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Biomedical subjects

S Murota

Publications and source records attributed to S Murota.

At least 199 records · Page 11Linked to original sources
PubMed1983

Identification of the mRNA and polypeptide subunit for prostaglandin endoperoxide synthase from mouse mastocytoma P-815 cells.

Cloned mouse mastocytoma P-815.2-E-6 cells are barely able to synthesize prostaglandins because of a lack of prostaglandin endoperoxide synthase activity. However, the addition of sodium n-butyrate at 1 mM induces synthesis de novo of prostaglandins in this cell line. Employing this system, we could isolate an mRNA for prostaglandin endoperoxide synthase by a combination of cell-free translation and immunoprecipitation. The antibody, prepared in rabbit by injecting purified prostaglandin endoperoxide synthase from bovine vesicular gland, was shown to cross-react with the corresponding enzyme from 2-E-6 cells. The poly(A)-containing mRNA has a sedimentation coefficient of 17S and codes for a single polypeptide chain of Mr 62 000 as estimated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The Mr of the mouse polypeptide chain appears very similar to that of the purified carbohydrate-free prostaglandin endoperoxide synthase from sheep vesicular gland. These findings are a contribution to the isolation of the gene for prostaglandin endoperoxide synthase.

Animals↗
PubMed1983

Effects of purified eicosapentaenoic acid on arachidonic acid metabolism in cultured murine aortic smooth muscle cells, vessel walls and platelets.

The effects of highly purified eicosapentaenoic acid (97% pure) on the arachidonic acid cascade in isolated murine vascular cells and platelets were studied. The incorporation of eicosapentaenoic acid was not as active as that of arachidonic acid in platelets. The ratio of incorporation of eicosapentaenoic acid to arachidonic acid into platelet phospholipids was about 0.7. Analysis of the phospholipid fractions of platelets after labeling with 14C-eicosapentaenoic acid and 14C-arachidonic acid revealed that the incorporation of 14C-eicosapentaenoic acid into the fraction is significantly less than that of 14C-arachidonic acid, while the incorporation of both fatty acids into other phospholipid fractions was almost the same. On the other hand, no significant difference between either fatty acid in incorporation rate, kinetics or distribution in cellular phospholipids was found in cultured aortic smooth muscle cells. Following treatment with eicosapentaenoic acid, cells produced less prostacyclin from endogenous arachidonic acid than did control cells. This was not due to the decrease in fatty acid cyclooxygenase activity, but rather, due to the decrease in arachidonic acid content in cellular phospholipids. In addition, eicosapentaenoic acid was neither converted to prostaglandin I3 by the vascular cells nor to thromboxane A3 by platelets. Furthermore, similar results were also obtained by in vivo experiments in which rats were fed with eicosapentaenoic acid enriched diet.

Animals↗
PubMed1983

Effect of eicosapentaenoic acid on the platelet aggregation and composition of fatty acid in man. A double blind study.

Twelve volunteers (mean age, 60.7 +/- 4.2 years) were treated with placebo for the first week and then given partially purified eicosapentaenoic acid (EPA, 67% purity) at 2 g per day for 4 weeks. Significant decreases in ADP-, collagen- and adrenalin-induced platelet aggregation were observed at 2 and 4 weeks after EPA treatment, together with an increase in the plasma ratio of EPA to arachidonic acid and in platelet phospholipids. It was concluded that the administration of partially purified EPA was effective in decreasing platelet aggregation, possibly by changing the platelet ratio of EPA to arachidonic acid.

Anticoagulants↗
PubMed1983

Calcium dependency of aortic smooth muscle cell migration induced by 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid. Effects of A23187, nicardipine and trifluoperazine.

We have previously reported that 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), a 12-lipoxygenase product of arachidonic acid in platelets, is a potent chemoattractant for rat aortic smooth muscle cells. In the present study, the mechanism involved in 12-HETE-associated smooth muscle cell migration was investigated in relation to calcium mobilization in the cells. Migration of smooth muscle cells was measured by a filter membrane technique in modified Boyden chambers. Smooth muscle cell migration induced by 12-HETE increased with the increase of extracellular Ca2+ concentration and became maximal at the physiological Ca2+ concentration of 1.25 mM. The calcium ionophore A23187, at concentrations of 0.2 and 2.0 microM, significantly stimulated cell migration. Nicardipine, a potent calcium-entry blocker, significantly inhibited 12-HETE-associated smooth muscle cell migration at concentrations from 10(-9) to 10(-5) M. Concentrations of trifluoperazine from 10(-9) to 10(-5) M and W-7 at 10(-5) M, which are specific inhibitors of calmodulin, also significantly inhibited cell migration induced by 12-HETE. Cytochalasin B at 1.0 and 10 microM, and colchicine at 0.1 and 1.0 microM concentrations drastically inhibited cell migration, indicating that actin-containing microfilaments and microtubules are involved in smooth muscle cell migration. These findings indicated that the stimulation of smooth muscle cell migration by 12-HETE is a highly calcium-dependent process and suggest that 12-HETE might act at the initial stage of smooth muscle cell migration through enhancing calcium influx through plasma membrane and thus stimulating cell migration.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1983

Effects of nitrogen dioxide exposure on prostacyclin synthesis in lung and thromboxane A2 synthesis in platelets in rats.

Effects of nitrogen dioxide (NO2) exposure on prostacyclin (PGI2) synthesis in the rat lung and thromboxane A2 (TXA2) synthesis in the platelets were studied. Male Wistar rats were exposed to 10 ppm NO2 for 1, 3, 5, 7 and 14 days. PGI2 synthesizing activity of homogenized lung decreased. The damage of PGI2 synthesizing activity reaches its maximum at 3 days. At 14 days, PGI2 synthesizing activity returned to the normal level. The activity of PGI2 synthetase decreased significantly. The formation of lipid peroxides due to NO2 exposure may cause the depression of PGI2 synthesizing activity of lung. On the other hand, platelet TXA2 synthesizing activity increased. This increased TXA2 synthesizing activity lasted at least till 3 days. Then, it returned to the normal level. The counts of platelet were decreased significantly by 1, 3, 5 and 7 days NO2 exposure. Then the decreased counts of platelet returned to the normal level at 14 days NO2 exposure. These results indicate that the depression of PGI2 synthesizing activity of lung by NO2 exposure cause an increase in TXA2 synthesizing activity of platelets. It may contribute to induce platelet aggregation and to the observed decrease in the number of platelets during NO2 exposure.

Animals↗
PubMed1983

Augmentation of 12-lipoxygenase activity of lymph node and spleen T cells in autoimmune mice MRL/1.

To investigate the relationship between development of autoimmune disease and arachidonic acid metabolism, we compared lipoxygenase and cyclooxygenase activities of lymphoid cells in MRL/1 mice during the development of the disease. Lipoxygenase activity per cell of lymph mo mice, although cyclooxygenase activity was low in both cases. These phenomena did not depend on aging since age-matched MRL/n mice lacking the lpr gene (congenic with MRL/1 mice), and BALB/c mice did not exhibit any change in the enzyme activity with aging. The lipoxygenase activity of 4 mo MRL/1 lymph node cells came mainly from nylon wool nonadherent cells. Lipoxygenase activity of nylon wool nonadherent spleen cells in MRL/1 mice was also higher in 4 mo mice than that in 1.5 mo mice, whereas we could not detect any difference in nylon wool adherent cells. The major lipoxygenase product was 12-hydroxyeicosatetraenoic acid (12-HETE), which was identified by a high performance liquid chromatography (HPLC). Our data suggest that change in arachidonic acid metabolism in T cells may be related to development of autoimmune disease in MRL/1.

Age Factors↗
PubMed1983

Dietary arachidonic acid supplementation increases thromboxane synthesizing activity in platelets from diabetics.

Thromboxane A2 has been implicated in thrombotic vascular diseases. The diabetic population has a high incidence of vascular complications. In the present study, activity of thromboxane synthetase in platelets of type 2 (non-insulin dependent) diabetic patients was studied. Thromboxane synthesizing activity in diabetic platelets was significantly higher than that in controls (p less than 0.05). An effect of dietary arachidonic acid on thromboxane synthesizing activity in platelets was also studied. Dietary arachidonic acid supplementation containing 1.7g of arachidonic acid per day for 1 week caused a significant increase of arachidonic acid content in plasma total lipids (p less than 0.05), but the increase of arachidonic acid in plasma total lipids did not correlate with the level of arachidonic acid in platelet phospholipids. Thromboxane synthesizing activity in platelets from the diabetics and the controls was augmented by a diet enriched in arachidonic acid from 2.20 +/- 0.15 to 2.56 +/- 0.20 and from 1.71 +/- 0.23 to 2.01 +/- 0.34, respectively. The increase in diabetic subjects was statistically significant (p less than 0.05). These results suggest that fatty acids of dietary origin are important factors in development of thrombotic vascular diseases.

Adult↗
PubMed1983

Increased arachidonic acid content in platelet phospholipids from diabetic patients.

Platelet abnormalities have been suggested to be linked with vascular diseases. Diabetes mellitus has a high incidence of vascular complications. In the present study gas-liquid chromatography analyses were carried out to investigate the fatty acid composition in platelet phospholipids of Type-2 (non-insulin-dependent) diabetic patients. Fatty acid composition in platelet phospholipids was different between diabetics and age-matched control subjects. Arachidonic acid increased significantly (p less than 0.01), and both eicosatrienoic acid and eicosapentaenoic acid decreased in diabetes mellitus. A good correlation was found between fatty acid content of platelet phospholipids and that of plasma total lipids in the fatty acids examined, except for arachidonic acid. The levels of arachidonic acid in diabetic platelet phospholipids was disproportionately high to these in plasma total lipids. These results suggest that the system for arachidonic acid incorporation into platelet phospholipids is specifically accelerated in the diabetic metabolic state.

8,11,14-Eicosatrienoic Acid↗
PubMed1983

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.

Animals↗
PubMed1982

Predominant synthesis of 5-hydroxyeicosatetraenoic acid by a cloned mastocytoma P-815 line, 2-E-6 cells.

Since mouse mast tumor P-815 cells produce the slow reacting substance of anaphylaxis, their 5-lipoxygenase activity was examined by determining the conversion of arachidonic acid to 5-hydroxyeicosatetraenoic acid (HETE). Mast tumor cells from mouse ascites fluid synthesized 12-HETE as a major and 5-HETE as a minor metabolite. Once the cells were transferred to an in vitro culture system, the predominant synthesis of 12-HETE was abolished and synthesis of 5-HETE was greater than that of 12-HETE. 2-E-6 cells, obtained by cloning the tumor cells, synthesized a negligible amount of 12-HETE, but produced a large amount of 5-HETE. When the 2-E-6 cells were inoculated into mice and harvested again from the ascites fluid, their ratio of 5-HETE to 12-HETE synthesis was similar to that of normal mouse peritoneal cells; that is, 12-HETE synthesis was much greater than 5-HETE synthesis. It is concluded that the predominant synthesis of 12-HETE in mast tumor cells was derived from natural peritoneal cells, which have very high 12-lipoxygenase activity. The cloned mastocytoma, 2-E-6 cells, should be useful in investigating regulation of 5-lipoxygenase activity.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1982

Enhancement of 5-lipoxygenase activity in mastocytoma P-815 cells by n-butyrate treatment.

Cloned mastocytoma P-815, 2-E-6 cells were used to investigate regulation of 5-lipoxygenase activity. 2-E-6 cells had high 5-lipoxygenase activity with slight 12-lipoxygenase activity. The 5-lipoxygenase activity was increased over 5-fold by treatment of the cells with 1 mM n-butyrate for 18 h. the most effective dose range being 0.1-5.0 mM. Treatment with n-butyrate for 18 h was more effective than treatment for 40 h. Addition of n-butyrate to an untreated cell homogenate had no stimulatory effect. The enhancement of 5-lipoxygenase activity by n-butyrate was accompanied by new synthesis of protein(s). 12-Lipoxygenase activity was not increased so much as 5-lipoxygenase activity by the treatment. This is the first report of stimulation of 5-lipoxygenase activity in cultured cells. The different responses of the two lipoxygenases to n-butyrate treatment strongly suggest that 5-lipoxygenase is a different enzyme from 12-lipoxygenase.

Animals↗
PubMed1982

Effects of reduced glutathione on the 12-lipoxygenase pathways in rat platelets.

Arachidonic acid is converted into several more polar products in addition to 12-l-hydroperoxyeicosa-5,8,10,14-tetraenoic acid (12-HPETE) and 12-l-hydroxyeicosa-5,8,10,14-tetraenoic acid (12-HETE) by the cytosol fractions of rat platelets. The more polar products are formed via the lipoxygenase pathways in the same way as are 12-HPETE and 12-HETE, since their formation is not inhibited by indomethacin but by eicosa-5,8,11,14-tetraynoic acid (ETYA). The presence of 0.5-1.5mm-reduced glutathione (GSH) in the reaction mixture prevents the formation of the more polar products and produces 12-HETE as the only metabolite from arachidonic acid by the 12-lipoxygenase pathway. l-Cysteine has the same effect as GSH. However, oxidized glutathione (GSSG) and l-cystine are not able to prevent the formation of the more polar products. The results indicate that 12-HPETE peroxidase in the 12-lipoxygenase pathway is a GSH-dependent peroxidase and the more polar products might be formed from the non-enzymic breakdown of the primary 12-lipoxygenase product of 12-HPETE, owing to insufficient capability of the subsequent peroxidase system to completely reduce 12-HPETE to 12-HETE. Thus the presence of GSH in the reaction mixture offers a convenient and precise cell-free assay system for 12-lipoxygenase in rat platelets. Routine assays of 12-lipoxygenase are carried out in the presence of 1mm-GSH in the reaction mixture. The synthesis of 12-HETE by 12-lipoxygenase is linear during the first 4 min of incubation at 37 degrees C, and has a pH optimum of 7.7. The 12-lipoxygenase reaches half-maximal activity at an arachidonate concentration of 20mum. Fractionation of cell homogenates indicates that the cytosol fraction possesses almost all the 12-lipoxygenase activity, whereas the microsomal fraction exhibits little enzyme activity.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1982

Effect of growth stage on histone H1 metabolism in human diploid fibroblasts.

Metabolism of histone H1 at different stages of cell growth was investigated in order to get a better understanding of the role of histone H1 in the cell growth of human diploid fibroblasts. Histone H1 content exhibited some fluctuation during the culture stage of cell growth. When cells entered confluent phase, the ratio of histone H1 to total histones decreased significantly. Histone H1 had a turnover half-life of 80 hours whereas nucleosomal histones did not significantly turn-over regardless of the growth stage. DNA synthesis was drastically diminished with increased cell density whereas histone synthesis was less sensitive to contact-inhibition. The gradual decline of histone H1 content with increased cell density suggests that its degradation is slightly superior to its residual synthesis. When the confluent cells were seeded at low density, cell proliferation resumed and histone H1 was synthesized and deposited to chromatin in a greater amount than nucleosomal histones, thus resulting in an abrupt increase of histone H1 content. The possible role of histone H1 metabolism in normal cells is discussed.

Cell Division↗