• 약학회지
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• 제22권3호
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• pp.120-127
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• 1978

We have studied the effect of ouabain, tool ginseng saponin, panax saponin C (protopanaxatriol derivative) and ginsenoside $Rb_{1}$ (protopanaxadiol derivative) on $Na^+,K^+$-ATPase and $Mg^{++}$-ATPase activities were determined by the method of Robinson and ATPase activities were determined by the method of King. The $Na^+,K^+$-ATPase activities were inhibitied by 90.1% and 51.1% respectively at the concentration of $10^{-3}M$ and $10^{-4}M$ ouabain. The results are consistent with those of Robinson. The $Na^+,K^+$-ATPase activities were increased by 14.3% and 10.0% respectively at the concentration of $10^{-4}$g/ml and $10^{-5}$g/ml total ginseng saponin. Panax saponin C obtained by the method of Han and ginsenoside $Rb_{1}$ obtained by the method of Shibata were used. The $Na^+,K^+$-ATPase activities were increased in the presence of panax saponin C and the increased activity with panax saponin C was greater than that with total ginseng saponin. On the other hand ginsenoside $Rb_{1}$ showed an inhibitory effect on $Na^+,K^+$-ATPase. Total ginseng saponin, panax saponin C and ginsenoside $Rb_{1}$ had no effect on $Mg^{++}$-ATPase. Therefore, it may be concluded that total ginseng saponin has dual effects on microsomal $Na^+,K^+$-ATPase, that is, panax saponin C exhibits stimulatory action, whereas ginsenoside $Rb_{1}$ shows inhibitory action.

• 대한약리학회지
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• 제13권2호
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• pp.1-9
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• 1977

최근 강심작용 약물로 보고된 부자 부타놀 분획의 가토 심방근에서의 자극빈도에 따른 강심효과를 분석한 결과는 다음과 같다. 1) 가토 심방근의 수축력은 자극빈도에 따라 polyphasic한 수축양상을 보였으며 rested-state contraction은 $120{\sim}400sec$의 interval에서 나타났으며 이후 10sec의 간격까지는 급격한 수축력의 감소를 보이는, 축적NIEA가 PIEA의 양을 능가하는 양상을 보였으며 이 이하의 자극 빈도에서는 수축력의 증가를 보였고 대부분 0.25 sec이하의 빈도의 자극으로는 수축력의 감소를 보였다. 2) Ouabain은 저 농도에서는 interval-strength curve에 대하여 rested-state contraction의 증가와 전 자극빈도에서 비슷한 수축력의 증가를 나타내었으며, 고농도에서는 rested-state contraction의 증가와 축적 NIEA의 소멸 속도를 증가시켰으나 이는 rested-state contraction을 나타내는 시간의 단축에 따른 이차적 효과로 판정하였다. 또한 ouabain 자극빈도에 따른 강심 효과의 양상은 세포외 calcium 농도를 증가시킨 때와 유사한 양상이었다. 3) Norepinerhrine은, rested-state contraction에는 거의 영향을 주지 않았으며, 고 빈도에서 더욱 현저한 강심 작용을 나타내었고 축적 PIEA의 양의 증가와 매 beat 당 산출하는 PIEA의 양의 증가를 나타내었다. 4) 부자 부타놀 분획은 norepinephrine과 유사하고 빈도에서 수축력의 증가가 현저 하였으며, 수축력의 증가는 용량 반응 상관을 보였으며, PIEA 축적량 및 매 beat 당 산출하는 PIEA를 증가 시켰다. 따라서 고빈도에서의 강력한 수축력의 증가는 PIEA에 대한 효과에 의할 것으로 사료하였다.

• 대한핵의학회지
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• 제32권2호
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• pp.121-128
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• 1998

목적 : 생물학적 성질이 potassium과 유사하고 핵의학분야에서 널리 이용되고 있으며 쉽게 구할 수 있는 T1-201을 이용하여 $Na^+-K^+$ ATPase의 활성도를 측정할 수 있는지를 알아보고자, 배양한 백서 대동맥평활근세포와 사람의 제대동맥 평황근세포에서 $Na^+-K^+$ ATPase의 활성도를 측정하곤 기존의 Rb-86으로 측정한 방법과 비교하였다. 또한 T1-201로 측정한 활성도에 대한 포도당, 인슐린 및 PMA의 영향을 알아보고자 하였다. 대상 및 방법: Sprague-Dawley 백서의 흉부대동맥과 인체태반의 제대동맥에서 얻은 평활근세포를 배양하여 사용하였고. ouabain첨가로 억제되는 Rb-86과 T1-201의 섭취율을 $Na^+-K^+$ ATPase에 의한 섭취율로 계산하여 활성도로 간주하였다. 결과: 배양된 백서대동맥 평활근세포에서 $Na^+-K^+$ ATPase 활성도는 고포도당배양액(22 mM) 상태에서 생리적 농도의 저포도당배양액(5 mM) 상태에 비하여 평균 28%의 저하를 보였으며, 인슐린 100 nM을 첨가하였을 때는 50%의 증가를 보였다. PKC효소를 활성화시키는 PMA는 20%의 증가를 나타내었다. 인체제대동맥의 평활근세포에서도 유사한 변화를 보였다. T1-201로 측정한 $Na^+-K^+$ ATPase의 활성도치의 변화는 Rb-86을 이용한 측정에서도 동일하게 나타났다. 결론: T1-201을 이용하여 측정한 $Na^+-K^+$ ATPase 황성도는 Rb-86을 이용한 측정치와 유사하여, T1-201은 세포막 $Na^+-K^+$ ATPase 활성도의 측정에 사용할 수 있으리라 판단된다. 인슐린과 PKC효소 자극제는 $Na^+-K^+$ ATPase 활성도를 증가시키고, 고농도의 포도당은 활성도를 감소시키는 것으로 사료된다.

• The Korean Journal of Physiology
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• 제17권2호
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• pp.125-133
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• 1983

Red cell glycolytic intermediates, metabolites and metabolic ratios were studied. Glycolytic intermediates were measured in neutralized perchloric acid extracts of red cell suspensions after 3 hr incubation at $37^{\circ}C$ in the presence and absence of saponin. Adenosine triphosphate(ATP), adenosine diphosphate(ADP), pyruvate and lactate were measured by enzymatic procedures involving stoichiometric oxidation or reduction of a pyridine nucleotide. Glucose was determined using glucose oxidase after zinc hydroxide extraction. The redox state was calculated from the lactate dehydrogenase equilibrium. Adenosine triphosphatase activity(ATPase) was measured by determining the amount of phosphate released from ATP by washed erythrocyte membranes(ghost) during 20 min. incubation. Both total hydrolysis and the amount of hydrolysis that occured in the presence of ouabain were measured. The second measurement yields Mg-ATPase and represents nonspecific ATPase activity of the membranes. The difference between total and Mg-ATPase activity can be attributed to Na-K-ATPase. For the measurement of sodium fluxes, human erythrocytes were preincubated in $^{22}Na$ for 3 hr at $37^{\circ}C$, washed and suspended in a tracer-free medium. The amount of $^{22}Na$ transported out of cells at any time was determined by analysis of supernatant samples taken at various time after addition of the labeled cells to isotope-free medium. The cells and medium were separated and the radioactivity appearing in the medium was measured. From the total radioactivity in the suspension and the radioactivity appearing in the medium at known time, the rate constant for sodium release was computed. The results are summarized as follows: 1) ATP and ATP/ADP were found to increase at every concentration of saponin tested whereas ADP declined at every cone. of saponin. The increase in pyruvate and lactate were observed at every cone, of saponin and thus $NAD^+/NADH$ computed from pyruvate/lactate also increased. Glucose utilization was stimulated by saponin. 2) $Na^+-K^+-ATPase$ activities showed a biphasic response to saponin, first increasing in lower concentration and then decreasing in higher concentration of saponin. 3) The efflux of sodium was significantly increased by saponin in the range of 5 to 10 mg%. The stimulatory effect of saponin on the rate constants for active(ouabain-sensitive) sodium efflux was inhibited by addition of ouabain.

• The Korean Journal of Physiology
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• 제29권1호
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• pp.69-80
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• 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.

• The Korean Journal of Physiology
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• 제23권2호
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• pp.351-361
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• 1989

The contraction of renal arterial strip by no.epineph.me (NE) or 40 mM $K^+$ were Significantly attenuated after histamine $(10^{-5}\;M)-induced$ contraction. The mechanisms of this phenomenon were investigated in the helical strips of isolated renal artery with the measurement of isometric tension. The arterial strip was immersed in the tris-buffered Tyrode's solution which was equilibrated with 100% $O_2\;at\;35^{\circ}C$. The contraction was induced by NE or 40 mM $K^+$ during the recovery from the histamine-induced contraction which lasted for 15 minutes. The contraction by NE was also attenuated in the $Ca^{2+}-free$ Tyrode's solution and the increase of contraction by addition of 2 mM $Ca^{2+}$ was attenuated as well. This attenuation phenomenon was not observed in the presence of low concentration $(3{\times}10^{-7}\;M)$ of histamine. This attenuation was not affected by destruction of endothelium, pretreatment with papaverine or propranolol. This attenuation was partially inhibited by pretreatment of ouabain or in low $K^+(0.5 mM)$ Tyrode's solution. But the attenuation in the $Ca^{2+}-free$ Tyrode's solution was not inhibited. Furthermore this attenuation was completely blocked by pretreatment of djphenhydramine $(H_1-receptor blocker)$ and potentiated by pretreatment of cimetidine $(H_2-receptor\;blocker)$. This attenuation Phenomenon was disappeared after recovery of 1 hour. From the above results, it is suggested that the attenuation phenomenon may be resulted partially from the activation of $Na^+-K^+$ exchange pump and partially from the depletion of intracellular $Ca^{2+}$ pool after the histamine-induced contraction mediated through $H_1-receptor$ function.

• The Korean Journal of Physiology
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• 제30권2호
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• pp.197-208
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• 1996

Endosomes lower their internal pH by an ATP-driven proton pump, which is critical to dissociation of many receptor-ligand complexes, the first step in the intracellular sorting of internalized receptors and ligands. Endosomes are known to exhibit n great range of pH values that can vary between 5.0 and 7.0 within a single cell although the factors that regulate endosomal pH remain uncertain. To evaluate the morphological and topological differences of endosomes in the different stages, confocal microscopy was used. The early endosomes labeled with fluorescein isothiocyanate-dextran for 10 min at $37^{\circ}C$ were identifiable at the peripheral and tubule-vesicular endosome compartment. In contrast, the late endosomes formed by 10 min pulse and 20 min trace were located deeper in the cytoplasm and showed more vesicular features than early endosomes. For the purpose of determining whether ATP-dependent acidification was heterogeneous and whether the differences in acidification were attributed to differences in the activity of $Na^{+}-K^{+}$-ATPase and/or $Cl^{-}$ channel, endocytic compartments were fractionated into subpopulation using percoll gradient and measured ATP-dependent acidification. While all fractions exhibited ATP-dependent acidification activity, both the initial rate of acidification and extent of proton translocation were lower in early endosomes and gradually increased in late endosomes. Phosphorylation by PKA and ATP enhanced ATP-dependent acidification in both early and late endosomes, hut there was no difference in the degree of enhancement by phosphorylation between two subpopulations. When ATP-dependent acidification was determined in the presence or absence of vanadate ($Na_{3}VO_{4}$) or ouabain, only early endosomes exhibited the vanadate or ouabain dependent stimulation of acidification activity, suggesting the inhibition of $Na^{+}-K^{+}$-ATPase. Therefore, it seems probable that the inhibition of early endosome acidification by $Na^{+}-K^{+}$-ATPase observed in vitro at least in part plays a physiological role in controlling the acidification of early endosomes in vivo.

• 대한약리학회지
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• 제26권2호
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• pp.113-120
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• 1990

전기장 자극으로 수축을 유발한 흰쥐 좌심방에서, 자극 빈도 변경에 따른 수축 운동의 변동에 미치는, 여러 경로를 통한 수축 유발 calcium의 영향을 검색하므로, 각 경로가 심근 수축에 미치는 영향을 추구하였다. 흰쥐 좌심방은 자극 빈도를 급격하게 낮추므로, 특징적인 삼단계 변동을 나타내었다. 즉, 처음의 급격한 수축 장력증가와, 두번째 일시적인 빠른 장력감소, 이어서 세번째로 수축장력의 유지단계로 나타났으며, 이때 수축장력은 고빈도 자극시의 2배 정도가 되었다. Caffeine처치는 이와같은 자극빈도 하강에 따른 수축 장력의 증가를 현저하게 억압하였다. Verapamil은 고빈도 자극시 수축 운동을 완전히 소실시켰으나, 저빈도 자극으로 변경시에는 verapamil 존재하에서도 수축 운동이 소생되었다. 한편 ouabain처치나 영양액내 sodium 배제시에는 저빈도 자극으로 나타나는 특징적인 두번째 단계의 변동이 소실되었다. 이러한 결과로 보아, 심근막의 calcium통로는 세포내 유지에 필수불가결한 경로이며, 심근 수축 유발 calcium의 주된 기원은 근 소포체로 부터 유리되는 것으로 믿어진다. 또한 sodium-calcium교환은 세포내 sodium농도의 변동에 따라 수축 유발 calcium양 형성에 조절 인자로서의 기능을 갖는 것으로 추측된다.

• 대한치과교정학회지
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• 제19권1호
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• pp.61-75
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• 1989

Recently, indirect evidences suggest that Na-Ca exchange mechanism is involved in bone resorption. To study this suggestion, effects of several drugs which increase the intracellular sodium concentration by different mechanisms on the PTH-induced bone resorption were analysed employing organ culture. Ulnae and radii were removed from 19-day fetal rats, prelabelled by subcutaneous injection of $200{\mu}\;Ci^{45}CaC1_2$ on the 17th day of gestation, and then explanted on the membrane filters in organ culture dishes. For studying the effects of amiloride, ouabain, monensin, and veratridine on the PTH-induced bone resorption, control group was cultured in BGJb media containing PTH (0.4U/ml) while experimental group was cultured in BGJb media containing PTH and drugs. The effects of drugs on the PTH-induced bone resorption were observed by the ratios of $\%-release$ of $^{45}Ca$ between paired control and experimental groups. The results were as follows: 1. $^{45}Ca$ release was significantly increased by PTH (0.4U/ml) at 48 and 72 hours of culture. 2. Amiloride, at concentration of $500{\mu}M$, significantly inhibited the PTH-induced bone resorption after 48 and 72 hours of culture. 3. Ouabain, at concentration of 0.1 mM, presented significant inhibition of PTH-induced bone resorption after 48 and 72 hours of culture, and at 0.5mM and 1mM, presented significant inhibition of PTH-induced bone resorption after 72 hours of culture. 4. Monensin, at concentration of 500nM, significantly inhibited PTH-induced bone resorption after 72 hours of culture. 5. Veratridine, at concentration of 0.5mM, presented significant inhibition of PTH-induced bone resorption after 48 and 72 hours of culture, and at 1mM, presented significant inhibition of PTH-induced bone resorption after 72 hours of culture. Taken altogether, these results suggest that Na-Ca exchange mechanism play a role in PTH-induced bone resolution.

• 대한약리학회지
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• 제12권1호
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• pp.7-14
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• 1976

Aconiti tuber butanol fraction shows positive inotropic effect on the isolated atrium of rabbit heart. To investigate the mechanism, the effect on microsomal ATPase activity of rabbit heart is observed. The microsomal fraction which contains the $Na^+$- and $K^+$-activated ATPase in the presence of $Mg^{++}$ is isolated from the left ventricle of rabbit heart. The microsomal ATPase activity is maximally stimulated at $Na^+$ and $K^+$ concentration of 100 mM and 10 mM respectively. Microsomal $Na^+-K^+$-activated ATPase is inhibited by ouabain and Aconiti tuber butanol fraction. Ouabain and Aconiti tuber butanol fraction depress $Na^+$-stimulation on microsomal ATPase activity, and the inhibitory effects are not completely reversed at $Na^+$ concentration of 300 mM. Also, $K^+$-stimulation on microsomal ATPase activity is inhibited by ouabin and Aconiti tuber butanol fraction and the inhibitions are not compeletely reversed at $K^+$ concentration of 30 mM. It is, therefore, suggested that the inhibitory effect of Aconiti tuber butanol fraction on the microsomal ATPase activity may contribute to leading to the positive inotropic effect.