Hsin-Hsiung Tai

Gossypol, a potent inhibitor of arachidonate 5- and 12-lipoxygenases

Gossypol inhibited 5- and 12-lipoxygenases of rat basophilic leukemia (RBL-1) cells with ID50 of 0.3 microM and 0.7 microM, respectively. Nearly two orders of magnitude of higher concentration of gossypol was required to inhibit prostaglandin synthetase. The inhibition was of a non-competitive type with respect to arachidonate.

Monoglyceride and diglyceride lipases from human platelet microsomes

In the present study, we have characterized the properties of both diglyceride lipase (lipoprotein lipase, EC 3.1.1.24) and monoglyceride lipases (acylglycerol lipase, EC 3.1.1.23) in an attempt to assess the potential roles of these two enzymes in the release of arachidonate in activated human platelets. Diglyceride lipase exhibited maximal activity at pH 3.5, whereas monoglyceride lipase showed optimal activity at pH 7.0. Neither of the lipases were inhibited by EDTA or stimulated by Ca2+, Mg2+ or Mn2+. Both enzymes, however, were strongly inhibited by Hg2+ and Cu2+, indicating the involvement of sulfhydryl groups in catalytic activity. This suggestion was further supported by their sensitivity toward sulfhydryl inhibitors, with monoglyceride lipase being more susceptible to inhibition. Both lipases were found to be inhibited to a different degree by a variety of antiplatelet drugs blocking aggregation and arachidonate release. Kinetic studies indicated that dichotomous metabolism of diacylglycerol to monoacylglycerol and to phosphatidic acid could occur concurrently, since the apparent Km values for diglyceride lipase and for diglyceride kinase were comparable. Further studies showed that the specific activity of monoglyceride lipase was at least 100-fold higher than that of diglyceride lipase, indicating that the rate-limiting step in the release of arachidonate was the reaction catalyzed by diglyceride lipase.

Hypoxia stimulates prostacyclin generation by dug lung in vitro

We have investigated the possibility that pulmonary biosynthesis of prostacyclin and thromboxane A2 (TXA2) may be affected by variations in PO2. Fresh lung homogenated from 8 dogs were incubated for 1 hour at 37 degrees C in Krebs-Ringer solution, at high (492 mmHg) or low (53 mmHg) PO2. After incubation, the stable metabolites of prostacyclin (6-keto-PGF1) and of TXA2 (TXB2) were measured by radioimmunoassay. The basal (pre-incubation) levels of these metabolites, measured in tubes in which biosynthesis was arrested by the addition of indomethacin (10 g/ml), were 0.76 +/- 0.15 and 0.76 +/- 0.11 ng/mg wet wt. for 6-keto-PGF1 alpha, in high and low PO2, respectively, and 0.97 +/- 0.09 and 0.82 +/- 0.15 x 10(-1) ng/mg wet wt. for TXB2, in high and low PO2 respectively. Synthesis during incubation in other tubes was estimated by subtracting basal values from those measured at the end of incubation. More 6-keto-PGF1 alpha was formed in homogenates exposed to low PO2 (3.01 +/- 0.45) than in those exposed kept at high PO2 (1.89 +/- 0.37, p less than 0.001), but equal amounts of TXB2 were bound under both conditions. These results suggest that hypoxia may stimulate pulmonary prostacyclin synthesis; a pulmonary vasodilator, prostacyclin may help modulate hypoxic vasoconstriction in the lung.

Induction of fatty acid cyclooxygenase in rat aortic smooth muscle cells by estradiol

We have previously reported that estradiol stimulates prostacyclin biosynthesis in cultured rat aortic smooth muscle cells by increasing fatty acid cyclooxygenase activity, and induction of new protein biosynthesis is involved since the effect of estradiol is blocked by cycloheximide treatment. In order to see whether estradiol stimulates de novo biosynthesis of fatty acid cyclooxygenase in cells, a specific radioimmunoassay of fatty acid cyclooxygenase was used to measure the enzyme. Estradiol significantly increased immunoreactive fatty acid cyclooxygenase in cells. Consequently, increase in cyclooxygenase activity results in the production of more prostaglandin endoperoxides for final increased synthesis of prostacyclin in these cells.

Stimulation of 12-lipoxygenase activity in rat platelets by 17β-estradiol

The effects of estradiol on the endogenous fatty acid (specifically, arachidonic acid) composition of cellular phospholipid fractions and the 12-lipoxygenase activity in rat platelets in vivo were studied. Estradiol had no significant effect on the endogenous fatty acid composition of cellular phospholipid fractions. However, estradiol significantly increased 12-lipoxygenase activity in platelets in a dose-dependent manner. The stimulatory effect of estradiol on platelet lipoxygenase was blocked by the anti-estrogen nafoxidine hydrochloride which was injected simultaneously together with estradiol in vivo, suggesting that the effect in target cells was due directly to estradiol.

Selective inhibition of 5-lipoxygenase pathway in rat pulmonary alveolar macrophages by cigarette smoking

Pulmonary alveolar macrophages from sham or cigarette-smoke-exposed rats were examined for their ability to transform exogenously added arachidonate to metabolites of lipoxygenase and cyclooxygenase pathways. Synthesis of 5-HETE and leukotriene B4 was selectively inhibited by cigarette smoke exposure, whereas the formation of prostaglandin E2 and thromboxane B2 remained unchanged. Selective inhibition of the lipoxygenase pathway was further reflected by the reduced content of leukotriene B4 in bronchoalveolar fluid of smoke-exposed rats. These results suggest that lipoxygenase-derived products may play a unique role in smoking-induced pulmonary diseases.

Changes in arachidonate metabolism in aortas and platelets in aging rats

Prostacyclin formation by aortas, thromboxane biosynthesis and 12-lipoxygenase pathway in platelets as a function of age was investigated. Our results indicate an age-related decrease in prostacyclin production by aortas, while no significant change in thromboxane biosynthesis in platelets from mature (12 month old) to senescent (24 month old) rats was observed. Evidence is also presented indicating a decrease in 12-L-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE) peroxidase activity in platelets as a function of age.

Does protein kinase C activation mediate thrombin-induced arachidonate release in human platelets?

Thrombin stimulated rapid formation of diacylglycerol, inositol 1,4,5-trisphosphate (IP3) and thromboxane B2 (TXB2) in human platelets. Formation of diacylglycerol and IP3 appeared to precede that of TXB2. Activation of protein kinase C by diacylglycerol combining with Ca+2 mobilization by IP3 has been implicated in mediating arachidonate release. However, addition of the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) to platelet suspension did not inhibit thrombin-stimulated arachidonate release and TXB2 synthesis, whereas addition of the Ca+2 antagonist, 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (TMB-8) or the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) abolished arachidonate release. The correlation of IP3 production with arachidonate release on increasing the concentrations of thrombin was further examined. IP3 production reached near maximum at 0.2 U/ml, whereas TXB2 synthesis continued to increase at 1 U/ml. These results suggest that protein kinase C activation may not mediate arachidonate release and that Ca+2 mobilization by IP3 may only partially account for arachidonate release in platelets stimulated with relatively high concentrations of thrombin.

Increase in thromboxane B2 and decrease in prostaglandin E2 and 6-ketoprostaglandin F1α release into rat bronchoalveolar fluid as a consequence of cigarette smoking

Rats were exposed to cigarette smoke once daily for 4 to 8 weeks. Bronchoalveolar lavage fluid was obtained from each animal and assayed for immunoreactive PGE2, TXB2 and 6-Keto-PGF1 alpha. Significant increase in TXB2 and decrease in PGE2 and 6-Keto-PGF1 a release into bronchoalveolar fluid as a consequence of cigarette smoking were observed. These changes of arachidonate metabolites in lung alveoli may account in part for bronchoconstriction induced by cigarette smoking.

Induction of a decrease in renal NAD+-dependent 15-hydroxyprostaglandin dehydrogenase activity by estradiol in rats

The effect of estradiol administration on renal and pulmonary NAD+-dependent 15-hydroxyprostaglandin dehydrogenase activities in rats was studied. Estradiol induced a significant decrease in renal but not in pulmonary enzyme activity. Kinetic parameters for the renal enzyme from control and treated groups were compared. An identical apparent Km for prostaglandin E2 was obtained for the enzyme from both groups. Vmax in the treated group was progressively decreased and plateaued 1 day after estradiol injection. The estradiol-induced decrease in renal enzyme activity was blocked by an anti-estrogen, nafoxidine, suggesting that the effect of estradiol was a was a receptor-mediated event. The decrease in renal prostaglandin catabolic enzyme activity induced by estradiol may result in prolonging the half-life of circulating prostacyclin and may account, in part, for the anti-thrombogenic effect of estradiol.