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Biomedical subjects

M Mitsuka

Publications and source records attributed to M Mitsuka.

11 recordsLinked to original sources
PubMed1997

Isolation and conformational analysis of fragment peptide corresponding to the heparin-binding site of hepatocyte growth factor.

Hepatocyte growth factor (HGF) is a potent mitogen for hepatocytes. The mitogenic activity of HGF is mediated by its binding to a high-affinity receptor, c-Met. Heparan sulfate is an initial binding site for HGF, based on its relative abundance on the cell surface. The binding of HGF to heparin or heparin-like molecules may induce oligomerization of HGF and facilitate c-Met-dependent mitogenesis [Zioncheck et al. (1995) J. Biol.Chem. 270, 16871-16878]. Thus, heparin binding is important for the biological activity of HGF. To identify the heparin-binding site of HGF, we isolated fragment peptides corresponding to the site by limited proteolysis and chemical degradation of recombinant human HGF (rhHGF). The heparin-binding ability of the peptides was expressed as their elution positions on heparin-affinity column chromatography with NaCl gradient elution. Because all of the heparin-binding peptides obtained in this study were isolated from the N-terminal hairpin-loop region (PyrGlu32-Asn127) of HGF, the region was identified as the heparin-binding site of HGF. One of the isolated peptides, Phe42-Glu111, containing the N-terminal hairpin-loop structure, was considered a suitable model peptide for the heparin-binding site of HGF. From the observation using circular dichroism spectroscopy, it was indicated that the secondary structure of the peptide changed from a random structure to a beta-sheet-like structure upon heparin binding. In addition, oligomerization of HGF in the presence of heparin was observed by dynamic light scattering. Based on our evidence, it is considered that the conformational change in the heparin-binding site may induce the oligomerization of HGF.

Amino Acid Sequence↗
PubMed1997

Delayed neuronal death prevented by inhibition of increased hydroxyl radical formation in a transient cerebral ischemia.

The salicylate-trapping method was used to detect hydroxyl radicals by measurement of stable adduct dihydroxybenzoic acid (DHBA). Ten minutes of forebrain ischemia followed by reperfusion induced the increase in DHBA in rat hippocampal perfusates. Postischemic treatment with a free radical scavenger, 3-methyl-1-phenyl-2-pyrazolin-5-one (MCI-186), significantly reduced the increase in DHBA and suppressed delayed neuronal death in the hippocampal CA1 region.

Animals↗
PubMed1994

Syntheses and pharmacological activities of novel optically active inhibitors of acyl-CoA: cholesterol O-acyltransferase: EAB-309 ((R)-N-2-(1,3-benzodioxol-4-yl)heptyl-N'-2,6-diisopropylphenylurea) and its enantiomer.

Novel and potent ACAT (acyl-CoA: cholesterol O-acyltransferase) inhibitors, (R)-N-2-(1,3-benzodioxol-4-yl)heptyl-N'-2,6-diisopropylphenylur ea (2a, EAB-309), and its enantiomer 2b (EAB-310), were prepared from 4-(1,3-benzodioxole)carbaldehyde (7) via optically active (R or S)-2-(1,3- benzodioxol-4-yl)heptanoic acid (12a or 12b). Compound 2a showed potent inhibitory effects on ACATs in vitro, and lowered plasma cholesterol in vivo. The IC50 value for inhibition of rat hepatic microsomal ACAT was 5 nM. The ED30 values of hypolipidemic activities in hamster and rat models were 0.25 and 0.75 mg/kg p.o., respectively. The results indicate that 2a has potential to be a novel hypocholesterolemic and antiatherosclerotic agent. The activities of 2a in vitro and in vivo were only several times more potent than those of the enantiomer 2b. Modeling studies suggested that the three-dimensional structures of the two enantiomers are similar to each other.

Animals↗
PubMed1993

Na(+)-H+ exchange inhibitors decrease neointimal formation after rat carotid injury. Effects on smooth muscle cell migration and proliferation.

The presence of multiple growth stimuli at the sites of vascular injury following angioplasty suggests that therapies targeted toward common growth pathways will be more effective than therapies that inhibit only a single growth factor. We tested this hypothesis using amiloride and ethyl isopropyl amiloride (EIPA), which are inhibitors of the Na(+)-H+ exchanger, whose activity is required in many cells for proliferation and migration. In the rat carotid injury model, EIPA (100 micrograms/h for 15 days) significantly decreased intimal area and the ratio of intimal to medial area, whereas amiloride (25 micrograms/h) showed an inhibitory trend that was similar to that observed for captopril (80 mg/kg per day) and heparin (25 U/h). EIPA and amiloride inhibited rat vascular smooth muscle cell DNA synthesis, with IC50 values of 8.8 and 82.2 microM, respectively. Using platelet-derived growth factor as a chemoattractant, EIPA caused a concentration-dependent inhibition of migration (IC50, approximately 60 microM). Because amiloride and EIPA have nonspecific effects on cellular function (especially inhibition of tyrosine kinases), we sought to characterize the specific role of the Na(+)-H+ exchanger in vascular smooth muscle cell proliferation and migration. We generated a Na(+)-H+ exchanger-deficient mutant cell line [RNHE(-)]. Studies with these cells suggested that the inhibitory effects of EIPA and amiloride were mediated only in part via Na(+)-H+ exchange because (1) RNHE(-) cells grew well at pH 6.8 to 7.5 in bicarbonate-containing medium, and (2) there was no difference in migration in response to platelet-derived growth factor in the RHNE(-) cells. In summary, these data indicate that amiloride and EIPA inhibit neointimal formation in the rat carotid after injury. However, the mechanism of inhibition is likely to involve cellular events other than Na(+)-H+ exchange, such as an effect on tyrosine kinases.

Amiloride↗
PubMed1991

Long-term regulation of Na(+)-H+ exchange in vascular smooth muscle cells: role of protein kinase C.

Regulation of intracellular pH (pHi) plays an important role in vascular smooth muscle cell (VSMC) contractile tone and growth. We have shown that pHi in proliferating VSMC is more alkaline (7.25) than in growth-arrested cells (7.10). To study the Na(+)-H+ exchanger in the growth-dependent regulation of VSMC pHi, ethylisopropylamiloride (EIPA)-sensitive Na+ influx was measured. Exposure of growth-arrested VSMC to 10% serum initially increased Na+ influx (145% of baseline at 30 min), which then decreased (52% of baseline at 24 h). Serum-induced alterations in the kinetic properties of the Na(+)-H+ exchanger were studied by analysis of its external Na+ binding site properties. Exposure of growth-arrested VSMC to 10% serum for 24 h increased the Km for external Na+ from 54 to 380 mM, with a change in the Vmax from 155 to 199 nmol Na+.mg protein-1.min-1. The change in Km was due to activation of protein kinase C (PKC). Phorbol 12,13-dibutyrate caused a 48% decrease in EIPA-sensitive influx, the inactive 4 alpha-phorbol 12,13-didecanoate had no effect, and the PKC inhibitor sphingosine reversed the effect. Therefore, the Na(+)-H+ exchanger in VSMC is regulated in a growth-dependent manner via PKC.

Animals↗
PubMed1990

Hypertrophy and hyperplasia cause differing effects on vascular smooth muscle cell Na+/H+ exchange and intracellular pH.

Mitogens and vasoconstrictors stimulate many of the same early intracellular signals (e.g. phospholipase C and protein kinase C activation) in vascular smooth muscle cells (VSMC). Despite these shared signals, angiotensin II is not mitogenic for cultured VSMC. The nonmitogenic effect of angiotensin II suggests that other intracellular signals associated with growth should differ between mitogens and vasoconstrictors. Because of the importance of intracellular pH (pHi) in growth, we compared the effects of 10% calf serum, 10 ng/ml platelet-derived growth factor, and 100 nM angiotensin II on pHi and Na+/H+ exchange. All agonists stimulated a rapid (less than 1 min) rise in pHi mediated by Na+/H+ exchange. However, exposure of growth-arrested VSMC to these agonists for 24 h caused significant differences in pHi: 7.18 (10% serum), 7.16 (platelet-derived growth factor), 6.99 (angiotensin II), and 7.08 (0.4% serum). Na+/H+ exchange activity was measured in acid-loaded cells by the ethyl isopropyl amiloride-sensitive influx of Na+ and efflux of H+. Both techniques showed that exposure to 10% serum caused approximately 45% decrease in Na+/H+ exchange activity without significant change in angiotensin II-treated cells. Thus, although the rapid changes in pHi and Na+/H+ exchange function are the same for angiotensin II and mitogens, the long term effects differ. The data suggest that differences in pHi regulatory mechanisms are important in determining whether an agonist causes VSMC hypertrophy or hyperplasia.

Amiloride↗
PubMed1989

An automated analysis of chemotaxis in vitro using a computer-assisted scanning densitometer.

The quantitation of chemotaxis in vitro was developed with a computer-assisted scanning densitometer. The method of estimating the number of cells on a filter was based on the photo-reflection from the nuclei of stained cells. Samples obtained from a 48-well micro chemotaxis assembly were successfully analyzed by this method. This assay system could quantitate chemotaxis much faster and more accurately than by cell counting under the microscope. It was sensitive enough to determine the responsiveness of SMCs and fibroblasts to various chemoattractants. This system could be applied to medical and biological screening tests for drugs and clones in laboratories.

Animals↗
PubMed1984

Increase of carbamylcholine-induced 22Na+ influx into pheochromocytoma PC12h cells by nerve growth factor.

Carbamylcholine (CCh)-induced 22Na+ influx into clonal rat pheochromocytoma PC12h cells increased remarkably by culturing the cells in the presence of nerve growth factor (NGF) at a concentration of 50 ng/ml. After 2-4 days in culture with NGF, the CCh-induced 22Na+ influx into cells was enhanced 3- to 5-fold compared to the influx into NGF-untreated cells. No increase of CCh-induced 22Na+ influx was seen prior to 15 h. The ED50 value for NGF to increase the responsiveness was 4.6 ng/ml. This effect could not be mimicked by the addition of 1 mM dibutyryl cyclic AMP. Besides NGF, epidermal growth factor increased the CCh-induced 22Na+ influx into cells to a lower extent that NGF did. Insulin and dexamethasone had no effect. By contrast, the amount of [125I] alpha-bungarotoxin binding to PC12h cells was not changed when cultured in the presence of NGF. It is concluded that the functional nAChR and alpha-bungarotoxin binding sites of PC12h cells are controlled by different mechanisms.

Animals↗
PubMed1983

Selective loss of acetylcholine sensitivity in a nerve cell line cultured in hormone-supplemented serum-free medium.

When clonal rat pheochromocytoma PC12h cells were cultured in a hormone-supplemented serum-free medium, the carbamylcholine-elicited catecholamine release from cells cultured in serum-free medium was completely abolished. On the other hand, the high potassium-induced catecholamine release was not changed, even in PC12h cells cultured in serum-free medium. The lack of carbamylcholine sensitivity was confirmed directly by measuring carbamylcholine-induced 22Na influx, which was completely abolished in PC12h cells cultured in serum-free medium. The loss of carbamylcholine-induced 22Na influx seemed to obey nearly first-order kinetics and was fully restored upon a re-exposure to serum. The half-time for the loss was about 1 day, and the cultivation for 5 days in serum-free medium caused a 95% decrease of the nicotinic sensitivity in PC12h cells. The carbamylcholine-induced 45Ca influx into cells also was lost due to the serum-free cultivation. By contrast, the binding of alpha-bungarotoxin, which is an antagonist of nicotinic acetylcholine receptor in muscular cells, remained and did not change in PC12h cells cultured, even in the serum-free medium. In addition, veratridine-dependent 22Na influx and high potassium-induced 45Ca influx into cells, and high potassium-induced 86Rb efflux from cells cultured in the serum-free medium were also perfectly preserved. These results suggest that PC12h cells cultured in the serum-free medium seemed to be a useful model for comparing the differential mechanisms between acetylcholine sensitivity and other membranous functions on this cell.

Acetylcholine↗