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Biomedical subjects

S Murota

Publications and source records attributed to S Murota.

At least 19 recordsLinked to original sources
PubMed1998

NO is not involved in the simvastatin induced cell division and differentiation in PC12 cells.

Simvastatin, a potent 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor has been reported to inhibit cell division and induce neurite-like outgrowth in PC12 cells [Sato-Suzuki, I. and Murota, S., Neurosci. Lett., 220 (1996) 21-24]. In the present paper, we examined whether the induced nitric oxide (NO) in the simvastatin-treated PC12 cells is involved in the growth arrest and differentiation as reported in nerve growth factor (NGF) treated PC12 cells. Treatment of PC12 cells with simvastatin caused peripherin formation and enhanced NO production just like NGF-treated PC12 cells. Different from NGF, however, NO synthase inhibitors could not affect the growth arrest and differentiation in simvastatin-treated PC12 cells. In conclusion, NO had nothing to do with cell division and differentiation in simvastatin-treated PC12 cells.

Animals↗
PubMed1998

Different substrate utilization between prostaglandin endoperoxide H synthase-1 and -2 in NIH3T3 fibroblasts.

Recent studies suggested that prostaglandin endoperoxide H synthase- and prostaglandin endoperoxide H synthase-2 (PGHS-1 and PGHS-2) utilize different pools of arachidonic acid for synthesizing prostanoids. Using cultured murine NIH3T3 fibroblasts, we investigated the mechanism for the different utilization of arachidonic acid between PGHS-1 and -2. Histofluorescence staining for PGHS activity in intact cells demonstrated that quiescent 3T3 cells expressed only PGHS-1 activity and serum-activated 3T3 cells pretreated with aspirin expressed only PGHS-2 activity. Endogenous arachidonic acid released by calcium ionophore A23187 was not converted by PGHS-1 but exclusively converted by PGHS-2. In the cell free system, the kinetics of PGHS-1 were not so much different from those of PGHS-2. However, in intact cells, arachidonic acid at concentrations lower than 2.5 microM was converted by PGHS-2 alone but not by PGHS-1. Our findings indicated that this small amount of arachidonic acid as released by some stimuli is converted exclusively by PGHS-2. Furthermore, treating the PGHS-2-expressing cells with sodium selenite or ebselen, reducing agents of intracellular peroxides, only decreased PGHS-2 activity. We speculate that only PGHS-2 has been activated by intracellular peroxides and subsequently, it can convert the arachidonic acid released endogenously.

3T3 Cells↗
PubMed1998

Arachidonic acid pretreatment enhances smooth muscle cell migration via increased Ca2+ influx.

It is well known that vascular smooth muscle cell (SMC) migration is an initial step in atheromatous plaque formation. In the present study, we investigated the effects of arachidonic acid (AA, 20:4 n-6) on bovine carotid artery SMC migration using the modified Boyden chamber technique. SMCs pretreated with 2.5 microg/ml of AA for 2 days, showed an enhanced migration response to fetal bovine serum. AA pretreatment (0.5-5.0 microg/ml) increased fetal bovine serum-induced SMC migration dose-dependently, and maximum stimulation was observed at a concentration of 2.5 microg/ml. However, AA pretreatment did not enhance fetal bovine serum-induced endothelial cell migration. Using lipid analysis, we found that AA was substantially incorporated into cellular phospholipids. When SMC migration was induced by platelet derived growth factor (PDGF)-BB, instead of serum, the stimulative effect of AA pretreatment was retained. SMCs pretreated with AA showed greater mobilization of intracellular Ca2+ in response to PDGF-BB than SMCs without AA pretreatment (controls). Nifedipine, a Ca2+ channel blocker, and glycoletherdiamine-tetraacetic acid (EGTA) had no effect on PDGF-induced migration of controls but both drugs reduced the enhanced PDGE-induced migration of AA-pretreated SMCs to the control level. Baicalein, an inhibitor of 12-lipoxygenase, reduced PDGF-BB-induced migration of both control and AA pretreated SMCs, however the AA-pretreated cells still showed enhanced migration compared to control cells. These findings suggest that AA accelerates SMC migration in the thickening of the intima during atheroma formation, via stimulation of extracellular Ca2+ influx.

Animals↗
PubMed1998

Expression of IL-6 receptor and GP130 in mouse bone marrow cells during osteoclast differentiation.

Interleukin-6 (IL-6) has been postulated as a possible mediator of bone loss after estrogen deficiency, and its signal is transduced via glycoprotein 130 (gp130) after binding IL-6 receptor (IL-6R) in the membrane of target cells. In this study, the expression of IL-6R and gp130 in bone marrow cells during osteoclast differentiation was investigated. Mouse bone marrow cells were isolated and cultured with or without 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3]. During the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs), IL-6R and gp130 expression in the mononuclear cells, stromal cells, and TRAP-positive MNCs were quantitated, using a laser cytometer with a fluorescence confocal microscopy. With 1,25(OH)2D3 stimulus, the level of gp130 significantly increased, but that of IL-6R did not in the stromal cells. In contrast, the levels of both gp130 and IL-6R significantly increased in the mononuclear cells by the treatment with 1,25(OH)2D3. The high expression of both gp130 and IL-6R was observed in the TRAP-positive mononuclear cells. Moreover, both IL-6R and gp130 were expressed in the TRAP-positive MNCs and isolated murine osteoclasts. The treatment of TRAP-positive MNCs with IL-6 caused enhancement of the resorbing activity in a dose-dependent manner, and the effect was prevented by a neutralizing antibody against IL-6R. These data suggest that gp130 and IL-6R, as well as IL-6, are involved in the formation and activation of osteoclasts.

Acid Phosphatase↗
PubMed1998

Involvement of cholesterol in osteoclast-like cell formation via cellular fusion.

Osteoclasts, the bone resorbing cells, are formed from hematopoietic precursors via plasma membrane fusion. To investigate the possibility that cholesterol is involved in cellular fusion events, we examined the effects of the depletion of low density lipoproteins (LDL) and the effects of a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor on osteoclast-like cell formation. Tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs), osteoclast-like cells, were formed as a result of the coculture of mouse spleen cells with mouse stromal cells TMS-14 in the presence of 1alpha, 25-dihydroxyvitamin D3 (1, 25 VD3). The depletion of LDL suppressed the formation of TRAP-positive MNCs, but the effect was abrogated by the addition of LDL. This inhibitory effect was not observed following the depletion of LDL in the early stages of the culture (day 0-3). The decrease in the number of TRAP-positive MNCs resulting from LDL depletion was accompanied by an increase in the number of TRAP-positive mononuclear cells. Furthermore, the formation of MNCs was also suppressed by the addition of simvastatin, an HMG-CoA reductase inhibitor. The inhibitory effect of simvastatin on the generation of TRAP-positive MNCs was dose and time dependent. However, treatment with simvastatin did not affect the increase in TRAP activities. These results suggest that cholesterol in the membranes of monocytes is involved in osteoclast-like cell formation via cellular membrane fusion events.

Acid Phosphatase↗
PubMed1997

Cationic amino acid transporter-2 mRNA induction by tumor necrosis factor-alpha in vascular endothelial cells.

Nitric oxide (NO) synthesis may be coupled to the activity of the cellular L-arginine transporter, namely the cationic amino acid transporter. The present study examined tumor necrosis factor (TNF)-alpha-induced alterations in the gene expression of the cationic amino acid transporter (CAT) and NO production in human umbilical vein endothelial cells. In quiescent endothelial cells, CAT-1 mRNA expression, determined by reverse transcription-polymerase chain reaction, was dominant to that of CAT-2. TNF-alpha (10 ng/ml for 1-24 h) induced a time-dependent increase in CAT-2 but not CAT-1 expression. Moreover, TNF-alpha (1-30 ng/ml) treatment for 6 h induced a concentration-dependent increase in CAT-2 mRNA expression. The upregulation of CAT-2 expression by TNF-alpha was associated with enhanced nitrite accumulation in the culture medium (70% increase compared with vehicle-treated cells at 24 h). Thus, induction of the cationic amino acid transporter may constitute one mechanism for the TNF-alpha-induced NO production in human umbilical vein endothelial cells.

Amino Acid Transport Systems, Basic↗
PubMed1997

A biotinylated perfringolysin O derivative: a new probe for detection of cell surface cholesterol.

theta-Toxin is a cholesterol-binding, pore-forming cytolysin of Clostridium perfringens. To detect cell surface cholesterol, we prepared a theta-toxin derivative, BC theta by biotinylation of a protease-nicked theta-toxin, which has the same binding affinity for cholesterol as theta-toxin without cytolytic activity. Human erythrocytes, V79 cells and human umbilical vein endothelial cells (HUVEC), were stained with BC theta coupled with FITC-avidin, and then the cells were analyzed by either flow cytometry or laser confocal microscopy. The fluorescence intensity increased in both intact and briefly fixed cells when treated with BC theta. BC theta-treated V79 cells were stained by neither trypan blue nor propidium iodide, indicating that BC stained just the outer surface of the plasma membrane of vital cells. Treatment of the cells with digitonin, a cholesterol-sequestering reagent, decreased the fluorescence intensity to the background level, indicating that BC theta staining is specific for cholesterol. The fluorescence intensity of erythrocytes pre-permeabilized with a small amount of theta-toxin increased more than ten-fold, suggesting higher cholesterol contents in the inner layer of the plasma membrane. When cells were cultured with cholesterol-depleted medium, the fluorescence intensity stained by BC theta decreased remarkably in V79 cells, but did not change in HUVEC. This indicates that cell surface cholesterol may be provided in different ways with these two cell lines. These results suggest that BC theta can be a useful probe for visualizing cell surface cholesterol and for evaluating the effects of cellular events on the topology and distribution of cholesterol.

Animals↗
PubMed1997

Involvement of prostaglandin endoperoxide H synthase-2 in osteoclast formation induced by parathyroid hormone.

Prostaglandin (PG)E2 is one of the most important endogenous bone resorbing factors. In the previous study, we demonstrated that osteoclast formation induced by IL-1 beta was mediated by PGE2 produced by induced prostaglandin endoperoxide H synthase-2 (PGHS-2) in osteoclastic cells. In the same bone marrow culture system, indomethacin also suppressed the osteoclast formation induced by PTH. The inhibition was abolished by exogenously added PGE2 at dose as low as 3 x 10(-9) M, which was too low to elevate the intracellular cAMP and calcium levels and also it was too low to cause osteoclast formation by itself. In order to estimate what kind of cell produced such small amount of PGE2 in the PTH treatment, we carried out antibody staining of PGHS-1&PGHS-2 and PGHS activity in the intact bone marrow cells. PTH was found to induce PGHS activity in tartrate-resistant acid phosphatase (TRACP) positive mononuclear cells and the PGHS activity was inhibited by NS-398, a specific inhibitor of PGHS-2. Immunocytochemical staining supported the expression of PGHS-2 in TRACP-positive mononuclear cells. These findings suggest that PGHS-2 induced by PTH may regulate osteoclast formation by different mechanism from that induced by IL-1 beta.

Acid Phosphatase↗
PubMed1997

Effect of IL-6 on tumor cell invasion of vascular endothelial monolayers.

The effect of interleukin-6 (IL-6) on the invasive capacity of B16-F1 mouse melanoma cells into vascular endothelial monolayers was examined, and an in vitro assay system for the quantitative determination of tumor cell invasiveness, using confocal microscopy with a fluorescence image analyzer, was developed. First, the invasive capacity of B16-F1 mouse melanoma cells against bovine vascular endothelial monolayers was estimated; then, the gap junctional intercellular communication (GJIC) of endothelial cells was examined. Treatment of endothelial cells with IL-6 resulted in a remarkable increase in the invasion of tumor cells into the endothelial monolayer, which was found to be significant from 25 ng/ ml, and peaked at levels of more than 50 ng/ml. This stimulatory effect of IL-6, which was observed from 3 h after the initiation of treatment and lasted for up to 24 h, was abolished by the addition of the anti-IL-6 antibody. Although phase-contrast microscopy did not reveal any morphological changes in the endothelial cells following treatment with 25-200 ng/ml IL-6 for 24 h, the GJIC was observed to be significantly decreased. These findings indicate that the invasive capacity of tumor cells into endothelial cells is affected by IL-6.

Analysis of Variance↗
PubMed1997

Re-expression of functional P-selectin molecules on the endothelial cell surface by repeated stimulation with thrombin.

P-selectin (GMP-140, PADGEM, CD62P) is a cell adhesion receptor which is believed to play an important role in inflammatory diseases by supporting leucocyte rolling. P-selectin is located on the granule membrane of Weibel-Palade bodies in resting endothelial cells and is expressed on the cell surface during cellular activation with various stimulators such as thrombin. Thereafter, P-selectin is internalized and sorted to the Golgi region and Weibel-Palade bodies again. However, whether P-selectin is re-expressed upon subsequent cellular stimulation has, to date, been unclear. To address this question, we measured the cellular content and surface expression of P-selectin, using indirect immunofluorescence and confocal laser cytometry. Surface expression of P-selectin reached a maximum < 2 min after thrombin stimulation and declined to basal levels after 180 min. Rechallenge with thrombin induced rapid surface re-expression of P-selectin, which was independent of de novo protein synthesis, since cycloheximide did not inhibit re-expression. Moreover, re-expressed P-selectin supported the adherence of HL60 promyelocytic cells. These results clearly demonstrated that functional P-selectin molecule was recycled after repeated stimulation with thrombin, raising the possibility that P-selectin is involved in chronic inflammation.

Endothelium, Vascular↗
PubMed1997

[Studies on 5-lipoxygenase inhibitors. Synthesis of and structure--activity relationship in p-hydroxyarylalkenylbenzoazoles].

In a previous paper we reported that 2-(p-hydroxyarylbutadienyl)benzoxazoles are highly potent 5-lipoxygenase inhibitors. We synthesized their ethenyl homologues and benzothiazole derivatives, and evaluated their 5-lipoxygenase inhibitory activity in vitro with cell-free rat basophilic leukemia (RBL-1). In most cases the replacement of benzoxazolyl with benzothiazolyl resulted in an enhancement of the activity. All compounds with butadienyl spacers tested herein exhibited strong inhibitory activities. While most of the ethenyl homologues showed weaker activities than their corresponding butadienyl homologues, some ethenyl compounds in the benzothiazole derivatives were found to be as potent as their corresponding butadienyl homologues. The inhibitory activity was also affected by the variation in the p-hydroxyaryl functionality.

Animals↗
PubMed1997

Enhancement of membrane fluidity in cholesterol-poor endothelial cells pre-treated with simvastatin.

We examined the membrane fluidity of the cholesterol-poor bovine carotid artery endothelial cells (BAEC). Cholesterol-poor BAEC were obtained by treating the cells with 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors under 10% low density lipoprotein (LDL)-deficient serum condition for 2 days. Simvastatin reduced the intracellular cholesterol content significantly at a concentration of 0.1 microgram/ml. The reduction in the cholesterol content was accompanied by the enhancement of the cell membrane fluidity which was measured by a photobleaching technique. Additional data suggested that the reduction in cholesterol content referred to the reduction in the proliferation of BAEC.

Animals↗
PubMed1996

Simvastatin inhibits the division and induces neurite-like outgrowth in PC12 cells.

Simvastatin, a potent 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor. inhibited cell division in a dose dependent fashion and induced neurite-like outgrowth in PC12 cells. The neurite-like outgrowth was detectable at 0.5 microg/ml of simvastatin 24 h after the treatment. The responses to simvastatin were completely prevented by incubating the cells with mevalonate. In contrast to simvastatin, pravastatin, a similar HMG-CoA reductase inhibitor but lipophobic, had no effect on the cells. The results provide new possibilities for the central nervous system (CNS) side effects of simvastatin therapy.

Acyl Coenzyme A↗