• 대한약리학회지
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• 제31권3호
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• pp.279-284
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• 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
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• 제9권2호
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• pp.103-108
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• 2005

To study the direct effect of somatostatin (SS) on calcium channel current ($I_{Ba}$) in guinea-pig gastric myocytes, $I_{Ba}$ was recorded by using whole-cell patch clamp technique in single smooth muscle cells. Nicardipine ($1{\mu}M$), a L-type $Ca^{2+}$ channel blocker, inhibited $I_{Ba}$ by $98{\pm}1.9$% (n=5), however $I_{Ba}$ was decreased in a reversible manner by application of SS. The peak $I_{Ba}$ at 0 mV were decreased to $95{\pm}1.5$, $92{\pm}1.9$, $82{\pm}4.0$, $66{\pm}5.8$, $10{\pm}2.9$% at $10^{-10}$, $10^{-9}$, $10^{-8}$, $10^{-7}$, $10^{-5}$ M of SS, respectively (n=3∼6; $mean{\pm}SEM$). The steady-state activation and inactivation curves of $I_{Ba}$ as a function of membrane potentials were well fitted by a Boltzmann equation. Voltage of half-activation ($V_{0.5}$) was $-12{\pm}0.5$ mV in control and $-11{\pm}1.9$ mV in SS treated groups (respectively, n=5). The same values of half-inactivation were $-35{\pm}1.4$ mV and $-35{\pm}1.9$ mV (respectively, n=5). There was no significant difference in activation and inactivation kinetics of $I_{Ba}$ by SS. Inhibitory effect of SS on $I_{Ba}$ was significantly reduced by either dialysis of intracellular solution with $GDP_{\beta}S$, a non-hydrolysable G protein inhibitor, or pretreatment with pertussis toxin (PTX). SS also decreased contraction of guinea-pig gastric antral smooth muscle. In conclusion, SS decreases voltage-dependent L-type calcium channel current ($VDCC_L$) via PTXsensitive signaling pathways in guinea-pig antral circular myocytes.

• The Korean Journal of Physiology and Pharmacology
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• 제20권3호
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• pp.315-324
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• 2016

Human cardiac fibroblasts (HCFs) have various voltage-dependent $K^+$ channels (VDKCs) that can induce apoptosis. Hydrogen peroxide ($H_2O_2$) modulates VDKCs and induces oxidative stress, which is the main contributor to cardiac injury and cardiac remodeling. We investigated whether $H_2O_2$ could modulate VDKCs in HCFs and induce cell injury through this process. In whole-cell mode patch-clamp recordings, application of $H_2O_2$ stimulated $Ca^{2+}-activated$ $K^+$ ($K_{Ca}$) currents but not delayed rectifier $K^+$ or transient outward $K^+$ currents, all of which are VDKCs. $H_2O_2-stimulated$ $K_{Ca}$ currents were blocked by iberiotoxin (IbTX, a large conductance $K_{Ca}$ blocker). The $H_2O_2-stimulating$ effect on large-conductance $K_{Ca}$ ($BK_{Ca}$) currents was also blocked by KT5823 (a protein kinase G inhibitor) and 1 H-[1, 2, 4] oxadiazolo-[4, 3-a] quinoxalin-1-one (ODQ, a soluble guanylate cyclase inhibitor). In addition, 8-bromo-cyclic guanosine 3', 5'-monophosphate (8-Br-cGMP) stimulated $BK_{Ca}$ currents. In contrast, KT5720 and H-89 (protein kinase A inhibitors) did not block the $H_2O_2-stimulating$ effect on $BK_{Ca}$ currents. Using RT-PCR and western blot analysis, three subtypes of $K_{Ca}$ channels were detected in HCFs: $BK_{Ca}$ channels, small-conductance $K_{Ca}$ ($SK_{Ca}$) channels, and intermediate-conductance $K_{Ca}$ ($IK_{Ca}$) channels. In the annexin V/propidium iodide assay, apoptotic changes in HCFs increased in response to $H_2O_2$, but IbTX decreased $H_2O_2$-induced apoptosis. These data suggest that among the VDKCs of HCFs, $H_2O_2$ only enhances $BK_{Ca}$ currents through the protein kinase G pathway but not the protein kinase A pathway, and is involved in cell injury through $BK_{Ca}$ channels.

• The Korean Journal of Physiology and Pharmacology
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• 제14권6호
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• pp.419-425
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• 2010

Mast cells are activated by specific allergens and also by various nonspecific stimuli, which might induce physical urticaria. This study investigated the functional expression of temperature sensitive transient receptor potential vanilloid (TRPV) subfamily in the human mast cell line (HMC-1) using whole-cell patch clamp techniques. The temperature of perfusate was raised from room temperature (RT, $23{\sim}25^{\circ}C$) to a moderately high temperature (MHT, $37{\sim}39^{\circ}C$) to activate TRPV3/4, a high temperature (HT, $44{\sim}46^{\circ}C$) to activate TRPV1, or a very high temperature (VHT, $53{\sim}55^{\circ}C$) to activate TRPV2. The membrane conductance of HMC-1 was increased by MHT and HT in about 50% (21 of 40) of the tested cells, and the I/V curves showed weak outward rectification. VHT-induced current was 10-fold larger than those induced by MHT and HT. The application of the TRPV 4 activator $3{\alpha}$-phorbol 12,13-didecanoate ($4{\alpha}$ PDD, $1\;{\mu}M$) induced weakly outward rectifying currents similar to those induced by MHT. However, the TRPV3 agonist camphor or TRPV1 agonist capsaicin had no effect. RT-PCR analysis of HMC-1 demonstrated the expression of TRPV4 as well as potent expression of TRPV2. The $[Ca^{2+}]_c$ of HMC-1 cells was also increased by MHT or by $4{\alpha}$ PDD. In summary, our present study indicates that HMC-1 cells express $Ca^{2+}$-permeable TRPV4 channels in addition to the previously reported expression of TRPV2 with a higher threshold of activating temperature.

• The Korean Journal of Physiology and Pharmacology
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• 제14권3호
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• pp.119-125
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• 2010

We investigated the effects of a hot-water extract of Artemisia iwayomogi, a plant belonging to family Compositae, on cardiac ventricular delayed rectifier $K^+$ current ($I_K$) using the patch clamp technique. The carbohydrate fraction AIP1 dose-dependently increased the heart rate with an apparent $EC_{50}$ value of $56.1{\pm}5.5\;{\mu}g/ml$. Application of AIP1 reduced the action potential duration (APD) in concentration-dependent fashion by activating $I_K$ without significantly altering the resting membrane potential ($IC_{50}$ value of $APD_{50}$: $54.80{\pm}2.24$, $IC_{50}$ value of $APD_{90}$: $57.45{\pm}3.47\;{\mu}g/ml$). Based on the results, all experiments were performed with $50\;{\mu}g/ml$ of AIP1. Pre-treatment with the rapidly activating delayed rectifier $K^+$ current ($I_{Kr}$) inhibitor, E-4031 prolonged APD. However, additional application of AIP1 did not reduce APD. The inhibition of slowly activating delayed rectifier $K^+$ current ($I_{Ks}$) by chromanol 293B did not change the effect of AIP1. AIP1 did not significantly affect coronary arterial tone or ion channels, even at the highest concentration of AIP1. In summary, AIP1 reduces APD by activating $I_{Kr}$ but not $I_{Ks}$. These results suggest that the natural product AIP1 may provide an adjunctive therapy of long QT syndrome.

• The Korean Journal of Physiology and Pharmacology
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• 제21권3호
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• pp.309-316
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• 2017

Transient receptor potential vanilloid 3 (TRPV3) is a non-selective cation channel with modest permeability to calcium ions. It is involved in intracellular calcium signaling and is therefore important in processes such as thermal sensation, skin barrier formation, and wound healing. TRPV3 was initially proposed as a warm temperature sensor. It is activated by synthetic small-molecule chemicals and plant-derived natural compounds such as camphor and eugenol. Schisandra chinensis (Turcz.) Baill (SC) has diverse pharmacological properties including antiallergic, anti-inflammatory, and wound healing activities. It is extensively used as an oriental herbal medicine for the treatment of various diseases. In this study, we investigated whether SC fruit extracts and seed oil, as well as four compounds isolated from the fruit can activate the TRPV3 channel. By performing whole-cell patch clamp recording in HEK293T cells overexpressing TRPV3, we found that the methanolic extract of SC fruit has an agonistic effect on the TRPV3 channel. Furthermore, electrophysiological analysis revealed that ${\gamma}$-schisandrin, one of the isolated compounds, activated TRPV3 at a concentration of $30{\mu}M$. In addition, ${\gamma}$-schisandrin (${\sim}100{\mu}M$) increased cytoplasmic $Ca^{2+}$ concentrations by approximately 20% in response to TRPV3 activation. This is the first report to indicate that SC extract and ${\gamma}$-schisandrin can modulate the TRPV3 channel. This report also suggests a mechanism by which ${\gamma}$-schisandrin acts as a therapeutic agent against TRPV3-related diseases.

• The Korean Journal of Physiology and Pharmacology
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• 제8권4호
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• pp.219-225
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• 2004

The pelvic ganglia provide autonomic innervations to the various urogenital organs, such as the urinary bladder, prostate, and penis. It is well established that both sympathetic and parasympathetic synaptic transmissions in autonomic ganglia are mediated mainly by acetylcholine (ACh). Until now, however, the properties of ACh-induced currents and its receptors in pelvic ganglia have not clearly been elucidated. In the present study, biophysical characteristics and molecular nature of nicotinic acetylcholine receptors (nAChRs) were studied in sympathetic and parasympathetic major pelvic ganglion (MPG) neurons. MPG neurons isolated from male rat were enzymatically dissociated, and ionic currents were recorded by using the whole cell variant patch clamp technique. Total RNA from MPG neuron was prepared, and RT-PCR analysis was performed with specific primers for subunits of nAChRs. ACh dose-dependently elicited fast inward currents in both sympathetic and parasympathetic MPG neurons $(EC_{50};\;41.4\;{\mu}M\;and\;64.0\;{\mu}M,\;respectively)$. ACh-induced currents showed a strong inward rectification with a reversal potential near 0 mV in current-voltage relationship. Pharmacologically, mecamylamine as a selective antagonist for ${\alpha}3{\beta}4$ nAChR potently inhibited the ACh-induced currents in sympathetic and parasympathetic neurons $(IC_{50};\;0.53\;{\mu}M\;and\;0.22\;{\mu}M,\;respectively)$. Conversely, ${\alpha}-bungarotoxin$, ${\alpha}-methyllycaconitine$, and $dihydro-{\beta}-erythroidine$, which are known as potent and sensitive blockers for ${\alpha}7$ or ${\alpha}4{\beta}2$ nAChRs, below micromolar concentrations showed negligible effect. RT-PCR analysis revealed that ${\alpha}3$ and ${\beta}4$ subunits were predominantly expressed in MPG neurons. We suggest that MPG neurons have nAChRs containing ${\alpha}3$ and ${\beta}4$ subunits, and that their activation induces fast inward currents, possibly mediating the excitatory synaptic transmission in pelvic autonomic ganglia.

• The Korean Journal of Physiology and Pharmacology
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• 제22권2호
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• pp.173-182
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• 2018

Recent studies have provided several lines of evidence that peripheral administration of oxytocin induces analgesia in human and rodents. However, the exact underlying mechanism of analgesia still remains elusive. In the present study, we aimed to identify which receptor could mediate the analgesic effect of intraperitoneal injection of oxytocin and its cellular mechanisms in thermal pain behavior. We found that oxytocin-induced analgesia could be reversed by $d(CH_2)_5[Tyr(Me)^2,Dab^5]$ AVP, a vasopressin-1a (V1a) receptor antagonist, but not by $desGly-NH_2-d(CH_2)_5[D-Tyr^2,Thr^4]OVT$, an oxytocin receptor antagonist. Single cell RT-PCR analysis revealed that V1a receptor, compared to oxytocin, vasopressin-1b and vasopressin-2 receptors, was more profoundly expressed in dorsal root ganglion (DRG) neurons and the expression of V1a receptor was predominant in transient receptor potential vanilloid 1 (TRPV1)-expressing DRG neurons. Fura-2 based calcium imaging experiments showed that capsaicin-induced calcium transient was significantly inhibited by oxytocin and that such inhibition was reversed by V1a receptor antagonist. Additionally, whole cell patch clamp recording demonstrated that oxytocin significantly increased potassium conductance via V1a receptor in DRG neurons. Taken together, our findings suggest that analgesic effects produced by peripheral administration of oxytocin were attributable to the activation of V1a receptor, resulting in reduction of TRPV1 activity and enhancement of potassium conductance in DRG neurons.

• 한국산학기술학회논문지
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• 제16권3호
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• pp.1964-1971
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• 2015

사람의 골수 또는 제대정맥에서 유래된 중간엽 줄기 세포 (hBM-MSC 또는 hUC-MSC)는 임상적 치료 적용에 매우 유용한 세포유형으로 알려져 왔다. 우리는 이러한 세포에서 two-pore 도메인 포타슘 (K2P)채널을 조사하였다. K2P 채널은 다양한 세포유형들에서 안정막 전위를 형성하는데 중요한 역할을 한다. 그들 중 TREK1은 수소, 저산소증, 다불포화 지방산, 항우울제 및 신경전달물질들의 표적이다. 우리는 RT-PCR 분석과 팻취고정기법을 이용하여 hBM-MSCs와 hUC-MSC가 기능적인 TREK1 채널을 발현하는지 조사했다. hBM-MSCs와 hUC-MSCs에서 100 pS 단일 채널 전도도를 가진 포타슘채널이 발견되었고, 그 채널은 세포막 신전 (-5 mmHg ~ -15 mmHg), 아라키도닉산 ($10{\mu}M$), 세포내 산성화 (pH 6.0)에 의해 활성화 되었다. 이러한 전기생리학적 성질은 TREK1과 유사하였다. 우리의 결과는 안정막 전위에 기여하는 TREK1 채널이 hBM-MSC와 hUC-MSC에 기능적으로 존재하고 있음을 제시한다.

• The Korean Journal of Physiology and Pharmacology
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• 제8권6호
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• pp.335-338
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• 2004

There are numerous studies on transepithelial transports in duct cells including $Cl^-$ and/or $HCO_3^-$. However, studies on transepithelial $K^+$ transport of normal duct cells in exocrine glands are scarce. In the present study, we examined the characteristics of $K^+$ currents in single duct cells isolated from guinea pig pancreas, using a whole-cell patch clamp technique. Both $Cl^-$ and $K^+$ conductance were found with KCI rich pipette solutions. When the bath solution was changed to low $Cl^-$, reversal potentials shifted to the negative side, $-75{\pm}4\;mV$, suggesting that this current is dominantly selective to $K^+$. We then characterized this outward rectifying $K^+$ current and examined its $Ca^{2+}$ dependency. The $K^+$ currents were activated by intracellular $Ca^{2+}$. 100 nM or 500 nM $Ca^{2+}$ in pipette significantly (P<0.05) increased outward currents (currents were normalized, $76.8{\pm}7.9\;pA$, n=4 or $107.9{\pm}35.5\;pA$, n=6) at +100 mV membrane potential, compared to those with 0 nM $Ca^{2+}$ in pipette $(27.8{\pm}3.7\;pA,\;n=6)$. We next examined whether this $K^+$ current, recorded with 100 nM $Ca^{2+}$ in pipette, was inhibited by various inhibitors, including $Ba^{2+}$, TEA and iberiotoxin. The currents were inhibited by $40.4{\pm}%$ (n=3), $87.0{\pm}%$ (n=5) and $82.5{\pm}%$ (n=9) by 1 mM $Ba^{2+}$, 5 mM TEA and 100 nM iberiotoxin, respectively. Particularly, an almost complete inhibition of the current by 100 nM iberiotoxin further confirmed that this current was activated by intracellular $Ca^{2+}$. The $K^+$ current may play a role in secretory process, slnce recycling of $K^+$ is critical for the initiation and sustaining of $CI^-$ or $HCO_3^-$ secretion in these cells.