• 약학회지
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• 제30권3호
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• pp.149-156
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• 1986

Many experiments have showed that the sodium and potassium ion transporting system and the Na, $^+K^+$-ATPase activity of membrane fragments are inhibited by digitalis glycosides and that the pump may be associated with the pharmacological receptor for the drugs. The aim of our investigation is to elucidate the ouabain binding sites occupation in heart following infusion of ouabain to intact animals by the $^3H$-ouabain binding assay. Lethal dose and 26 percent of lethal dose of ouabain were infused to intact rabbit through ear vein. Microsomal fraction was fractionated from ouabain treated rabbit heart. $^3H$-ouabain binding to these fraction in vitro was studied by the Schwartz's method. $^3H$-ouabain binding to heart microsomal fraction was also studied following infusion of ginseng ethanol extract and caffeine to rabbits respectively. 1) The infusion of lethal dose ouabain (113$\mu\textrm{g}$/kg) inhibited the specific $^3H$-ouabain binding to rabbit heart microsomal fraction to the level of 60% (p<0.01) of control group and the infusion of 26% of lethal dose of ouabain led to the level of 79% (p<0.01) of the control group. 2) Time course of binding of 0.4$\mu{M}$ $^3H$-ouabain to microsomal fraction from rabbit heart following infusion of lethal and 26% of lethal dose of ouabain showed dose dependence at various incubation time. 3) Compared with control, only slight change of $K_d$ and $B_{max}$ was detected in in vitro $^3H$-ouabain binding after infusion of ginseng ethanol extract (300mg/kg) to rabbit. 4) In caffeine infusion group, $^3H$-ouabain binding yielded nearly the same results as control group.

• 약학회지
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• 제28권3호
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• pp.129-138
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• 1984

Specific [$^{3}H$] ouabain binding and $Ca^{2+}$ -uptake were measured to elucidate the role of high affinity [$^{3}H$] ouabain binding site in rat cardiac myocytes which contain 65% of rod cells. High affinity [$^{3}$H] ouabain binding site, which is about 3% of total pump sites, with apparent dissociation constant ($K_{D}$) of $1.1{\times}10^{-7}M$ and maximum binding site concentration (Bmax) of 1.2 pmol/mg protein ($1.754{\times}10^{5}cells$) were identified. At the concentration of $10^{-7}M$ to $10^{-4}M$, ouabain produced concentration dependent increase in $Ca^{2+}$-uptake of myocytes. The effect of ouabain on $Ca^{2+}$-uptake was not effected by membrane depolarization (elevated K+ in incubation medium) or verapamil. These results suggest that in rat ventricular myocytes the ouabain receptor complex to high affinity site may increase Na+ - $Ca^{2+}$ exchange across the sarcolemmal membrane by inhibition of Na+, K+ - ATPase.

• Biomolecules & Therapeutics
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• 제2권2호
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• pp.114-119
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• 1994

[$^3$H]Ouabain binding parameters ( $K_{D}$ and $B_{max}$) to control rat ventricular strips and Langendorff preparations which were not previously exposed to ouabain were compared with those to both preparations that had been first exposed to a complete ouabain dose range of dose-response curve (10$^{-8}$ to 10$^{4}$M). In rat ventricular strips and Langendorff perfused heart preparations, cumulative dose-response curves of ouabain revealed biphasic positive inotropic effects, a "low-dose" effect and a "high-dose" effect with E $d_{50}$ values of 0.5 $\mu$M and 35 $\mu$M ouabain, respectively. The "low-dose" effect in ventricular strip disappeared or was diminished significantly when the ouabain dose-response curve was repeated after the washout of the effects of the first dose-response curve, whereas there were no significant differences in the maximal "high-dose"effect in both exposures to oubain. However, both of the control and ouabain-preexposed Langendorff perfused hearts revealed the same low-dose effects. The $K_{D}$ value for [$^3$H] ouabain binding and the ouabain binding site concentration ( $B_{max}$) estimated by [$^3$H]ouabain displacement assay in control preparations were 230 nM and 2 pmol/mg protein, respectively. [$^3$H]Ouabain binding parameters were not changed by repeated exposure to high concentrations of ouabain. These results suggest that digitalis receptor desensitization in the rat ventricular strip may due to the change of post-receptor events induced by ouabain binding to a high affinity site ($\alpha$$_2$isoform).).).).).

• 약학회지
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• 제29권2호
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• pp.76-89
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• 1985

The effects of ginseng saponin on the activity, phosphorylation, [$^{3}$H] ouabain binding and light scattering (disruption) of purified $Na^{+}$ ,$K^{+}$ -ATPase isolated from the outer medulla of sheep kidney were compared to those of gypsophila saponin, sodium dodecylsulfate (SDS), and Triton X-100 on the same parameters. $Na^{+}$ , $K^{+}$ -ATPase activity, phosphorylation, and [$^{3}H$] ouabain binding were inhibited by ginseng saponin (triol>total>diol), SDS, or Triton X-100, but increased by gypsophila saponin. Low doses of ginseng saponin (3.mu.g saponin/.mu.g protein) decreased phosphorylation sites and ouabain binding site concentration (Bmax) without any change of turnover number and affinity for ouabain binding which were decreased by high dose of ginseng saponin (over 10.mu.g saponin/.mu.g protein), SDS or Triton X-100. On the other hand, gypsophila saponin increased the affinity without any change of Bmax for ouabain binding. Inhibition of $Na^{+}$ ,$K^{+}$ -ATPase activity by ginseng saponin and SDS or Triton X-100 appeared before and after decrease in light scattering, respectively. These data suggest that ginseng saponins (total, diol, triol saponin) inhibit $Na^{+}$ , $K^{+}$ -ATPase activity by specific direct and general detergent action at low and high concentrations, respectively, and this inhibitory action of ginseng sapornin to $Na^{+}$ , $K^{+}$ -ATPase is not general action of all saponins.

• 약학회지
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• 제40권6호
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• pp.734-738
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• 1996

The development of the alpha2 isoform of (Na,K)ATPase which is high affinity ouabain receptors was studied in the differentiating nonfusing muscle cell line BC3H-1. T he differentiation process of BC3H-1 cell line was confirmed by 2-dexy-D-[$^3$H] glucose uptake experiment and the quantity of the expression of ${\alpha}_2$ isoform was measured using a whole cell [$^3$H] ouabain-binding assay. Undifferentiated growing BC3H-1 cells, myoblasts, exhibited low levels of insulin-stimulated glucose uptake and [$^3$H] ouabain-binding sites. In contrast, differentiated BC3H-1 cells, myocytes, had a 5.6-fold increase in insulin-stimulated glucose uptake and 5-fold increase in [$^3$H] ouabain-binding sites. Scatchard analysis showed that myocytes developed more [$^3$H] ouabain-binding sites than myoblasts vath a dissociation constant (kd) of 6${\times}10^{-8}$M and capacity of 6.l${\times}10^{-5}$ sites/cell. Therefore. it seems that myoblasts express low levels of ${\alpha}_2$ subunit and probably the majority of ${\alpha}_1$ subunit, whereas myocytes express high levels of ${\alpha}_2$ isoform. The results indicate that the expression of ${\alpha}_2$ isoform is developmentally regulated during differentiation and that BC3H-1 culture system provides an excellent model for the study of differentiation and mechanism of (Na,K)ATPase action in muscle which requires electrical excitability.

• Archives of Pharmacal Research
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• 제19권5호
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• pp.381-385
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• 1996

Rat ventricles respond with a biphasic positive inotropic effect to ouabain, low-dose and high-dose effects but rat atria with only a monophasic high dose effect. In an effect to understand the difference in response to ouabain of two tissues between rat atria and ventricles the levels of the $a_{2}$ -isoform of the $Na^{+}$, $K^{+}$-ATPase which has higher affinity for ouabain than the $a_{1}$-iso-form were determined by a $[^{3}H]$ouabain binding assay. The yield of protein per gram wet weight was about 47 mg for atria and 100 mg for ventricles. The $K_{d}$ values of ouabain for the high-affinity ouabain binding site $(a_{2} -isoform)$ were nearly the same (230 nM) in the atria and ventricles. However, the numbers of the $a_{2}$-isoform $(B_{max})$ per mg protein were approximately half in the atria. When the binding data were expressed in unit per gram tissue wet weight, the numbers of $a_{2}$ -isoform in the atria was about 25% of that in the ventricles. THese results demonstrate that the $a_{2}$ -isoform of the $Na^{+}$, $K^{+}$-ATPase in the rat atria could be detected by $[^{3}H]$ouabain binding assay and the levels of this isoform are too low to show the low-dose effect of ouabain.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1994년도 춘계학술대회 and 제3회 신약개발 연구발표회
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• pp.301-301
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• 1994

〔$^3$H〕Ouabain binding parameters(K$\_$D/ and B$\_$max/,) in homogenates prepared fpom control rat ventricular strip and Langendorff preparations which were not previously exposed to ouabain were compared to those in homogenates from ventricular strip and Langendorff preparations that had been first exposed to a complete ouabain dose-response curve(10$\^$-7/M to 10$\^$-4/ M). In rat ventricular strips and Langendorff perfused rat heart preparations, cumulative dose-response cruves of ouabain revealed biphasic positive inotropic effects, a "low-dose" and a "high-dose" effect with ED$\_$50/ values of 0.5${\mu}$M and 35${\mu}$M ouabain, respectively- The "low-dose" effect in rat ventricular strips disappeared or was diminished significantly when the ouabain dose-response curve wag repeated after the washout of the effects of the first curve, whereas the maximal "high-dose" effect was identical in both exposures to oubain. However, there was no change in the "low-dose" effects in both sets of the Langendorff perfused hearts. The contractile activity of the pre-exposed strips did not indicate the presence of residual ouabain since their basal contractile force was decreased 10% compared to initial control. 〔$^3$H〕Ouabain binding parameters, K$\_$D/ and B$\_$max/, were not changed comparing homogenate of control ventricular strips with that of strips pre-exposed to ouabain. These results suggest that digitalis receptor desensitization in the rat ventricular strip may due to the change of post-receptor events induced by ouabain binding to a high affinity site(${\alpha}$$_2$ isoform).

• 대한약리학회지
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• 제27권1호
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• pp.33-43
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• 1991

고혈압백서(1-kidney, 1-clip-hypertensive rat)의 적혈구에서 노화 과정에 따른 Na, K-ATPase의 변동을 관찰하고저 노화적혈구를 분리한다음 세포막에서의 Na-pump 활성도 및 ouabain의 결합실험과 Rb의 세포내 유입실험을 시행하여 다음과 같은 결과를 얻었다. 1. 본 실험에 사용한 고혈압 백서의 혈압은 수축기 및 이완기 혈압이 165.5/119.0 mmHg로 유의하게 증가 하였다. 노화 적혈구의 평균용적(MCV)과 세포막 단백질 함량은 감소되고 혈색소치는 증가되었다. 2. 110 mM NaCl 및 10 mM KCI 존재하에서의 적혈구 세포막 Na, K-ATPase활성도는 대조군에 비해 고혈압군에서 억제 되었으며 양군 모두에서 노화에 의해 그활성도가 감소되었다. 3. 4 mM RbCl존재하에서 Ouabain에 의해 억제되는 Rb의 유입은 정상 및 고혈압군의 노화적혈구에서 약간 감소되었으며 고혈압군의 young erythrocyte에서는 오히려 약간 증가 되었다. 4. 16 mM RbCl 존재하에서 Ouabain에 의해 억제되는 Rb의 유입은 양군의 노화 적혈구에 서는 각군의 young erythrocyte에 비해 약 30-50% 감소되었으며, 고혈압군에서는 특히 young erythrocyte에서 정상군의 young erythrocyte에 비해 유의하게 감소되었다. 5. $0.13{\times}10^{-6}M$과 $1{\times}10^{-6}M$에서의 ouabain binding은 정상군의 노화적혈구에서는 young erythrocyte에 비해 약간 감소되었으나 고혈압군의 노화적혈구에서는 유의하게 감소되었다. 6. $6{\times}10^-6}M$과 $64{\times}10^-6M$ 에서의 ouabain binding은 양군의 노화 적혈구에서는 약간 감소되었지만 유의성은 없었으며 고혈압군의 young erythrocyte 및 노화적혈구에서는 정상군의 young erythrocyte및 노화 적혈구에 비해 유의하게 감소되었다. 이상의 결과로부터 (1) 고혈압쥐의 young erythrocyte에서는 low affinity의 Na-pump수의 감소및 molecular activity의 증가, (2) 정상쥐의 노화 적혈구에서는 molecular activity의 저하, (3) 고혈압쥐의 노화적혈구에서는 molecular activity의 저하 및 high affinity와 low affinity의 Na-pump수의 저하등에 의하여 Na-pump의 기능이 변동될 수 있을 것으로 추측된다.

• Archives of Pharmacal Research
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• 제16권4호
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• pp.305-311
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• 1993

$Na^+,K^+$-ATPase activity, $Na^+$-dependent phosphorylation, and $[^3H]$ ouabain binding in sarcolemma prepared from 4 week old spontaneously hypertensive rat(SHR) ventricles were compared to the same parameters in sarcolemma from age matched nomotensive Wister-Kyoto (WKY) rat ventricles to examine whether the reduced myocardial $Na^+$-pump activity in SHR is an inherited enzymatic defect or a second phenomenon due to sustained hypertension. The total body weights, ventricular weights, and blood pressures were the same for SHR and WKY. No significant differences in sarcolemmal protein content and protein recovery were noted between the two groups. Sarcolemma isolated from SHR ventricles showed significantly less $Na^+,K^+$-ATPase activity ande number of phosphorylation sites when compared to sarcolemma from the WKY ventricles. Equilibrium binding of $[^3H]$ouabain and the tumover number of myocardial $Na^+,K^+$-ATPase, however, were the same for both groups. These reults indicate that the low affinity $(\alpha,\;or\;\alpha^1)\;\alpha$ isoform is the same in ventricles of SHR and WKY. The reduced amount of isoform of the $Na^+,K^+$-ATPase inprehypetensive SHR ventricles may play some role in the development of hypertension.

• Archives of Pharmacal Research
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• 제9권1호
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• pp.29-38
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• 1986

The effects of ginseng saponins on the sarcolemmal $Na^{+}$, $K^{+}$-ATPase were compared to gypsophila saponin, sodium dodecylsulfate (SDS), and Triton X-100 to elucidate whether the effects are due to the membrane distruption, using a highly enriched preparation of cardiac sarcolemma prepared from dog ventricular myocardium. About 26% and 29% of vesicles in the preparation, enriched in ouabain-sensitive $Na^{+}$, $K^{+}$-ATP ase, $\beta$-adrenergic and muscarinic receptors are rightside-out and inside-out orientation, respectively. Ginseng saponins (triol>total> diol) inhibited $Na^{+}$, $K^{+}$-ATP ase activity, $Na^{+}$, $K^{+}$-ATPase activity and [$^{3}$H]ouabain binding of sarcolemmal vesicles. However, gypsophila saponin, SDS (0.4$\mu$g/$\mu$g protein) and Triton X-100 (0.6 $\mu$g/$\mu$g protein) caused about 1.35 and 1.40-fold increase in $Na^{+}$, $K^{+}$-ATPase activity and [$^{3}$H] oubain binding, respectively. Especially, the activating effect of gypsophila saponin on membrane Na+, K+ ATPase was detected at gypsophila saponin to sarcolemmal protein ratios as high as 100. Low dose of ginseng saponin (3$\mu$g/$\mu$g protein) decreased the phosphorylation sites and the concentration of ouabain binding sites (Bmax) without affecting the turnover number and affinity for ouabain binding, while gypsophila saponin, SDS(0.4 ug/ug protein), ahd Triton X-100 (0.6$\mu$g/$\mu$g protein) increased the Bmax. The results suggest that ginseng saponins cause a decrease in the number of active sites by interacting directly with $Na^{+}$, $K^{+}$-ATPase before disruption of membrane barriers of sarcolemmal vesicles.