• The Korean Journal of Physiology and Pharmacology
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• 제20권6호
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• pp.629-639
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• 2016

The present study was designed to investigate the characteristics of gintonin, one of components isolated from Korean Ginseng on secretion of catecholamines (CA) from the isolated perfused model of rat adrenal gland and to clarify its mechanism of action. Gintonin (1 to $30{\mu}g/ml$), perfused into an adrenal vein, markedly increased the CA secretion from the perfused rat adrenal medulla in a dose-dependent fashion. The gintonin-evoked CA secretion was greatly inhibited in the presence of chlorisondamine ($1{\mu}M$, an autonomic ganglionic bloker), pirenzepine ($2{\mu}M$, a muscarinic $M_1$ receptor antagonist), Ki14625 ($10{\mu}M$, an $LPA_{1/3}$ receptor antagonist), amiloride (1 mM, an inhibitor of $Na^+/Ca^{2+}$ exchanger), a nicardipine ($1{\mu}M$, a voltage-dependent $Ca^{2+}$ channel blocker), TMB-8 ($1{\mu}M$, an intracellular $Ca^{2+}$ antagonist), and perfusion of $Ca^{2+}$-free Krebs solution with 5mM EGTA (a $Ca^{2+}$chelater), while was not affected by sodium nitroprusside ($100{\mu}M$, a nitrosovasodialtor). Interestingly, LPA ($0.3{\sim}3{\mu}M$, an LPA receptor agonist) also dose-dependently enhanced the CA secretion from the adrenal medulla, but this facilitatory effect of LPA was greatly inhibited in the presence of Ki 14625 ($10{\mu}M$). Moreover, acetylcholine (AC)-evoked CA secretion was greatly potentiated during the perfusion of gintonin ($3{\mu}g/ml$). Taken together, these results demonstrate the first evidence that gintonin increases the CA secretion from the perfused rat adrenal medulla in a dose-dependent fashion. This facilitatory effect of gintonin seems to be associated with activation of LPA- and cholinergic-receptors, which are relevant to the cytoplasmic $Ca^{2+}$ increase by stimulation of the $Ca^{2+}$ influx as well as by the inhibition of $Ca^{2+}$ uptake into the cytoplasmic $Ca^{2+}$ stores, without the increased nitric oxide (NO). Based on these results, it is thought that gintonin, one of ginseng components, can elevate the CA secretion from adrenal medulla by regulating the $Ca^{2+}$ mobilization for exocytosis, suggesting facilitation of cardiovascular system. Also, these findings show that gintonin might be at least one of ginseng-induced hypertensive components.

• The Korean Journal of Physiology and Pharmacology
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• 제14권1호
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• pp.29-35
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• 2010

We have shown that myosin light chain kinase (MLCK) was required for the off-contraction in response to the electrical field stimulation (EFS) of feline esophageal smooth muscle. In this study, we investigated whether protein kinase C (PKC) may require the on-contraction in response to EFS using feline esophageal smooth muscle. The contractions were recorded using an isometric force transducer. On-contraction occurred in the presence of $N^G$-nitro-L-arginine methyl ester (L-NAME), suggesting that nitric oxide acts as an inhibitory mediator in smooth muscle. The excitatory composition of both contractions was cholinergic dependent which was blocked by tetrodotoxin or atropine. The on-contraction was abolished in $Ca^{2+}$-free buffer but reappeared in normal $Ca^{2+}$-containing buffer indicating that the contraction was $Ca^{2+}$ dependent. 4-aminopyridine (4-AP), voltage-dependent $K^+$ channel blocker, significantly enhanced on-contraction. Aluminum fluoride (a G-protein activator) increased on-contraction. Pertussis toxin (a $G_i$ inactivator) and C3 exoenzyme (a rhoA inactivator) significantly decreased on-contraction suggesting that Gi or rhoA protein may be related with $Ca^{2+}$ and $K^+$ channel. ML-9, a MLCK inhibitor, significantly inhibited on-contraction, and chelerythrine (PKC inhibitor) affected on the contraction. These results suggest that endogenous cholinergic contractions activated directly by low-frequency EFS may be mediated by $Ca^{2+}$, and G proteins, such as Gi and rhoA, which resulted in the activation of MLCK, and PKC to produce the contraction in feline distal esophageal smooth muscle.

• 한국의학물리학회지:의학물리
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• 제11권1호
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• pp.29-38
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• 2000

지금까지 각종 신경전달물질의 칼슘통로 억제 효과는 일반적으로 protein kinase 의 관여없이 G-protein mediated, membrane-delimited mechanism$^{1)}$ 으로 설명되어왔다. 그러나 최근들어 protein kinase C (PKC)의 활성화가 몇몇 신경전달물질에 의한 칼슘통로 억제효과를 야기하는 중요한 세포내 기전으로 보고되고 있다 그러므로 본 연구에서는 흰쥐 교감신경뉴론을 대상으로 하여 whole cell patch clamp technique을 사용하여 칼슘전류를 기록하고, 세포밖에 norepinephrine (NE)과 함께 PKC agonist 인 phorbol-12, 13-dibutyrate (PDBu)을 투여하면서 PDBu 전 처치로 인하여 NE 에 의한 칼슘전류 억제에 어떤 변화가 초래되는 지를 분석함으로써, 신경전달물질의 칼슘전류 억제효과시 PKC의 역할을 밝히고자 하였다. PDBu (500 nM) 처치는 칼슘전류의 크기를 증가시켰으며 이는 막전압 의존성을 보여 -10 mV ~ +10 mV 의 저분극 자극시 가장 크게 전류크기가 증가하였다. 또한 PDBu 처치는 tail current 의 deactivation을 느리게 하였다. PDBu 는 NE 에 의하여 활성화되는 pertussis toxin 예민성 G protein pathway를 통한 칼슘전류 억제를 감소시켰다. 비특이적인 protein kinase 길항제인 staurosporine (1 $\mu$M) 을 전처치 하고 PDBu를 투여하면 PDBu의 칼슘전류 크기 증가 효과가 소실되었으며 또한 NE에 의한 칼슘전류 억제를 해제하는 PDBu 의 조절효과도 소실되었다. 이상의 결과로부터 Protein Kinase C 가 활성되면 G protein을 경유하여 나타나는 칼슘전류 억제 효과가 소실된다고 결론지을 수 있다. Protein Kinase C 에 의하여 인산화되는 부위가 G-protein 인지 혹은 칼슘통로인 지에 관한 해답을 얻기 위하여는 추후 연구가 진행되어야 한다.

• The Korean Journal of Physiology and Pharmacology
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• 제18권4호
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• pp.341-346
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• 2014

Lubiprostone is a chloride ($Cl^-$) channel activator derived from prostaglandin $E_1$ and used for managing constipation. In addition, lubiprostone affects the activity of gastrointestinal smooth muscles. Interstitial cells of Cajal (ICCs) are pacemaker cells that generate slow-wave activity in smooth muscles. We studied the effects of lubiprostone on the pacemaker potentials of colonic ICCs. We used the whole-cell patch-clamp technique to determine the pacemaker activity in cultured colonic ICCs obtained from mice. Lubiprostone hyperpolarized the membrane and inhibited the generation of pacemaker potentials. Prostanoid $EP_1$, $EP_2$, $EP_3$, and $EP_4$ antagonists (SC-19220, PF-04418948, 6-methoxypyridine-2-boronc acid N-phenyldiethanolamine ester, and GW627368, respectively) did not block the response to lubiprostone. L-NG-nitroarginine methyl ester (L-NAME, an inhibitor of nitric oxide synthase) and 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, an inhibitor of guanylate cyclase) did not block the response to lubiprostone. In addition, tetraethylammonium (TEA, a voltage-dependent potassium [$K^+$] channel blocker) and apamin (a calcium [$Ca^{2+}$]-dependent $K^+$ channel blocker) did not block the response to lubiprostone. However, glibenclamide (an ATP-sensitive $K^+$ channel blocker) blocked the response to lubiprostone. Similar to lubiprostone, pinacidil (an opener of ATP-sensitive $K^+$ channel) hyperpolarized the membrane and inhibited the generation of pacemaker potentials, and these effects were inhibited by glibenclamide. These results suggest that lubiprostone can modulate the pacemaker potentials of colonic ICCs via activation of ATP-sensitive $K^+$ channel through a prostanoid EP receptor-independent mechanism.

• 고려인삼학회:학술대회논문집
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• 고려인삼학회 2006년도 춘계학술대회
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• pp.76-76
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• 2006

• The Korean Journal of Physiology and Pharmacology
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• 제14권1호
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• pp.21-28
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• 2010

Phenolic compounds affect intracellular free $Ca^{2+}$ concentration ($[Ca^{2+}]_i$) signaling. The study examined whether the simple phenolic compound octyl gallate affects ATP-induced $Ca^{2+}$ signaling in PC12 cells using fura-2-based digital $Ca^{2+}$ imaging and whole-cell patch clamping. Treatment with ATP ($100\;{\mu}M$) for 90 s induced increases in $[Ca^{2+}]_i$ in PC12 cells. Pretreatment with octyl gallate (100 nM to $20\;{\mu}M$) for 10 min inhibited the ATP-induced $[Ca^{2+}]_i$ response in a concentration-dependent manner ($IC_{50}=2.84\;{\mu}M$). Treatment with octyl gallate ($3\;{\mu}M$) for 10 min significantly inhibited the ATP-induced response following the removal of extracellular $Ca^{2+}$ with nominally $Ca^{2+}$-free HEPES HBSS or depletion of intracellular $Ca^{2+}$ stores with thapsigargin ($1\;{\mu}M$). Treatment for 10 min with the L-type $Ca^{2+}$ channel antagonist nimodipine ($1\;{\mu}M$) significantly inhibited the ATP-induced $[Ca^{2+}]_i$ increase, and treatment with octyl gallate further inhibited the ATP-induced response. Treatment with octyl gallate significantly inhibited the $[Ca^{2+}]_i$ increase induced by 50 mM KCI. Pretreatment with protein kinase C inhibitors staurosporin (100 nM) and GF109203X (300 nM), or the tyrosine kinase inhibitor genistein ($50\;{\mu}M$) did not significantly affect the inhibitory effects of octyl gallate on the ATP-induced response. Treatment with octyl gallate markedly inhibited the ATP-induced currents. Therefore, we conclude that octyl gallate inhibits ATP-induced $[Ca^{2+}]_i$ increase in PC12 cells by inhibiting both non-selective P2X receptor-mediated influx of $Ca^{2+}$ from extracellular space and P2Y receptor-induced release of $Ca^{2+}$ from intracellular stores in protein kinase-independent manner. In addition, octyl gallate inhibits the ATP-induced $Ca^{2+}$ responses by inhibiting the secondary activation of voltage-gated $Ca^{2+}$ channels.

• The Korean Journal of Physiology
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• 제23권2호
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• pp.263-275
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• 1989

Mechanical contractions and electrical activities of the fundic longitudinal and antral circular muscle fibers were investigated in order to elucidate topical differences of gastric motility. K-induced contracture was produced by exposure of muscle strips to high K Tyrode solution. Membrane potential and mechanical contraction were simultaneously recorded by conventional glass microelectrode method and single sucrose-gap technique. All experiments were performed in tris-buffered Tyrode solution which was aerated with $100%\;O_2\;and\;kept\;35^{\circ}C$. The results obtained were as follows: 1) The resting membrane potential of circular muscle cells in the antral region was about 10 mV more negative than that in the fundic region. 2) The membrane potentials decreased almost linearly as the extracellular KCI concentration was increased both in antral circular muscle cells and in fundic longitudinal muscle cells. 3) The thresholdal K concentration of K-contracture was 15 mM (membrane potential, -48 mV) for the antral circular muscle strip and 20 mM for the fundic longitudinal muscle cells. 4) The ratio of membrane permeability coefficient for $Na^+\;and\;K^+,\;P_{Na}/P_K\;({\alpha})$ was 0.065 for antral circular muscle cells and was 0.108 for fundic longitudinal muscle cells. 5) K-contracture of antral and fundic smooth muscle strips showed the contracture composed of phasic and tonic components. The amplitude of the phasic component increased sigmoidally in a dose-dependent manner, whereas that of the tonic component was maximal at a concentration of 40 mM KCI and at the concentrations above or below 40 mM KCI the amplitude was reduced. 6) The inverse relationship between the amplitude of tonic component and extracellular KCI concentration in the range of 40 to 150 mM KCI was more prominent in the antral circular muscle strip than in the fundic longitudinal muscle strip, where the amplitude of the tonic component decreased less steeply and was maintained higher at the same high K concentrations. 7) The tonic component was totally dependent on the external $Ca^{2+}$ and completely abolished by verapamil, while tile phasic component was far less dependent on the external $Ca^{2+}$ and partially suppressed by verapamil. From the above results, the following conclusions could be made. 1) The phasic component of K-contracture is produced both by intracellular $Ca^{2+}$ mobilization and by $Ca^{2+}$-influx from outside, while the tonic component is generated and maintained by the $Ca^{2+}-influx$ through the potential-dependent $Ca^{2+}$ channel. 2) The mechanism of reducing the free $Ca^{2+}$ concentration in the myoplasm seems to be more developed in the antral circular muscle than in the fundic longitudinal muscle. 3) The lower resting membrane potential of the fundic longitudinal muscle cell reflects a relatively high $P_{Na}/P_K$ ratio of about 0.108.

• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 2003년도 정기총회 및 학술발표회
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• pp.46-46
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• 2003

Salviae Miltiorrhizae Radix has been used for treatment of cardiovascular diseases in oriental medicine. To investigate the possible involvement of cardiac ion channel in this effect, we examined electrophysiological effects of the extract of Salviae Miltiorrhizae Radix on action potentials and ionic currents in rat ventricular myocytes. The extracts of Salviae Miltiorrhizae Radix were fractionated into nine fractions, and the effect of each fraction on action potential was tested. The fraction containing monomethyl lithospermic acid-A (LSA-A) induced a significant prolongation of action potential duration (APD). LSA-B which is a major component of Salviae Miltiorrhizae Radix, however, did not cause a significant effect. In voltage clamp experiments, the effects of LSA-A on K currents, Ca currents and Na currents were tested. Neither K currents nor L-type Ca currents were affected by LSA-A. On the contrary, LSA-A significantly slowed down the inactivation kinetics of the Na current with no effect on the fast component of the inactivation process. The amplitude of the peak current and the voltage-dependence of activation were not changed by LSA-A. The effect of LSA-A on Na current was abolished when high concentration of $Ca^{2+}$ buffer (10 mM BAPTA) was included in the pipette solution or when Ca2+ current was blocked by nicardipine (1 $\mu$M) in the bath solution.n.

• 대한수의학회지
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• 제37권2호
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• pp.331-338
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• 1997

최근 동물의 진통 및 진정을 목적으로 널리 사용되고 있는 imidazole 유도체인 clonidine, medetomidine, etomidate 등의 약물과 xylazine의 효과를 발정정지기의 척출 돼지 자궁근에서 검토하였다. Clonidine($10^{-8}{\sim}10^{-6}M$)이나 medetomidine($10^{-8}{\sim}10^{-6}M$)은 xylazine과 비슷한 정도로 용량의존적인 자궁근의 수축을 일으켰다. Clonidine, medetomidine, xylazine 등의 $EC_{50}$는 각각 24.7nM, 19.9nM, 45.1nM이었다. 그러나 etomidate는 $10^{-6}M$ 미만의 농도에서 반응이 거의 없었으며, $10^{-6}M$ 이상에서 수축반응을 일으켰다. 이들 agonists의 효과는 yohimbine($10^{-8}{\sim}10^{-6}M$), idazoxan($10^{-7}{\sim}10^{-5}M$), tolazoline($10^{-7}{\sim}10^{-5}M$) 등의 ${\alpha}_2-adrenoceptor$ antagonists에 의해서 차단되었으나, ${\alpha}_1-adrenoceptor$ antagonist인 prazosin ($10^{-6}M$)에 의해서는 차단되지 않았다. 또한 $Ca^{2+}-free$ medium이나 verapamil($10^{-5}M$)의 전처치에 의해서 이들 agonist의 효과가 완전히 차단되었다. 결론적으로 발정정지기의 돼지 자궁근에서 clonidine, medetomidine, etomidate, xylazine 등은 ${\alpha}_2-adrenoceptors$의 흥분을 통해 자궁근의 수축을 일으키며, 이 효과는 voltage-dependent $Ca^{2+}$ channels을 통한 extracellular $Ca^{2+}$ influx의 증가에 의한 것으로 추론하였다.

• The Korean Journal of Physiology
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• 제26권2호
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• pp.113-122
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• 1992

It is well known that chromaffin cells of adrenal medulla secrete catecholamine in response to sympathetic nerve activation and the influx of $Ca^{2+}$ through the voltage dependent $Ca^{2+}$ channels (VDCC) in the cell membrane do a major role in this secretory process. In this study, we explored the effect of divalent cations on VDCC of rat chromaffin cells. Rat (Sprague-Dawley rat, 150-250 gm) chromaffin cells were isolated and cultured. Standard giga seal, whole cell recording techniques were employed to study $Ca^{2+}$ current with external and internal solutions that could effectively isolate VDCC currents $(NMG\;in\;external\;and\;TEA\;and\;Cs^{2+}\;in\;internal\;solution)$. The voltage dependence and the inactivation time course of VDCC in our cells were identical to those of bovine chromaffin cells. A persistent inward current was first activated by depolarizing step pulse from the holding potential (H.P.) of -80 mV to -40 mV, increased to maximum amplitude at around +10 mV, and became smaller with progressively higher depolarizing pulses to reverse at around +60 mV. The inactivation time constant $(\tau)$, fitted from the long duration test potential (2 sec) was $1295.2{\pm}126.8$ msec $(n=20,\;1\;day\;of\;culture,\;mean\;{\pm}S.E.M.)$ and the kinetic parameters were not altered along the culture duration. Nicardipine $(10\;{\mu}M)$ blocked the current almost completely. Among treated divalent cations such as $Cd^{2+},\;Co^{2+},\;Ni^{2+},\;Zn^{2+}\;and\;,Mn^{2+},\;Cd^{2+}$ was the most potent blocker on VDCC. When the depolarizing step pulse from -80 mV to 10 mV was applied, the equilibrium dissociation constant $(K_d)$ of $Cd^{2+}\;was\;39\;{\mu}M,\;K_d\;of\;Co^{2+}\;was\;100\;{\mu}M\;and\;K_d\;of\;Ni^{2+}];was];780{\mu}M.$ The principal findings of this study are as follows. First, the majority of $Ca^{2+}$ channels in rat chromaffin cells are well classified to L-type $Ca^{2+}$ channel in the view of kinetics and pharmacology. Second, all divalent cations tested could block the $Ca^{2+}$ current and the most potent blocker among the tested was $Cd^{2+}$.