Yasunobu Suketa

Stimulatory effect of zinc on bone formation in tissue culture

The present investigation was undertaken to clarify the in vitro effect of zinc on bone metabolism in tissue culture. Calvaria were removed from weanling rats (3-week-old males) and cultured for periods up to 96 hr in Dulbeccos Modified Eagle Medium (high glucose, 4500 mg/dl) supplemented with antibiotics and bovine serum albumin. The experimental cultures contained 10(-7) to 10(-3) M zinc sulfate. All cultures were incubated at 37 degrees in 5% CO2/95% air. Zinc uptake by bone was increased significantly in cultures with concentrations of zinc greater than 10(-6) M. Bone calcium content was increased significantly by the presence of 10(-4) M zinc. This increase was blocked by the presence of 10(-6) M cycloheximide. Bone alkaline phosphatase activity was elevated in the presence of zinc (10(-6) to 10(-3) M), but the effect was inhibited by 10(-7) M cycloheximide or 10(-8) M actinomycin D. Zinc (10(-4) M) also significantly increased ATPase activity in the bone, whereas it did not alter significantly by pyrophosphatase, acid phosphatase and beta-N-acetylglucosaminidase activities. Furthermore, bone collagen content was raised by 10(-6) to 10(-4) M zinc. This elevation was prevented by 10(-7) cycloheximide or 10(-8) M actinomycin D. Bone DNA content and [3H]thymidine incorporation by the bone were not altered significantly by 10(-4) M zinc. These findings indicate that the zinc had a direct stimulatory effect on bone mineralization in vitro, and that bone protein synthesis was a necessary component of this response. Zinc may stimulate bone formation in tissue culture.

Zinc stimulation of bone protein synthesis in tissue culture: Activation of aminoacyl-tRNA synthetase

The present investigation was undertaken to clarify the effect of zinc on bone protein synthesis in tissue culture. Calvaria were removed from 3-week-old male rats and cultured for periods up to 96 hr in Dulbeccos Modified Eagle Medium (high glucose, 4500 mg/dl) supplemented with antibiotics and bovine serum albumin. The calvaria were incubated at 37 degrees in 5% CO2/95% air in the medium containing 10(-6)-10(-4) M zinc. Zinc content in bone cells was increased when the culture was treated with 10(-5) and 10(-4) M zinc for 48 hr. When calvaria cultured in the presence of 10(-4) M zinc were pulsed with [14C]uridine, the incorporation of [14C]uridine into the bone RNA was not increased significantly. In the pulse with [3H]leucine, the presence of 10(-5) to 10(-4) M zinc in the medium caused a significant increase in the incorporation of [3H]leucine into the acid-insoluble residues of bone tissue. This increase was blocked completely by treatment with 10(-7) M cycloheximide, an inhibitor of protein synthesis. When [3H]leucine was added into the reaction mixture containing the 5500 g supernatant fraction of the homogenate prepared from calvaria cultured in the presence of 10(-4) M zinc, the in vitro protein synthesis was increased about 2-fold. The activity of [3H]leucyl-tRNA synthetase in the 105,000 g supernatant fraction (cytosol) of the bone homogenate was increased about 2-fold by the culture with 10(-4) M zinc. The presence of 10(-4) M dipicolinate, a specific chelator of zinc, in the culture medium negated the effect of zinc on [3H]leucyl-tRNA synthetase activity. The addition of 10(-7) to 10(-6) M zinc into the reaction mixture containing enzyme extracts obtained from uncultured rat calvaria caused a 2-fold increase of [3H]leucyl-tRNA synthetase activity. These results clearly indicate that zinc induces the stimulation of protein synthesis at the translational level in bone cells. The present study further supports the view that zinc increases protein synthesis in bone cells and that the metal induces bone formation.

Nitric oxide blocks the cell cycle of mouse macrophage-like cells in the early G2+M phase

The effects of nitric oxide produced by macrophage‐like cells (Mml) on the cell cycle were investigated. Mml cells lost proliferative activity in the presence of interleukin‐6 (IL‐6) and a subpopulation accumulated in the G2+ M phase. This level increased in proportion to the incubation time. The DNA content of the cells was slightly lower than that of Mml cells treated with vinbrastine or demecolcine, drugs which block the cell cycle in the M phase. The peak of the early G2+M phase was reduced by treatment with N G‐mono‐methyl‐l‐arginine. However, after treatment with exogenous nitric oxide or sodium nitroprusside, the G0/G1 phase increased, but the early‐G2+M and the S phase decreased. The flow cytometry pattern in IL‐6‐treated Mml was the same as that of cytochalasin B‐treated Mml. These data suggest that endogenous nitric oxide affects the microfilament system of IL‐6‐treated Mml cells and blocks the cell cycle in the early G2+M phase.

Up-regulation of Sodium-dependent Glucose Transporter by Interaction with Heat Shock Protein 70

Heat shock stress induces some heat shock proteins, including Hsp70, and activates sodium-dependent glucose transport in porcine renal LLC-PK1 cells, but its mechanisms have not been described in detail. We investigated whether sodium-dependent glucose transporter (SGLT1) interacts with Hsp70 to increase SGLT1 activity. Heat shock stress increased SGLT1 activity without changing SGLT1 expression. The increase of SGLT1 activity was completely inhibited by an anti-transforming growth factor-β1 (TGF-β1) antibody. Instead of heat shock stress, TGF-β1 increased SGLT1 activity dose- and time-dependently without changing SGLT1 expression. We found that the amount of Hsp70 immunoprecipitated from TGF-β1-treated cells with an anti-SGLT1 antibody was higher than that of the control cells. Transfection of an anti-Hsp70 antibody into the cells inhibited the increase of SGLT1 activity. With confocal laser microscopy, both SGLT1 and Hsp70 was localized near the apical membrane in the TGF-β1-treated cells, and an anti-Hsp70 antibody disturbed this localization. Furthermore, we clarified that an anti-Hsp70 antibody inhibited interaction of SGLT1 with Hsp70 in vitro. These results suggest that Hsp70 forms a complex with SGLT1 and increases the expression level of SGLT1 in the apical membrane, resulting in up-regulation of glucose uptake.

Reorganization of ZO‐1 by sodium‐dependent glucose transporter activation after heat stress in LLC‐PK1 cells

Heat stress (HS) induces activation of high‐affinity sodium‐dependent glucose transporter (SGLT1) in porcine renal LLC‐PK1 cells. In this study, we investigated the roles of SGLT1 activation in reorganization of zonula occludens‐1 (ZO‐1), a cytosolic tight junction (TJ) protein, after HS. HS (42°C, 3 h) caused decrease in transepithelial electrical resistance (TER). Subsequent incubation at 37°C for 12 h increased TER above pre‐HS level. The treatment of phloridzin, a potent SGLT1 inhibitor, or the replacement of glucose with a nonmetabolizable glucose analog blocked the recovery of TER and increased the transepithelial flux of FITC‐dextran (4,000 Da). Immunofluorescent staining of ZO‐1 showed that HS diffused ZO‐1 from cell contact to cytosolic sites. Furthermore, the fraction of ZO‐1 was distributed from the Triton X‐100 insoluble to the Triton X‐100 soluble pool. After incubation at 37°C for 12 h, cell contact and ZO‐1 extractability with Triton X‐100 returned to pre‐HS conditions, but the recovery was completely prevented by phloridzin. Tyrosine kinases activity was increased by HS that was inhibited by phloridzin. Genistein and CGP77675, tyrosine kinases inhibitors, blocked the recovery of TER and increased the transepithelial flux of FITC‐dextran. Furthermore, these inhibitors prevented the recovery of cell contact and ZO‐1 extractability with Triton X‐100 as same as phloridzin. These findings suggested that the activation of SGLT1 reorganized ZO‐1 mediated by elevation of tyrosine kinases activity after heat injury.

Isolation, physicochemical properties, and amino acid composition of a cadmium- binding protein from cadmium- treated chlorellaellipsoidea

Abstract A purified cadmium-binding protein (Ch-CdBP) in Cd-treated Chlorella ellipsoiden was obtained by gel filtration, ion-exchange chromatography, and high performance liquid chromatography The Ch-CdBP was elucidated to be a monomer of about 9000 daltons as the molecular weight by SDS gel-electrophoresis. The isoelectric point of the protein was round to be about 4.0 by disk isoelectric focusing. The Ch-CdBP was estimated to have 69 amino acid residues which consisted of nine kinds of amino acids such as aspartic and glutamic acids, serine, threonine, glycine, alanine, half-cystine, lysine, and arginine. Thus, Cd-CdBP characterized as an acidic metalloprotein with high contents ot glutamic acid and half-cystine residues but not bisulfide bond.

Inhibitory mechanisms of antibody production by nitrogen oxides released from activated macrophages during the immune response: relationship to energy consumption.

We investigated the relationship between the sensitivity of mouse splenocytes in immune response to nitrogen oxides and energy consumption rate of the cells. Macrophage‐like cells (Mm 1) pretreated with lL‐6 served as the source of the nitrogen oxides. The antibody production of both 2,4,6‐trinitrophenyl‐keyhole limpet haemocyanin‐primed splenocytes and B cell hybridomas was markedly reduced; about 20–40% of splenocytes and B cell hybridomas were killed by co‐culture with IL‐6‐treated Mm 1. Cell viability and antibody production were completely restored by the addition of NG‐monomethyl L‐arginine to the culture medium. The cytotoxicity of the nitrogen oxides was correlated with the distance between effector and target cells. Under conditions of low cytotoxicity, antibody production by B cell hybridomas was suppressed by the nitrogen oxides, this suppression not being correlated with the reduction in cell growth. The sensitivity of the target cells differed in co‐cultures of antigen‐primed splenocytes and B cell hybridomas with IL‐6‐treated Mm 1. The nitric oxide‐sensitivity of the cells corresponded to their 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide reducing activity and ATP consumption rate. These findings suggest that nitrogen oxides act as regulatory molecules in immune response in three ways: cytostasis, reduction of cell growth and suppression of antibody synthesis.

Chicken DT40 cells stably transfected with the rat P2X7 receptor ion channel: a system suitable for the study of purine receptor-mediated cell death

We have generated and characterised a clone of chicken DT40 lymphocytes stably transfected with the rat P2X(7) receptor (rP2X(7)). Successful transfection was confirmed by Western blotting. Under voltage clamp, P2X(7)-expressing cells responded to ATP and dibenzoyl-ATP (Bz-ATP) (a more potent P2X(7) receptor agonist) with a rapidly activating and sustained inward current. The EC(50) values for these agonists were 305 and 15 microM, respectively. Bz-ATP evoked Ca(2+) and Mn(2+) influx into transfected cells as determined by Fura-2 spectrofluorimetry. Responses to Bz-ATP were inhibited by pre-treatment of cells with oxidised ATP. Treatment of cells with Bz-ATP for up to 24hr produced time- and concentration-dependent cell death. This was associated with an increase in caspase-3-like activity, exposure of phosphatidylserine on the outside of cell membrane and DNA cleavage, indicating death by apoptosis. Pre-treatment with Z-VAD-fmk, a pan-caspase inhibitor, reduced the DNA fragmentation and phosphatidylserine externalisation, but did not affect overall rates of cell death at 24hr, implicating caspase-independent mechanisms. The properties of rP2X(7) receptors expressed in DT40 cells are similar to those described for other expression systems. Because DT40 cells lack functionally detectable endogenous P2 receptors and are highly amenable to genetic manipulation, stably transfected DT40 cells provide a novel and potentially useful model system in which to investigate the intracellular signal transduction pathways associated with P2X(7) receptor stimulation, in particular those involved in induction of cell death.

Changes in urinary ion excretion and related renal enzyme activities in fluoride-treated rats

Fluoride (NaF, 50 mg/kg po) administration to rats caused an increased urinary excretion of inorganic phosphate, calcium, magnesium, potassium, and sodium associated with polyuria. The renal enzyme activities of Na+ and K+-stimulated adenosine triphosphatase [(Na+ + K+)-ATPase], Mg2+ and Ca2+-stimulated adenosine triphosphatase [(Mg2+ + Ca2+)-ATPase], acid phosphatase, and alkaline phosphatase were decreased by single oral doses of fluoride. A decrease in renal (Na+ + K+)-ATPase activity was associated with an increase in urine volume and a decrease in serum sodium concentration.

Differential regulation of Na+,K+-ATPase and the Na+-coupled glucose transporter in hypertensive rat kidney

Several Na(+) transporters are functionally abnormal in the hypertensive rat. Here, we examined the effects of a high-salt load on renal Na(+),K(+)-ATPase and the sodium-coupled glucose transporter (SGLT1) in Dahl salt-resistant (DR) and salt-sensitive (DS) rats. The protein levels of Na(+),K(+)-ATPase and SGLT1 in the DS rat were the same as those in the DR rat, and were not affected by the high-salt load. In the DS rat, a high-salt load decreased Na(+),K(+)-ATPase activity, and this decrease coincided with a decrease in the apparent Mechaelis constant (K(m)) for ATP, but not with a change of maximum velocity (V(max)). On the contrary, a high-salt load increased SGLT1 activity in the DS rat, which coincided with an increase in the V(max) for alpha-methyl glucopyranoside. The protein level of phosphorylated tyrosine residues in Na(+),K(+)-ATPase was decreased by the high-salt load in the DS rat. The amount of phosphorylated serine was not affected by the high-salt load in DR rats, and could not be detected in DS rats. On the other hand, the amount of phosphorylated serine residues in SGLT1 was increased by the high-salt load. However, the phosphorylated tyrosine was the same for all samples. Therefore, we concluded that the high-salt load changes the protein kinase levels in DS rats, and that the regulation of Na(+),K(+)-ATPase and SGLT1 activity occurs via protein phosphorylation.