• The Korean Journal of Physiology
• /
• v.18 no.2
• /
• pp.163-169
• /
• 1984

The effect of vanadate on the optimum pH of Na-K-ATPase was investigated. The results were as follows: 1) The optimum PH of Na-K-ATPase was shifted from PH 7.4 to 6.8 at 10 mM K by $5{\times}10^{-6}M$ vanadate. 2) The ratio of Na-K-ATPase activity at pH 6.8 and 7.4 increased with increasing vanadate concentration. 3) Inspite of the presence of $5{\times}10^{-6}M$ vanadate Na-K-ATPase activity at pH 7.4 was higher than that at pH 6.8 below 50 mM $Na^+$, and the ratio of Na-K-ATPase activity at pH 7,4 and 6.8 was higher than that of the control. 4) Na-K-ATPase activity at pH 7.4 was higher than that at pH 6.8 below 7mM $K^+$. 5) Optimum pH of Na-K-ATPase activity was shifted from pH 7.4 to 6.8 by $10^{-5}M$ vanadate at 5 mM $K^+$. 6) $K^+$-pNPPase activity increased with lowering of pH, and the degree of inhibition of $K^+$-pNPPase activity by $10^{-7}$M vanadate was decreased with lowering of pH. These results suggest that vanadate shifts the optimum pH of Na-K-ATPase activity to more acidic PH than PH 7.4. This effect may not be caused by the decrease in the inhibitory potency of vanadate itself to Na-K-ATPase by the change of medium pH, but mainly by the alteration of Na-and K-binding site, which appears in the presence of vanadate only.

• Molecules and Cells
• /
• v.40 no.12
• /
• pp.925-934
• /
• 2017

The Cdc6 protein is essential for the initiation of chromosomal replication and functions as a licensing factor to maintain chromosome integrity. During the S and G2 phases of the cell cycle, Cdc6 has been found to inhibit the recruitment of pericentriolar material (PCM) proteins to the centrosome and to suppress centrosome over-duplication. In this report, we analyzed the correlation between these two functions of Cdc6 at the centrosome. Cdc6 depletion increased the population of cells showing centrosome over-duplication and premature centrosome separation; Cdc6 expression reversed these changes. Deletion and fusion experiments revealed that the 18 amino acid residues (197-214) of Cdc6, which were fused to the Cdc6-centrosomal localization signal, suppressed centrosome over-duplication and premature centrosome separation. Cdc6 mutant proteins that showed defective ATP binding or hydrolysis did not exhibit a significant difference in suppressing centrosome over-duplication, compared to the wild type protein. In contrast to the Cdc6-mediated inhibition of PCM protein recruitment to the centrosome, the independence of Cdc6 on its ATPase activity for suppressing centrosome over-duplication, along with the difference between the Cdc6 protein regions participating in the two functions, suggested that Cdc6 controls centrosome duplication in a manner independent of its recruitment of PCM proteins to the centrosome.

• Journal of Acupuncture Research
• /
• v.17 no.3
• /
• pp.208-218
• /
• 2000

This study was undertaken to determine if Salviae Radix (SR) exerts protective effect against oxidant-induced inhibition of phosphate uptake in renal proximal tubular cells. Membrane transport function and cell death were evaluated by measuring phosphate uptake and trypan blue exclusion, respectively, in opossum kidney (OK) cells, an established proximal tubular cell line. $H_2O_2$ was used as a model oxidant. $H_2O_2$ inhibited the phosphate uptake in a dose-dependent manner over the concentration range of 0.1-0.5 mM. Similar fashion was observed in cell death. However, the phosphate uptake was more vulnerable to $H_2O_2$ than cell death, suggesting that $H_2O_2$-induced inhibition of phosphate uptake is not totally attributed to cell death. Decreasedphosphate uptake was associated with ATP depletion and inhibition of $Na^+$-pump activity as determined by direct inhibition of $N^+-K^+$-ATPase activity. When cells were treated with $H_2O_2$ in the presence of 0.05% SR, the inhibition of phosphate uptake and cell death induced by $H_2O_2$ was significantly attenuated. SR restored ATP depletion and decreased $Na^+-K^+$-ATPase activity, and this is likely responsible for the protective effect of SR on decreased phosphate uptake. The protective effect of SR was similar to the $H_2O_2$ scavenger catalase. SR reacts directly with $H_2O_2$ to reduce the effective concentration of the oxidant. The iron chelator deferoxamine prevented the inhibition of phosphate uptake and cell death induced by $H_2O_2$, suggesting that $H_2O_2$-induced cell injury is resulted from an iron-dependent mechanism. These results indicate that SR exerts the protective effect against $H_2O_2$-induced inhibition of phosphate uptake by reacting directly with $H_2O_2$ like the $H_2O_2$scavenger enzyme catalase, in OK cells. However, the underlying mechanism remains to be explored.

• Applied Biological Chemistry
• /
• v.42 no.2
• /
• pp.116-122
• /
• 1999

In order to characterize the property of $Ca^{2+}$ transport in plant cell, microsomes were prepared from the roots of tomato and microsomal $^{45}Ca^{2+}$ uptake was measured. When 1 mM vanadate, a selective inhibitor of P-type ATPases, 50 mM $NO_3^-$, a specific inhibitor of vacuolar $H^{+}-ATPase$, and both of these inhibitors were treated, the microsomal $^{45}Ca^{2+}$ uptakes were inhibited by 20, 33 and 47%, respectively. The inhibitory effects of these two inhibitors were investigated by using a protonophore, gramicidin. When the chemical gradient of $H^{+}$ was relieved by gramicidin, the uptake was decreased by 30%, implying the presence of $Ca^{2+}/H^+$ antiporter in the microsomal membrane. In the $^{45}Ca^{2+}$ uptake experiment, the effect of gramicidin was independent of vanadate-induced inhibition. However, when the activity of vacuolar $H^{+}-ATPase$ was inhibited by $NO_3^-$, the effect of gramicidin was severely decreased. Meanwhile, thapsigargin, a specific antagonist of ER/SR-type $Ca^{2+}-ATPase$, inhibited the microsomal $^{45}Ca^{2+}$ uptake and the maximum inhibitory effect was obtained at $10\;{\mu}M$. The effect of thapsigargin was blocked by $NO_3^-$ and gramicidin, but not by vanadate. These results imply that vanadate directly inhibits the activity of $Ca^{2+}-ATPase$; however, $NO_3^-$ and thapsigargin block the activity of $Ca^{2+}/H^+$ antiporter by inhibiting the vacuolar $H^{+}-ATPase$. In conclusion, the microsomal $^{45}Ca^{2+}$ uptakes are mediated by two major enzymes, $Ca^{2+}-ATPase$ and $Ca^{2+}/H^+$ antiporter in tomato root tissue.

• Biomolecules & Therapeutics
• /
• v.23 no.1
• /
• pp.53-59
• /
• 2015

In this study, we investigated the inhibitory activities on gastritis and gastric ulcer using liriodendrin which is a constituent isolated from Kalopanax pictus. To elucidate its abilities to prevent gastric injury, we measured the quantity of prostaglandin $E_2$ ($PGE_2$) as the protective factor, and we assessed inhibition of activities related to excessive gastric acid be notorious for aggressive factor and inhibition of Helicobacter pylori (H. pylori) colonization known as a cause of chronic gastritis, gastric ulcer, and gastric cancer. Liriodendrin exhibited higher $PGE_2$ level than rebamipide used as a positive control group at the dose of $500{\mu}M$. It was also exhibited acid-neutralizing capacity (10.3%) and $H^+/K^+$-ATPase inhibition of 42.6% ($500{\mu}M$). In pylorus-ligated rats, liriodendrin showed lower volume of gastric juice ($4.38{\pm}2.14ml$), slightly higher pH ($1.53{\pm}0.41$), and smaller total acid output ($0.47{\pm}0.3mEq/4hrs$) than the control group. Furthermore liriodendrin inhibited colonization of H. pylori effectively. In vivo test, liriodendrin significantly inhibited both of HCl/EtOH-induced gastritis (46.9 %) and indomethacin-induced gastric ulcer (46.1%). From these results, we suggest that liriodendrin could be utilized for the treatment and/or protection of gastritis and gastric ulcer.

• The Korea Journal of Herbology
• /
• v.30 no.5
• /
• pp.15-21
• /
• 2015

Objectives : Methicillin-resistantStaphylococcus aureus(MRSA) is a human pathogen. New antibacterial agents are needed to treat MRSA-related infections. This study investigated the antibacterial activity of EtOH 70% extracts ofTonghyeonipal-dan(THD) which prescription is composed of oriental medicine against MRSA.Methods : The antibacterial activity of THD was evaluated against MRSA strains by using the Disc diffusion method, broth microdilution method, Checkerboard dilution test, and Time-kill test; its mechanism of action was investigated by bacteriolysis, detergent or ATPase inhibitors were used.Results : The minimum inhibitory concentration (MIC) of THD is 1,000~2,000 μg/mL against MRSA. In the checkerboard dilution test, fractional inhibitory concentration index (FICI) of THD in combination with antibiotics indicated synergy or partial synergism againstS. aureus. Furthermore, a time-kill assay showed that the growth of the tasted bacteria was considerably inhibited after 24 h of treatment with the combination of THD with selected antibiotics. For measurement of cell membrane permeability, THD 500 μg/mL along with concentration of Triton X-100 (TX) and Tris-(hydroxymethyl) aminomethane (TRIS) were used. In the other hand, N,N-dicyclohexylcarbodimide (DCCD) and Sodium azide (NaN3) were used as an inhibitor of ATPase. TX, TRIS, DCCD and NaN3 cooperation againstS. aureusshowed synergistic action.Conclusions : Accordingly, antimicrobial activity of THD was affected by cell membrane and inhibitor of ATPase were assessed. These results suggest that THD has antibacterial activity, and that THD extract offers great potential as a natural antibiotic against MRSA.

• The Korean Journal of Physiology
• /
• v.29 no.1
• /
• pp.69-80
• /
• 1995

Renal proximal tubular hypertrophy and hyperfunction are known to be early manifestations of experimental and human diabetes. As the hypertrophy and hyperfunction have been suggested to be central components in the progression to renal failure, an understanding of their underlying causes is potentially important for the development of therapy. A primary rabbit kidney proximal tubule cell culture system was utilized to evaluate the possibility that the renal proximal tubular hypertrophy and hyperfunction observed in vivo in diabetes mellitus, can be attributed to effects of elevated glucose levels on membrane transport systems. Primary cultures of rabbit proximal tubules, which achieved confluence at 10 days, exhibited brush-border characteristics typical of proximal tubular cells. Northern analysis indicated $2.2{\sim}2.3$ and 2.0 kb Na/glucose cotransporter RNA species appeared in fresh and cultured proximal tubule cells after confluence, repectively. The cultured cells showed reduced Na/glucose cotransporter activity compared to fresh proximal tubules. Primary cultured proximal tubule cells incubated in medium containing 20 mM glucose have reduced ${\alpha}-MG$ transport compared to cells grown in 5 mM glucose. In the proximal tubule cultures incubated in medium containing 5 mM or 20 mM glucose, phlorizin at 0.5 mM inhibited 0.5 mM ${\alpha}-MG$ uptake by 84.35% or 91.85%, respectively. The uptake of 0.5 mM ${\alpha}-MG$ was similarly inhibited by 0.1 mM ouabain (41.97% or 48.03% inhibition was observed, respectively). In addition, ${\alpha}-MG$ uptake was inhibited to a greater extent when $Na^{+}$ was omitted from the uptake buffer (81.86% or 86.73% inhibition was observed, respectively). In cell homogenates derived from the primary cells grown in 5 mM glucose medium, the specific activity of the Na/K-ATPase $(6.17{\pm}1.27\;{\mu}mole\;Pi/mg\;protein/hr)$ was 1.56 fold lower than the values in cell homogenates treated with 360 mg/dl D-glucose, 20 mM $(9.67{\pm}1.22\;{\mu}mole\;Pi/mg\;protein/hr)$. Total $Rb^{+}$ uptake occurred at a significantly higher rate (1.60 fold increase) in primary cultured rabbit kidney proximal tubule cell monolayers incubated in 20 mM glucose medium $(10.48{\pm}2.45\;nM/mg\;protein/min)$ as compared with parallel cultures in 5 mM glucose medium. $Rb^{+}$ uptake rate in 5 mM glucose medium was reduced by 28% when the cultures were incubated with 1 mM ouabain. The increase of the $Rb^{+}$ uptake by rabbit kidney proximal tubule cells in 20 mM glucose could be attributed primarily to an increase in the rate of ouabain-sensitive $Rb^{+}$ uptake $(5\;mM\;to\;20\;mM;\;4.68{\pm}0.85\;to\;8.38{\pm}1.37\;nM/mg\;protein/min)$. In conclusion, the activity of the renal proximal tubular Na,K-ATPase is elevated in high glucose concentration. In contrast, the activity of the Nafglucose cotransport system is inhibited.

• Proceedings of the Korean Society of Toxicology Conference
• /
• 2005.05a
• /
• pp.208-208
• /
• 2005

• Journal of Acupuncture Research
• /
• v.19 no.2
• /
• pp.211-220
• /
• 2002

Objective : This study was undertaken to determine whether Scutellaria baicalensis Georgi extract (SbG) exerts the protective effect against oxidant-induced alterations in organic cation transport in the renal proximal tubule. Methods : Organic cation transport was estimated by examining alterations in tetraethylammonium (TEA) uptake in rabbit renal cortical slices. The slices were treated with 0.2 mM tBHP for 60 min at $37^{\circ}C$. tBl-IP caused an inhibition in TEA uptake by renal cortical slices. Such an effect was accompanied by depressed Na+-K+-ATPase activity and ATP depletion. Result : SbG prevented tBHP-induced inhibition of TEA uptake in a dose-dependent manner at the concentration ranges of 0.05-0.1%. SbG also prevented H2O2-induced reduction in TEA uptake. tBHP-induced inhibition of Na+-K+-ATPase activity and ATP depletion were significantly prevented by 0.05% SbG. Oxidants increased LDH release, which was blocked by SbG. Oxidants caused a significant increase in lipid peroxidation and its effect was prevented by SbG. Conclusion : These results suggest that SbG prevents oxidant-induced alterations in organic cation transport in rabbit renal cortical slices. Such protective effects of SbG may be attributed to inhibition of peroxidation of membrane lipid.

• The Korean Journal of Ecology
• /
• v.20 no.2
• /
• pp.83-94
• /
• 1997

Stomatal closing by ozone and water stress could reduce further ozone injury by inhibition of ozone influx to the tissue. Direct effect of ozone on stomata can be explained from two aspects which are a stimulation of stomatal closing and an inhibition of stomatal opening. An increase of $Ca^{2+}$ influx into cytoplasm by ozone could stimulate potassium efflux ion channel and inhibits inward potassium ion channels. By this mechanism ozone could induce stomatal closing. On the other hand, ozone could inhibit stomatal opening by affecting the activity of $H^{+}$ dependent ATPase of the membrane in guard cells. This would inhibit proton efflux which precede stomatal opening. It is also possible that ozone could reduce the activity of photosynthesis in guard cells which lead to affect the production of osmotically active sugars and energy. Indirect effect of ozone to stomata is through the effect of $CO_2$ elevation as a result of damage of the photozynthetic machinery. This indirect effect is slower than the direct effect.