Some natural compounds isolated from Chinese plants were found to have strong inhibitory activity for 5-lipoxygenase and were thus able to inhibit leucotriene synthesis. Among these compounds, caffeic acid was selected for study; more than 100 of its derivatives were synthesized and tested for 5-lipoxygenase inhibitory activity. Two derivatives, TMK-919 and TMK-920, proved to be 100 times more potent than the parent compound and were able to inhibit the homologous PCA reaction significantly in rats at a dose of 10 mg/kg, i.v.

Animals↗

PubMed1985

[Prostaglandins and related substances: metabolic pathways].

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Animals↗

PubMed1984

Kallikrein stimulates prostacyclin production in bovine vascular endothelial cells.

Cultured endothelial cells isolated from bovine carotid aorta produce prostacyclin (prostaglandin I2) and a small amount of prostaglandin E2. The effects of kallikrein (EC 3.4.21.8) on the release of prostacyclin from the cells were studied with the radioimmunoassay technique. Kallikrein stimulated the release of prostacyclin in a dose-dependent manner. The maximal stimulation reached up to 9.2-fold at 0.1 micrograms/ml of kallikrein. The effect was not associated with the activation of the fatty acid cyclooxygenase, but with the stimulation of arachidonic acid release. But kallikrein itself did not have phospholipase activity. On the other hand, at the same doses, kallikrein failed to induce platelet aggregation or enhance platelet aggregation induced by collagen. Our findings suggest that the vasodilator effect of kallikrein is mediated in part by prostacyclin production. Furthermore, we investigated the possibility that the stimulatory effect of kallikrein on prostacyclin production in endothelial cells is associated with kinin formation. Bradykinin and lysylbradykinin (kallidin) also stimulated the release of prostacyclin, but the effects were far less than that of kallikrein. And the stimulation due to the addition of both kallikrein and bradykinin on prostacyclin and arachidonic acid release was not competitive or additive, but synergistic. Moreover, even if fetal calf serum was incubated with kallikrein, bradykinin was not detected at all. When kallikrein was pre-incubated with aporotinin, which is an inactivator of kallikrein, the effect of kallikrein was completely abolished. These findings suggest that the stimulatory effect of kallikrein on the release of prostacyclin from vascular cells is possibly not due to kinin formation, but to other substance(s) produced by this serine proteinase.

Animals↗

PubMed1984

Caffeic acid is a selective inhibitor for leukotriene biosynthesis.

.eukotrienes are significantly involved in immunoregulation and in a variety of diseases, including asthma, inflammation and various allergic conditions. They are initially biosynthesized by 5-lipoxygenase from arachidonic acid, which can also be metabolized to prostaglandin endoperoxide by cyclooxygenase. The specific inhibitors for 5-lipoxygenase would be useful not only as tools for investigating the regulation mechanism of leukotriene biosynthesis, but also as drugs for clinical use. Although recently a few selective inhibitors have been reported, most of them are difficult to obtain, since they are new compounds. We found that caffeic acid, which is one of the most common reagents, is a selective inhibitor for 5-lipoxygenase and therefore for leukotriene biosynthesis. The inhibitory effect of its methyl ester on 5-lipoxygenase (ID50 = 4.8 X 10(-7) M) was stronger than that of caffeic acid itself (ID50 = 3.7 X 10(-6) M). Caffeic acid inhibited 5-lipoxygenase in a non-competitive manner. Caffeic acid and its methyl ester did not inhibit prostaglandin synthase activity at all, at least up to 5 X 10(-4) M, but rather stimulate at higher doses. The biosynthesis of leukotriene C4 and D4 in mouse mast tumor cells was also inhibited completely with 10(-4) caffeic acid. Besides, caffeic acid had little effect on arachidonic acid metabolism in platelet at less than 1 X 10(-5) M, but at higher doses it showed a definite inhibitory effect, i.e., thromboxane B2, HHT (12(S)-hydroxy-5,8,10-heptadecatetraenoic acid) and 12-HETE (12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid) syntheses were inhibited 33, 40 and 80% at 1 X 10(-4) M, respectively. Platelet aggregation induced by arachidonic acid was also inhibited by caffeic acid at high dose, while platelet aggregation induced by ADP is not influenced by caffeic acid at all. The observations on caffeic acid and its derivatives may contribute to leukotriene research.

Animals↗

PubMed1984

Increased arachidonic acid incorporation into platelet phospholipids in type 2 (non-insulin-dependent) diabetes.

Arachidonic acid uptake activity was measured in platelets obtained from 27 Type 2 diabetic patients and 18 age-matched control subjects. In both groups after 1 h incubation almost all the incorporated 14C-arachidonic acid was located in the phospholipids of the platelets. Arachidonic acid was predominantly incorporated into phosphatidylcholine. The radioactivity incorporated into platelets increased linearly with incubation time, up to 90 min. The linear increase was observed at arachidonic acid concentrations of 0.1-1.0 micrograms/ml in both groups. The rate of incorporation of radioactivity in diabetic platelets was about 1.4 times higher than that in control platelets at all arachidonic acid concentrations studied. The arachidonic acid uptake activity of diabetic platelets (577 +/- 26 ng/60 min per 10(9) platelets) was significantly higher than that in control platelets (410 +/- 26 ng/60 min per 10(9) platelets). No significant correlations were found between the arachidonic acid uptake activity and fasting plasma glucose, total cholesterol or triglyceride levels. The arachidonic acid uptake activity of platelets was significantly higher in diabetic patients with proliferative retinopathy than in those with little or no background retinopathy. In addition, there were no significant differences between control and diabetic subjects in the uptake activity of platelets for linoleic acid and oleic acid. These data may explain the elevated arachidonic acid content in diabetic platelet phospholipids and enhancement of thromboxane synthesis in diabetes.

Aged↗

PubMed1984

A proposed method for exploring anti-aggregatory effects of eicosapentaenoic acid in the rat.

Eicosapentaenoic acid (EPA) has been reported to be anti-aggregatory in humans, but it is difficult to confirm that anti-aggregatory effect in rat. To induce an anti-aggregatory effect of EPA even in rats, an attempt was made to modify fatty acid composition of the diet, i.e., a diet low in linoleic acid, which is a precursor of arachidonic acid (AA) in vivo. A group of rats eating EPA with butter, which contained low linoleic acid, as a lipid source for 12 consecutive days revealed significant inhibition of platelet aggregation induced by collagen. The inhibition was complete at 3.0 micrograms of collagen, a dose which caused complete aggregation in rats which received EPA with corn oil. In these experiments, the ratio of EPA to AA in platelet phospholipids was 0.038 for corn oil, 0.306 for corn oil plus EPA, 0.050 for butter and 0.703 for butter plus EPA. These results indicated that if one blocks the in vivo supply of AA, it causes an increase in EPA to AA ratio in platelet phospholipids, and consequently leads to an inhibition of platelet aggregation even in the rat.

Animals↗

PubMed1984

Heparan sulfate enhances growth of transformed human cells.

Previous studies showed that cell surface heparan sulfate (HS) is involved in density-dependent growth regulation of normal human cells. In this study the effects of HS on proliferation of transformed cells were studied in vitro. Exogenously added HS prepared from normal tissues (rat kidney and bovine kidney) enhanced growth of transformed human and animal cells (gamma ray- or virus-transformed WI-38, and HeLa cells and chemically induced mouse hepatoma cells), but inhibited that of normal human and animal cells (WI-38, 3T3, and rabbit liver cells). HS was less effective on growth of both normal and transformed human cells at higher cell density. Although the exogenous HS did not bind to cells tightly, HS was found to affect cell growth not by modulation of growth-related substances in the medium, but through contact with the cell surface. HS preparation from tumour cells (mouse hepatoma cells) exerted similar effects on cell growth. Heparin, structurally similar to HS, inhibited growth of both normal and transformed human cells. These findings suggest that: 1. HS plays a particular function in contact regulation of cell proliferation. 2. Transformation-related changes in the structure of HS molecules do not much affect the function of HS. 3. The cellular transformation, however, is accompanied by alteration in the growth-regulating system sensitive to extracellular HS.

Animals↗

PubMed1983

Estradiol suppresses electrically-induced arterial thrombosis by increasing prostaglandin biosynthesizing activity of the aorta in rats.

The anti-thrombotic effect of estradiol was studied using an electrically induced thrombus model in rats. Estradiol treatment significantly inhibited the thrombus formation induced by electric shock. In the meantime, a stimulation of prostacyclin biosynthesis by estradiol in aortas was found. This is the first report showing the correlation between the effects of the estradiol on the thrombus formation in an experimental model and on the prostacyclin production by aortas in the same animal system.

Animals↗

PubMed1983

Stimulation of eicosapentaenoic acid metabolism in washed human platelets by 12-hydroperoxyeicosatetraenoic acid.

Washed human platelets were not able to convert eicosapentaenoic acid (EPA) to thromboxane B3 (TXB3) and 12-hydroxyeicosapentaenoic acid (AA) to washed human platelets induced conversion of EPA to TXB3 and 12-HEPE. Esculetin, a specific inhibitor of 12-lipoxygenase, prevented the effect of AA, but cyclooxygenase inhibitor did not. The conversion of AA to TXB2 was not affected by the same dose of esculetin. These data suggest that products of AA formed by 12-lipoxygenase in human platelets have stimulatory effects on EPA metabolism. When AA was preincubated with washed human platelets, its effect on EPA conversion was reduced, suggesting that a labile product of AA formed by 12-lipoxygenase is involved in the facilitation of EPA metabolism. Addition of 12-hydroperoxyeicosatetraenoic acid directly to washed human platelets caused dose-dependent synthesis of TXB3 and 12-HEPE, while addition of 12-hydroxyeicosatetraenoic acid had no effect. Thus, 12-hydroperoxyeicosatetraenoic acid formed from AA promotes the metabolism of EPA in washed human platelets.

Arachidonic Acid↗

PubMed1983

Inhibitory effect of esculetin on 5-lipoxygenase and leukotriene biosynthesis.

Esculetin (6,7-dihydroxycoumarin) showed inhibitory effects on 5- and 12-lipoxygenases of cloned mastocytoma cells. The half-inhibition dose (ID50) for each enzyme was 4 X 10(-6) and 2.5 X 10(-6) M, respectively. No inhibition at all, but rather a stimulating effect on prostaglandin synthesis, was observed, especially at higher doses of esculetin. Leukotriene synthesis by mouse mast tumour cells was also reduced in the presence of esculetin in parallel with 5-lipoxygenase inhibition. From these results, esculetin may be useful as a tool for research on regulatory mechanisms or physiological functions of leukotrienes.

Animals↗

PubMed1983

Effects of eicosapentaenoic acid on arachidonic acid metabolism in cultured vascular cells and platelets: species difference.

The metabolism of eicosapentaenoic acid (EPA) in cultured vascular smooth muscle cells isolated from human, rat, rabbit and miniature pig and bovine endothelial cells were studied. EPA was not able to be converted to any prostaglandins (PGs) in murine and porcine smooth muscle cells. However, in human and rabbit smooth muscle cells and bovine endothelial cells EPA was easily converted to delta 17-6-ketoPGF1 alpha, which is a stable metabolite of PGI3. Cyclooxygenase and 12-lipoxygenase activities in platelets isolated from human citrated blood were almost completely inhibited by EPA at the dose over 4 micrograms. But in platelets isolated from rat the inhibitory effects of EPA on arachidonic acid (AA) metabolism were much smaller than that in human platelets. In rat, EPA was not only being converted to no PGI3, but also being a blocker to PGI2 synthesis in vascular cells. Moreover, the rat EPA has much less activity in inhibiting thromboxane A2 (TXA2) synthesis in platelets. On the contrary, in human EPA was not only easily converted to PGI3 in vascular cells, but also blocking TXA2 synthesis in platelets. Thus, anti-aggregatory effects of EPA was positive in human and negative in rat perhaps due to species difference in sensitivity to EPA.

6-Ketoprostaglandin F1 alpha↗

PubMed1983

Selective inhibition of 5-lipoxygenase by natural compounds isolated from Chinese plants, Artemisia rubripes Nakai.

Three of four natural compounds, which are caffeic acid, eupatilin and 4'-demethyleupatilin, isolated from Chinese plant, Artemisia rubripes Nakai selectively inhibited 5-lipoxygenase of cultured mastocytoma cells. Half-inhibition doses (ID50) for caffeic acid, eupatilin and 4'-demethyleupatilin were 3.7, 14 and 18 X 10(-6) M, respectively. The inhibition by caffeic acid was non-competitive types. Prostaglandin synthase activities were little inhibited by eupatilin and 4'-demethyleupatilin, but rather stimulated by caffeic acid. The formation of leukotriene C4 and D4 by mast tumor cells was almost completely suppressed by these compounds at 10(-4) M.

Animals↗

PubMed1983

Aortic smooth muscle cell migration caused by platelet-derived growth factor is mediated by lipoxygenase product(s) of arachidonic acid.

The relation between platelet-derived growth factor (PDGF)-induced smooth muscle cell migration, measured in Boyden chambers, and cellular arachidonic acid cascade was studied by using rat aortic smooth muscle cells. Partially purified PDGF stimulated cell migration significantly at a concentration of 1.33-133.0 micrograms/ml. Treatment of the cells with 10(-4)M of 5,8,11,14-eicosatetraynoic acid, an inhibitor of lipoxygenase and cyclooxygenase, and 10(-4)M of caffeic acid, a specific inhibitor of lipoxygenase, caused a significant suppression of PDGF-induced cell migration. Treatment with indomethacin, an inhibitor of cyclooxygenase, did not affect cell migration. These data indicate the involvement of a lipoxygenase product(s) of arachidonic acid in PDGF-associated smooth muscle cell migration.

Animals↗