• Title, Summary, Keyword: whole cell patch clamp

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The Effect of Tyrosine Kinase Inhibitors on the L-type Calcium Current in Rat Basilar Smooth Muscle Cells

  • Bai, Guang-Yi;Yang, Tae-Ki;Gwak, Yong-Geun;Kim, Chul-Jin
    • Journal of Korean Neurosurgical Society
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    • v.39 no.3
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    • pp.215-220
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    • 2006
  • Objective : Tyrosine kinase inhibitors may be useful in the management of cerebral vasospasm. It has not yet been reported whether L-type $Ca^{2+}$ channels playa role in tyrosine kinase inhibitors-induced vascular relaxation of cerebral artery. This study was undertaken to clarify the role of L-type $Ca^{2+}$ channels in tyrosine kinase inhibitors-induced vascular relaxation, and to investigate the effect of tyrosine kinase inhibitors on L-type $Ca^{2+}$ channels currents in freshly isolated smooth muscle cells from rat basilar artery. Methods : The isolation of rat basilar smooth muscle cells was performed by special techniques. The whole cell currents were recorded by whole cell patch clamp technique in freshly isolated smooth muscle cells from rat basilar artery. Results : Patch clamp studies revealed a whole-cell current which resembles the L-type $Ca^{2+}$ current reported by others. The amplitude of this current was decreased by nimodipine and increased by Bay K 8644. Genistein[n=5], tyrphostin A-23[n=3]. A-25[n=6] $30{\mu}M$ reduced the amplitude of the L -type $Ca^{2+}$ channel current in whole cell mode. In contrast, diadzein $30{\mu}M$ [n=3]. inactive analogue of genistein, did not decrease the amplitude of the L-type $Ca^{2+}$ channels current. Conclusion : These results suggest that tyrosine kinase inhibitors such as genistein, tyrphostin A-23, A-25 may relax cerebral vessel through decreasing level of intracellular calcium, [$Ca^{2+}$]i, by inhibition of L-type $Ca^{2+}$ channel.

Effects of Samchulkunbi-tang in Cultured Interstitial Cells of Cajal of Murine Small Intestine

  • Kim, Jung Nam;Kwon, Young Kyu;Kim, Byung Joo
    • Journal of Physiology & Pathology in Korean Medicine
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    • v.27 no.1
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    • pp.112-117
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    • 2013
  • We studied the modulation of pacemaker activities by Samchulkunbi-tang (SCKB) in cultured interstitial cells of Cajal (ICC) from murine small intestine with the whole-cell patch-clamp technique. Externally applied SCKB produced membrane depolarization in the current-clamp mode. The pretreatment with $Ca^{2+}$-free solution and thapsigargin, a $Ca^{2+}$-ATPase inhibitor in endoplasmic reticulum, abolished the generation of pacemaker potentials and suppressed the SCKB-induced action. The application of flufenamic acid (a nonselective cation channel blocker) abolished the generation of pacemaker potentials by SCKB. However, the application of niflumic acid (a chloride channel blocker) did not inhibit the generation of pacemaker potentials by SCKB. In addition, the membrane depolarizations were inhibited by not only GDP-${\beta}$-S, which permanently binds G-binding proteins, but also U-73122, an active phospholipase C inhibitor. These results suggest that SCKB modulates the pacemaker activities by nonselective cation channels and external $Ca^{2+}$ influx and internal $Ca^{2+}$ release via G-protein and phospholipase C-dependent mechanism. Therefore, the ICC are targets for SCKB and their interaction can affect intestinal motility.

Effects of Phenylephrine on the Excitability of Medial Vestibular Nuclear Neurons in Rats

  • Jeong, Han-Seong;Huh, Hae-Ryong;Jang, Myung-Joo;Hong, Seol-Hee;Jang, Su-Jeong;Park, Jin;Lee, Seung-Han;Kim, Jae-Ha;Park, Jong-Seong
    • The Korean Journal of Physiology and Pharmacology
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    • v.10 no.3
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    • pp.131-135
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    • 2006
  • Coeruleo-vestibular pathway which connects locus coeruleus and vestibular nuclei is noradrenergic. This study was designed to elucidate the effects of phenylephrine on the spontaneous activity of acutely isolated medial vestibular nuclear neurons of rat by whole-cell patch-clamp technique. Sprague-Dawley rats, aged 14 to 16 days, were used. After enzymatic digestion, dissociated medial vestibular neurons were transferred to a recording chamber mounted on an inverted microscope, and spontaneous action potentials were recorded by standard patch-clamp techniques. In current-clamp mode, the frequency of spontaneous action potential of medial vestibular nuclear neurons was decreased by phenylephrine (n=15). Phenylephrine increased the amplitude of afterhyperpolarization without changes in the resting membrane potential and spike width. In voltage-clamp mode, the whole potassium currents of the medial vestibular nuclear neurons were increased by phenylephrine (n=12). These experimental results suggest that ${\alpha}-receptor$ mediates the inhibitory effects on the neuronal activity of the medial vestibular nuclear neuron.

Effect of Fluid Pressure on L-type $Ca^{2+}$ Current in Rat Ventricular Myocytes (백서 심실 근세포 L형 $Ca^{2+}$ 전류에 대한 유체압력의 효과)

  • Lee Sun-Woo;Woo Sun-Hee
    • YAKHAK HOEJI
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    • v.50 no.2
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    • pp.111-117
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    • 2006
  • Cardiac chambers serve as mechanosensory systems during the haemodynamic or mechanical disturbances. To examine a possible role of fluid pressure (FP) in the regulatien of atrial $Ca^{2+}$ signaling we investigated the effect of FP on L-type $Ca^{2+}$ current $(I_{Ca})$ in rat ventricular myocytes using whole-cell patch-clamp technique. FP $(\sim40cm\;H_2O)$ was applied to whole area of single myocytes with electronically controlled micro-jet system. FP suppressed the magnitude of peak $I_{Ca}$ by $\cong25\%$ at 0 mV without changing voltage dependence of the current-voltage relationship. FP significantly accelerated slow component in inactivation of $I_{Ca}$, but not its fast component. Analysis of steady-state inactivation curve revealed a reduction of the number of $Ca^{2+}$ channels available for activity in the presence of FP. Dialysis of myocytes with high concentration of immobile $Ca^{2+}$ buffer partially attenuated the FP-induced suppression of $I_{Ca}$. In addition, the intracellular $Ca^{2+}$ buttering abolished the FP-induced acceleration of slow component in $I_{Ca}$ inactivation. These results indicate that FP sup-presses $Ca^{2+}$ currents, in part, by increasing cytosolic $Ca^{2+}$ concentration.

Inhibition of Pacemaker Activity of Interstitial Cells of Cajal by Hydrogen Peroxide via Activating ATP-sensitive $K^+$ Channels

  • Choi Seok;Parajuli Shankar Prasad;Cheong Hyeon-Sook;Paudyal Dilli Parasad;Yeum Cheol-Ho;Yoon Pyung-Jin;Jun Jae-Yeoul
    • The Korean Journal of Physiology and Pharmacology
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    • v.11 no.1
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    • pp.15-20
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    • 2007
  • To investigate whether hydrogen peroxide($H_2O_2$) affects intestinal motility, pacemaker currents and membrane potential were recorded in cultured interstitial cells of Cajal(ICC) from murine small intestine by using a whole-cell patch clamp. In whole cell patch technique at $30^{\circ}C$, ICC generated spontaneous pacemaker potential under current clamp mode(I=0) and inward currents(pacemaker currents) under voltage clamp mode at a holding potential of -70 mV. When ICC were treated with $H_2O_2$ in ICC, $H_2O_2$ hyperpolarized the membrane potential under currents clamp mode and decreased both the frequency and amplitude of pacemaker currents and increased the resting currents in outward direction under voltage clamp mode. Also, $H_2O_2$ inhibited the pacemaker currents in a dose-dependent manner. Because the properties of $H_2O_2$ action on pacemaker currents were same as the effects of pinacidil(ATP-sensitive $K^+$ channels opener), we tested the effects of glibenclamide(ATP-sensitive $K^+$ channels blocker) on $H_2O_2$ action in ICC, and found that the effects of $H_2O_2$ on pacemaker currents were blocked by co- or pre- treatment of glibenclamide. These results suggest that $H_2O_2$ inhibits pacemaker currents of ICC by activating ATP-sensitive $K^+$ channels.

Glycine- and GABA-mimetic Actions of Shilajit on the Substantia Gelatinosa Neurons of the Trigeminal Subnucleus Caudalis in Mice

  • Yin, Hua;Yang, Eun-Ju;Park, Soo-Joung;Han, Seong-Kyu
    • The Korean Journal of Physiology and Pharmacology
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    • v.15 no.5
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    • pp.285-289
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    • 2011
  • Shilajit, a medicine herb commonly used in Ayurveda, has been reported to contain at least 85 minerals in ionic form that act on a variety of chemical, biological, and physical stressors. The substantia gelatinosa (SG) neurons of the trigeminal subnucleus caudalis (Vc) are involved in orofacial nociceptive processing. Shilajit has been reported to be an injury and muscular pain reliever but there have been few functional studies of the effect of Shilajit on the SG neurons of the Vc. Therefore, whole cell and gramicidin-perfotrated patch clamp studies were performed to examine the action mechanism of Shilajit on the SG neurons of Vc from mouse brainstem slices. In the whole cell patch clamp mode, Shilajit induced short-lived and repeatable inward currents under the condition of a high chloride pipette solution on all the SG neurons tested. The Shilajit-induced inward currents were concentration dependent and maintained in the presence of tetrodotoxin (TTX), a voltage gated $Na^+$ channel blocker, CNQX, a non-NMDA glutamate receptor antagonist, and AP5, an NMDA receptor antagonist. The Shilajit-induced responses were partially suppressed by picrotoxin, a $GABA_A$ receptor antagonist, and totally blocked in the presence of strychnine, a glycine receptor antagonist, however not affected by mecamylamine hydrochloride (MCH), a nicotinic acetylcholine receptor antagonist. Under the potassium gluconate pipette solution at holding potential 0 mV, Shilajit induced repeatable outward current. These results show that Shilajit has inhibitory effects on the SG neurons of Vc through chloride ion channels by activation of the glycine receptor and $GABA_A$ receptor, indicating that Shilajit contains sedating ingredients for the central nervous system. These results also suggest that Shilajit may be a potential target for modulating orofacial pain processing.

GABAA Receptor- and Non-NMDA Glutamate Receptor-Mediated Actions of Korean Red Ginseng Extract on the Gonadotropin Releasing Hormone Neurons

  • Cho, Dong-Hyu;Bhattarai, Janardhan Prasad;Han, Seong-Kyu
    • Journal of Ginseng Research
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    • v.36 no.1
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    • pp.47-54
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    • 2012
  • Korean red ginseng (KRG) has been used worldwide as a traditional medicine for the treatment of various reproductive diseases. Gonadotropin releasing hormone (GnRH) neurons are the fundamental regulators of pulsatile release of gonadotropin required for fertility. In this study, an extract of KRG (KRGE) was applied to GnRH neurons to identify the receptors activated by KRGE. The brain slice patch clamp technique in whole cell and perforated patch was used to clarify the effect of KRGE on the membrane currents and membrane potentials of GnRH neurons. Application of KRGE (3 ${\mu}g$/${\mu}L$) under whole cell patch induced remarkable inward currents (56.17${\pm}$7.45 pA, n=25) and depolarization (12.91${\pm}$3.80 mV, n=4) in GnRH neurons under high $Cl^-$ pipette solution condition. These inward currents were not only reproducible, but also concentration dependent. In addition, inward currents and depolarization induced by KRGE persisted in the presence of the voltage gated $Na^+$ channel blocker tetrodotoxin (TTX), suggesting that the responses by KRGE were postsynaptic events. Application of KRGE under the gramicidin perforated patch induced depolarization in the presence of TTX suggesting its physiological significance on GnRH response. Further, the KRGE-induced inward currents were partially blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; non-NMDA glutamate receptor antagonist, 10 ${\mu}M$) or picrotoxin (PIC; $GABA_A$ receptor antagonist, 50 ${\mu}M$), and almost blocked by PIC and CNQX mixture. Taken together, these results suggest that KRGE contains ingredients with possible GABA and non-NMDA glutamate receptor mimetic activity, and may play an important role in the endocrine function of reproductive physiology, via activation of $GABA_A$ and non-NMDA glutamate receptors in GnRH neurons.

Physiological and Pharmacological Characterization of Glutamate and GABA Receptors in the Retina

  • Yang, Xiong-Li;Shen, Ying;Han, Ming-Hu;Lu, Tao
    • The Korean Journal of Physiology and Pharmacology
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    • v.3 no.5
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    • pp.461-469
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    • 1999
  • Glutamate and ${\gamma}-aminobutyric$ acid (GABA) are major excitatory and inhibitory neurotransmitters in the vertebrate retina, respectively. Using the whole-cell patch clamp technique and a rapid solution changer, glutamate and GABA receptors have been extensively investigated in carp retina. Glutamate receptors on both horizontal and amacrine cells may be an AMPA preferring subtype, which predominantly consists of flop splice variants. $GABA_A$ and $GABA_C$ receptors coexist in bipolar cells and they both show significant desensitization. Kinetics analysis demonstrated that activation, deactivation and desensitization of the $GABA_C$ receptor-mediated response of these cells are overall slower than those of the $GABA_A$ response. Endogenous modulator $Zn^{2+}$ in the retina was found to differentially modulate the kinetic characteristics of the $GABA_C$ and $GABA_A$ responses.

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Effect of Metabolic Inhibition on Inward Rectifier K Current in Single Rabbit Ventricular Myocytes (토끼 단일 심근세포에서 대사억제시 Inward Rectifier$(I_{K1})$의 변화)

  • Chung, Yu-Jeong;Ho, Won-Kyung;Earm, Yung-E
    • The Korean Journal of Physiology and Pharmacology
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    • v.1 no.6
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    • pp.741-748
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    • 1997
  • In the present study, we have investigated the effect of metabolic inhibition on the inward rectifier K current ($I_{K1}$). Using whole cell patch clamp technique we applied voltage ramp from +80 mV to -140 mV at a holding potential of -30 mV and recorded the whole cell current in single ventricular myocytes isolated from the rabbit heart. The current-voltage relationship showed N-shape (a large inward current and little outward current with a negative slope) which is a characteristic of $I_{K1}$. Application of 0.2 mM dinitrophenol (DNP, an uncoupler of oxidative phosphorylation as a tool for chemical hypoxia) to the bathing solution with the pipette solution containing 5 mM ATP, produced a gradual increase of outward current followed by a gradual decrease of inward current with little change in the reversal potential (-80 mV). The increase of outward current was reversed by glibenclamide ($10\;{\mu}M$), suggesting that it is caused by the activation of $K_{ATP}$. When DNP and glibenclamide were applied at the same time or glibenclamide was pretreated, DNP produced same degree of reduction in the magnitude of the inward current. These results show that metabolic inhibition induces not only the increase of $K_{ATP}$ channel but also the decrease of $I_{K1}$. Perfusing the cell with ATP-free pipette solution induced the changes very similar to those observed using DNP. Long exposure of DNP (30 min) or ATP-free pipette solution produced a marked decrease of both inward and outward current with a significant change in the reversal potential. Above results suggest that the decrease of $I_{K1}$ may contribute to the depolarisation of membrane potential during metabolic inhibition.

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Protein Kinase Modulates the $GABA_c$ Currents in Cone-horizontal Cell Axon-terminals Isolated from Catfish Retina

  • Paik, Sun-Sook;Lee, Sung-Jong;Jung, Chang-Sub;Bai, Sun-Ho
    • Proceedings of the Korean Biophysical Society Conference
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    • pp.54-54
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    • 1999
  • Protein kinase modulation of gamma-aminobutyric acid C (GABA$_{c}$) currents in freshly dissociated catfish retinal cone-horizontal cell axon-terminals was studied under voltage clamp with the use of the whole cell patch-clamp technique. Responses to pulses of GABA were monitored in intracellular application of adenosin 3',5'-cycle monophophate (cAMP)-dependent protein kinase (PKA) and protein kinase C (PKC) activators, and their inhibitors or inactive analogues.(omitted)d)

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