Hypotensive action of arachidonic acid, slow acting analphylactic substance (SRS-A) and thromboxane A2 in rats]

In the rat, diethylcarbamazine, imidazole and L 8027 do not modify the hypotensive activity of PGE2 and arachidonic acid. The formation of SRS-A from arachidonic acid does not compete with the synthesis of PG in the cardiovascular system of the rat. The thromboxane A2 does not participate in the hypotensive activity of arachidonic acid in the rat.     

Inhibition of the prostaglandin-liberating activity of bradykinin in the rat]

In the rat, hydrocortisone, dexamethasone and chloroquine do not inhibit the hypotensive activity of arachidonic acid, which is due to endogenous prostaglandin synthesis. They reduce or suppress the secondary hypotensive phase induced by bradykinin. These substances inhibit the prostaglandin releasing activity of bradykinin but do not interfere with prostaglandin synthesis.     

Effect of oxamethacin and sulindac on prostaglandin synthesis]

1. Indomethacin and oxamethacin reduce the formation of malonaldehyde from arachidonic acid by rat platelets in vitro. Sulindac has no influence on this formation. 2. Indomethacin, oxamethacin and sulindac inhibit the action of arachidonic acid on the rat stomach strips in vitro. 3. These three anti-inflammatory drugs suppress the hypotensive activity of arachidonic acid in the rat in vivo. 4. The comparison of the inhibitory activities of these drugs in vivo and in vitro shows that sulindac is converted to a potent anti-prostaglandin-synthetase metabolite and that a part of the action of oxamethacin depends on its conversion to indomethacin in the rat.     

Hypotensive effects in cats caused by horseradish peroxidase mediated by metabolites of arachidonic acid

Horseradish peroxidase (HRP) given intravenously was shown to possess a marked hypotensive effect in cats. The effect was not noticeably influenced by antihistamines. Indomethacin and acetylsalicylic acid completely blocked the hypotensive effect of HRP. It is concluded that the effect of HRP on blood pressure is due to stimulation of the synthesis of metabolites of arachidonic acid.     

Role of platelets in arterial hypotension induced by arachidonic acid and in carrageenan induced edema in the rat]

When rat platelets are incubated in vitro in the presence of aspirin, the formation of malonaldehyde from arachidonic acid is inhibited. The production of malonaldehyde reflects the synthesis of prostaglandins and associated compounds. The same inhibition is found when the platelets originate from rats pretreated with aspirin. Small doses of aspirin are active in vitro and 10-20 mg/kg in vivo. This dosage of aspirin does not affect the hypotensive activity of arachidonic acid nor the oedematous properties of carrageenan in the rat. These two effects are thus independent from the prostaglandins formed in the platelets.     

Changes, induced by certain flavonoids, of the hypotensive effects of arachidonic acid]

In the rat, silybine and Z 12007, a derivative of rutoside, increase the vasodepressive activities of arachidonic acid, a prostaglandin precursor. They reduce the activity of PGE2. Quercetine also increases the hypotensive action of arachidonic acid. These three flavonoids are supposed to increase the prostaglandin biosynthesis.     

Cardiovascular reactivity to arachidonic acid, prostaglandin E2, prostacyclin and bradykinin in carrageenan treated rats

In the rat, during carrageenan-induced peritonitis, the hypotensive activity of arachidonic acid, which depends on PG biosynthesis, is increased during the first hours, and thereafter is not modified. The vasodilator action of PGE2 is reduced during the first day while the actions of PGI2 and bradykinin are not modified. The concentrations of plasma albumin and haptoglobin, two inhibitors of PG biosynthesis are reduced during the first hours. Thereafter the concentration of haptoglobin is increased by 100% while the concentration of albumin remains diminished. During this peritonitis, no plasmatic inhibitory influence on PG synthesis is seen. The anti-inflammatory action of carrageenans does not depend on PG synthesis inhibition.     

Relationship between lipemia and sensitivity to arachidonic acid in rabbits]

The hypotensive effect of arachidonic acid in the rabbit increases simultaneously with the fall of the plasmatic unesterified fatty acids (NEFA), after treatment with nicotinic acid or heparin. In the case of nicotinic acid sensitization, which occurs in 10 minutes, the fall of NEFA occurs immediately. The sensitization by heparin appears only after 40 minutes following the injection; during this latency, the NEFA are enhanced; after the fall of NEFA, the rabbit becomes more sensitive to arachidonic acid. Mechanisms of heparinic sensitization are discussed.     

Effect of 14,15-epoxyeicosatrienoic acid infusion on blood pressure in normal and hypertensive rats

Intravenous (IV) and intraarterial (IA) infusion of 14,15-epoxyeicosatrienoic acid (14,15-EET) produced a dose-dependent decrease in mean arterial blood pressure (MAP) in normal and spontaneously hypertensive rats (SHR). The hypotensive effect of 14,15-EET was observed from 1 microgram/kg to 10 micrograms/kg with a maximum reduction in MAP as much as 45 +/- 6 mmHg in both normal and SHR. In normal rats the hypotensive effect was found to be more pronounced when 14,15-EET was infused IA than IV. This suggests that 14,15-EET may be metabolized as it passes through the lungs. However, in SHR there was no difference in MAP when 14,15-EET was infused either IA or IV. This indicates that there is a differential removal of the epoxide across the pulmonary circulation. Administration of indomethacin failed to inhibit the hypotensive action of 14,15-EET, suggesting that it may not be a cyclo-oxygenase dependent mechanism. However, the PAF antagonist of BN-52021 inhibited the hypotensive action of 14,15-EET. This therefore, suggests that the release of PAF may be involved in the hypotensive action of this epoxide of arachidonic acid.     

Platelet stimulation in rats by lambda carrageenan and fibrinogen

Defibrinogenation by defibrase does not modify the hypotensive activity of lambda carrageenan in the Brown Norway rat. Defibrinogenation by defibrase and kininogen depletion by ellagic acid do not change this activity in the Wistar rat. This hypotensive action of lambda carrageenan which depends on platelet stimulation, is unaffected by the lack of fibrinogen.     

Effects of arachidonic acid and indomethacin on the in vitro release of prostaglandins by aortic strips of spontaneously hypertensive rats

Aortic strips removed from spontaneously hypertensive (SH) rats and preincubated with arachidonic acid (1.0 X 10(-5) g/ml) for 15 min produced two times more prostaglandin (PG) like material than aortae unexposed to the precursor of PG biosynthesis. The stimulating effect of arachidonic acid was largely inhibited by indomethacin (1.0 X 10(-5) g/ml). Also, the release of PG-like material by aortic strips derived from SH rats treated with an intravenous injection of indomethacin (10 mg/kg) was inhibited by 74% compared with the control tissues. These results raised the possibility that the in vivo conversion of arachidonic acid by large arteries of SH rats may contribute to the hypotensive effect of this PG precursor in SH rats.     

Phospholipase A2 in macrophage plasma membrane releases arachidonic acid from phosphatidylinositol

A high level of arachidonic acid release from [2-14C]arachidonylphosphatidylinositol (PI) was observed at neutral pH (6.0-7.0) in the presence of purified plasma membranes of guinea pig peritoneal macrophages. This activity was at least 10-fold higher than that with arachidonylphosphatidylcholine (PC) or phosphatidylethanolamine (PE) as substrate. The accumulation of [14C]diacylglycerol and [14C]phosphatidic acid was not detected at any time, and arachidonic acid release from [14C]arachidonyldiacylglycerol was not detectable either. The data suggest that arachidonic acid release from PI may not occur via the phospholipase C pathway. In this paper, we demonstrate the possibility that arachidonic acid release from PI at neutral pH in the macrophage plasma membrane is dependent on the action of phospholipase A2 (EC 3.1.1.4) -like activity. The maximum arachidonic acid release was dependent upon both pH and substrate. Particularly, the activity of arachidonic acid release from PI at neutral pH was very high compared with that from PC or PE. We suggest that phosphatidylinositol phospholipase A2 (EC 3.1.1.52) may play an important role in providing arachidonic acid for subsequent metabolic activity in the macrophages.     

Different substrate utilization between prostaglandin endoperoxide H synthase-1 and -2 in NIH3T3 fibroblasts

Recent studies suggested that prostaglandin endoperoxide H synthase-1 and prostaglandin endoperoxide H synthase-2 (PGHS-1 and PGHS-2) utilize different pools of arachidonic acid for synthesizing prostanoids. Using cultured murine NIH3T3 fibroblasts, we investigated the mechanism for the different utilization of arachidonic acid between PGHS-1 and -2. Histofluorescence staining for PGHS activity in intact cells demonstrated that quiescent 3T3 cells expressed only PGHS-1 activity and serum-activated 3T3 cells pretreated with aspirin expressed only PGHS-2 activity. Endogenous arachidonic acid released by calcium ionophore A23187 was not converted by PGHS-1 but exclusively converted by PGHS-2. In the cell free system, the kinetics of PGHS-1 were not so much different from those of PGHS-2. However, in intact cells, arachidonic acid at concentrations lower than 2.5 μM was converted by PGHS-2 alone but not by PGHS-1. Our findings indicated that this small amount of arachidonic acid as released by some stimuli is converted exclusively by PGHS-2. Furthermore, treating the PGHS-2-expressing cells with sodium selenite or ebselen, reducing agents of intracellular peroxides, only decreased PGHS-2 activity. We speculate that only PGHS-2 has been activated by intracellular peroxides and subsequently, it can convert the arachidonic acid released endogenously.     

Stimulation of human platelet guanylate cyclase by fatty acids.

Guanylate cyclase from human platelets was over 90% soluble, even when assayed in the presence of Triton X-100. A time-dependent increase in activity occurred when the enzyme was incubated at 37 degrees and this spontaneous activation was prevented by dithiothreitol. Arachidonic acid stimulated the soluble enzyme activity approximately 2- to 3-fold. Linear double reciprocal plots of guanylate cyclase activation as a function of arachidonic acid concentration were obtained with a Ka value of 2.1 muM. A Hill coefficient of 0.98 was obtained indicating that one fatty acid binding site is present for each catalytic site. Concentrations of arachidonic acid in excess of 10 muM caused less than maximal stimulation. Dihomo-gamma-linolenic acid and two polyunsaturated 22 carbon fatty acids stimulated the activity of guanylate cyclase to the same degree as did arachidonic acid. The methyl ester of arachidonic acid was much less effective. Diene, monoene, and saturated fatty acids of various carbon chain lengths as well as prostaglandins E1, E2, and F2alpha, had little or no effect. These data indicate that the structural determined required for stimulation by fatty acids of soluble platelet guanylate cyclase is a 1,4,7-octatriene group with its first double bond in the omega6 position. This structural group is similar to the substrate specificity determinants of fatty acid cyclooxygenase, the first enzyme of the prostaglandin synthetase complex. However, conversion of arachidonic acid to a metabolite of the cyclooxygenase pathway did not appear to be required for activation of the cyclase since activation occurred in the 105,000 X g supernatant fraction and pretreatment of this fraction with aspirin did not alter the ability of arachidonic acid to activate guanylate cyclase. Kinetic studies showed that the stimulation of guanylate cyclase by arachidonic acid is primarily an effect on maximal velocity. Arachidonic acid did not alter the concentration of free Mn2+ required for optimal activity. It is concluded that the activity of the soluble form of guanylate cyclase in cell-free preparations of human platelets can be increased by a lipid-protein interaction involving specific polyunsaturated fatty acids.     

Activation of the phosphatidylinositol 3-kinase-Akt/protein kinase B signaling pathway in arachidonic acid-stimulated human myeloid and endothelial cells: involvement of the ErbB receptor family

Although arachidonic acid has been demonstrated to stimulate a wide variety of cellular functions, the responsible mechanisms remain poorly defined. We now report that arachidonic acid stimulated the activity of class Ia phosphatidylinositol 3-kinase (PI3K) in human umbilical vein endothelial cells, HL60 cells, and human neutrophils. Pretreatment of endothelial cells with AG-1478, an inhibitor of the ErbB receptor family, resulted in the suppression of PI3K activation by arachidonic acid. The fatty acid enhanced the tyrosine phosphorylation of ErbB4 but not of ErbB2 or ErbB3. The ability of arachidonic acid to stimulate PI3K activity in neutrophils was suppressed by indomethacin and nordihydroguaiaretic acid, inhibitors of the cyclooxygenases and lipoxygenases, respectively, but not by 17-octadecynoic acid, an inhibitor of omega-hydroxylation of arachidonic acid by cytochrome P450 monooxygenases. Consistent with this, the activity of PI3K in neutrophils was stimulated by 5-hydroxyeicosatetraenoic acid. Arachidonic acid also transiently stimulated the phosphorylation of Akt on Thr-308 and Ser-473. Although PI3K was not required for the activation of the mitogen-activated protein kinases, ERK1, ERK2, and p38, in arachidonic acid-stimulated neutrophils, the fatty acid acted via PI3K to stimulate the respiratory burst. These results not only define a novel mechanism through which some of the actions of arachidonic acid are mediated but also demonstrate that, in addition to ErbB1 (epidermal growth factor receptor), ErbB4 can also be transactivated by a non-epidermal growth factor-like ligand.     

Defining the role of protein kinase c in calcium-ionophore-(A23187)-mediated activation of phospholipase A2 in pulmonary endothelium.

We sought to investigate the mechanisms by which the calcium ionophore A23187 triggers arachidonic acid release in bovine pulmonary endothelial cells and to test the hypothesis that protein kinase C is involved in this process. Our results indicate that the mechanism by which A23187 increases phospholipase A2 activity and arachidonic acid release in bovine pulmonary arterial endothelial cells depends upon the concentration studied. At concentrations of 1 microM and 2.5 microM, A23187 increases phospholipase A2 activity and arachidonic acid release without stimulating protein kinase C. At concentrations of 5-12.5 microM, A23187 increases arachidonic acid release and phospholipase A2 activity in conjunction with a dose-dependent activation of membrane-bound protein kinase C. To test the hypothesis that these doses of A23187 increase phospholipase A2 activity by stimulating protein kinase C, we studied the effect of prior treatment with the protein kinase C inhibitor sphingosine. Sphingosine inhibits the increase in phospholipase A2 activity and arachidonic acid release caused by A23187 over the range 5-12.5 microM. To investigate further the potential role of protein kinase C, we studied the effects of the inactive phorbol ester 4 alpha-phorbol 12 beta-myristate 13 alpha-acetate (4 alpha-PMA) and an active phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (4 beta PMA). Neither 4 alpha-PMA nor 4 beta-PMA affected basal arachidonic acid release. 4 alpha-PMA also did not augment the effects of A23187. In contrast, 4 beta-PMA significantly augments the increase in phospholipase A2 activity and arachidonic acid release caused by lower doses of A23187. Under these conditions, sphingosine completely inhibits the stimulatory effects of 4 beta-PMA on protein kinase C translocation, phospholipase A2 and arachidonic acid release. Thus, at low doses (1 microM and 2.5 microM) A23187 increases phospholipase A2 activity and arachidonic acid release by a mechanism that does not involve protein kinase C. At these A23187 doses, activating membrane-bound protein kinase C with 4 beta-PMA causes a synergistic increase in phospholipase A2 activity and arachidonic acid release. At higher doses (5-12.5 microM), A23187 acts in large part by stimulating protein kinase C translocation. Overall, our results indicate that activating membrane-bound protein kinase C by itself is an insufficient stimulus to increase phospholipase A2 activity and arachidonic acid release in pulmonary endothelial cells, but activating protein kinase C can substantially augment the increase in phospholipase A2 activity and arachidonic acid caused by a small increase in intracellular calcium.     

Antagonism of PAF-induced death in mice

The ability of three platelet activating factor (PAF) antagonists, BN52021, L652, 731 and 48740RP, and the leukotriene antagonist FPL55712 to block iv PAF-induced death was tested in mice. PAF-induced sudden death was been previously characterized as a model of systemic anaphylaxis and circulatory shock related its hypotensive actions. Of the drugs, BN52021 and L652, 731 provided dose-dependent protection against PAF toxicity, whereas the others had no effect. 48740RP was, however active against PAF-induced rabbit platelet aggregation. BN52021 was inactive in three other mouse sudden death models in which arachidonic acid, U46619 or collagen combined with epinephrine is injected iv to provoke a thrombotic/ischemic sudden death. In contrast, the TXA₂ antagonist SQ29548 inhibited the acute toxicity of two of these latter challenges (arachidonic acid and thromboxane agonist U46619), but was inactive against PAF lethality.These results suggest that PAF toxicity in mice is a specific model for PAF agonism, and is not mediated by TXA₂ or peptido-leukotrienes. Further, PAF-induced mortality should be a simple and useful technique for testing potential PAF antagonists for activity by various routes of administration.     

The effects of arachidonic and other fatty acids on canine neutrophil metabolism

The effect of arachidonic acid on the metabolic activity and chemiluminesence of canine neutrophils was investigated to gain further insight into its role in the neutrophil metabolic burst. Arachidonic acid was found to stimulate metabolic activity and luminol-augmented chemiluminescence. The increased metabolic activity was detected by both oxygen uptake measurements and assays of hexose monophosphate shunt activity. An inhibitor of lipoxygenase and cyclooxygenase,5, 8, 11, 14-eicosatetraynoic acid prevented the hexose monophosphate shunt response to arachidonic acid. Aspirin or indomethacin, blockers of cyclooxygenase, inhibited chemiluminescence but failed to block the metabolic response to arachidonic acid. Since superoxide dismutase and 2-deoxyglucose, a blocker of glucose metabolism, inhibited the chemiluminescent response of neutrophils to arachidonic acid, it is likely that oxygen radicals produced via the hexose monophosphate shunt are required for the chemiluminescent reaction. In addition it was found that inhibition of cyclooxygenase activity blocked chemiluminescence but not the metabolic stimulation induced by sodium fluoride, suggesting that the chemiluminescence stimulated by sodium fluoride is associated with endogenous fatty acid stores. From these studies it can be concluded that arachidonic acid products of the cyclooxygenase pathway do not play a significant role in the metabolic response of neutrophils when arachidonic acid or sodium fluoride is the stimulant while the lipoxygenase pathway appears to be involved. The metabolic response is not linked to the chemical reaction that causes neutrophil, chemiluminesence, although the chemiluminescent response depends on hexose monophosphate shunt activity and presumably the oxygen radicals that ultimately result from that process.     

Inhibition of Choline Acetyltransferase Activity by Serum Albumin Modified with Octanoic Acid and Other Fatty Acids

In this study, we examined the effect of fatty acids on choline acetyltransferase (ChAT) activity. ChAT is unstable in a solution of low protein concentration, so serum albumin (BSA) is usually added to stabilize the enzyme. However, we found that ChAT from bovine caudate nucleus rapidly lost its activity when diluted with a buffer containing commercial preparations of BSA. This effect was caused by octanoic acid, which was found in the gas chromatography/mass spectrometry system of lipid extract in commercial BSAs. The inhibition of ChAT activity by octanoic acid depended on the concentrations of the octanoic acid and of the albumin. We also found that ChAT activity was decreased by some long-chain fatty acids, arachidonic acid having exhibited the strongest effect. The extent to which arachidonic acid inhibited ChAT activity depended on the molar ratio of arachidonic acid and albumin, rather than upon the concentration of arachidonic acid. The effect of octanoic acid and arachidonic acid on ChAT activity appeared to increase in the presence of albumin.     

Origin of the arachidonic acid released post-mortem in rat forebrain.

To determine the origins of the arachidonic acid released post-mortem in brain tissue, [3H]arachidonic acid was injected by the intracerebro-ventricular route and radioactivity monitored in complex lipids and free arachidonic acid at various times after decapitation. The specific activity of the released arachidonic acid was close to that in the total phospholipid fraction and much lower than that of the neutral lipids. The phospholipid with the closest specific activity to the free arachidonic acid recovered at the end of the post-mortem period was phosphatidylinositol. Phosphatidylcholine showed a small but significant decrease in radioactivity post-mortem and could contribute 37% of the arachidonic acid released to the free fatty acid fraction. Arachidonic acid released in rat forebrain after decapitation thus comes from a mixture of phospholipids with phosphatidylinositol and phosphatidylcholine being the major source. Phosphatidylserine and phosphatidic acid did not make important contributions to the free arachidonic acid. In the microsomal fraction, the specific activity of the free arachidonic acid was very close to that in phosphatidylinositol.