Administration Of Arachidonic Acid Research Articles

Effects of arachidonic acid plus zinc on glucose disposal in genetically diabetic (ob/ob) mice.

The present study is designed to determine whether arachidonic acid (AA) plus zinc improves clinical signs of diabetes in genetically diabetic ob/ob mice. In the first study, effects of acute administration of AA plus zinc on glucose disposal were determined in ob/ob and lean mice (n = 6 each). In the second study, ob/ob and lean mice were treated with increasing doses of AA plus zinc for 2 weeks (n = 5 each). Postprandial and fasting blood glucose concentrations, three-hour-area-average above fasting glucose concentration (TAFGC), water and food intake, body weight and plasma insulin concentrations were measured. Acute administration of AA plus zinc significantly increased glucose disposal in ob/ob mice. In the second study, postprandial and fasting blood glucose concentrations, TAFGC, and water and food intake in ob/ob mice treated with AA plus zinc for 2 weeks were significantly decreased compared with those in mice given no AA. Plasma insulin concentrations in both lean and ob/ob mice were not changed by AA treatment in drinking water. AA plus zinc in drinking water is effective in decreasing blood glucose levels in obese mice. These results indicate that use of these compounds should be considered as a dietary supplement to control hyperglycaemia in patients with type II diabetes.

Effects of selective cyclooxygenase-2 inhibition on vascular responses and thrombosis in canine coronary arteries.

Prostanoid synthesis via the action of cyclooxygenase-2 (COX-2) is a component of the inflammatory response. Prostacyclin, a product of COX-2 in vascular endothelium, has important physiological roles, such as increasing blood flow to injured tissues, reducing leukocyte adherence, and inhibiting platelet aggregation. We examined the possibility that selective COX-2 inhibition could suppress the protective effects of prostacyclin, resulting in an alteration of the hemostatic balance and vascular tone. Circumflex coronary artery thrombosis was induced in dogs by vascular electrolytic injury. Orally administered celecoxib (COX-2 inhibition) or high-dose aspirin (HDA) (COX-1 and COX-2 inhibition) did not alter time to occlusive thrombus formation compared with controls (celecoxib 77.7+/-7.2 minutes, HDA 72.0+/-18.5 minutes, control 93.0+/-21.8 minutes). Oral HDA with an endothelial recovery period (HDA-ER) (COX-1 inhibition) produced a significant increase in time to vessel occlusion (257.0+/-41.6 minutes). The observed increase in time to occlusion was abolished when celecoxib was administered to animals dosed with HDA-ER (80.7+/-20.6 minutes). The vasomotor effect of endothelium-derived prostacyclin was examined by monitoring coronary flow during intracoronary administration of arachidonic acid or acetylcholine. In celecoxib-treated animals, vasodilation in response to arachidonic acid was reduced significantly compared with controls. The results indicate important physiological roles for COX-2-derived prostacyclin and raise concerns regarding an increased risk of acute vascular events in patients receiving COX-2 inhibitors. The risk may be increased in individuals with underlying inflammatory disorders, including coronary artery disease.

Suppression of sodium current by arachidonic acid in atrial myocytes from patients with coronary heart disease.

This study was designed to examine the effects of arachidonic acid (AA) on atrial myocytes from patients with coronary heart disease. The patch clamp technique was used to record sodium current in human atrial myocytes, before and after administration of intracellular AA. The suppression of sodium current induced by AA was voltage- and dose-dependent, with an IC50 of 10.3 microM. The activation curves of relative conductance in absence versus presence of AA, 10 microM, nearly overlapped. The 50% channel activation was at 40.8 +/- 2.7 mV in the control state versus 42.5 +/- 3.1 mV in presence of AA (n = 10, P > 0.05). AA at 10 microM shifted the steady-state inactivation relationship significantly, from 94.5 +/- 3.4 mV to 116.6 +/- 4.1 mV (n = 11, P < 0.01) at the 50% channel inactivation point. The 50% recovery time from the inactivation state was significantly longer in the presence of 10 microM AA (27.3 +/- 1.7 ms), than in the control state (5.9 +/- 0.4 ms n = 8, P < 0.01). In conclusion, AA suppressed the sodium current and prolonged the duration of recovery from inactivation in atrial myocytes from patients with coronary heart disease.

Arachidonic acid rescues hippocampal long-term potentiation blocked by group I metabotropic glutamate receptor antagonists

Although there is evidence that group I metabotropic glutamate receptors participate in long-term potentiation, the role of these receptors remains unclear. Among antagonists of group I metabotropic glutamate receptors, the mGluR5-selective 6-methyl-2-(phenylethynyl)-pyridine inhibited long-term potentiation in the CA1 region of hippocampal slices from 30-day-old rats, whereas (RS)-1-aminoindan-1,5-dicarboxylic acid and cyclopropan[b]chromen-1a-carboxylic acid ethylester, which are more selective for mGluR1, failed to inhibit long-term potentiation. Evidence also indicates that arachidonic acid is required for long-term potentiation, as inhibition of phospholipase A 2 blocks long-term potentiation. Administration of arachidonic acid immediately after tetanic stimulation restored long-term potentiation that had been inhibited by group I antagonists. Furthermore, arachidonic acid overcame inhibition of long-term potentiation by xestospongin C, an inositol triphosphate receptor channel blocker, or by thapsigargin, an agent that depletes intracellular calcium stores. However, arachidonic acid did not restore long-term potentiation blocked by N-methyl- d-aspartate receptor antagonists. Although it has been assumed that the source of the arachidonic acid necessary for long-term potentiation is N-methyl- d-aspartate receptor activation, our results suggest that during long-term potentiation group I metabotropic glutamate receptors cause arachidonic acid release by mobilization of intracellular calcium.

PLA(2) dependence of diaphragm mitochondrial formation of reactive oxygen species.

Contraction-induced respiratory muscle fatigue and sepsis-related reductions in respiratory muscle force-generating capacity are mediated, at least in part, by reactive oxygen species (ROS). The subcellular sources and mechanisms of generation of ROS in these conditions are incompletely understood. We postulated that the physiological changes associated with muscle contraction (i.e., increases in calcium and ADP concentration) stimulate mitochondrial generation of ROS by a phospholipase A(2) (PLA(2))-modulated process and that sepsis enhances muscle generation of ROS by upregulating PLA(2) activity. To test these hypotheses, we examined H(2)O(2) generation by diaphragm mitochondria isolated from saline-treated control and endotoxin-treated septic animals in the presence and absence of calcium and ADP; we also assessed the effect of PLA(2) inhibitors on H(2)O(2) formation. We found that 1) calcium and ADP stimulated H(2)O(2) formation by diaphragm mitochondria from both control and septic animals; 2) mitochondria from septic animals demonstrated substantially higher H(2)O(2) formation than mitochondria from control animals under basal, calcium-stimulated, and ADP-stimulated conditions; and 3) inhibitors of 14-kDa PLA(2) blocked the enhanced H(2)O(2) generation in all conditions. We also found that administration of arachidonic acid (the principal metabolic product of PLA(2) activation) increased mitochondrial H(2)O(2) formation by interacting with complex I of the electron transport chain. These data suggest that diaphragm mitochondrial ROS formation during contraction and sepsis may be critically dependent on PLA(2) activation.

The Characterization of Human Amnion Epithelial and Mesenchymal Cells: the Cellular Expression, Activity and Glucocorticoid Regulation of Prostaglandin Output

The amnion, a single layer of epithelial cells (EC) overlying layers of mesenchymal cells (MC) has been identified as a source of intrauterine prostaglandins (PG). The objectives of the present study were: (1) to establish a technique for the isolation and culture of pure amnion EC and MC preparations, (2) to characterize the cellular expression of PGHS-II and PGHS activity within these separated amnion cells and (3) to characterize the pattern of glucocorticoid stimulation of these separated amnion cells. Term gestation human amnion was collected after elective caesarean section or vaginal delivery. A trypsin digestion was used to isolate EC and a mechanical digestion and collagenase dispersion was used to isolate MC. Following 48 or 96h in culture, cells were incubated for 24h in the presence or absence of 1μm arachidonic acid and treated with cortisol (F: 10–1000n m) or 1μm dexamethasone (DEX). Cell types were identified by immunohistochemistry (IHC). Immunoreactive PGHS-II (ir-PGHS-II) and glucocorticoid receptor (ir-GR) were localized by IHC. PGHS activity was measured as PGE2output determined by radioimmunoassay. Mean PGE2production by MC at 72h was 22-fold greater (P< 0.05) and at 120h was 32-fold greater (P< 0.03) than PGE2output by EC. Administration of arachidonic acid stimulated a 5.0-fold increase in PGE2output (P< 0.0002) by EC after 72h and a 3.6-fold increase (P< 0.05) after 120h but did not alter MC PGE2output. Despite exogenous substrate, EC PGE2output remained significantly less than PGE2output by MC. There was no difference in PG production by EC and MC with the onset of labour. Ir-GR expression was found in both EC and MC. F and/or DEX with and without arachidonic acid (AA) stimulated PGE2output by EC. Only DEX and not F increased PGE2output by MC. These data suggest that relatively pure EC and MC preparations can be established from amnion. PG output and its regulation appears to differ within these two amnion cell types, dependent upon (1) substrate availability and (2) the regulation of PGHS activity.

Induction of Apoptosis by Arachidonic Acid in Human Retinoblastoma Y79 Cells: Involvement of Oxidative Stress

Arachidonic acid administration caused apoptosis in Y79 cells, as shown by typical morphological changes, phosphatidylserine externalization, chromatin condensation, processing and activation of caspase-3 and cleavage of the endogenous caspase substrate poly-(ADP-ribose)-polymerase. Arachidonic acid also caused lamin B cleavage, suggesting caspase-6 activation. Arachidonic acid treatment was accompanied by increased formation of the lipid peroxidation end products malondialdehyde and 4-hydroxy-2-nonenal, lowering in reduced glutathione content and in mitochondrial membrane potential. Inhibiting glutathione synthesis sensitized Y79 cells to apoptosis-inducing stimuli, whilst replenishing reduced glutathione attenuated arachidonic acid toxicity. Similar findings were obtained using hydroperoxyeicosatetranoic acids (oxygenated metabolites of arachidonic acid which deplete the reduced glutathione pool) and nordihydroguaretic acid, a general inhibitor of lipooxygenase pathway, which may also trigger rapid depletion of reduced glutathione. Melittin, which is known to activate phospholipase A2, also potently induced apoptosis. Arachidonic acid toxicity was inversely related to cell density. This could depend on an increased production of molecules with antiapoptotic effect; insulin-like growth factors could most likely be one of these molecules. These results propose a role for oxidative stress in the cytotoxicity induced by arachidonic acid in Y79 cells and suggest that these cells could be protected from such toxicity as long as sufficient levels of reduced glutathione and survival factors are present.

Role of arachidonic acid in leukotriene B 4-induced guinea-pig eosinophil homotypic aggregation

The activation of eosinophils with the lipid mediator, leukotriene B 4, induces their homotypic aggregation. Upon activation with leukotriene B 4, eosinophils release a significant amount of arachidonic acid, a process dependent on the activation of phospholipase A 2. Here, we have evaluated whether arachidonic acid could induce aggregation of eosinophils and whether the release of arachidonic acid mediated the aggregation induced by leukotriene B 4. The exogenous administration of arachidonic acid induced a concentration-dependent eosinophil homotypic aggregation. Pretreatment of eosinophils with a 5-lipoxygenase inhibitor or a leukotriene B 4 receptor antagonist abrogated arachidonic-acid-induced aggregation. Arachidonic acid induced a significant increase in leukotriene B 4 levels and desensitised leukotriene B 4-induced aggregation in a dose-dependent manner. Moreover, this desensitisation was effectively reversed by a 5-lipoxygenase inhibitor. However, arachidonic acid failed to induce a rise in intracellular Ca 2+ in eosinophils and failed to desensitise these cells to rises in intracellular Ca 2+ induced by leukotriene B 4. Pretreatment of eosinophils with the phospholipase A 2 inhibitor, mepacrine, inhibited the aggregation responses induced by 1 nM leukotriene B 4 by approximately 50% but had no significant effect on the other concentrations of leukotriene B 4 tested (0.1 to 100 nM). In conclusion, arachidonic acid stimulates eosinophil aggregation indirectly via the release of leukotriene B 4. Although a significant amount of arachidonic acid is released in response to activation of eosinophils with leukotriene B 4, the arachidonic acid released does appear to play a major role in mediating leukotriene B 4-induced eosinophil aggregation.

Extracellular and intracellular arachidonic acid-induced contractions in rat aorta

Arachidonic acid induced contractions of de-endothelized rat aortic rings. A more potent effect was obtained after intracellular administration of arachidonic acid using liposomes. Contractions induced by extracellular arachidonic acid were inhibited similarly to phenylephrine-induced contractions by the L-type Ca 2+ channel blocker, methoxyverapamil (D600), and the calmodulin inhibitor, calmidazolium. In contrast, contractions induced by arachidonic acid-filled liposomes were not affected by these compounds. Indomethacin did not affect the contractions induced by either extra- or intracellular arachidonic acid, whereas nordihydroguaiaretic acid relaxed contractions induced by extracellular arachidonic acid but not those induced by arachidonic acid-filled liposomes. Apart from a relaxing effect on contractions induced by extracellular arachidonic acid or by phenylephrine, protein kinase C inhibition with 1-(5-isoquinolinesulphonyl-2-methylpiperazine (H7)) had an even more prominent relaxing effect on contractions induced by arachidonic acid-filled liposomes. Therefore, arachidonic acid exerts a contractile effect on rat aorta, and this effect is regulated differently depending on the site of application.

Selective inhibition of cyclooxygenase-2 by NS-398 in endotoxin shock rats in vivo.

The role of cyclooxygenase (COX)-2 was examined using a rat endotoxin shock model and the potency and selectivity of NS-398, a COX-2 selective inhibitor in vitro, for COX-2 activity was examined in vivo. Male Wistar rats (weighing 140-180 g) were used. Lipopolysaccharide (LPS, 1 mg/kg, i.v.) was administered to rats (LPS-treated rats) and expression of COX-1 mRNA and COX-2 mRNA in the aorta and peripheral blood leukocytes was examined by RT-PCR. COX activity was assessed by measuring the plasma 6-keto prostaglandin (PG) F1 alpha, PGE2 and thromboxane (TX)B2 30s after administration of arachidonic acid (AA, 3 mg/kg, i.v.), NS-398 (0.3-100 mg/kg, p.o.) or indomethacin (0.3-3 mg/kg, p.o.) was administered 1 h before the AA injection. COX-2 mRNA was detectable in the aorta and peripheral blood leukocytes at least from 3 to 9 h after the LPS injection but not in non-LPS-treated rats. Plasma 6-keto PGF1 alpha, PGE2 and TXB2 levels after AA injection into LPS-treated rats were significantly enhanced compared to findings in non-LPS-treated rats. NS-398 showed significant inhibition of the increase in PGs in LPS-treated rats, the ED50 values being 0.35 mg/kg for 6-keto PGF1 alpha, 1.5 mg/kg for PGE2 and < 0.3 mg/kg for TXB2. NS-398 even at 100 mg/kg did not significantly suppress the increased PGs levels in non-LPS-treated rats. In contrast, indomethacin significantly inhibited plasma PGs levels after AA injection into LPS-treated rats and non-LPS-treated rats. The ED50 values in LPS-treated rats, determined by 6-keto PGF1 alpha, PGE2 and TXB2 production, were 1.0, 1.3 and 2.3 mg/kg and those in non-LPS-treated rats were 0.42, 0.24 and 0.93 mg/kg, respectively. In a rat endotoxin shock model, expression of COX-2 plays a role in an increase in COX activity. NS-398 showed preferential inhibitory effects on COX-2 activity in vivo. This approach is useful to directly analyze the inhibitory activity of NSAIDs for COX-1 and COX-2 in vivo.

Clearance and metabolism of arachidonic acid by C6 glioma cells and astrocytes.

Effects of increased levels of arachidonic acid (AA) were analyzed in vitro by employment of C6 glioma cells and astrocytes from primary culture. The cells were suspended in a physiological medium added with arachidonic acid (AA) in a concentration range from 0.01 to 0.5 mM. The concentration profiles of the fatty acid and AA-metabolites were subsequently followed for 90 min. AA was measured by gas chromatography, whereas the AA-metabolites PGF2 alpha and LTB4 by radioimmunoassay (RIA). Following administration of AA at 0.05 or 0.1 mM the medium was completely cleared from the fatty acid within 10 to 15 min. However, when 0.5 mM were added, AA concentrations of 0.36 +/- 0.055 mM were found at 20 min, while 0.275 +/- 0.045 mM at 90 min. Addition of AA (0.1 mM) to cell-free medium was also associated with a steady decline of its concentration, although the decrease was markedly delayed as compared to the clearance in the presence of glial cells. AA was subjected to dose-dependent metabolisation in the cell suspension as demonstrated by the production of PGF2 alpha and LTB4. Following addition of 0.01 or 0.5 mM, concentrations of PGF2 alpha increased to a 1.9- or 4.9-fold level within 10 min, whereas those of LTB4 rose to a 1.3- or 33.7-fold level. This was attenuated or completely blocked, respectively, by the cyclo- and lipoxygenase inhibitor BW 755C. Formation of both metabolites from AA was also observed when studying astrocytes from primary culture. The current findings demonstrate an impressive efficacy of C6 glioma cells and astrocytes to clear arachidonic acid from the suspension medium and to convert the lipid compound into prostaglandins and leukotrienes. Uptake and metabolisation of AA by the glial elements may play an important role in vivo, for example in cerebral ischemia.

Neutrophil release of arachidonic acid, oxidants, and proteinases: causally related or independent

This investigation examined the concept that arachidonic acid (AA) serves as a second messenger in stimulation of the respiratory burst and degranulation of polymorphonuclear neutrophils (PMN). The main support for this idea is from observations that reagent AA, added to cell suspensions, stimulates the respiratory burst and degranulation and these events are blocked by PLA 2 inhibitors. We verified that exogenously-added AA stimulated release of O 2 −, myeloperoxidase (MPO), and lysozyme (LZ), but this required amounts of AA which approximated the critical micellar concentration. This suggested that such administration of AA might act as an extracellular agonist, similar to particulate stimuli, rather than acting as a second messenger as might occur following mobilization of AA from cellular membranes. To investigate the role of fatty acids released by hydrolysis of cellular phospholipids, exogenously-added group I, II or III PLA 2's were used to mobilize fatty acids from cellular membranes. Mole quantities of cell-associated free fatty acids were measured by negative ion chemical ionization gas chromatography/mass spectrometry. AA mobilization in response to exogenous PLA 2 was dose-(0.1 to 10 U/ml PLA 2) and time-dependent (peak at 1 to 2 min with a reduction by 4 min). Resting neutrophils contained < 10 pmol free AA/10 7 PMN; the receptor-mediated agonist N-formyl-methionyl-leucyl-phenylalanine (fMLP) alone did not increase these values. Exogenously-added PLA 2 generated large quantities of free AA in control and fMLP-treated cells (462 ± 122 and 2097 ± 176 pmol/10 7 PMN, respectively); however, this did not induce O 2 −, nor did it augment the level of O 2 − stimulated by fMLP. Also, PLA 2 caused no degranulation and did not alter degranulation induced by fMLP. PLA 2 also did not alter O 2 − or degranulation responses in primed PMN. The data indicate that mobilization of AA from cellular phospholipids neither stimulates nor modulates the respiratory burst or degranulation of PMN.

Differential influence of arachidonic vs. eicosapentaenoic acid on experimental pulmonary hypertension.

The impact of the 2- and 3-series prostanoid precursors arachidonic acid (AA) and eicosapentaenoic acid (EPA) on experimental pulmonary hypertension was investigated. The model of buffer-perfused rabbit lungs was stimulated by infusion of Escherichia coli hemolysin (HlyA), which is known to provoke sustained thromboxane (Tx)-mediated pulmonary hypertension. Release of di- and trienoic Tx into the recirculating perfusate was quantified by a post-high-performance liquid chromatography enzyme-linked immunosorbent assay technique. HlyA at 0.08 hemolytic unit/ml caused a sustained rise in pulmonary arterial pressure (PAP; maximum increase 14 +/- 2 mmHg) accompanied by progressive TxB2 liberation (maximum perfusate concn 33 +/- 4 pg/ml, baseline < 2 pg/ml). Between 5 and 30 nM, AA provoked a transient monophasic rise in PAP (maximum pressor response 1.5-15 mmHg) and concomitant TxB2 release (peak concn 2-30 pg/ml). Simultaneous administration of HlyA and AA exhibited additive effects with regard to mediator release and pressor responses. EPA at 200-2,000 nM caused a transient rise in PAP similar to that provoked by 5-30 nM AA (maximum pressor response 3-18 mmHg). This was accompanied by liberation of TxB2 (peak concn 16 +/- 5 and 28 +/- 4 pg/ml after 1,000 and 2,000 nM EPA) and TxB3 (peak concn 9 +/- 4 and 30 +/- 3 pg/ml). Combined application of HlyA and EPA resulted in approximate addition of the TxB2 release reaction to each single compound, and TxB3 liberation more than doubled (maximum concn 59 +/- 12 pg/ml). The pressor responses to HlyA-EPA (200-2,000 nM) did not, however, surpass those to HlyA-AA (5-30 nM).(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacological characterization of cinnamophilin, a novel dual inhibitor of thromboxane synthase and thromboxane A2 receptor

1. The pharmacological effects of cinnamophilin, a new lignan, isolated from Cinnamomum philippinense, was determined in vitro in human platelet, rat isolated aorta and guinea-pig isolated trachea and in vivo in mice and guinea-pigs. 2. Cinnamophilin inhibited dose-dependently human platelet-rich plasma (PRP) aggregation induced by arachidonic acid (AA), collagen and U-46619 with IC50 of 5.0 +/- 0.4, 5.6 +/- 0.6 and 3.0 +/- 0.4 microM, respectively. The second wave of ADP- or adrenaline-induced platelet aggregation was inhibited by cinnamophilin, while the first wave was only slightly inhibited by cinnamophilin above 30 microM. 3. Cinnamophilin was found to be a thromboxane A2 (TXA2) receptor blocking agent in human platelet, rat aorta and guinea-pig trachea as revealed by its competitive antagonism of U-46619-induced aggregation of human-PRP, contraction of rat aortic rings and guinea-pig tracheal rings with pA2 values of 7.3 +/- 0.2, 6.3 +/- 0.1 and 5.2 +/- 0.2, respectively. 4. [3H]-inositol monophosphate formation and the rise of intracellular Ca2+ caused by U-46619 in human platelet was suppressed by cinnamophilin (10 microM). 5. Cinnamophilin induced a dose-dependent inhibition of thromboxane B2 (TXB2) formation, while the prostaglandin E2 (PGE2) formation was increased. Cinnamophilin did not affect unstimulated platelet adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels. When the platelets were challenged with AA, a dose-dependent rise in cyclic AMP was observed. Dazoxiben (a pure TX synthase inhibitor) and SQ 29548 (a pure TXA2 receptor antagonist) did not affect cyclic AMP levels in AA-treated platelets. 6. A high concentration of cinnamophilin (100 MicroM), failed to attenuate the contractile response of rat aorta to endothelin-l, angiotensin II, 5-hydroxytryptamine or noradrenaline. Contraction of tracheal rings induced by histamine, carbachol or KCl was also not inhibited by cinnamophilin (100 MicroM).7. Thirty min after intraperitoneal (i.p.) administration of cinnamophilin (100 microg kg-1), tail bleeding time of mice was prolonged more markedly than with indomethacin, dazoxiben or SQ 29548.8. Intravenous administration of AA (50 microg kg-1) to guinea-pig induced bronchoconstriction. Cinnamophilin(0.1 mg kg-1, i.v.) was administered 1 min before AA, the bronchoconstriction response to AA was abolished.9. It is concluded that cinnamophilin is a novel dual TX synthase inhibitor and TXA2 receptor antagonist and that it may be a useful tool for the investigation and treatment of diseases involving TXA2 disorders.

The mechanism of coronary collateral vasoconstriction in response to cyclooxygenase blockade.

The present study was performed to examine the mechanism by which cyclooxygenase blockade produces vasoconstriction in well-developed coronary collateral vessels. Eight dogs were studied 4 to 6 months after occlusion of the left anterior descending coronary artery (LAD) had been performed to stimulate collateral vessel growth. At the time of study, the LAD was cannulated at the site of occlusion for measurement of retrograde blood flow as an index of collateral blood flow. Levels of 6-ketoprostaglandin F1 alpha were 32 +/- 13% higher in blood diverted from the collateral-dependent LAD than in aortic blood (P < .05); the increase in this stable product of prostacyclin metabolism indicated production of prostacyclin across the coronary collateral system. Administration of arachidonic acid into the left main coronary artery to reach collateral vessels entering the LAD resulted in a 21 +/- 6% increase in retrograde flow (P < .01), demonstrating cyclooxygenase activity with production of vasodilator prostaglandins in the collateral system. Ibuprofen (10 mg/kg IV) caused a 55 +/- 7% decrease in retrograde flow (P < .03), suggesting that cyclooxygenase blockade inhibited tonic production of vasodilator prostaglandins in the collateral system. In contrast, neither thromboxane synthase inhibition with dazmegrel nor thromboxane receptor blockade with SQ 30741 caused a significant change in collateral flow, thus failing to support thromboxane-induced collateral constriction. After cyclooxygenase blockade, prostacyclin infused into the left main coronary artery was able to restore retrograde flow to the preibuprofen level.(ABSTRACT TRUNCATED AT 250 WORDS)

Peroxidation of linoleic, arachidonic and oleic acid in relation to the induction of oxidative DNA damage and cytogenetic effects.

In the present study, the possible role of the polyunsaturated fatty acids linoleic and arachidonic acid in the chemical induction of carcinogenesis has been investigated. Analysis of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) levels in 2'-deoxyguanosine (dG) and isolated DNA has demonstrated that linoleic and arachidonic acid are capable of inducing this specific genotoxic damage. This effect appears to be related to the degree of fatty acid unsaturation, since it was not induced by monounsaturated oleic acid. Enzymatic peroxidation of linoleic and arachidonic acid resulted in a significant increase in oxidative DNA damage. Studies on the interference of radical scavengers with the induction of 8-oxodG in combination with electron spin resonance spectroscopy demonstrated that the superoxide anion was generated during peroxidation of these fatty acids and that singlet oxygen is most likely involved in the formation of oxidative DNA damage. The level of oxidative damage in dG and single-stranded DNA was higher as compared to that in native DNA after equimolar treatment. Exposure of human lymphocytes to linoleic or arachidonic acid did not result in a significant increase in levels of 8-oxodG. This may indicate that the rate of intracellular peroxidation is relatively low and/or that nuclear DNA in intact cells is effectively protected against genetic damage induced by reactive oxygen species. It is therefore concluded that relatively short periods of linoleic or arachidonic acid administration are not likely to impose a direct genotoxic risk. It can, however, not be excluded that chronic exposure to polyunsaturated fatty acids induces oxidative DNA damage or is related to cancer risk by epigenetic mechanisms, as is also indicated by the observed cytotoxic effects of linoleic and arachidonic acid.

Relevance of vasoactive mediators for the blood pressure effects of intravenous arachidonic acid injection in rats

Vasopressor response and release of eicosanoids following intravenous injection of arachidonic acid (AA) were examined in normotensive rats. AA administration caused a rapid initial fall of arterial pressure followed by a brief rise and a subsequent prolonged fall in anesthetized rats. Immediately after AA injection the blood levels of TXB 2 and 6-keto-PGF 1α, the stable metabolites of TXA 2 and prostacyclin, rose, from 1.52 ± 0.23 ng/ml to 176.4 ± 42.6 ng/ml and from 4.05 ± 0.67 ng/ml to 171.4 ± 31.2 ng/ml, respectively. Blood pressure behaviour and eicosanoid blood level were influenced by different inhibitors and antagonists of vasoactive mediators. The cyclooxygenase inhibitor acetylsalicylic acid completely eliminated the second blood pressure depression after AA injection and simultaneously diminished TXB 2 and 6-keto-PGF 1α formation in murine blood, whereas the TXA 2 receptor antagonist BM 13.177 prevented the return of the blood pressure to preinjection level after the initial brief fall in arterial pressure. Although the TXA 2 synthase inhibitor HOE 944 markedly inhibited TXB 2 formation, no influence on AA-induced blood pressure changes could be registered. The receptor antagonist of platelet activating factor BN 52021 and the serotonin and histamine receptor antagonist cyproheptadine also reduced TXB 2 amounts, in murine blood without any effects on blood pressure behaviour.

Interaction between cortisol and arachidonic acid on the secretion of LH from ovine pituitary tissue

In several species, glucocorticoids act directly on the pituitary gonadotroph to suppress the gonadotrophin-releasing hormone (GnRH)-induced secretion of the gonadotrophins, especially LH. A mechanism for this action of these adrenal steroids has not been established, but it appears that the glucocorticoids influence LH release by acting on one or more post-receptor sites. This study investigated whether glucocorticoids disrupt GnRH-induced LH release by altering the liberation of arachidonic acid from plasma membrane phospholipids, a component of GnRH-induced LH release. Using perifused ovine pituitary tissue, it was established that exposure of gonadotrophs to 1-1000 nmol cortisol/l for 4 h or longer significantly reduced GnRH-stimulated LH release with the maximal inhibitory effect being observed after 6 h of exposure to cortisol. This suppressive effect of cortisol could be reversed by administration of arachidonic acid, which in its own right could stimulate LH release from ovine pituitary tissue. Furthermore, the inhibitory effect of cortisol on GnRH-stimulated LH release could be directly correlated with decreased pituitary responsiveness to GnRH-stimulated arachidonic acid liberation, consistent with our hypothesis that glucocorticoids can suppress GnRH-induced secretion of LH by reducing the amount of arachidonic acid available for the exocytotic response of GnRH.

Hypoxia-induced change in prostanoids production and coronary flow in isolated rat heart

The contribution of prostanoids to the change in coronary flow induced by hypoxia was examined in Langendorff-perfused rat heart. In the coronary effluent, 5 prostanoids, i.e., prostaglandins (PGs) D 2, E 2, and F 2 α , 6-keto PGF 1α and thromboxane (TX) B 2, were quantified by GC/MS, whereas PGA 2 , B 2 and E 1 were not detected under any conditions. During hypoxia, coronary flow initially increased to 189.5 ± 17.8% of the control, and at the same time release of all PGs, except for TXB 2 , increased significantly (6-keto PGF 1α : from 3.57 ± 0.98 to 5.54 ± 1.25 pmol/min·g, D 2: from 1.47 ± 0.26 to 2.22 ± 0.26 pmol/min·g, E 2: from 0.27 ± 0.08 to 0.96 ± 0.21 pmol/min·g, F 2 α : from 0.23 ± 0.09 to 0.48 ± 0.13 pmol/min·g, TXB 2 : from 0.61 ± 0.10 to 0.58 ± 0.15 pmol/min·g). During the later phase (10–20 min) of hypoxia, coronary 1 2 ow decreased without concomitant decrease in the release of PGs. The administration of indomethacin (10 μ m) and aspirin (1 m m) did not affect the normoxic coronary flow. However, during the early phase of hypoxia, they significantly suppressed the increase in coronary flow. Administration of arachidonic acid (1 mg/l) increased PG release 6.4–12.5-fold and increased coronary flow to 176.1 ± 6.5% of the control level. In the presence of arachidonic acid, there was a good correlation between the coronary flow and the amount of released vasodilative PGs ( PGE 2 and 6-keto PGF 1α ), suggesting the contribution of these PGs to coronary vasoregulation. On the other hand, when hearts were made hypoxic in the presence of arachidonic acid, percentage increase in PG release was much reduced, and similarly, coronary flow was not elevated. These results indicate that the increase in coronary flow during the early phase of hypoxia is mediated, at least in part, by the increased release of vasodilative PGs.

Leukotrienes in brain: natural occurrence and induced changes

Peptidoleukotrienes (SP-LTs) (both total product and individual LTC 4 and LTE 4) and LTB 4 were measured by radioimmunoassay in cerebrospinal fluid (CSF) collected from the third ventricle of conscious cats. Total SP-LT was expressed as LTE 4 after treating samples with crude γ-glutamyltranspeptidase. Prostaglandin (PG) E 2 and throm☐ane (TX) B 2, the stable metabolite of TXA 2, were also assayed in part of the experiments. Under basal conditions, SP-LT and LTC 4 were consistently measurable (respectively, 327 ± 14and244 ± 41pg/ml), while native LTE 4 was below the threshold of the assay (60–280 pg/ml) in most cases. LTB 4 was barely detectable (30 ± 2pg/ml) or not detectable at all. PGE 2 was normally less abundant than TXB 2 (31 ± 4vs281 ± 47pg/ml). Intracerebroventricular (i.c.v.) administration of arachidonic acid (40 μg) caused a 4-fold increase in SP-LT levels which was relatively small and transient compared to PGE 2 (76-fold) and TXB 2 (23-fold) while there was no change in either native LTE 4 or LTB 4. A similar response was obtained with platelet-activating factor (PAF, 1 μg i.c.v.), though SP-LT elevation (4-fold) was more persistent. A further rise in SP-LT (9-fold) was noted when PAF administration was preceded by indomethacin (500 μg i.c.v.), whereas PAF effect was reversed by pretreatment with either the PAF antagonist, BN52021 (1 μg i.c.v.), or the 5-lipoxygenase inhibitors, U-60, 257 (75 μg i.c.v.) and L-651, 392 (10 mg/kg p.o.). PAF was also effective in causing a 3-fold rise in LTC 4. Unlike PAF, pyrogens (endotoxin i.c.v. or i.v.; interleukin-1 i.v.) at doses above threshold for fever had no effect on LT levels in CSF, both in the absence and presence of indomethacin pretreatment. We conclude that SP-LTs are a normal constituent of CSF, LTC 4 being the major species. The response to PAF accords with a pathogenetic role of the compounds in inflammatory processes and the reactive changes to injury. No evidence was obtained for the involvement of SP-LTs in the central mechanism of fever.