• 대한한의학회지
• /
• 제43권1호
• /
• pp.33-41
• /
• 2022

Objectives: Drug-induced blockade of the human ether-à-go-go related gene (hERG) potassium ion channel causes acquired long QT syndrome, which is known to cause cardiac arrhythmias and be fatal. To establish safety evidence of herbal formulae, we evaluated the effects of 31 herbal formulae on hERG channel activity. Methods: The current through hERG channel was measured by changing the membrane voltage before and after treatment with 31 herbal formulae in HEK 293 cell overexpressing hERG channel using a whole-cell patch clamp system. The current-voltage curves and the activity curves were fitted, and the hERG activity and 50% inhibitory concentration (IC₅₀) according to each herbal formula were calculated. Results: Chokyungjongok-tang, Oncheong-eum, and Cheongsangbangpung-tang strongly inhibited the hERG activity, with IC₅₀ values of 67.67, 141.2, and 296.3 ㎍/mL, respectively. Yeonkyopaedok-san, Eunkyo-san, Ukgan-san gajinphibanha, Daegunjoong-tang (except Oryzae gluten), Insamyangyoung-tang, Banhahubak-tang, SokyungHwalhyul-tang, Jodeung-san, Hyeonggaeyeongyo-tang, and Bangkeehwangkee-tang weakly inhibited hERG activity, with IC₅₀ values ranging from 400 to 1000 ㎍/mL. The other 18 herbal formulae showed very weak hERG activity inhibition of less than 50% at the highest concentration (1000 ㎍/mL). Conclusion: This study provided safety information on cardiotoxicity by cardiac arrhythmia risk assessment of herbal formulae, and is expected to be a reference data for predicting the safety and risk of herbal formulae.

• The Korean Journal of Physiology and Pharmacology
• /
• 제28권4호
• /
• pp.335-344
• /
• 2024

Diphenyleneiodonium (DPI) has been widely used as an inhibitor of NADPH oxidase (Nox) to discover its function in cardiac myocytes under various stimuli. However, the effects of DPI itself on Ca²⁺ signaling and contraction in cardiac myocytes under control conditions have not been understood. We investigated the effects of DPI on contraction and Ca²⁺ signaling and their underlying mechanisms using video edge detection, confocal imaging, and whole-cell patch clamp technique in isolated rat cardiac myocytes. Application of DPI suppressed cell shortenings in a concentration-dependent manner (IC₅₀ of ≅0.17 µM) with a maximal inhibition of ~70% at ~100 µM. DPI decreased the magnitude of Ca²⁺ transient and sarcoplasmic reticulum Ca²⁺ content by 20%-30% at 3 µM that is usually used to remove the Nox activity, with no effect on fractional release. There was no significant change in the half-decay time of Ca²⁺ transients by DPI. The L-type Ca²⁺ current (I_Ca) was decreased concentration-dependently by DPI (IC₅₀ of ≅40.3 µM) with ≅13.1%-inhibition at 3 µM. The frequency of Ca²⁺ sparks was reduced by 3 µM DPI (by ~25%), which was resistant to a brief removal of external Ca²⁺ and Na⁺. Mitochondrial superoxide level was reduced by DPI at 3-100 µM. Our data suggest that DPI may suppress L-type Ca²⁺ channel and RyR, thereby attenuating Ca²⁺-induced Ca²⁺ release and contractility in cardiac myocytes, and that such DPI effects may be related to mitochondrial metabolic suppression.

• The Korean Journal of Physiology and Pharmacology
• /
• 제10권1호
• /
• pp.31-38
• /
• 2006

Fluoxetine, widely used for the treatment of depression, is known to be a selective serotonin reuptake inhibitor (SSRI), however, there are also reports that fluoxetine has direct effects on several receptors. Employing whole-cell patch clamp techniques in rat brain slice, we studied the effects of fluoxetine on corticostriatal synaptic transmission by measuring the change in spontaneous excitatory postsynaptic currents (sEPSC). Acute treatment of rat brain slice with fluoxetine ($10{\mu}M$) significantly decreased the amplitude of sEPSC ($8.1{\pm}3.3$%, n=7), but did not alter its frequency ($99.1{\pm}4.7$%, n=7). Serotonin ($10{\mu}M$) also significantly decreased the amplitude ($81.2{\pm}3.9$%, n=4) of sEPSC, but did not affect its frequency ($105.8{\pm}8.0$, n=4). The effect of fluoxetine was found to have the same trend as that of serotonin. We also found that the inhibitory effect of fluoxetine on sEPSC amplitude ($93.0{\pm}1.9$%, n=8) was significantly blocked, but not serotonin ($84.3{\pm}1.6$%, n=4), when the brain slice was incubated with p-chloroamphetamine ($10{\mu}M$), which depletes serotonin from the axon terminals and blocks its reuptake. These results suggest that fluoxetine inhibits corticostriatal synaptic transmission through postsynaptic, and that these effects are exerted through both serotonin dependent and independent mechanism.

• 한국생물물리학회:학술대회논문집
• /
• 한국생물물리학회 2003년도 정기총회 및 학술발표회
• /
• pp.23-23
• /
• 2003

It has been suggested that the impairment of smooth muscle cell (SMC) function by alterations in the $Ca^{2+}$-activated $K^{+}$ ( $K_{Ca}$ ) channels accounts for the reduction in coronary reserve during left ventricular hypertrophy (LVH). However, this hypothesis has not been fully investigated. The main goal of this study was to assess whether the properties of $K_{Ca}$ channels in coronary SMCs were altered during LVH. New Zealand white rabbits (0.8-1.0 kg) and Sprague-Dawley rats (300-400 g) were randomly selected to receive either an injection of isoproterenol (300 $\mu\textrm{g}$/kg body weight) or an equal volume of 0.9％ saline (1 mL/kg body weight). The animals developed LVH 10 days after injection. In patch-clamp experiments, the unitary current amplitude and open probability for the $K_{Ca}$ channels were significantly reduced in LVH patches compared with control patches. The concentration-response curve of the $K_{Ca}$ channel to [C $a^{2+}$]$_{i}$ was shifted to the right. Inhibition of the $K_{Ca}$ channels with TEA was more pronounced in LVH cells than in the control cells. The whole-cell currents of $K_{Ca}$ channels were reduced during LVH. Western blot analysis indicated no differences in $K_{Ca}$ channel expression between the control and LVH coronary SM membranes. In contraction experiments, the effect of a high $K^{+}$concentration on the resting tension of the LVH coronary artery was greater than on that of the control. The effect of TEA on the resting tension of the LVH coronary artery was reduced as compared with the effect on the control. Our findings imply a novel mechanism for reduced coronary reserve during LVH.ing LVH.

• 대한수의학회지
• /
• 제38권2호
• /
• pp.280-289
• /
• 1998

Voltage-sensitive ion channels contribute to establishment of the cell excitablity and the generation of the cellular function. At hamster oocytes in the primitive stage during developing process, an inward current elicited by voltage pulses was found to be carried mainly by $Ca^{2+}$. Even at present, $Ca^{2+}$ channels serve as the most probable route to pass this inward current but there is no evidence of the presence of this channels in eggs. To date, both the characteristic properties and the physiological role in the early stage of development remain unclear. Here we examined the characteristic properties of the inward current and changes in this currents at unfertilized oocytes, fertilized zygotes and two-cell embryos using whole-cell voltage clamp technique. The inward current carried reportedly by $Ca^{2+}$ was remained following removing external $Ca^{2+}$ but completely abolished by further replacement of impermeants such as tetramethylammonium ion ($TMA^+$) or $choline^+$ instead of $[Na^+]_0$. Tetrodotoxin did not affect on this inward current remained at $[Ca^{2+}]_0$-free condition. Removal of $Na^+$ ion out of the experimental solution clearly decreased the current. After adding 2mM $Ca^{2+}$ to the $Na^+$-free media, the inward current was restored. Interestingly, this current carried by either $Ca^{2+}$ or $Na^+$ was decreased by the reduction of intracellular $Cl^-$ concentration, or by $Cl^-$ channel blockers such as niflumic acid, DIDS and SITS. When $Cl^-$ concentration was lowered without changes in other ionic components, this inward current was reduced. At fertilized oocytes and two-cell embryos, the inward current carried by $Ca^{2+}$ and $Na^+$ was severely reduced. Also $Cl^-$ component could not be observed. From these results, the inward current is composed of $Ca^{2+}$, $Na^+$ and $Cl^-$ component, suggesting that the channel carrying this inward current is not selective specifically to $Ca^{2+}$. During early stage of development, the voltage-sensitive ion current seems not to contribute essentially to the cell cleavage and differentiation. The loss of $Cl^-$ component after fertilization suggests that $Cl^-$ may play a role in maintaining the viability of unfertilized ova.

• 한국생물물리학회:학술대회논문집
• /
• 한국생물물리학회 2003년도 정기총회 및 학술발표회
• /
• pp.39-39
• /
• 2003

In our previous study, WEHI-231, an immature B cell line, showed intractable increase in [C $a^{2+}$]$_{c}$ after the B-cell receptor (BCR) ligation and treatment with 2-aminoethoxydiphenylborate (2-APB), which was never observed in Bal-17, a mature B cell line (Nam et al., 2003, FEBS Lett). In this study, a whole cell voltage clamp study revealed a specific expression of a novel type of $K^{+}$ current, namely voltage-independent background-type $K^{+}$ channels (IK-bg), in WEHI-231 cells. IK-bg was dramatically increase by the application of 2-APB (50 $\square$M), which induced severe hyperpolarization of WEHI-231 from -45 ㎷ to -90 ㎷, When dialyzed with $Mg^{2+}$ and ATP-free pipette solution, a spontaneous development of IK-bg and membrane hyperpolarization were observed. IK-bg was insensitive to classical $K^{+}$ channel blockers (TEA, glibenclamide, $Ba^{2+}$(1 mM)), whereas blocked by quinine and quinidine in a voltage-dependent manner ($IC_{50}$/=6~9 $\square$M at +60㎷). Phorbol myrstate, a PKC activator, decreased the amplitude of IK-bg. Extracellular acidification (pH 6.5) slightly inhibited IK-bg. Arachidonic acid, riluzole, or hyposmotic stress could not affect the IK-bg after the full development by the intracellular dialysis with Mg-ATP-free solution. In a cell-attached mode of single channel recording from WEHI231, we found two types of voltage-independent $K^{+}$ channels with unitary conductance of 300 pS and 120 pS, respectively. Both channels showed very short mean open times and their open probabilities were increase by the application of 2-APB. In Bal-17 cells, no such $K^{+}$ current was observed in 50 cells tested. In summary, WEHI-231 immature B cells express background $K^{+}$ channels. The pharmacological properties and the large unitary conductance suggest that novel types of two-pore domain $K^{+}$ channels (2-P-K channels) might be expressed in WEHI-231, which may provide an intriguing targets of signal transduction in the immature B lymphocytes.e B lymphocytes.

• The Korean Journal of Physiology
• /
• 제27권1호
• /
• pp.93-105
• /
• 1993

The present study was performed to investigate the properties of ionic currents elicited by voltage pulses in the unfertilized eggs of mouse and hamster by using the whole cell voltage clamp techniques and to find out if there are any differences in properties between eggs of the two rodents. In addition, the modulatory effect of G proteins and protein kinase C (PKC) on the ionic channels were observed. The inward current in hamster eggs was shown to be due to $Ca^{2+}\;current\;(i_{ca})$). The current voltage relations of these currents in hamster egg were analogous to those in mouse eggs. The amplitude of $i_{ca}$ in the hamster egg was larger than that in the mouse egg ($-3.12{\pm}1.07\;nA\;vs.\;-1.71{\pm}0.71\;nA,\;mean{\pm}\;SD$). These results suggest that the $Ca^{2+}$ channels in both kinds of eggs have similar channel properties but their density, and/or conduct ance per unit area is higher in hamster eggs than in mouse eggs. Outward currents in eggs of both mouse and hamster were carried by $K^+$. In hamster eggs, they appeared to comprise at least two components; a transient outward component ($i_{to}$) and a steady state component ($i_{\infty}.$ The $i_{to}$ was found to be dependent on intracellular $Ca^{2+}$ concentration; whereas on the other hand $i_{\infty}\;was\;Ca^{2+}$-independent. $Ca^{2+}$ currents were increased in eggs treated with GTP (or $GTP{\gamma}S$) or fluoroaluminate ($AIF_4^-$). In the hamster egg these increments were antagonized by GDP (or $GDP{\beta}S$) application. In contrast to the enhancement of $i_{ca},\;i_k$ was reduced following GTP (or $GTP{\gamma}S$) perfusion in mouse eggs. The transient component ($i_{to}$) in hamster eggs was increased by adding GTP but decreased by phorbol ester, TPA or dioctanoyl glycerol (DOG). Simultaneous application of $GTP{\gamma}S$ and DOG suppressed $i_{to}$ more effectively than a single application or DOG or TPA. From the above results, we have shown that ionic currents elicited by voltage pulses existed in the unfertilized eggs of mouse and hamster. There are at least two types of currents, $i_{ca}\;and\;i_k$ in mouse eggs, while three types, $i_{ca},\;Ca^{2+}$-dependent $i_k$ and $Ca^{2+}$-independent $i_k$ exist in hamster eggs. ionic channels in these eggs may be regulated either directly by GTP and PKC or indirectly by the substances linked with GTP and PKC.

• 대한약리학회지
• /
• 제31권3호
• /
• pp.279-284
• /
• 1995

본 연구의 목적은 외부에서 nitric oxide (NO)를 투여 하였을때 심근 수축력, 심박동수의 변화 및 혈관 평활근에 대한 효과를 비교함으로서 NO에 대한 이들 장기의 민감도가 서로 같은지 또는 상이한지를 알아보고자 하였다. 본 실험에서는 PIANO 방법에 의한 근장력의 변화와 아울러 심근에서의 $Ca^{2+}$ current를 측정하였다. 랫트의 심방근에 대한 PIANO $(STZ,\;100\;{\mu}M)$는 심근수축력 및 심박동수에 전혀 변화를 주지 않았지만 혈관 평활근에서는 강한 이완 작용을 나타내었다. 한편, 8-Br-cGMP도 고농도 $(100\;{\mu}M)$에서만 심근 수축력을 억제하였다. 토끼의 심방근세포에서 Whole cell voltage patch clamp를 사용시 bradykinin, SNP, 8-Br-cGMP 및 PIANO는 $Ca^{2+}$ current를 억제하였다. 이러한 사실은 외부에서 공급되는 NO에 대한 심근과 혈관 평활근의 반응에는 민감도의 차이가 있음을 암시하며 더 나아가 심근의 경우에도 NO 반응에는 종 (species)간의 차이와 동일 종이라 하더라도 세포(cell)와 장기(tissue)에 차이가 있을 가능성을 제시하였다.

• The Korean Journal of Physiology and Pharmacology
• /
• 제13권6호
• /
• pp.437-442
• /
• 2009

A non-steroidal anti-inflammatory drug (NSAID) has many adverse effects including cardiovascular (CV) risk. Diclofenac among the nonselective NSAIDs has the highest CV risk such as congestive heart failure, which resulted commonly from the impaired cardiac pumping due to a disrupted excitationcontraction (E-C) coupling. We investigated the effects of diclofenac on the L-type calcium channels which are essential to the E-C coupling at the level of single ventricular myocytes isolated from neonatal rat heart, using the whole-cell voltage-clamp technique. Only diclofenac of three NSAIDs, including naproxen and ibuprofen, significantly reduced inward whole cell currents. At concentrations higher than $3\;{\mu}M$, diclofenac inhibited reversibly the $Na^+$ current and did irreversibly the L-type $Ca^{2+}$ channels-mediated inward current $(IC_{50}=12.89\pm0.43\;{\mu}M)$ in a dose-dependent manner. However, nifedipine, a well-known L-type channel blocker, effectively inhibited the L-type $Ca^{2+}$ currents but not the $Na^+$ current. Our finding may explain that diclofenac causes the CV risk by the inhibition of L-type $Ca^{2+}$ channel, leading to the impairment of E-C coupling in cardiac myocytes.

• The Korean Journal of Physiology and Pharmacology
• /
• 제22권5호
• /
• pp.539-546
• /
• 2018

Botulinum toxin type A (BoNT/A) has been used therapeutically for various conditions including dystonia, cerebral palsy, wrinkle, hyperhidrosis and pain control. The substantia gelatinosa (SG) neurons of the trigeminal subnucleus caudalis (Vc) receive orofacial nociceptive information from primary afferents and transmit the information to higher brain center. Although many studies have shown the analgesic effects of BoNT/A, the effects of BoNT/A at the central nervous system and the action mechanism are not well understood. Therefore, the effects of BoNT/A on the spontaneous postsynaptic currents (sPSCs) in the SG neurons were investigated. In whole cell voltage clamp mode, the frequency of sPSCs was increased in 18 (37.5%) neurons, decreased in 5 (10.4%) neurons and not affected in 25 (52.1%) of 48 neurons tested by BoNT/A (3 nM). Similar proportions of frequency variation of sPSCs were observed in 1 and 10 nM BoNT/A and no significant differences were observed in the relative mean frequencies of sPSCs among 1-10 nM BoNT/A. BoNT/A-induced frequency increase of sPSCs was not affected by pretreated tetrodotoxin ($0.5{\mu}M$). In addition, the frequency of sIPSCs in the presence of CNQX ($10{\mu}M$) and AP5 ($20{\mu}M$) was increased in 10 (53%) neurons, decreased in 1 (5%) neuron and not affected in 8 (42%) of 19 neurons tested by BoNT/A (3 nM). These results demonstrate that BoNT/A increases the frequency of sIPSCs on SG neurons of the Vc at least partly and can provide an evidence for rapid action of BoNT/A at the central nervous system.