Possible inhibitory function of endogenous 15-hydroperoxyeicosatetraenoic acid on prostacyclin formation in bovine aortic endothelial cells

Arachidonic acid is metabolized via the cyclooxygenase pathway to several potent compounds that regulate important physiological functions in the cardiovascular system. The proaggregatory and vasoconstrictive thromboxane A2 produced by platelets is opposed in vivo by the antiaggregatory and vasodilating activity of prostacyclin (prostaglandin I2) synthesized by blood vessels. Furthermore, arachidonic acid is metabolized by lipoxygenase enzymes to different isomeric hydroxyeicosatetraenoic acids (HETE's). This metabolic pathway of arachidonic acid was studied in detail in endothelial cells obtained from bovine aortae. It was found that this tissue produced 6-ketoprostaglandin F1 alpha as a major cyclooxygenase metabolite of arachidonic acid, whereas prostaglandins F2 alpha and E2 were synthesized only in small amounts. The monohydroxy fatty acids formed were identified as 15-HETE, 5-HETE, 11-HETE and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT). The latter two compounds were produced by cyclooxygenase activity. Nordihydroguaiaretic acid (NDGA), a rather selective lipoxygenase inhibitor and antioxidant blocked the synthesis of 15- and 5-HETE. It also strongly stimulated the cyclooxygenase pathway, and particularly the formation of prostacyclin. This could indicate that NDGA might exert its effect on prostacyclin levels by preventing the synthesis of 15-hydroperoxyeicosatetraenoic acid (15-HPETE), a potent inhibitor of prostacyclin synthetase. 15-HPETE could therefore act as an endogenous inhibitor of prostacyclin production in the vessel wall.     

Experimental atherosclerosis in rabbits: platelet aggregation, thromboxane A2 generation and anti-aggregatory potency of prostacyclin.

Experimental atherosclerosis in rabbits was associated with increased aggregation of their platelets to arachidonic acid, and with increased generation of thromboxane A2 by their platelet-rich plasma. A heightened susceptibility of platelets to the anti-aggregatory action of prostacyclin against the ADP-induced aggregation was also observed. It is concluded that in advance atherosclerosis the platelet system is hypersensitive to biologically active metabolites of arachidonic acid.     

Macrophage eicosanoid formation is stimulated by platelet arachidonic acid and prostaglandin endoperoxide transfer

The present study was designed to determine whether platelets transfer arachidonic acid or prostaglandin endoperoxide intermediates to macrophages which may be further metabolized into cyclooxygenase products. Adherent peritoneal macrophages were prepared from rats fed either a control diet or an essential fatty acid-deficient diet, and incubated with a suspension of washed rat platelets. Macrophage cyclooxygenase metabolism was inhibited by aspirin. In the presence of a thromboxane synthetase inhibitor, 7-(1-imidazolyl)heptanoic acid, immunoreactive 6-ketoprostaglandin F1 alpha formation was significantly increased 3-fold. Since this increase was greater (P less than 0.01) than that seen with either 7-(1-imidazolyl)heptanoic acid-treated platelets or aspirin-treated macrophages alone, these results indicate that shunting of endoperoxide from platelets to macrophages may have occurred. In further experiments, macrophages from essential fatty acid-deficient rats were substituted for normal macrophages. Essential fatty acid-deficient macrophages, depleted of arachidonic acid, produced only 2% of the amount of eicosanoids compared to macrophages from control rats. When platelets were exposed to aspirin, stimulated with thrombin, and added to essential fatty acid-deficient macrophages, significantly more immunoreactive 6-ketoprostaglandin F1 alpha was formed than in the absence of platelets. This increased macrophage immunoreactive 6-ketoprostaglandin F1 alpha synthesis, therefore, must have occurred from platelet-derived arachidonic acid. These data indicate that in vitro, in the presence of an inhibition of thromboxane synthetase, prostaglandin endoperoxides, as well as arachidonic acid, may be transferred between these two cell types.     

Experimental atherosclerosis in rabbits: Platelet aggregation, thromboxane A2 generation and anti-aggregatory potency of prostacyclin

Experimental atherosclerosis in rabbits was associated with increased aggregation of their platelets to arachidonic acid, and with increased generation of thromboxane A₂ by their platelet-rich plasma. A heightened susceptibility of platelets to the anti-aggregatory action of prostacyclin against the ADP-induced aggregation was also observed. It is concluded that in advanced atherosclerosis the platelet system is hypersensitive to biologically active metabolites of arachidonic acid.     

Acrolein: a potent modulator of lung macrophage arachidonic acid metabolism

Resting rat pulmonary alveolar macrophages exposed to acrolein were stimulated to synthesize and release thromboxane B2 and prostaglandin E2 in a dose-dependent manner. Zymosan-activated pulmonary alveolar macrophages released approximately twice as much prostaglandin E2 as thromboxane B2, whereas acrolein-activated pulmonary alveolar macrophages released 4-5 times less prostaglandin E2 than thromboxane B2. In the zymosan-stimulated pulmonary alveolar macrophages, acrolein also induced a reversal in the relative amounts of prostaglandin E2 and thromboxane B2 synthesized and released into the culture medium. This reversal was achieved by a dose-dependent reduction in prostaglandin E2 synthesis. Although phagocytosis was also inhibited in a dose-dependent manner, the reduction in prostaglandin E2 appeared to be partially independent of particle ingestion since thromboxane B2 synthesis was not affected by low doses of acrolein. In fact, high doses induced a slight enhancement in thromboxane B2 synthesis. These results suggest that acrolein selectively inhibited the enzyme, prostaglandin endoperoxide E isomerase, necessary for the conversion of the endoperoxide to prostaglandin E2. Sulfhydryl reagents such as N-ethylmaleimide and 5,5'-dithiobis (2-nitrobenzoic acid) mimicked acrolein's effects, and reduced glutathione afforded protection against the effects of acrolein. These results indicated the possible involvement of acrolein's sulfhydryl reactivity in the inhibition of the isomerase enzyme. Propionaldehyde had no effect on macrophage arachidonic acid metabolism whereas crotonaldehyde mimicked the effects of acrolein. Pulmonary macrophages were unable to reverse the acrolein effects on arachidonate metabolite synthesis after 6 h in an acrolein-free environment. These data indicated the necessity of the unsaturated carbon bond for the acrolein effects on arachidonic acid metabolism and the relative irreversibility of acrolein's reaction with the macrophage.     

Peroxidase-dependent deactivation of prostacyclin synthetase.

A study of the enzymes of the arachidonic acid cascade revealed a high sensitivity of prostacyclin synthetase and a complete resistance of thromboxane A2 synthetase to time-dependent destruction by an oxidant [Ox] released during the peroxidase-catalyzed reduction of hydroperoxy fatty acids. The destructive action of [Ox] derived from prostaglandin G1 (PGG1), 15-hydroperoxy-PGE1, 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid, and 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid upon prostacyclin synthetase was prevented by 2-aminomethyl-4-t-butyl-6-iodophenol. On the other hand, deactivation resulting from PGG2 metabolism was neither time-dependent nor sensitive to 2-aminomethyl-4-t-butyl-6-iodophenol. The possibility that the action of [Ox] may alter the arachidonic acid cascade in favor of thromboxane A2 is discussed in view of its possible implications in inflammatory and other pathological processes.     

Arachidonic acid metabolism and prostaglandin production by primate preimplantation blastocysts.

Preimplantation embryos of many species are known to synthesize prostaglandins. These tissue hormones are believed to influence embryonic metabolism, as well as embryo-maternal interaction during implantation although their putative role(s) remains obscure. Here, prostaglandin production by blastocysts from cynomolgus monkeys (Macaca fascicularis) was examined qualitatively during in vitro culture. Tritium labelled arachidonic acid was metabolized to 6 keto-prostaglandin F1 alpha, 2,3-dinor-prostaglandin F1 alpha and thromboxane B2, as characterized by HPLC separation. Also, 6-keto-prostaglandin F1 alpha, and thromboxane B2 as characterized by HPLC separation. Also, 6-keto-prostaglandin F1 alpha and thromboxane B2 were identified by specific RIA's. Our data suggest that the main arachidonic acid metabolites produced by blastocysts of cynomolgus monkeys are prostacyclin and thromboxane.     

Control of prostanoid synthesis: role of reincorporation of released precursor fatty acids

Prostanoid synthesis is limited by the availability of free arachidonic acid. This polyunsaturated fatty acid is liberated by phospholipases and usually is an intermediate of the deacylation-reacylation cycle of membrane phospholipids. In rat peritoneal macrophages, ethylmercurisalicylate (merthiolate) or N-ethylmaleimide (NEM) dose dependently inhibited the incorporation of arachidonic acid into cellular phospholipids, at lower concentrations specifically into phosphatidylcholine. Furthermore, merthiolate could be shown to be a rather selective inhibitor of lysophosphatidylcholine acyltransferase. In contrast, phospholipase A2 activity was not affected over a wide dose range. Consequently, macrophages showed a large increase in prostanoid synthesis (prostaglandin E, prostacyclin and thromboxane) in the presence of both lysophosphatide acyltransferase inhibiting agents. Similar results were obtained with human platelets, in which merthiolate increased the release of thromboxane. Addition of free arachidonic acid also enhanced prostanoid synthesis in macrophages. At optimal concentrations, merthiolate had no further augmenting effect. It is concluded that the rate of prostanoid synthesis is not only controlled by phospholipase A2 activity, but rather by the activity of the reacylating enzymes, mainly lysophosphatide acyltransferase.     

The adenylate cyclase of rheumatoid synovial fluid macrophages is more sensitive for dl-5E, 19,14-di dehydro-carbo-prostacyclin, a stable prostacyclin analogue, than for prostaglandin E2

dl-5E, 19,14-di dehydro-carbo-prostacyclin (DDH-carbo PGI2), a stable prostacyclin (PGI2) derivative, but not prostaglandin (PG) E2, stimulated the adenylate cyclase of synovial fluid macrophages, isolated from rheumatoid patients with an active synovitis, in a dose dependent manner (10-1000 ng/ml). DDH-carbo PGI2 also stimulated synovial macrophage cAMP synthesis when injected into the knee joint. Exogenous arachidonic acid (AA) had little effect on cyclic-AMP (cAMP) formation or PGI2 release (assayed as 6ketoPGF1 alpha). It stimulated, however, the release of PGE2 and, to a lesser extent, thromboxane (Tx) A2 (measured as TxB2).     

Effects of OKY 1581 on bronchoconstrictor responses to arachidonic acid and PGH2

The influence of OKY 1581, a thromboxane synthase inhibitor, on airway responses to arachidonic acid and endoperoxide, [prostaglandin (PG) H2], were investigated in anesthetized, paralyzed, mechanically ventilated cats. Intravenous injections of arachidonic acid and PGH2 caused dose-related increases in transpulmonary pressure and lung resistance and decreases in dynamic and static compliance. OKY 1581 significantly decreased airway responses to arachidonic acid but not to PGH2. Sodium meclofenamate, a cyclooxygenase inhibitor, abolished airway responses to arachidonic acid but had no effect on airway responses to PGH2. OKY 1581 or meclofenamate has no effect on airway responses to PGF2 alpha, PGD2, or U 46619, a thromboxane mimic. In microsomal fractions from the lung, OKY 1581 inhibited thromboxane formation without decreasing prostacyclin synthesis or cyclooxygenase activity. These studies show that OKY 1581 is a selective thromboxane synthesis inhibitor in the cat lung and suggest that a substantial part of the bronchoconstrictor response to arachidonic acid is due to thromboxane A2 formation. Moreover, the present data suggest that airway responses to endogenously released and exogenous PGH2 are mediated differently and that a significant part of the response to exogenous PGH2 may be due to activation of an endoperoxide/thromboxane receptor, since responses to PGH2 are blocked by the thromboxane receptor antagonist SQ 29548.     

Thromboxane-induced pulmonary vasoconstriction: involvement of calcium

Infusion of tert-butyl hydroperoxide (t-bu-OOH) or arachidonic acid into rabbit pulmonary arteries stimulated thromboxane B2 (TxB2) production and caused pulmonary vasoconstriction. Both phenomena were blocked by cyclooxygenase inhibitors or a thromboxane synthase inhibitor. The increase in pulmonary arterial pressure caused by either t-bu-OOH or arachidonic acid infusion correlated with the concentration of TxB2 in the effluent perfusate. The concentration of TxB2 in the effluent perfusate, however, was always 10-fold greater after arachidonic acid infusion. In the rabbit pulmonary vascular bed lipoxygenase products did not appear involved in the vasoactive response to t-bu-OOH or exogenous arachidonic acid infusion. Calcium entry blockers or a calcium-free perfusate prevented the thromboxane-induced pulmonary vasoconstriction. Calmodulin inhibitors also blocked the pulmonary vasoconstriction induced by t-bu-OOH without affecting the production of TxB2 or prostacyclin. These results suggest that thromboxane causes pulmonary vasoconstriction by increasing cytosol calcium concentration.     

Manipulation of platelet aggregation by prostaglandins and their fatty acid precursors: pharmacological basis for a therapeutic approach

Addition of the one-, two- or three- series endoperoxide to human platelet-rich plasma tend to suppress aggregation, through the action of their respective non-enzymatic breakdown products PGE1, PGD2, or PGD3 all of which elevate cyclic AMP levels. On the other hand, these stable primary products do not arise in appreciable amounts from intrinsic endoperoxides generated from either endogenous or exogenous free fatty acids. 5,8,11,14,17-Eicosapentaenoic acid (EPA) suppresses arachidonic acid (5,8,11,14-eicosatetraenoic acid) conversion by cyclooxygenase (as well as lipoxygenase) to aggregatory metabolites in platelets. Exogenously added EPA was capable of inhibiting PRP aggregation induced either by exogenous or endogenous (released by ADP or collagen) arachidonate. The hypothetical combination of an EPA-rich diet and a thromboxane synthetase inhibitor might abolish production of the pro-aggregatory species, thromboxane A2, and enhance formation of the anti-aggregatory metabolite, prostacyclin. Whereas EPA is not detectably metabolized by platelets, dihomo-gamma-linolenic acid (8,11,14-eicosatrienoic acid) is primarily converted by cyclooxygenase and thromboxane synthetase into the inactive metabolite, 12-hydroxyheptadecadienoic (HHD) acid. Pretreatment of human platelet suspensions with the thromboxane synthetase inhibitor imidazole unmasks the aggregatory property of PGH1 and DLL which was partially compromised by the PGE1 formed. The combination of the thromboxane synthetase inhibitor and an adenylate cyclase inhibitor unmasks a complete irreversible aggregation by DLL or PGH1. The basis of a dietary strategy that replaces AA with DLL must rely on the production by the platelet of an inactive metabolite (HHD) rather than thromboxane A2.     

Effect of toxins on arachidonic acid metabolism in rat cultured pulmonary alveolar macrophages

The action of a trichothecene (T-2), microcystin-LR and saxitoxin on arachidonic acid metabolism in cultured rat alveolar macrophages was studied. Pulmonary macrophages exposed to T-2 trichothecene were stimulated to synthesize and release large amount of thromboxane B2 (TxB2) and 6-Keto F1 alpha. Microcystin-LR induced significant release of prostaglandins F2 alpha (140%), PGE2 (175%) and TxB2 (169%) compared to controls. Saxitoxin induced TxB2 release by 37%. Arachidonic acid release was stimulated by all three toxins. The release of arachidonic acid and its metabolites in alveolar macrophages exposed to T-2 toxin was partially blocked by fluocinolone (1 microM). These results suggest that macrophages synthesize and release inflammatory mediators in response to toxin exposure, and fluocinolone may protect against T-2 toxicosis.     

A comparative study of thromboxane and prostacyclin release from ex vivo and cultured bovine vascular endothelium

Thromboxane B2, 6-keto-Prostaglandin F1 alpha, and Prostaglandin E2 release have been quantitated from cultured adult bovine endothelial cell monolayers and from ex Vivo vascular segments employing specific radioimmunoassays and thin layer chromatography. Release of all three prostaglandins was demonstrable from both endothelial cell systems under basal conditions and following exposure to the ionophore A23187 and arachidonic acid. In culture, the quantity of 6-keto-PGF1 alpha released was diminished compared to amounts released from the vessel segments while thromboxane B2 and prostaglandin E2 release were similar in the two endothelial model systems. However, the amount of thromboxane B2 assayed was small and the quantity of thromboxane A2 it represents is probably of little in vivo significance compared to prostacyclin.     

Control of prostanoid synthesis: Role of reincorporation of released precursor fatty acids

Prostanoid synthesis is limited by the availability of free arachidonic acid. This polyunsaturated fatty acid is liberated by phospholipases and usually is an intermediate of the deacylation-reacylation cycle of membrane phospholipids. In rat peritoneal macrophages, ethylmercurisalicylate (merthiolate) or N-ethylmaleimide (NEM) dose dependently inhibited the incorporation of arachidonic acid into cellular phospholipids, at lower concentrations specifically into phosphatidylcholine. Furthermore, merthiolate could be shown to be a rather selective inhibitor of lysophosphatidylcholine acyltransferase. In contrast, phospholipase A₂ activity was not affected over a wide dose range. Consequently, macrophages showed a large increase in prostanoid synthesis (prostaglandin E, prostacyclin and thromboxane) in the presence of both lysophosphatide acyltransferase inhibiting agents. Similar results were obtained with human platelets, in which merthiolate increased the release of thromboxane. Addition of free arachidonic acid also enhanced prostanoid synthesis in macrophages. At optimal concentrations, merthiolate had no further augmenting effect. It is concluded that the rate of prostanoid synthesis is not only controlled by phospholipase A₂ activity, but rather by the activity of the reacylating enzymes, mainly lysophosphatide acyltransferase.     

Arachidonic acid metabolites and an early stage of pulmonary hypertension in chronically hypoxic newborn pigs

Our purpose was to determine whether production of arachidonic acid metabolites, particularly cyclooxygenase (COX) metabolites, is altered in 100-400-microm-diameter pulmonary arteries of piglets at an early stage of pulmonary hypertension. Piglets were raised in either room air (control) or hypoxia for 3 days. A cannulated artery technique was used to measure responses of 100-400-microm-diameter pulmonary arteries to arachidonic acid, a prostacyclin analog, or the thromboxane mimetic. Radioimmunoassay was used to determine pulmonary artery production of thromboxane B(2) (TxB(2)) and 6-keto-prostaglandin F(1alpha) (6-keto-PGF(1alpha)), the stable metabolites of thromboxane and prostacyclin, respectively. Assessment of abundances of COX pathway enzymes in pulmonary arteries was determined by immunoblot technique. Arachidonic acid induced less dilation in pulmonary arteries from hypoxic than in pulmonary arteries from control piglets. Pulmonary artery responses to prostacyclin and were similar for both groups. 6-Keto-PGF(1alpha) production was reduced, whereas TxB(2) production was increased in pulmonary arteries from hypoxic piglets. Abundances of both COX-1 and prostacyclin synthase were reduced, whereas abundances of both COX-2 and thromboxane synthase were unaltered in pulmonary arteries from hypoxic piglets. At least partly due to altered abundances of COX pathway enzymes, a shift in production of arachidonic acid metabolites, away from dilators toward constrictors, may contribute to the early phase of chronic hypoxia-induced pulmonary hypertension in newborn piglets.     

Changes in prostacyclin and thromboxane biosynthesis and their catabolic enzyme activity in kidneys of aging rats

The effects of aging on the prostacyclin and thromboxane biosynthesis and prostaglandin catabolic enzyme activity in rat kidney were investigated. The prostacyclin biosynthesis, using arachidonic acid as substrate, was the greatest in young kidneys (2 months old) and then progressively decreased in mature (12 months old) and old (24 months old) kidneys, while thromboxane biosynthetic activity showed no significant change as a function of age. When prostaglandin H2 was used as substrate, the prostacyclin and thromboxane biosynthesis showed similar results as when arachidonic acid was used as substrate; the prostacyclin biosynthesis progressively decreased and thromboxane biosynthesis showed no significant change as a function of age. The fatty acid cyclooxygenase in kidney was measured by a specific radioimmunoassay. No significant change in renal fatty acid cyclooxygenase as a function of age was found. Thus, we concluded that the progressive decrease in renal prostacyclin biosynthesis as a function of age is due to a defect in prostacyclin synthetase in aged kidneys. The prostaglandin catabolic enzyme, NAD+-dependent 15-hydroxyprostaglandin dehydrogenase, in kidneys was also investigated. The enzyme activity progressively decreased as a function of age, which suggested a decrease in the metabolism of thromboxane A2 in aged kidneys. The present results, indicating a decrease in renal prostacyclin biosynthesis and renal 15-hydroxyprostaglandin dehydrogenase activity with aging, might contribute to a plausible explanation of the progressive decrease in renal functions in the elderly.     

Arachidonic acid is preferentially metabolized by cyclooxygenase-2 to prostacyclin and prostaglandin E2

The two cyclooxygenase isoforms, cyclooxygenase-1 and cyclooxygenase-2, both metabolize arachidonic acid to prostaglandin H2, which is subsequently processed by downstream enzymes to the various prostanoids. In the present study, we asked if the two isoforms differ in the profile of prostanoids that ultimately arise from their action on arachidonic acid. Resident peritoneal macrophages contained only cyclooxygenase-1 and synthesized (from either endogenous or exogenous arachidonic acid) a balance of four major prostanoids: prostacyclin, thromboxane A2, prostaglandin D2, and 12-hydroxyheptadecatrienoic acid. Prostaglandin E2 was a minor fifth product, although these cells efficiently converted exogenous prostaglandin H2 to prostaglandin E2. By contrast, induction of cyclooxygenase-2 with lipopol- ysaccharide resulted in the preferential production of prostacyclin and prostaglandin E2. This shift in product profile was accentuated if cyclooxygenase-1 was permanently inactivated with aspirin before cyclooxygenase-2 induction. The conversion of exogenous prostaglandin H2 to prostaglandin E2 was only modestly increased by lipopolysaccharide treatment. Thus, cyclooxygenase-2 induction leads to a shift in arachidonic acid metabolism from the production of several prostanoids with diverse effects as mediated by cyclooxygenase-1 to the preferential synthesis of two prostanoids, prostacyclin and prostaglandin E2, which evoke common effects at the cellular level.     

A selective defect in arachidonic acid release from macrophage membranes in high potassium media

Murine peritoneal macrophages cultured in minimal essential medium (alpha-MEM; 118 mM Na+, 5 mM K+) released arachidonic acid (20:4) from phospholipids on encountering a phagocytic stimulus of unopsonized zymosan. In high concentrations of extracellular K+ (118 mM), 3H release from cells prelabeled with [3H]20:4 was inhibited 80% with minimal reduction (18%) in phagocytosis. The inhibitory effect of K+ on 20:4 release was fully reversed on returning cells to medium containing Na+ (118 mM). Preingestion of zymosan particles by macrophages maintained in high K+ medium resulted in cells being "primed" for 20:4 release, which was only effected (without the further addition of particles) by changing the medium to one containing Na+. In contrast, 20:4 release from cells stimulated with the calcium ionophore A23187 was unimpaired by the elevated K+ medium, suggesting no direct effect of high K+ on the phospholipase. Macrophages stimulated with zymosan in alpha-MEM metabolized the released 20:4 to prostacyclin, prostaglandin E2 (PGE2), and leukotriene C (LTC). The smaller quantity of released 20:4 in high K+ medium was recovered as 6-Keto-PGF1 alpha, the breakdown product of prostacyclin, and PGE2. No LTC was synthesized. In high K+, resting (no zymosan) macrophages synthesized hydroxyeicosatetraenoic acids from exogeneously supplied 20:4 in proportions similar to cells maintained in alpha-MEM. These findings and the similarity of products (including LTC) produced by {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 stimulated cells in alpha-MEM and high K+ medium indicated that the cyclooxygenase and lipoxygenase pathway enzymes were not directly inhibited by high extracellular K+. We conclude that high concentrations of extracellular K+ uncouple phagocytosis of unopsonized zymosan from the induction of the phospholipase responsible for the 20:4 cascade and suggest that the lesion is at the level of signal transduction between the receptor-ligand complex and the phospholipase.     

Synthesis and metabolism of leukotrienes by human endothelial cells: influence on prostacyclin release

The synthesis and metabolism of leukotrienes (LTs) by endothelial cells was investigated using reverse-phase high-performance liquid chromatography. Cells were incubated with [14C]arachidonic acid. LTA4 or [3H]LTA4 and stimulated with ionophore A23187. The cells did not synthesize leukotrienes from [14C]arachidonic acid. LTA4 and [3H]LTA4 were converted to LTC4, LTD4, LTE4 and 5,12-diHETE. Endothelial cells metabolized [3H]LTC4 to [3H]LTD4 and [3H]LTE4. The metabolism of [3H]LTC4 was inhibited by L-serine-borate complex, phenobarbital and acivicin in a concentration-related manner, with maximal inhibition occurring at a concentration of 0.1 M, 0.01 M and 0.01 M, respectively. LTC4, LTB4 and LTD4 stimulated the synthesis of prostacyclin, measured by radioimmunoassays as 6-keto-PGF1 alpha. The stimulation by LTC4 was greater than that by LTD4 or LTB4. LTE4, 14,15-LTC4 and 14,15-LTD4 failed to stimulate the synthesis of prostacyclin. LTD4 and LTB4 also stimulated the release of PGE2, whereas LTC4 did not. Serine-borate and phenobarbital inhibited LTC4-stimulated synthesis of prostacyclin in a concentration-related manner. They also inhibited the release of prostacyclin by histamine, A23187 and arachidonic acid. Acivicin had no effect on the release of prostacyclin by LTC4, histamine or A23187. Furthermore, FPL-55712, an LT receptor antagonist, inhibited LTC4-stimulated prostacyclin synthesis but had no effect on histamine-stimulated release of prostacyclin or PGE2. Indomethacin inhibited both LTC4- and histamine-stimulated release. The results show that (a) endothelial cells metabolize LTA4, LTC4 and LTD4 but do not synthesize LTs from arachidonic acid; (b) LTC4 act directly at the leukotriene receptor to stimulation prostacyclin synthesis; (c) the presence of the glutathione moiety at the C-6 position of the eicosatetraenoic acid skeleton is necessary for leukotriene stimulation of prostacyclin release; and (d) the metabolism of LTC4 to LTD4 and LTE4 does not appear to alter the ability of LTC4 to stimulate the synthesis of PGI2.