• Archives of Pharmacal Research
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• 제21권3호
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• pp.315-319
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• 1998

Gastric smooth muscle of cats was used to investigate the involvement of protein kinase in vanadate-induced contraction. Vanadate caused a contraction of cat gastric smooth muscle in a dose-dependent manner. Vanadate-induced contraction was totally inhibited by 2 mM EGTA and 1.5 mM $LACI_3$ and significantly inhibited by $10\mu$M verapamil and $1\mu$M nifedipine, suggesting that vanadate-induced contraction is dependent on the extracellular $Ca^{2+}$ concentration, and the influx of extracellular $Ca^{2+}$ was mediated through voltage-dependent $Ca^{2+}$ channel. Both protein kinase C inhibitor and tyrosine kinase inhibitor significantly inhibited the vanadate-induced contraction and the combined inhibitory effect of two protein kinase inhibitors was greater than that of each one. But calmodulin antagonists did not have any influence on the vanadate-induced contraction. On the other hand, both forskolin ($1\mu$M) and sodium nitroprusside ($1\mu$M) significantly inhibited vanadate-induced contraction. Therefore, these results suggest that both protein kinase C and tyrosino kinase are involved in the vanadate-induced contraction which required the influx of extracellular $Ca^{2+}$ in cat gastric smooth muscle, and that the contractile mechanism of vanadate may be different from that of agonist binding to its specific receptor.

• The Korean Journal of Physiology
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• 제18권2호
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• pp.139-150
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• 1984

Vanadate의 회장 평활근에 대한 수축작용이 Na-K-ATPase를 억제하기 때문인지를 구명하기 위하여 Na-K-ATPase를 억제하는 ouabain과의 작용의 차이를 관찰한 결과 다음과 같은 결론을 얻었다. 1) Ouabain에 의해 나타나는 수축은 2중 peak를 나타내었으나 vanadate 의해서는 단일 Peak만을 보였다. 2) Ouabain에 의한 수축은 atropine$(2{\times]10^{-6}M)$에 의해 강력하게 억제되었으나 vanadate의 작용은 영향을 받지 않았다. 3) Ouabain에 의한 수축은 vanadate에 비해 외부의 $Ca^{++}4농도 및 Ca-길항제에 대해 민감하게 영향을 받았다. 4) 용액내 $Na^+$이 없을때 혹은 고농도의 $K^+$존재하에서 ouabain에 의한 수축반응은 거의 나타나지 않았으나 vanadate에 의한 수축은 영향을 받지 않았다. 5) Vanadate에 의한 수축은 ouabain 존재시에 더욱 증가되었다. 6) 3시간동안 incubation한 결과 vanadate는 ouabain과 달리 세포내 $Na^+$의 농도에 영향을 미치지 못하였다. 이상의 결과로 보아 ouabain과 vanadate는 서로 다른 기전에 의해 회장 평활근에서 수축반응을 유발시키는 것으로 추측된다.

• The Korean Journal of Physiology
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• 제18권2호
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• pp.125-137
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• 1984

Vanadate의 수축에 이용되는 $Ca^{2+}$의 동원 경로와 Na-Pump억제가 vanadate의 수축에 어떤 영향을 미치는 지를 밝히기 위해 본 실험을 시행하여 다음과 같은 곁과를 얻었다. 1) 흰쥐의 자궁근에서는 vanadate는 수축을 일으켜 $5{\times}10^{-4}M$에서 최대수축을 나타내었으며 사람의 자궁근이 흰쥐의 자중근에 비해 vanadate에 더 민감한 반응을 보였다. 2) Vanadate에 의한 수축은 $Ca^{2+}$제거에 의해 완전히 억제되지 않았고 사람의 자궁근이 외부 $Ca^{2+}$의 농도변화에 더 민감한 반응을 보였다. 3) Vanadate에 의한 수축은 verapamil농도를 증가시킴에 따 억제되었으며 100k에 극한 수축을 완전 억제시키는$3{\times}10^{-5}M$ verapamil 존재하에서도 최대수의 40%정도가 남아있었고, 이 크기는 $Ca^{2+}$없는 용액에서의 수축의 크기와 유사하였다. 4) Na-pump억제시 vanadate의 수축은 증가하였고 이 현상은 $3{\times}10^{-5}M$ verapamil 존재하에서도 나타났다. 5) $Ca^{2+}$없는 ouabain용액에서 전처치후에 vanadate에 의한 수축은 증가하지 않았으나 외부내 $Ca^{2+}$을 부가할 나타나는 반음은 대조군에 비해 현저히 증가하였다. 6) Verapamil 존재시 vanadate에 의한 $Ca^{45}$유입은 완전히 억제되었으나 ouabain으로 처리한 후는 verapamil 존재하에서도 vanadate가 현저히 $Ca^{45}$유입을 일으켰다. 7) Ouabain이나 K 없는 용액으로 치리시간이 증가함에 따라 vanadate에 의한 수축의 증가정도는 더욱 더 현저하였다. 8) Ouabain 전처치시 증가된 vanadate에 의한 수축은 $10^{-4}M$ papaverine에 의해 현저히 억제되었다. 9) Acetylcholine에 의한 수축은 verapamil 존재하에서도 Na-pump억제 시간이 증가함에 따라 증가하였다. 이상의 결과로 볼 때 vanadate에 대해 사람의 자궁근이 흰쥐의 자궁근에 비해 더 민감한 반응을 보이고 vanadate에 의한 수축에는 외부와 내부 $Ca^{2+}$이 모두 이용되며 Na-pump 억제시 여러가지 근수축물질이 verapamil에 의해 억제되지 않는 $Ca^{2+}$유입을 일으키며 이 유입경로의 성질은 확실히 알 수 없으나 Papaverine에 의해 억제되며 막전위의 변화와 관련이 있는 것으로 생각된다.

• Journal of Chest Surgery
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• 제21권2호
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• pp.291-298
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• 1988

The effect of Vanadate on the isometric contraction, membrane potential and intracellular calcium ion activities of rabbit myocardial cells were investigated, using calcium selective microelectrode, filled with neutral calcium ion carrier, ETH-1001. The resting tension, the membrane potential and the intracellular calcium ion activities were recorded in normal Tyrode solution and compared with those in the contracture induced by 10 mM Vanadate. The following results were obtained: 1. The dose-response relationship between the contraction of Vanadate and twitch tension showed near-maximum response in 5mM with no corresponding changes in action potential. 2. The resting tension increased up to the amplitude of a control twitch in 10mM Vanadate with resting membrane potential, hyperpolarized. 3. Increase in intracellular calcium ion activities proceeded the contracture by 10mM Vanadate which were restored to the control level in accordance with a decrease of intracellular calcium ion activities. 4. The amplitude of contractures by 10mM Vanadate were 90-120% of the control twitch tension in which the intracellular calcium ion activities were increased about 70 times from p Ca, 7.1 in the control to p Ca, 5.8 in contractures.

• The Korean Journal of Physiology
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• 제22권2호
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• pp.189-206
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• 1988

The effects of ouabain on the contractile and electrical activities were investigated in the isolated preparations of guinea-pig taenia coli, and compared with those of vanadate. Spontaneous contractions were recorded with force transducer, and electrical activites were measured by use of suction electrode, or single sucrose-gap technique. The contractions were induced by the electrical stimulation for 5 seconds every 1 minute with alternating current (60 Hz, 3.0 V/cm) through the platinum electrodes located in parallel with the long axis of the preparation. All experiments were performed in tris-buffered Tyrode solution which was aerated with $100%{\;}O_2$ and kept at $35^{\circ}C$. The results obtained were as follows: 1) Responses of spontaneous contractions to ouabain were concentration-dependent; $10^{-7}M$ ouabain caused a rise of basal tone. Above the concentration of $10^{-6}M$ ouabain, an initial increase followed by a decrease in tension was observed. 2) A continuous spike discharge was induced by the administration of $10^{-7}M$ ouabain. Above $10^{-6}M$ ouabain, a transient initial increase followed by a decrease in spike frequency and amplitude was produced, and finally membrane potential was sustained at a certain level without a spike discharge. 3) The characteristic response to $10^{-7}M$ ouabain was not blocked by the pretreatment with $10^{-7}M$ atropine. 4) The electrically induced contractions were completely suppressed at the concentration of $2{\times}10^{-7}M$ ouabain. These contractions were blocked more rapidly in paralled with the increase in ouabain concentration. 5) Effects of vanadate on the spontaneous activities were quite different from those of ouabain; $10^{-6}M$ vanadate increased the amplitude of contractions and $10^{-5}M$ vanadate increased slightly both amplitude and frequency of spontaneous contractions. $10^{-4}M$ vanadate showed irregular phasic contractions superimposed on the increased basal tone. 6) $10^{-5}M$ vanadate depolarized the membrane potential and shortened the interval between the bursts of spike discharge, whereas $10^{-4}M$ vanadate induced continuous spike discharge with membrane depolarization. 7) Vanadate caused a characteristic inhibitory response to the contractions induced by electrical stimulation; An initial rapid inhibition of tension development and then gradual recovery to a certain level. From the above results, the following conclusions could be made: 1) The rise of basal tone at $10^{-7}M$ ouabain is due to continuous spike discharge without a silent period. The continuous spike discharge is likely to be associated with a slight membrane depolarization caused by the blockage of Na pump. 2) The biphasic response induced by above $10^{-6}M$ ouabain seems to occur by the different mechanisms. The initial increase in tension is associated with depolarization along with an increase in spike frquency, whereas the subsequent relaxation occurs through a non-electrical mechanism. 3) The characteristic response to $10^{-7}M$ ouabain is resulted not from the action on intrinsic nerve terminal, but from its direct action on the membrane of smooth muscle cells. 4) The phasic contractions superimposed on the increased basal tone at the concentration of $10^{-4}M$ vanadate is resulted from the continuous spike discharge with membrane depolarization, of which mechanism remains unknown. 5) The inhibitory action of ouabain on the electrically induced contractions suggests that the increasein intracellular Na in some way inhibits the electrically induced $Ca^{2+}$ influx. The mechanism of vanadate action on the induced contractions remains unknown.

• 한국응용과학기술학회지
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• 제24권1호
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• pp.61-66
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• 2007

X-ray diffraction studies have been made to investigate the effects of binding of ADP, ADP+Vi, ADP+AIF4, $ADP+BeF_3$ on the structure of glycerinated rabbit skeletal muscle in the rigor state. Although these phosphate analogs are known to bind actively cycling myosin heads, it is not clear whether they can bind to the attached heads in the rigor muscle. We have found that these analogs can bind to the myosin heads attached to actin filaments in the rigor state. The present results indicate that (1) bound myosin heads altered their conformation in the proximal end toward the plane perpendicular to the fiber axis when MgADP bound to them, and (2) myosin heads were dissociated substantially (up to 50%) from actin filaments but still remained in the vicinity of actin filaments when MgADP and metallofluorides (AIF4 and BeF3) or vanadate bound to them. We detected new conformations of myosin heads attached to actin filaments when they had MgADP or ADP.Pi analogs. We report here these findings on the effects of MgADP and MgADP+phosphate analogs to the rigor crossbridges.