to that already described for polymorphonuclear cells (PMN). The effect of AA was concentration-dependent and was shared only by the structurally related di-homo-gamma-linolenic acid among the other fatty acids tested. A number of agents able to induce platelet aggregation such as ADP, collagen, serotonin and a stable prostaglandin analogue all failed to stimulate MNL or PMN aggregation. Inhibitors of cyclo-oxygenase activity such as acetylsalicylic acid and indomethacin not only did not prevent AA-induced aggregation, but even potentiated it. In contrast, both nordihydroguaiaretic acid and BW 755C, two inhibitors of cyclo-oxygenase and lipoxygenase, strongly prevented MNL aggregation. Thus AA seems to aggregate MNL through the mediation of lipoxygenase products. This is supported by the observation that leukotriene B4 (LTB4) also induced MNL aggregation. When highly purified lymphocyte and monocyte preparations were assessed separately, the latter responded to AA similarly to mixed MNL whereas lymphocyte aggregation was inconsistent, small and reversible even at high concentrations of AA. Although the pathophysiological significance of the MNL aggregation described here is still obscure, assembly of these cells--particularly monocytes--at the site of injury might be a crucial event.

Cooperative effects of 1-O-alkyl-2-O-acetyl- [formula omitted]-glycero-3-phosphocholine (PAF-acether) and exogenous arachidonic acid in stimulation of human blood platelets

Cooperative effects of PAF-acether and arachidonic acid in blood platelet activation were studied in human platelet-rich plasma. Using a combination of low concentrations of PAP-acether and subthreshold amounts of exogenous arachidonic acid an enhancement of aggregation and an increased formation of malondialdehyde are obtained. Both effects are completely suppressed either by acetylsalicylic acid or by use of PAF-acether desensitized platelets, indicating that cyclooxygenase products and intact receptors for PAP-acether are of primary importance in the observed synergism. Stimulating activity of PAF-acether on the formation of cyclooxygenase products is also seen with use of higher concentrations of arachidonic acid during mild stimulation of human platelets. This effect of PAP-acether is dependent on the concentration used and is obviously due to changes in the platelet membrane as derived from experiments with lysed platelets. Enhanced capacity of platelets to synthesize endoperoxides and thromboxane in consequence of an in vivo release of PAF-acether into the circulation might be of pathobiochemical relevance in some circumstances.

Arachidonic acid stimulates the formation of 1,2-diacylglycerol and phosphatidic acid in human platelets. Degree of phospholipase C activation correlates with protein phosphorylation, platelet shape change, serotonin release, and aggregation.

Exogenous unlabeled arachidonic acid (AA) added to human platelets prelabeled with [3H]AA induces breakdown of [3H]phosphatidylinositol and the rapid and transient formation of [3H]1,2-diacylglycerol and [3H]phosphatidic acid (PA), indicating activation of phosphatidylinositol-specific phospholipase C. Formation of [3H]1,2-diacylglycerol and [3H]PA is inhibited by pretreatment of platelets with aspirin, which suggests that endoperoxides or thromboxane A2 are responsible for AA-induced stimulation of phospholipase C. Exogenous unlabeled AA also induces the formation of [32P]PA or [14C]PA in platelets that have been prelabeled with 32Pi or [14C]AA, respectively. Increased radioactivity in PA reflects increased content of PA as measured by the fatty acid composition of PA. The relation of PA production, which reflects stimulation of phospholipase C, to specific platelet responses was further investigated. Low concentrations of AA (0.05-0.2 microM) induces platelet shape change in parallel to formation of 50-100% PA and phosphorylation of a 40,000 molecular weight protein. Higher concentrations of AA (0.5-50 microM) stimulate the formation of a further amount of PA (200-250%), and phosphorylation of 40,000 molecular weight protein, platelet aggregation, and serotonin release. Indomethacin inhibits all these observed changes by inhibiting the conversion of AA by platelet cyclooxygenase. In contrast, prostacyclin blocks these responses without affecting conversion of AA by platelet cyclooxygenase and thromboxane synthetase. We conclude that formation of endoperoxides and thromboxane A2 is necessary but not sufficient for platelet activation by AA. Only if PA is formed are platelets activated. The results indicate a central role for the phospholipase C pathway in the process of platelet activation.

Regulation of arachidonate-induced platelet aggregation by the lipoxygenase product, 12-hydroperoxyeicosatetraenoic acid

The lipoxygenase product, 12-hydroperoxyeicosatetraenoic acid (12-HPETE) was biosynthesized, purified and incubated with washed human platelets. It inhibited arachidonic acid, azo-prostaglandin H 2 or U-46619-induced aggregation and secretion in a concentration-dependent fashion (IC 50 = 2–3 μM. Collagen-induced aggregation and secretion were also inhibited (IC 50 = 6 μM. 12-HPETE inhibited malondialdehyde and thromboxane B 2 formation in platelets stimulated with arachidonic acid or thrombin. While thrombin-induced aggregation was unaffected by 50 μM 12-HPETE, thrombin-induced secretion was inhibited. Inhibition of secretion by 12-HPETE was observed in platelets from untreated as well as aspirin-treated donors, indicating that 12-HPETE inhibits secretion independently of its ability to inhibit prostaglandin formation. Aggregation of washed human platelets by arachidonic acid yielded a bell-shaped concentration-response curve. Diminished aggregation at higher concentrations was associated with an increase in the ratio of lipoxygenase products to thromboxane B 2. The data suggest that 12-HPETE formation may regulate platelet aggregation and secretion and that its primary effect, at low concentrations, is inhibition of endoperoxide-induced responses. At higher concentrations 12-HPETE also inhibits arachidonic acid metabolism. Thus, the combined inhibitory effects on endoperoxidase-induced aggregation and thromboxane formation would explain the dimineshed aggregation observed in response to high concentrations of arachidonic acid.

Prostaglandin endoperoxides, thromboxane A2 and adenosine diphosphate in collagen-induced aggregation of rabbit platelets.

1 A bioassay technique is described for simultaneously monitoring rabbit platelet aggregation with measurement of thromboxane A(2) (TxA(2)) and prostaglandins released in response to collagen or arachidonic acid (AA).2 Five imidazole derivatives were examined as inhibitors of thromboxane synthetase and compared with the effect of the cyclo-oxygenase inhibitor indomethacin; 1-(7-carboxyheptyl) imidazole was identified as the most potent and selective inhibitor of thromboxane synthetase and was used with indomethacin to investigate the relative contribution of the prostaglandin endoperoxides prostaglandin G(2) (PGG(2))/PGH(2) and TxA(2) in mediating platelet aggregation induced by collagen or AA.3 Platelet aggregation induced by a low concentration of collagen was abolished by indomethacin and carboxyheptylimidazole whilst in response to a high concentration or collagen only partial inhibition of aggregation occurred.4 The contribution of adenosine diphosphate (ADP) released from platelets during collagen or AA-induced aggregation was examined using the substrate/enzyme complex creatine phosphate/creatine phosphokinase (CP/CPK). The CP/CPK complex abolished aggregation induced by a low dose of collagen whilst aggregation to a high dose of collagen was only partially inhibited.5 Aggregation induced by a high dose of collagen was abolished by a combination of CP/CPK with indomethacin or carboxyheptylimidazole.6 AA-induced aggregation was abolished by indomethacin. Carboxyheptylimidazole abolished aggregation induced by a low dose of AA but inhibition was surmounted with increasing concentrations of AA in the absence of TxA(2) formation.7 PGH(2)-induced aggregation was unaffected by indomethacin and only partially inhibited by carboxyheptylimidazole. AA or PGH(2)-induced platelet aggregation was unaffected by CP/CPK.8 In conclusion, aggregation of rabbit platelets induced by a low concentration of collagen was dependent on synergism between TxA(2) and ADP whilst at high concentrations of collagen, sufficient TxA(2) and ADP were released to induce aggregation independently of each other.9 The small amounts of prostaglandin endoperoxides produced from endogenous arachidonate have apparently no direct pro-aggregatory role. However, the relatively large amount which can be produced by a high concentration of exogenous AA when TxA(2) formation is prevented can cause aggregation of rabbit platelets.

Suppression of murine T-cell mitogenesis by metabolic products of arachidonic acid.

We examined the ability of arachidonic acid (AA) to inhibit mitogen stimulation of mouse splenic lymphocytes. Dose related suppression of [3H]-thymidine incorporation by cells stimulated with either phytohemagglutinin or concanavalin A occurred at concentrations of AA above 1 microgram/ml. AA-induced suppression required that AA be added to cultures within 6 hours of the addition of mitogen. Addition of AA at 24 hours resulted in diminished suppression, while delaying addition of AA until 48 hours had no effect on mitogen stimulation. Preincubation with AA did not alter mitogen stimulation when cells were washed free of AA. The addition of indomethacin or mefanamic acid to the cultures significantly reversed AA-induced suppression. Inhibitors of the lipoxygenase pathway or thromboxane synthesis had no effect, while an inhibitor of both thromboxane and prostacyclin synthesis partially prevented AA-induced suppression only at the highest concentration of AA. Removal of adherent cells resulted in a diminished capacity for AA to suppress mitogenesis and to stimulate PGE2 synthesis.

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 B 2 (TxB 2), the stable metabolites of prostacyclin (PGI 2) and TxA 2, 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 TxB 2 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.

Biosynthesis of Prostaglandin D2,15-ketoprostaglandin E2, and Hydroxy Fatty Acids by Ram Seminal Vesicle Microsomes

At high arachidonic acid concentrations (164 μM) and without exogenous cofactors, ram seminal vesicle microsomes produced prostaglandin E2 and two less polar products, identified as prostaglandin D2 and 15-ketoprostaglandin E2. The ratio of the biosynthetic products formed depended on the exogenous cofactor and on the arachidonic acid concentration. At high arachidonic acid concentrations (>150 μM), tryptophan, phenol, and glutathione stimulated prostaglandin E2 formation, but each affected the formation of the other prostaglandins differently. Ascorbic acid markedly stimulated hydroxy fatty acid formation. GLC-mass spectral analysis of the hydroxy fatty acid fraction indicated the presence of 11-hydroxy-5,8,12,14-eicosatetraenoic acid, 15-hydroxy-5,8,11,13-eicosatetraenoic acid, and 12-hydroxy-5,8,10-heptadecatrienoic acid. At low arachidonic acid concentrations (30 μM). glutathione still stimulated prostaglandin E2 biosynthesis, but the other cofactors stimulated 6-ketoprostaglandin F1α”︁ and hydroxy fatty acid formation.

Biochemical studies in Hermansky-Pudlak syndrome.

In order to find the basic defect in the Hermansky-Pudlak Syndrome (HPS), biochemical studies of platelets and leucocytes were undertaken. Glutathione levels of platelets were normal and regeneration of GSH similar to controls occurred after incubation with diamide (a specific agent for GSH oxidation). Phospholipid and fatty acid composition of HPS platelets was normal. The amount of peroxides found in platelet membranes was not elevated. A subnormal aggregation with arachidonic acid could be obtained in PRP using a high concentration of arachidonic acid (2 mM), but normal malondialdehyde levels were measured, suggesting a normal prostaglandin synthesis in HPS platelets. Glutathion peroxidase and p-phenylenediamide-mediated peroxidase (PPD-peroxidase) were normal in leucocytes of 1 HPS patient. Lysosomal enzymes as far as investigated were normal.

The effects of albumin bound fatty acids on the platelet inhibitory function of human endothelial cells.

This study was carried out to evaluate the effects of albumin-bound fatty acids on the anti-platelet effects of endothelial cells. Primary cultures of human endothelial cells (ECM), grown in confluent monolayers, were incubated with plasma or growth medium enriched with albumin-bound fatty acids (FA) for 2-20 h. The effects of ECM on ADP-induced platelet aggregation (PA) and collagen-induced PA and prostaglandin synthesis in platelet-rich plasma were tested. ECM released an inhibitor of platelet reactions which resembled the activity of PGI2 (prostacyclin). The inhibitory activity was increased by preincubation of ECM with arachidonic acid (AA). A moderate decrease of the activity was obtained by incubation with long-chain saturated, monoenoic and dienoic unsaturated fatty acids. A pronounced decrease of the inhibitor was obtained by incubation with di-homo-gamma-linolenic acid (DHLA). Paired combinations of AA with the other fatty acids in the incubation medium partially restored the inhibitory activity obtained by the separate FA. The stimulation of the inhibitor by AA was dose dependent and high concentrations of AA reduced this activity. The present study indicates that the quantity and quality of the plasma free fatty acids can affect the endothelial cells' ability to act as a non-thrombogenic surface.

Effect of substrate concentration on inhibition of prostaglandin synthetase of bull seminal vesicles by anti-inflammatory drugs and fenamic acid analogs

Although microsomes of bull seminal vesicle synthesize prostaglandins F 2α, E 2, and D 2 from arachidonic acid under suitable assay conditions, prostaglandin E 2 is the only significant product at either low concentration of arachidonic acid or high concentration of microsomes. Studies of inhibition of prostaglandin synthesis in vitro by anti-inflammatory drugs at both high (1 mM) and low (1 μM) concentrations of arachidonic acid, suggest three distinct mechanisms of inhibition. Benzydamine and flazalone are non-competitive or weakly competitive with arachidonic acid and, at high concentrations of arachidonic acid, they augment synthesis of prostaglandin E 2 while inhibiting production of prostaglandins F 2α and D 2. Niflumic acid and the arylacetic acids naproxen and ibuprofen are competitive inhibitors, inhibiting all products equally, but with 100–500-fold greater potency at the low substrate concentration. The fenamic acids, indomethacin, aspirin, and phenylbutazone also inhibit equally all prostaglandin products, but are only 20–50 times more potent at the low substrate concentration. Studies with analogs of the fenamic acids indicate that the diphenylamine portion of their structure is essential for inhibition of prostaglandin synthesis, whereas the o-carboxyl and m-alkyl substituents greatly enhance inhibitory potency.

The fatty acid composition of phospholipids and glycolipids in Jimpy mouse brain.

Abstract— Phospholipids and sphingolipids from brains of normal and Jimpy mice were isolated in a pure form by thin‐layer chromatographic procedures. The fatty acid composition of the major phospholipids, i.e. ethanolamine glycerophospholipids, serine glycerophospholipids, choline glycerophospholipids and inositol glycerophospholipids, as well as sphingomyelin, cerebrosides and sulphatides was determined by gas‐liquid chromatography. A specific fatty acid pattern for each of the four glycerophospholipids was found. The fatty acid composition of inositol glycerophospholipid, which has not previously been studied in mouse brain, was characterized by a high concentration of arachidonic acid. After 16 days of age, fatty acid analysis showed definite differences between the phospholipids from normal and mutant brains. A small increase of polyunsaturated fatty acids in glycerophospholipids of ethanolamine, serine and choline from the Jimpy central nervous system was found, which has been explained by the myelin deficiency. Sphingomyelin, cerebrosides and sulphatide analyses showed a wide distribution of saturated and mono‐unsaturated fatty acids in both normal and mutant mice. A reduction in the amount of long‐chain fatty acids was demonstrated in mutant brain sphingolipids; in sulphatides and cerebrosides, the amount of non‐hydroxy fatty acids was reduced to a greater extent than in sphingomyelin.The distribution of fatty acids in sphingolipids from the myelin and microsomal fractions was also investigated in both types of mice. Cerebrosides were characterized by a high content of long‐chain fatty acids in myelin as well as in microsomes. Sulphatides and sphingomyelin, on the other hand, showed a higher content of medium‐chain fatty acids in microsomes than in myelin. In the mutant brain, the amount of long‐chain fatty acids was reduced in both subcellular fractions. The deviation from normal in the pattern of fatty acid distribution in Jimpy brain is discussed in relation to the current concepts of glycolipid biosynthesis.