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Biomedical subjects

Y Amagai

Publications and source records attributed to Y Amagai.

9 recordsLinked to original sources
PubMed1990

Fibroblast growth factor-induced decrease in the phosphorylation of Nsp100 mediated through a calcium-dependent mechanism and blocked by lectins.

Separate treatment of PC12h cells with basic fibroblast growth factor (bFGF) and with epidermal growth factor (EGF) induced a selective decrease in the incorporation of radioactive phosphate into a 100,000-dalton soluble protein during phosphorylation with (gamma-32P)ATP of soluble extracts from the cells, as was seen previously with nerve growth factor (NGF). This 100,000-dalton soluble protein was designated in earlier studies as nerve growth factor-sensitive protein 100 (Nsp100). The inhibitory effects of bFGF and EGF on Nsp100 phosphorylation were prevented by pretreatment of PC12h cells with the calcium chelator, EGTA. Treatment of PC12h cells with the plant lectin wheat germ agglutinin (WGA), which binds to N-acetylglucosamine and sialic acid residues on glycoconjugates, blocked the inhibitory effects of bFGF, EGF, and NGF on Nsp100 phosphorylation. The blockage by WGA was reversed by the addition of the lectin-specific sugar N-acetylglucosamine to the PC12h cultures. Although pretreatment of PC12h cells with succinylated WGA, which has the ability to bind to N-acetylglucosamine but not to sialic acid residues, failed to block the inhibitory effect of NGF on Nsp100 phosphorylation as described previously, it did prevent the inhibitory effect of bFGF on this phosphorylation. These data suggest that in PC12h cells bFGF and EGF induce a decrease in the phosphorylation of Nsp100 mediated through a Ca2(+)-dependent mechanism, as in the case of NGF. Furthermore, the blockage of the bFGF-induced inhibition of Nsp100 phosphorylation by WGA and its succinylated form indicates that N-acetylglucosamine residues of bFGF receptor molecules might be involved in the mechanism by which bFGF inhibits the phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaloids↗
PubMed1989

Calcium action potential and prolonged afterhyperpolarization in developing myotubes of a mouse clonal myogenic cell line.

Under a high-Ca condition (greater than 5 mM), myotubes of a mouse myogenic cell line MC3T3-A1/M13 generated a long-lasting Ca action potential and a prolonged afterhyperpolarization (a.h.p.) during their in vitro development. The action potential was sensitive to Co or verapamil. Under a voltage-clamp condition, membrane depolarization more positive than -20 mV evoked a Ca-dependent inward current, which was apparently prolonged and responsible for the generation of the long-lasting action potential. The appearance of the Ca action potential preceded that of a Na spike by about 24 h, and it developed so that the maximum rate of rise became 26 +/- 4 V/s by day 7. Then this Ca-dependent potential faded as the myotubes matured, until the action potentials became solely Na-dependent. The a.h.p. was evoked accompanying the Ca action potential and was inhibited by quinine or quinidine, showing that it is operated by Ca-activated K channels. This channel developed together with the Ca channel and continued to exist during the myotube maturation process. These results indicate that the MC3T3-A1/M13 myotube at the initial stage of development has a highly developed Ca spike system that is due mostly to a high-threshold-type Ca channel.

Action Potentials↗
PubMed1989

A voltage-dependent calcium current in mouse MC3T3-E1 osteogenic cells.

MC3T3-E1 osteogenic cells in a growing state were voltage-clamped by the whole-cell patch-clamp method. The MC3T3-E1 cells exhibited a transient, fast-inactivating Ca inward current upon depolarizing pulses from a holding potential of -80 mV. This current had a threshold of activation of about -50 mV and was insensitive to the dihydropyridine, nifedipine. These results show that MC3T3-E1 cells have a voltage-dependent Ca channel corresponding to the "T-type."

Animals↗
PubMed1986

MC3T3-G2/PA6 preadipocytes support in vitro proliferation of hemopoietic stem cells through a mechanism different from that of interleukin 3.

Both MC3T3-G2/PA6 preadipocytes and interleukin 3 (IL 3) can support in vitro proliferation of mouse hemopoietic stem cells (CFU-S). We examined whether MC3T3-G2/PA6 cells produce IL 3 and whether a common mechanism might underlie the action of both of these agents. We used cultured mast cells, DA-1 cells, and FDC-P2 cells as the targets of IL 3 and conditioned medium (CM) of WEHI-3 cells as a source of IL 3. MC3T3-G2/PA6 CM did not support the growth of the above cells. IL 3 mRNA was not detected in the preadipocytes. Since CM obtained from the cocultures of bone marrow cells and MC3T3-G2/PA6 cells did not have a significant effect on the growth of the IL 3-dependent cells, none of the bone marrow cells seem to produce IL 3 under the influence of the preadipocytes. When the factor-dependent cells were cocultured with MC3T3-G2/PA6 cells, the former did not survive, whereas mast cells and DA-1 cells intimately associated with the preadipocytes. Even when bone marrow cells, mast cells, and MC3T3-G2/PA6 cells were cocultured, the number of CFU-S increased, but not that of mast cells. These results seem to exclude the possibility of the action of IL 3 in the microenvironment provided by MC3T3-G2/PA6 preadipocytes.

Adipose Tissue↗
PubMed1983

In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria.

We investigated the capacity of a clonal osteogenic cell line MC3T3-E1, established from newborn mouse calvaria and selected on the basis of high alkaline phosphatase (ALP) activity in the confluent state, to differentiate into osteoblasts and mineralize in vitro. The cells in the growing state showed a fibroblastic morphology and grew to form multiple layers. On day 21, clusters of cells exhibiting typical osteoblastic morphology were found in osmiophilic nodular regions. Such nodules increased in number and size with incubation time and became easily identifiable with the naked eye by day 40-50. In the central part of well-developed nodules, osteocytes were embedded in heavily mineralized bone matrix. Osteoblasts were arranged at the periphery of the bone spicules and were surrounded by lysosome-rich cells and a fibroblastic cell layer. Numerous matrix vesicles were scattered around the osteoblasts and young osteocytes. Matrix vesicles and plasma membranes of osteoblasts, young osteocytes, and lysosome-rich cells showed strong reaction to cytochemical stainings for ALP activity and calcium ions. Minerals were initially localized in the matrix vesicles and then deposited on well-banded collagen fibrils. Deposited minerals consisted exclusively of calcium and phosphorus, and some of the crystals had matured into hydroxyapatite crystals. These results indicate that MC3T3-E1 cells have the capacity to differentiate into osteoblasts and osteocytes and to form calcified bone tissue in vitro.

Alkaline Phosphatase↗
PubMed1983

Development of excitability during the in vitro differentiation of a newly established myogenic cell line.

Developmental changes in the membrane electrical properties during the differentiation of a newly established clonal myogenic cell line MC3T3-A1/M13 (M13) derived from newborn mouse calvaria were studied using the conventional intracellular recording method. M13 cells proliferated in vitro with a population doubling time of about 20 hr when they were cultured in alpha-MEM containing 10% newborn bovine serum at 37 degrees C. After they had achieved confluence and stopped growing, myotube formation by fusion of individual postmitotic mononucleated cells took place within 48 hr, and it advanced until 70-80% of the total number of nuclei were incorporated into such myotubes. Mononucleated M13 cells had a resting membrane potential (Em) of -22.5 +/- 1.7 mV (mean +/- S. D.) and responded passively to current stimuli, indicating that they are non-excitable. On the contrary, multinucleated myotubes had EmS ranging from -25 to -70 mV, depending on the stage of their development. Newly fused myotubes had relatively less negative EmS and showed no response, whereas myotubes later in development showed delayed rectification against depolarizing current pulses, proving the development of a voltage-sensitive outward current system. Further, mature myotubes had an Em of -58.5 +/- 3.2 mV and generated fast action potentials having a maximum rate of rise of 315 +/- 11 V/sec and a duration of 3.0 +/- 0.5 msec (measured at half-height). These action potentials were identified as tetrodotoxin-insensitive Na+ spikes. These results indicate that the membrane excitability of the M13 myogenic cell line develops well after the formation of myotubes, with an increase in Em as maturation proceeds.

Action Potentials↗
PubMed1982

Hormonal responsiveness of a preadipose cell line derived from newborn mouse calvaria.

We established a clonal preadispose cell line from newborn mouse calvaria. Cells of this cell line, designated MC3T3-G2/PA6, had the capacity to convert to adipose cells, to accumulate triglycerides in their cytoplasm, and to mature to differentiated fat cells in a resting state. This adipose conversion was markedly accelerated by addition of dexamethasone, which was the most potent inducer among the steroid hormones tested. The presence of dexamethasone was needed during the steroid hormones tested. The presence of dexamethasone was needed during logarithmic growth phase for maximal conversion. The frequency of adipose conversion was dependent on exposure time to the hormone, but cells already committed to differentiation continued to accumulate lipid and developed into mature adipose cell even in its absence. This indicates that the hormone accelerates the initiation of the adipose conversion, but is not required for the ongoing conversion process. In fact, it was rather inhibitory for the process of fat accumulation. Insulin alone slightly inhibited the adipose conversion, but its combination with dexamethasone neutralized the above inhibitory effect of dexamethasone. The responsiveness of this cell line is consistent with that observed for mouse bone marrow preadipocytes in primary culture but differs from that for preadipose cell lines derived from extramedullary tissues. These results strongly suggest that the MC3T3-G2/PA6 cell line was derived from bone marrow.

Adipose Tissue↗
PubMed1982

A new preadipose cell line derived from newborn mouse calvaria can promote the proliferation of pluripotent hemopoietic stem cells in vitro.

A clonal preadipose cell line MC3T3-G2/PA6, established from newborn mouse calvaria, responds to glucocorticoids and converts to adipose cells in a fashion similar to bone marrow preadipocytes. We investigated the effect of the cells on in vitro hemopoiesis of mouse bone marrow cells by cocultivation. When bone marrow cells were inoculated into confluent cultures of MC3T3-G2/PA6 cells (10(4)-10(6) cells/25-cm2 flask), the number of hemopoietic stem cells (CFU-S) significantly increased during 7-day cultivation in proportion to inoculum size. Under these conditions, active replication of CFU-S was maintained for several weeks until MC3T3-G2/PA6 cell layers detached from the substratum. This capacity of the MC3T3-G2/PA6 line was unique because other established cell lines, including the MTF preadipose line, failed to support CFU-S growth. When bone marrow cells were not allowed to contact the MC3T3-G2/PA6 cell layer, only a small number of CFU-S survived for 7 days. Moreover, MC3T3-G2/PA6 cell-conditioned medium did not show any growth-promoting activity for CFU-S. These results indicate that the MC3T3-G2/PA6 cell line has the ability to promote the proliferation of CFU-S through a short range cell-to-cell interaction by providing an in vitro microenvironment probably similar to that for in vivo hemopoiesis.

Adipose Tissue↗