• The Korean Journal of Physiology
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• v.30 no.1
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• pp.63-76
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• 1996

The aim of present study was to characterize phosphate uptake and to investigate the mechanism for the insulin and insulin-like growth factor(IGF) stimulation of phosphate uptake in primary cultured rabbit renal proximal tubule cells. Results were as follows : 1. The primary cultured proximal tubule cells had accumulated $6.68{\pm}0.70$ nmole phosphate/mg protein in the presence of 140 mM NaCl and $2.07{\pm}0.17$ nmole phosphate/mg protein in the presence of 140 mM KCl during a 60 minute uptake period. Raising the concentration of extracellular phosphate to 100 mM$(48.33{\pm}1.76\;pmole/mg\;protein/min)$ induced decrease in phosphate uptake compared with that in control cells maintained in 1 mM phosphate$(190.66{\pm}13.01\;pmole/mg\;protein/min)$. Optimal phosphate uptake was observed at pH 6.5 in the presence of 140 mM NaCl. Phosphate uptake at pH 7.2 and pH 7.9 decreased to $83.06{\pm}5.75%\;and\;74.61{\pm}3.29%$ of that of pH 6.5, respectively. 2. Phosphate uptake was inhibited by iodoacetic acid(IAA) or valinomycin treatment $(62.41{\pm}4.40%\;and\;12.80{\pm}1.64%\;of\;that\;of\;control,\;respectively)$. When IAA and valinomycin were added together, phosphate uptake was inhibited to $8.04{\pm}0.61%$ of that of control. Phosphate uptake by the primary proximal tubule cells was significantly reduced by ouabain treatment$(80.27{\pm}6.96%\;of\;that\;of\;control)$. Inhibition of protein and/or RNA synthesis by either cycloheximide or actinomycin D markedly attenuated phosphate uptake. 3. Extracellular CAMP and phorbol 12-myristate 13 acetate(PMA) decreased phosphate uptake in a dose-dependent manner in all experimental conditions. Treatment of cells with pertussis toxin or cholera toxin inhibited phosphate uptake. cAMP concentration between $10^{-6}\;M\;and\;10^{-4}\;M$ significantly inhibited phosphate uptake. Phosphate uptake was blocked to about 25% of that of control at 100 ng/ml PMA. 3-Isobutyl-1-methyl-xanthine(IBMX) inhibited phosphate uptake. However, in the presence of IBMX, the inhibitory effect of exogenous cAMP was not significantly potentiated. Forskolin decreased phosphate transport. Acetylsalicylic acid did not inhibit phosphate uptake. The 1,2-dioctanoyl-sn-glycorol(DAG) and 1-oleoyl-2-acetyl-sn- glycerol(OAG) showed a inhibitory effect. However, staurosporine had no effect on phosphate uptake. When PMA and staurosporine were treated together, inhibition of phosphate uptake was not observed. In conclusion, phosphate uptake is stimulated by high sodium and low phosphate and pH 6.5 in the culture medium. Membrane potential and intracellular energy levels are also an important factor fer phosphate transport. Insulin and IGF-I stimulate phosphate uptake through a mechanisms that involve do novo protein and/or RNA synthesis and decrease of intracellular cAMP level. Also protein kinase C(PKC) is may play a regulatory role in transducing the insulin and IGF-I signal for phosphate transport in primary cultured proximal tubule cells.

• The Korean Journal of Physiology and Pharmacology
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• v.16 no.6
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• pp.393-398
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• 2012

Mast cells are involved in allergic responses, protection against pathogens and autoimmune diseases. Dexamethasone (Dex) and other glucocorticoids suppress $Fc{\varepsilon}RI$-mediated release of inflammatory mediators from mast cells. The inhibition mechanisms were mainly investigated on the downstream signaling of Fc receptor activations. Here, we addressed the effects of Dex on Fc receptor expressions in rat mast cell line RBL-2H3. We measured mRNA levels of Fc receptors by real-time PCR. As expected, Dex decreased the mRNA levels of activating Fc receptor for IgE ($Fc{\varepsilon}R$) I and increased the mRNA levels of the inhibitory Fc receptor for IgG $Fc{\gamma}RIIb$. Interestingly, Dex stimulated transcriptions of other activating receptors such as Fc receptors for IgG ($Fc{\gamma}R$) I and $Fc{\gamma}RIII$. To investigate the mechanisms underlying transcriptional regulation, we employed a transcription inhibitor actinomycin D and a translation inhibitor cycloheximide. The inhibition of protein synthesis without Dex treatment enhanced $Fc{\gamma}RI$ and $Fc{\gamma}RIII$ mRNA levels potently, while $Fc{\varepsilon}RI$ and $Fc{\gamma}RIIb$ were minimally affected. Next, we examined expressions of the Fc receptors on cell surfaces by the flow cytometric method. Only $Fc{\gamma}RIIb$ protein expression was significantly enhanced by Dex treatment, while $Fc{\gamma}RI$, $Fc{\gamma}RIII$ and $Fc{\varepsilon}RI$ expression levels were marginally changed. Our data showed, for the first time, that Dex regulates Fc receptor expressions resulting in augmentation of the inhibitory receptor $Fc{\gamma}RIIb$.

• Journal of Life Science
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• v.13 no.4
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• pp.441-447
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• 2003

To examine the role of protein kinase C (PKC) in regulation of interleukin-1 beta (IL-1$\beta$)-induced iNOS expression, IL-1$\beta$-induced nitrite production was observed in cultured vascular smooth muscle (VSM) cells pretreated with phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-butyrate (PDB) as PKC activator; 4$\alpha$-phorbol-didecanoate (PDD) as PKC non-activator. Nitrite production induced by IL-1$\beta$ was increased by the presence of increasing concentration of PMA ranging from 2 to 200 nM. However, in VSM cells pretreated with PMA and PDB, IL-1$\beta$-induced $NO_2$ production was decreased in proportion to the duration of pretreatment, and most significantly decreased in pretreatment time of 24 hours. Using RT-PCR method, the expression of iNOS mRNA induced by IL-1$\beta$ was decreased in VSM cells pretreated with PMA 200 nM for 24 hours. These results suggest that decrease in IL-I$\beta$-induced nitrite production by the pretreatment of PMA result from inhibition of iNOS expression and the inhibition related to PMA-induced PKC down-regulation.

• Radiation Oncology Journal
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• v.15 no.3
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• pp.175-186
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• 1997

Purpose : To evaluate the qualitative immunologic changes by ionizing radiation. we studied the altered capacities of the macrophages and lymphocytes to produce cytokines in conjunction with resistance to Listeria monocytegenes (LM) infection in mice Materials and Methods : BALB/c mice and Listeria monocytogenes were used. The mice were infected intraperitoneally with $10^5LM$ at 1 day after irradiation (300cGy) and sacrificed at 1, 3, 5 days after infection, and then the numbers of viable LM per spleen in the irradiated and control group were counted. Tumor necrosis factor-alpha ($TNF-\alpha$), interferon-gamma ($IFN-\gamma$). interleukin-2 (IL-2), and nitric oxide (NO) were assessed after irradiation. Results : Under gamma-ray irradiation with a dose range of 100-850cGy, the number of total splenocytes decreased markedly in a dose-dependent manner, while peritoneal macrophages did so slightly Cultured peritoneal macrophages produced more $TNF-\alpha$ in the presence of lipopolysaccharide (LPS) during the 24 hours after in vitro irradiation, but their capacity of $TNF-\alpha$ Production showed a decreased tendency at 5 days after in vivo total body irradiation. With 100cGy and 300cGy irradiation, cultured peritoneal macrophages produced more NO in the presence of LPS during the 24 hours after in vitro irradiation than without irradiation. Activated splenocytes from irradiated mice (300cGy) exhibited a decreased capacity to Produce IL-2 and $IFN-\gamma$ with Concavalin-A stimulation at 3 days after irradiation. When BALB/c mice were irradiated to the total body with a dose of 300cGy, they showed enhanced resistance during early innate phase, but a significant inhibition of resistance to LM was found in the late innate and acquired T-cell dependent phases. Conclusion : These results su99es1 that increased early innate and decreased late innate and acquired immunity to LM infection by ionizing radiation (300cGy) may be related to the biphasic altered capacity of the macrophages to produce $TNF-\alpha$ and the decreased capacities of the lymphocytes to produce IL-2 and $IFN-\gamma$ in addition to a marked decrease in the total number of cells.

• Tuberculosis and Respiratory Diseases
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• v.42 no.4
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• pp.522-534
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• 1995

Background: In the pathogenesis of acute lung injury induced by lipopolysaccharide(LPS), oxygen radiclls are known to be involved in one part. Superoxide dismutase(SOD) protects oxygen radical-induced tissue damage by dismutating superoxide to hydrogen peroxide. In eukaryotic cells, two forms of SOD exist intracellularly as a cytosolic, dimeric copper/zinc-containing SOD(CuZnSOD) and a mitochondrial, tetrameric manganese-containing SOD(MnSOD). But there has been little information about SOD gene expression and its regulation in pulmonary alveolar macrophages(PAMs). The objective of this study is to evaluate the SOD gene expression induced by LPS and its regulation in PAMs of rat. Method: In Sprague-Dawley rats, PAMs obtained by broncholaveolar lavage were purified by adherence to plastic plate. To study the effect of LPS on the SOD gene expression of PAMs, they were stimulated with different doses of LPS($0.01{\mu}g/ml{\sim}10{\mu}g/ml$) and for different intervals(0, 2, 4, 8, 24hrs). Also for evaluating the level of SOD gene regulation actinomycin D(AD) or cycloheximide(CHX) were added respectively. To assess whether LPS altered SOD mRNA stability, the rate of mRNA decay was determined in control group and LPS-treated group. Total cellular RNA extraction by guanidinium thiocyanate/phenolfchlorofonn method and Northern blot analysis by using a $^{32}P$-labelled rat MnSOD and CuZnSOD cDNAs were performed. Results: The expression of mRNA in MnSOD increased dose-dependently, but not in CuZnSOD. MnSOD mRNA expression peaked at 8 hours after LPS treatment. Upregulation of MnSOD mRNA expression induced by LPS was suppressed by adding AD or CHX respectively. MnSOD mRNA stability was not altered by LPS. Conclusion: These findings show that PAMs of rat could be an important source of SOD in response to LPS, and suggest that their MnSOD mRNA expression may be regulated transcriptionally and require de novo protein synthesis without affecting mRNA stability.