Preventive effect of MCI-186 on 15-HPETE induced vascular endothelial cell injury in vitro

Using cultured bovine aortic endothelial cells, the effects of MCI-186, a radical scavenger, were studied on arachidonic acid metabolism and on the cell injury caused by 15-HPETE. MCI-186 at 3 X 10(-5) M enhanced prostacyclin production in the intact endothelial cells without affecting phospholipase A2. When endothelial cell homogenates were used as an enzyme source, it was found that MCI-186 stimulated the conversion of arachidonic acid to prostacyclin like phenol, perhaps by trapping OH radicals produced in the process of the conversion of PGG2 to PGH2. On the other hand, MCI-186 was found to inhibit lipoxygenase metabolism of arachidonic acid in cell free homogenates of rat basophilic leukemia cells. The lipoxygenase inhibition caused by 3 X 10(-5) M MCI-186 was almost equivalent to that caused by 3 X 10(-6) M BW 755C. MCI-186 remarkably protected against endothelial cell damage caused by 15-HPETE. 3 X 10(-5) M of 15-HPETE caused endothelial cell death in about 60% of the population: however, pretreatment of the cells with 10(-5) M of MCI-186 or concomitant addition of 10(-5) M of MCI-186 with 15-HPETE to the cultures prevented the cell death completely. These results suggest that MCI-186 may become an unique anti-ischemic drug.     

The existence of distinct classes of prostaglandin E2 receptors mediating adenylate cyclase and phospholipase C pathways in osteoblastic clone MC3T3-E1.

We have reported previously that PGE2 evoked an increase in intracellular calcium level ([Ca2+]i) in mouse osteoblastic cells (1). Here, we investigated the effects of PGE1 and PGF2 alpha on cAMP production and [Ca2+]i in comparison with those of PGE2. In osteoblastic clone, MC3T3-E1 cells, PGE1 stimulated cAMP production, but had no effect on [Ca2+]i, whereas PGF2 alpha evoked only [Ca2+]i increase. In contrast, PGE2 not only stimulated cAMP production, but also increased [Ca2+]i. From the Scatchard plot analysis of PGE2 it was confirmed that there were two classes of PGE2 binding sites (Kd value, 9.2 nM; binding site, 29 fmole/mg protein, and Kd value, 134 nM; binding site, 148 fmole/mg protein). As the increase in [Ca2+]i was caused by PGF2 alpha and PGE2, but not by PGE1, we investigated the displacement of [3H]-PGF2 alpha binding. The displacement capacity of unlabeled PGE2 was about 110 of that of PGF2 alpha, while that of PGE1 was very low even at 500-fold excess. These data indicate the possibility that the dual action of PGE2 is mediated by distinct receptor systems.     

Comparison of arachidonic acid metabolism between myofibroblasts and fibroblasts isolated from rat palatal mucoperiosteum

Myofibroblasts were cultured successfully from experimental wound tissue in rat palatal mucoperiosteum. Arachidonic acid metabolizing activity in cultured myofibroblasts was compared with that in fibroblasts cultured from normal mucoperiosteum. Prostaglandins biosynthesized from [14C]arachidonic acid in cell-free homogenates of both myofibroblasts and fibroblasts were prostaglandins D2, E2 and F2 alpha, and the activity producing each prostaglandin was not significantly different between the myofibroblasts and the fibroblasts, whereas smooth muscle cells, which are histologically similar to myofibroblasts, produced mainly 6-ketoprostaglandin F1 alpha, and relatively small amounts of prostaglandin E2. The release of arachidonic acid from cells prelabeled with [14C]arachidonic acid was compared among three types of cell. The calcium ionophore A23187 strongly enhanced arachidonic acid release in all three cell types. Bradykinin, 5-hydroxytryptamine and prostaglandin F2 alpha affected the stimulation of arachidonic acid release in the fibroblasts but were less or not effective in the myofibroblasts and smooth muscle cells. In addition, prostaglandin E2 biosynthesized in response to several stimuli was measured by radioimmunoassay. The content of prostaglandin E2 correlated closely with arachidonic acid release. In this study, we showed homogeneity between the myofibroblasts and fibroblasts in prostaglandin synthesizing activity and similarity in response to various stimuli between the myofibroblasts and smooth muscle cells, from the standpoint of arachidonic acid metabolism.     

Growth-coupled changes in glucosaminoglycans (heparan sulfate and hyaluronic acid) in normal and transformed human fibroblasts

Changes in glycosaminoglycans (GAGs) were investigated in relation to cell density, growth and transformation of human fibroblasts. Relative amounts (percentages of the total GAGs) of heparan sulfate (HS) increased and those of hyaluronic acid (HA) decreased in growth-reduced (serum-starved, exogenous HS-treated and dense) cultures of normal (WI-38) cells. In contrast, transformed (WI-38 CT-1) cells exerted such GAG changes only in serum-starved cultures, but not in HS-treated or dense cultures. These results indicate that the changes in glucosaminoglycans (G1cAGs) (HS and HA) is coupled exclusively with cell growth.     

Actions of exogenous heparan sulfate and hyaluronic acid on growth and thymidine incorporation of normal and transformed human fibroblasts. A comparison with the effects of high cell density and low serum concentration and a warning against thymidine incorporation as a measure of DNA synthesis

Treatment of normal human (WI-38) cells with exogenous heparan sulfate (HS) reduced cell growth and incorporation of radio-isotope-labeled thymidine (TdR) into DNA. In spite that growth of their transformants (WI-38 CT-1) was enhanced by HS treatment, transformed cells also decreased in TdR incorporation thereby. This peculiar observation was explained by a reduction of TdR uptake, leading to a decrease in specific radioactivity of newly synthesized DNA. The changes in cell growth and TdR incorporation by HS treatment were revealed to be similar to the changes with increasing cell density rather than by serum starvation.     

Regulation of cyclic GMP phosphodiesterase in the submandibular gland of adult rat

Cyclic GMP phosphodiesterase was investigated in the submandibular gland of the adult rat to elucidate the regulatory mechanisms of cGMP concentration in this gland. Ca2+ sensitivity was easily demonstrated as in other tissues using EGTA in the buffer for elution from DEAE-cellulose. The presence of inhibitor proteins for basal and calmodulin-activated portions of Ca2+-dependent phosphodiesterase was suggested. The inhibitor for the basal activity of Ca2+-dependent phosphodiesterase was deemed to be a heat-labile protein, which decreased Vmax, but had no effect on the Km value for cGMP. The presence of more than one kind of inhibitor for the calmodulin-activated portion of the Ca2+-dependent phosphodiesterase was also suggested. One of these, which was not absorbed on DEAE-cellulose, was a heat-labile protein which caused an increase of Km for cGMP, but no change of Vmax.     

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.     

The induction of recessive mutations in mouse primordial germ cells with N-ethyl-N-nitrosourea

A specific-locus test was carried out to examine the mutagenic activity of N-ethyl-N-nitrosourea (ENU) on mouse primordial germ cells (PGC). Embryos of C3H/He mice were treated transplacentally with 30 or 50 mg ENU per kg of maternal body weight on day 8.5, 10.5, or 13.5 of gestation (G8.5 day, G10.5 day, or G13.5 day). Male and female mice that had been treated with ENU in embryonic stages were mated with female or male tester PW mice to detect recessive mutations induced in PGC.

ENU induced recessive mutations at a relatively high rate in PGC at these developmental stages. The most sensitive stage was G10.5 day. On G8.5 day, the induced mutation rate in males and females was not significantly different. Cluster mutations, which originate from the limited number of PGC and cell killing, were more frequently induced at an earlier developmental stage. The induced mutation rate per unit dose of ENU (1 mg/kg) was higher in G8.5 and G10.5 day PGC than in stem-cell spermatogonia. It can be concluded that mouse PGC are more sensitive than stem-cell spermatogonia to the induction of recessive mutations by ENU.     

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.