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Biomedical subjects

K Sorimachi

Publications and source records attributed to K Sorimachi.

At least 55 records · Page 3Linked to original sources
PubMed1988

Induction of alkaline phosphatase activity by synergistic action of dibutyryl cyclic adenosine monophosphate, prednisolone, butyrate and sodium chloride in cultured cells.

Human urinary bladder carcinoma cells (JTC-32) retain a low alkaline phosphatase activity. Prednisolone or a hypertonic concentration of NaCl caused a moderate increase in the activity (10- to 15-fold of control), but dibutyryl cAMP or butyrate did not. Examination of the combined effect of these four agents revealed that they acted synergistically in any combination. When the cells were incubated with the four agents together, the enzyme activity increased 60- to 250-fold. Serum also contributed to this synergistic increase. These agents slightly inhibited cell growth and protein synthesis. The enzyme induction was completely inhibited by cycloheximide or actinomycin D. The synergistic effect of the four agents on the enzyme activity was also observed in other strains of carcinoma cells, human urinary bladder carcinoma cells (JTC-30) and monkey hepatocarcinoma cells (NCLP-6E). Thus, it is concluded that the coexistence of the four agents provides general and superior conditions for the induction of alkaline phosphatase in cultured carcinoma cells.

Alkaline Phosphatase↗
PubMed1988

The autorelease of alkaline phosphatase from the plasma membrane during the incubation of cultured liver cell homogenates.

When a rat hepatoma cell (R-Y121B) homogenate was incubated at 37 degrees C, 30-70% of the total alkaline phosphatase was released into the supernatant fluid from the precipitate fractions. The release reached a plateau level after 10 h of incubation at 37 degrees C. The optimum pH value for the release was 7.4. Alkaline phosphatase activity increased during the incubation of the cell homogenates, but this increase was independent of the enzyme release. Serum increased not only alkaline phosphatase activity in the cultured cells but also enzyme release in their homogenates. In addition, we examined a rat liver homogenate and the following 11 cell lines: 3 hepatoma cell lines, including the R-Y121B cell line, 4 liver cell lines, 2 human urinary bladder carcinoma cell lines, a kidney cell line, and a mouse adrenal tumor cell line. Only in the cultured liver cell line and hepatoma cell lines, 30-60% of the total enzyme was released into the soluble fraction from the precipitate fractions; the release was not observed in the other cell lines, nor in the rat liver homogenate. The release of alkaline phosphatase took place in both heat-stable and heat-labile alkaline phosphatases. Alkaline phosphatase, extracted from cell homogenates, showed two bands during polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The mobilities of the two bands changed inversely with or without sodium dodecyl sulfate. In general, the alkaline phosphatase which showed slow mobility with sodium dodecyl sulfate was more readily released from the plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1988

Requirement of serum pretreatment for induction of alkaline phosphatase activity with prednisolone, butyrate, dibutyryl cyclic adenosine monophosphate and NaCl in human liver cells continuously cultured in serum-free medium.

A cell line (HuL-1) derived from normal fetal human liver was adapted to grow continuously in a modified Eagle's minimum essential medium without serum or hormones. The population doubling time of this adapted cell line (HuL-1-317) was about 72 h and the modal number of chromosomes was 54. The morphology of HuL-1-317 cells was round in the absence of serum, but at 37 degrees C with the addition of serum (1-10%), the cells flattened. HuL-1-317 cells had a low level of alkaline phosphatase activity. However the enzyme activity was slightly enhanced by the combination of prednisolone, butyrate, dibutyryl cyclic adenosine monophosphate and a hypertonic concentration of NaCl after 3 days of incubation at 37 degrees C. The increase in alkaline phosphatase activity with the four agents was further amplified dose-dependently by the pretreatment of the cells with serum. The stimulatory effect of the serum was evident at concentrations as low as 1%, and was maximal at 20%. The half life of the effect of serum on alkaline phosphatase induction was 48 h at 37 degrees C. Serum alone could not enhance the enzyme activity without the four agents. The present results indicate that serum contributes to the regulation of alkaline phosphatase induction by the combination of prednisolone, butyrate, dibutyryl cyclic adenosine monophosphate and NaCl in fetal human liver cells (HuL-1-317).

Alkaline Phosphatase↗
PubMed1987

Activation of alkaline phosphatase with Mg2+ and Zn2+ in rat hepatoma cells. Accumulation of apoenzyme.

In Reuber rat hepatoma cells (R-Y121B), alkaline phosphatase activity increased without de novo enzyme synthesis (Sorimachi, K., and Yasumura, Y. (1986) Biochim. Biophys. Acta 885, 272-281). The enzyme was partially purified by butanol extraction from the particulate fractions. The incubation of the extracted alkaline phosphatase with the cytosol fraction induced a large increase in enzyme activity (5-10-fold of control). The dialyzed cytosol was more effective than the undialyzed cytosol during an early period of incubation at 37 degrees C. This difference between the dialyzed and the undialyzed cytosol fractions was due to endogenous Na+. For maximal activation of the enzyme, both Mg2+ above 1 mM and Zn2+ at low concentrations (below 0.01 mM) were needed, although Zn2+ at high concentrations (above 0.1 mM) showed an inhibitory effect. Zn2+ and Mg2+ alone slightly increased alkaline phosphatase activity. This activation of the enzyme was temperature dependent and was not observed at 0 or 4 degrees C. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the increase in alkaline phosphatase activity did not involve the fragmentation of the enzyme and that 65Zn2+ bound to it during enzyme activation with 65Zn2+ and Mg2+. The cytosol fraction not only supplied Zn2+ to the nascent enzyme but also increased the maximal enzyme activity more than did direct addition of metal ions. Ferritin and metallothionein contributed to the activation of alkaline phosphatase with the metal ions. Since the binding of Zn2+ and Mg2+ to the nascent alkaline phosphatase is disturbed in Reuber rat hepatoma cells (R-Y121B), the apoenzyme is accumulated inside the cells. The binding of Zn2+ and Mg2+ to the apoenzyme readily takes place in the cell homogenates accompanied by an increase in catalytic activity without new enzyme synthesis.

Alkaline Phosphatase↗
PubMed1987

Increase in insulin binding affinity by chloroquine in cultured rat hepatoma cells.

Chloroquine increased the binding of 125I-labeled insulin to rat hepatoma cells (R-Y121B) at 4 degrees C. The effect of chloroquine on insulin binding was amplified at 23 degrees C, and a large increase in cell-associated radioactivity was observed. Scatchard analysis showed that chloroquine increased the affinity of insulin for cells at both 4 degrees C and 23 degrees C, and that this increase eventually caused the accumulation of insulin internalized by cells at 23 degrees C.

Animals↗
PubMed1987

Increases in phenolic and nonphenolic ring deiodinase activity due to serum in monolayer liver cell cultures.

When monkey hepatocarcinoma cells (NCLP-6E) were treated with 10% of various serum preparations for 24 h at 37 degrees C, nonphenolic ring deiodinase activity increased 2.0- to 2.3-fold. Phenolic ring deiodinase activity also increased 0.9- to 2.1-fold. Dialysis of the sera enhanced the effect on deiodinase activities in some preparations, but reduced activity in other serum preparations. Similarly, a 1.3- to 3.1-fold increase in phenolic ring deiodinase activity was observed in rat hepatoma cells (R-Y121B). In this case, dialysis usually reduced the effect of the sera. It is concluded that both large molecule(s) (undialyzable) and small molecule(s) (dialyzable) in serum contribute to the regulation of phenolic and nonphenolic ring deiodinase activity in NCLP-6E and R-Y121B cells.

Animals↗
PubMed1987

Concanavalin A changes not only the number of insulin binding sites but also the binding affinity in rat hepatoma cells in culture.

When rat hepatoma cells (R-Y121B) were first incubated with labeled insulin, followed by concanavalin A, at 500 micrograms/ml at 23 degrees C, the total cell-associated radioactivity increased. At 4 degrees C, however, this increase was not observed. Scatchard analysis revealed that concanavalin A increased insulin binding affinity. The bound insulin was internalized together with concanavalin A. When the cells were incubated with concanavalin A prior to insulin addition, however, the total cell-associated radioactivity decreased at both temperatures. This was caused by the masking of insulin binding sites by the lectin.

Animals↗
PubMed1986

Regulation of alkaline phosphatase activity in rat hepatoma cells. Effects of serum proteins, cycloheximide, actinomycin D, chloroquine, dinitrophenol and potassium cyanide.

Alkaline phosphatase activity in rat hepatoma cells (R-Y121B) cultured in a monolayer at 0.5% serum was enhanced by serum, bovine serum albumin, casein and gamma-globulin, but ovalbumin, polyvinylpyrrolidone, dexamethasone, insulin and dibutyrylcyclic AMP showed little effect on alkaline phosphatase activity. In addition, cycloheximide, actinomycin D, chloroquine, dinitrophenol and potassium cyanide also increased the enzyme activity, although the incorporation of [14C]leucine into cellular proteins was almost completely inhibited in the presence of these cytotoxic substances. When R-Y121B cell homogenates were incubated at 37 degrees C, alkaline phosphatase activity increased in a pH-dependent manner: the maximal increase was observed at pH 7.1. The magnitudes of the increase differed among cell homogenates and a 4- to 10-fold increase was observed. Alkaline phosphatase in R-Y121B cells was apparently heat-stable, but that in the cells obtained from various treatments was heat labile and the latter activity decreased to less than 50% of the initial activity after 15 min of incubation at 56 degrees C. Alkaline phosphatase in the control and also in the treated cells was more sensitive to L-homoarginine than L-phenylalanine. The Lineweaver-Burk plot showed that the increases in the enzyme activity were accompanied by changes not only in V but also in Km for alkaline phosphatase reaction. Finally, it has been suggested that the increases in alkaline phosphatase activity under various conditions are due to the conversion of the molecule with a low enzyme activity to the molecule with a high enzyme activity in R-Y121B cells.

Alkaline Phosphatase↗
PubMed1985

Effects of cycloheximide on inverse regulation of phenolic and nonphenolic ring deiodinases in monkey hepatocarcinoma cells.

Both phenolic and nonphenolic ring deiodinase activities in monkey hepatocarcinoma cells (NCLP-6E) were increased by addition of serum in a concentration-dependent manner: the stimulatory effect of serum was evident at a concentration as low as 1.5%, and was maximal at 5%. Lineweaver-Burk analysis showed that the increases in the deiodinase activities are due to the increase in Vmax, but not in Km. The addition of cycloheximide at concentrations ranging from 0.1 to 50 micrograms/ml inhibited the stimulatory effect of serum on phenolic ring deiodinase activity progressively. On the other hand, nonphenolic ring deiodinase activity was increased as much as 4-fold by the addition of 0.5-5 micrograms/ml cycloheximide together with 0.5% serum; a high concentration of the drug, 50 micrograms/ml, however, did not elicit such an increase. Actinomycin D at 5 micrograms/ml completely abolished the increase in nonphenolic ring deiodinase activity by serum or cycloheximide. In addition, actinomycin D inhibited the increase in phenolic ring deiodinase activity by serum in a dose-dependent manner at concentrations ranging from 0.05 to 5 micrograms/ml. It is concluded that phenolic and nonphenolic ring deiodinases are regulated by different mechanisms in monkey hepatocarcinoma cells (NCLP-6E).

Animals↗
PubMed1985

Characterization of alkaline phosphatase in human urinary bladder carcinoma cell lines and enzyme regulation with prednisolone or sodium chloride.

In order to characterize newly established human urinary bladder carcinoma cell lines (JTC-29, JTC-30, JTC-31, JTC-32, JTC-33 and JTC-34) by Kakuya et al. [In Vitro, 19, 591-599 (1983)], we investigated alkaline phosphatase in these cell lines. Relatively high alkaline phosphatase activity was found in JTC-29 and JTC-32 cells, but the enzyme activity in the other cell lines was very low. Prednisolone induced alkaline phosphatase activity even at 0.005 micrograms/ml (14 nM) and the effect was maximal at 0.05 micrograms/ml. NaCl also induced alkaline phosphatase activity at 50 mM. A further increase in the activity was observed at 0.1 M NaCl, but at 0.2 M all cells came off from plastic culture dishes without an increase in the enzyme activity during the incubation for 24 h at 37 degrees C. Both actinomycin D and cycloheximide abolished the increases in the enzyme activity with prednisolone or NaCl. The Lineweaver-Burk plot showed that the increases in the enzyme activity are due to the increases in Vmax, but not in Km. Alkaline phosphatase in JTC-32 cells and prednisolone-treated JTC-32 cells was heat stable and 100% of the initial enzyme activity remained after 30 min of incubation at 56 degrees C. However, the enzyme in JTC-29 cells was heat labile and its activity was reduced to less than 50% after 20 min of incubation at 56 degrees C. L-Phenylalanine was the strongest inhibitor for the enzyme reactions in JTC-32 cell homogenates among L-phenylalanine, L-leucine and L-homoarginine, whereas L-homoarginine was the strongest for the enzyme reactions in JTC-29 cell homogenates.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1985

Inhibitory effect of chloroquine on concanavalin A internalization into cell monolayer culture.

When cultured rat hepatoma cells (R-Y121B) were incubated with insulin in the presence of chloroquine at 25 degrees C, chloroquine increased both insulin binding to the cell surface and its internalization into cells. In addition, in order to elucidate whether or not the effect of chloroquine on insulin receptors is specific, concanavalin A binding to the cells was examined in the presence or absence of chloroquine, since insulin receptors have concanavalin A binding sites. When the cells were incubated with concanavalin A in the presence of chloroquine, this drug decreased concanavalin A internalization in a dose-dependent manner: The inhibitory effect of chloroquine on concanavalin A internalization was evident at a concentration as low as 0.05 mM, and increased up to 0.5 mM. The effect of chloroquine was not related to the degradation of concanavalin A incubated with cells. When the cells were incubated with concanavalin A at 4 degrees C in the presence of chloroquine even at 0.5 mM, the inhibitory effect of chloroquine was not observed. It is concluded that the behavior of insulin receptors in the presence of chloroquine is different from that of concanavalin A receptors, although insulin receptors have concanavalin A binding sites. Chloroquine apparently increases insulin binding to cells in vitro, and this is due to the effect of chloroquine on insulin-receptor recyclization to which lysosome contributes [16, 31, 32]; this drug inhibits the function of lysosome which digests insulin-receptor complexes and eventually insulin bound to its receptors is accumulated in intracellular compartments.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Temperature- and dose-dependent internalization of concanavalin A in monolayer culture.

When rat hepatoma cells (R117-21B) were incubated for 20 h at 37 degrees C with 125I-labeled concanavalin A at low concentrations (0.5-10 micrograms/ml), only 20-30% of the cell-associated radioactivity was released by alpha-methyl-D-mannoside, but at high concentrations (50-500 micrograms/ml), 60-80% of the cell-associated radioactivity was released. At 4 degrees C, the cell-associated radioactivity decreased with the increase in concentration of concanavalin A, and more than 80% of the cell-associated radioactivity was released by alpha-methyl-D-mannoside. These results suggest that the amount of cell-associated concanavalin A is related to the physicochemical state of the plasma membrane, which can be altered by the incubation temperature or by the concentration of concanavalin A, the transitional concentration being 5-10 micrograms/ml.

Animals↗
PubMed1984

Concanavalin A can trap insulin and increase insulin internalization into cells cultured in monolayer.

When rat hepatoma cells (R-Y121B) were incubated with insulin at 37 degrees C, concanavalin A increased insulin internalization into cells. When R-Y121B cells were first incubated with labeled insulin at 4 degrees C then with concanavalin A at various concentrations at 37 degrees C, the total cellular radioactivity was much higher at high lectin concentrations than at low lectin concentrations. This increase was not only due to an increase in insulin internalization into cells but also to an increase in insulin binding to cell surfaces. Concanavalin A can trap insulin on the insulin receptors - a "trapping" effect. It has been concluded that insulin and concanavalin A binding sites are very close to each other on the insulin receptors.

Animals↗
PubMed1984

'Superactivation' of alkaline phosphatase activity by cycloheximide in rat hepatoma cell cultures.

We have found that rat hepatoma cells (R-Y121B) retain alkaline phosphatase activity, and that this enzyme activity is increased by cycloheximide. Actinomycin D also increased the enzyme activity. This increase due to actinomycin D was partially inhibited by cycloheximide. The characteristics of alkaline phosphatase of the cells treated or untreated with cycloheximide or actinomycin D were similar to each other; they were heat labile and the enzyme reaction was strongly inhibited by L-homoarginine, but weakly by L-phenylalanine. The increase in alkaline phosphatase activity with cycloheximide has been termed a 'superactivation' of alkaline phosphatase.

Alkaline Phosphatase↗
PubMed1983

The release of cytosolic proteins from cells treated with concanavalin A during scraping from plastic culture dishes.

When rat hepatoma cells (HTC and R117-21B), treated with concanavalin A (conA) at 37 degrees C, were scraped from plastic culture dishes with a silicone-rubber policeman, the cell membranes were broken and the cytoplasm was released. This phenomenon was also observed in cells treated with conA at 4 degrees C, even though it took a longer time to show the same effect. The effect of 10 micrograms/ml of conA on the release of the cellular proteins reached a plateau when the treatment was carried out at 37 degrees C. Ninety percent of this effect was abolished by 10 mM of alpha-methyl-D-mannoside. The effect was completely nullified by 100 mM. At 4 degrees C, however, even 100 mM of this sugar could not abolish this effect. The apparent decrease in the cellular proteins with conA after scraping was observed not only in the logarithmic phase, but also in the stationary phase of cell growth. The breakdown of plasma membranes with conA eventually caused decrease in tyrosine aminotransferase activity, even though the lectin induced the enzyme activity in cultured cells.

Animals↗
PubMed1983

Different effects of concanavalin A on insulin binding to cultured cells in monolayers: "blocking" and "trapping" effects of concanavalin A.

The effect of concanavalin A on insulin binding was studied in cells cultured in a monolayer. Preliminary incubation of concanavalin A with Reuber rat hepatoma cells ( R117 - 21B ) showed an inhibitory effect on insulin binding at 25 degrees C and 4 degrees C; a "blocking" effect. But, even at high concentrations (greater than 50 micrograms/ml), concanavalin A could not completely inhibit the specific binding of insulin to these cells. Under these conditions, the number of high affinity insulin-binding sites decreased when compared to the number of sites in the control. In contrast, when cells first were incubated with insulin then with insulin plus concanavalin A, the binding of insulin increased, even at high concentrations of the lectin. This is a "trapping" effect. This effect increased the number of insulin-binding sites as compared to the number in the control. The apparent equilibrium constant of insulin for the cells was changed slightly by the concanavalin A in R117 - 21B cells. The binding of concanavalin A to these cells also was investigated. Insulin did not affect its binding to the cells. This study shows that insulin-binding sites can be classified into two groups in which insulin binding is affected by concanavalin A or is independent of the lectin. The "blocking" effect of concanavalin A on insulin binding suggests that the binding sites of insulin and concanavalin A on the insulin receptors are very close.

Animals↗
PubMed1983

Up-regulation of insulin receptors with dexamethasone in cultured human urinary bladder carcinoma cells.

Cultured human urinary bladder carcinoma cells ( JTC -32) were used to investigate the regulation of insulin receptors by dexamethasone. When the cells were preincubated with dexamethasone at 37 degrees C, insulin binding sites increased up to 24 h. A large increase in insulin binding sites took place for 14 h of preincubation with dexamethasone. At lower concentrations of dexamethasone (less than 1 nM), no significant increase in insulin binding sites was observed, but the maximal increase was observed at more than 10 nM. Scatchard plots showed that dexamethasone increased the number of high affinity insulin binding sites (2.8 fold) without any change in the apparent equilibrium constant in JTC -32 cells. In addition, this steroid hormone also increased the number of low affinity insulin binding sites (1.6 fold) with a small change in the apparent equilibrium constant. Although insulin and dexamethasone did not affect the number of cells or the amount of cellular proteins per dish, dexamethasone plus insulin slightly increased them.

Cells, Cultured↗