Characterization of the stimulatory effects of PGF2α on the release of arachidonic acid

Fibroblasts derived from a rat carrageenin granuloma were cultured in the presence of radioactive arachidonic acid, palmitic acid and linoleic acid. More than 90% of each labeled fatty acid was incorporated into a phospholipid fraction by the cells in 18 hrs. Arachidonic acid was evenly incorporated into phosphatidylcholine and phosphatidylethanolamine, while both palmitic acid and linoleic acid were almost entirely incorporated into phosphatidylcholine. The position of phosphatidylcholine where the fatty acids were incorporated was different for each fatty acid. The ratio of the amount of fatty acid incorporated into the 2-position to the amount incorporated into the 1-position of phosphatidylcholine for each fatty acid was >90% for arachidonic acid, 2:1 for palmitic acid and 5:1 for linoleic acid. In the case of phosphatidylethanolamine, most arachidonic acid (>90%) was incorporated into the 2-position. PGF_2^α caused the stimulation of arachidonic acid release but not of palmitic acid and linoleic acid from pre-labeled fibroblasts.The serum in the medium was completely replaceable by bovine serum albumin. The effect of PGF_2^α increased with an increasing concentration of bovine serum albumin, suggesting that serum only acts as a ‘trap’ for released arachidonic acid. The effect of PGF_2^α was greater than bradykinin, and no synergistic effect was seen, although an additive effect was observed.The effect of PGF_2^α depended on the concentration of calcium ions under magnesium-supplemented conditions.     

Stimulation of prostaglandin cyclooxygenase and prostacyclin synthetase activities by estradiol in rat aortic smooth muscle cells

The effects of estradiol on the arachidonic acid pool and prostacyclin biosynthetic activity in rat aortic smooth muscle cells were studied. Estradiol has no significant effect on the distribution of [14C]arachidonic acid in cells with respect to prostacyclin production assay, the endogenous fatty acid (specifically, arachidonic acid) composition of cellular phospholipid fractions and cellular phospholipase (or/and lipase) activities. However, estradiol significantly stimulates both prostaglandin cyclooxygenase and prostacyclin synthetase activities of cells, and induction of new protein biosynthesis is involved in the effect of estradiol on the stimulation of prostacyclin biosynthetic activity.     

Macrophage as a target of quercetin glucuronides in human atherosclerotic arteries: implication in the anti-atherosclerotic mechanism of dietary flavonoids

Epidemiological studies suggest that the consumption of flavonoid-rich diets decreases the risk of cardiovascular diseases. However, the target sites of flavonoids underlying the protective mechanism in vivo are not known. Quercetin represents antioxidative/anti-inflammatory flavonoids widely distributed in the human diet. In this study, we raised a novel monoclonal antibody 14A2 targeting the quercetin-3-glucuronide (Q3GA), a major antioxidative quercetin metabolite in human plasma, and found that the activated macrophage might be a potential target of dietary flavonoids in the aorta. Immunohistochemical studies with monoclonal antibody 14A2 demonstrated that the positive staining specifically accumulates in human atherosclerotic lesions, but not in the normal aorta, and that the intense staining was primarily associated with the macrophage-derived foam cells. In vitro experiments with murine macrophage cell lines showed that the Q3GA was significantly taken up and deconjugated into the much more active aglycone, a part of which was further converted to the methylated form, in the activated macrophages. In addition, the mRNA expression of the class A scavenger receptor and CD36, which play an important role for the formation of foam cells, was suppressed by the treatment of Q3GA. These results suggest that injured/inflamed arteries with activated macrophages are the potential targets of the metabolites of dietary quercetin. Our data provide a new insight into the bioavailability of dietary flavonoids and the mechanism for the prevention of cardiovascular diseases.     

Induction of KDR expression in bovine arterial endothelial cells by thrombin: involvement of nitric oxide.

Thrombin, a multifunctional serine protease, is generated at the site with vascular injuries. It not only participates in the coagulation cascade, but also can induce a lot of events related to cell mitogenesis and migration. In this study, we investigated the effect of thrombin on endothelial cell proliferation induced by vascular endothelial growth factor (VEGF). Thrombin promoted proliferation of cultured bovine carotid endothelial cells in a time- and dose-dependent manner. Moreover, it drastically enhanced the cell growth stimulated by VEGF. This stimulatory effect was reduced by inhibitors of either protein kinase C (PKC) or mitogen-activated protein kinase kinase (MAPKK). Thrombin induced a significant increase in the level of mRNA of the kinase domain-containing receptor (KDR), but not tms-like tyrosine kinase (Flt-1), in a time-dependent manner, which reached the maximum after 24 h of stimulation. This increase coincides well with the KDR protein expression. The luciferase assay showed that thrombin induced an about 7.5-fold increase in the KDR promoter activity compared with the control. This enhanced KDR promoter activity was also abolished by inhibitors of either PKC or MAPKK. The deletion analyses indicated that the region between -115 and -97 (containing Sp1 binding region) within the KDR promoter gene was required for the enhanced KDR expression induced by thrombin and VEGF. Moreover, the nitric oxide synthase (NOS) inhibitor abolished both the accelerated cell proliferation and the increased KDR expression induced by thrombin and VEGF. This inhibition was abrogated by DETA NONOate, a NO donor with long half-life. These findings suggest that thrombin might potentiate the VEGF-induced angiogenic activity through increasing the level of the VEGF receptor KDR, in which production of NO is involved.     

Inhibitory effects of eicosapentaenoic acid (EPA) on the hypoxia/reoxygenation-induced tyrosine kinase activation in cultured human umbilical vein endothelial cells

We have previously reported that the n-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) inhibited the abnormal gap junctional intercellular communication (GJIC) induced by hypoxia/reoxygenation (H/R) via suppressing tyrosine kinase (TK) activation (Zhang et al., Prostaglandins Leukot Essent Fatty Acids, 1999; 61: 33-40). However, the mechanisms by which EPA-inhibited TK activation remained unidentified. In this study we investigated whether reactive oxygen species (ROS) and growth factor-receptor systems would contribute to the H/R-induced TK activation or not. The results showed that H/R-induced ROS production, which reached the peak after 30 min of reoxygenation. Pretreatment with 10 microM EPA significantly inhibited this ROS production. However, the TK inhibitor genistein (10 microM) failed to inhibit the generation of ROS, although it completely inhibited TK activation. On the other hand, the ROS inhibitor DMSO (0.5% v/v) showed little effect on TK activation while it significantly blocked ROS production. Further EPA and genistein, but not DMSO and superoxide dismutase (SOD, 300 U/ml), prevented cells from GJIC injury induced by H/R. Moreover, EPA protected against VEGF-induced reduction in GJIC and phosphorylation of connexin 43. These data suggest that growth factor, but not ROS, might be involved in the EPA-inhibited TK activation induced by H/R.     

Design and synthesis of orally bioavailable inhibitors of inducible nitric oxide synthase. synthesis and biological evaluation of dihydropyridin-2(1H)-imines and 1,5,6,7-tetrahydro-2H-azepin-2-imines

The process of discovery and biological evaluation of alpha,beta-unsaturated cyclic amidines, as selective inhibitors of inducible nitric oxide synthase (iNOS), is reported. Dihydropyridin-2(1H)-imines and 1,5,6,7-tetrahydro-2H-azepin-2-imines were synthesized and biologically evaluated both in vitro and in vivo using a nitric oxide synthase inhibition assay. Compounds 1, 5, 6, 8-12 and 16 exhibited potent inhibition of iNOS. Among these, compounds 6, 7, 10, 11 and 16 showed 5- to 19-fold isoform selectivity. Compounds 1, 6, 10, 11 and 16 also showed potent inhibitory activity in the NOx accumulation assay in mice. Compounds 1 and 6 showed excellent bioavailability (BA) in rats when administered orally. Full details are presented here, including the structure-activity relationship (SAR) studies, the chemistry of these compounds, and the pharmacokinetic data and the computer-aided docking study of 10 with hiNOS.     

Inhibitory effect of monoacylglycerol on fatty acid uptake into rat intestinal epithelial cells

We investigated the influence of glycerol esters on oleic acid uptake into IEC-6 cells. Monoolein, especially 2-monoacylglycerol, significantly inhibited the cellular uptake. Although diolein slightly inhibited the oleic acid uptake, triolein, glycerol and monooctanoate had no effect. These results suggest that after lipid digestion in the intestine, long-chain fatty acid uptake may be influenced by another digestive product, 2-monoacylglycerol.     

Effects of glucose on migration, proliferation and tube formation by vascular endothelial cells.

In order to elucidate the association between hyperglycemia and the vascular complications of diabetes, the effects of high glucose concentrations on the migration, proliferation and tube formation of bovine carotid artery endothelial cells were investigated. Cells treated with 16.7 and 33.3 mM glucose for 6 days showed 1.69- and 1.75-fold increase in serum-induced migration compared with cells treated with 5.6 mM glucose (p less than 0.05). The effect of glucose on cell proliferation was affected by serum concentration. When this was below 0.5%, a high glucose concentration stimulated cell growth to a maximum of 1.73 times that at a serum concentration of 0.05% (p less than 0.01) whereas at a serum concentration of 10%, growth was inhibited (p less than 0.05). Tube formation was studied by culturing the cells between two layers of collagen gel. Ultrastructurally, tubular structures were composed of one to several endothelial cells containing pinocytotic vesicles and cytoplasmic projections, and linked by junctional complexes. A basal lamina-like structure surrounded the abluminal surface. Treatment of the cells with 16.7 and 27.8 mM glucose for 4 days stimulated tubular elongation 1.85 and 1.71 times, respectively (p less than 0.01). Other osmogenic molecules such as mannitol and sucrose did not affect tube formation. These data imply that high glucose concentrations mimicking diabetic hyperglycemia may not inhibit the repair of endothelial injury and could act as a stimulator of neovascularization.