• 한국응용약물학회:학술대회논문집
• /
• 한국응용약물학회 1996년도 춘계학술대회
• /
• pp.175-175
• /
• 1996

The spinal dorsal horn is the area where primary afferent fibers terminate and cutaneous sensory information is Processed. A number of putative neurotransmitter substances, including excitatory and inhibitory amino acids and peptides, are present in this region and sites and cellular mechanisms of their actions have been a target of numerous studies. In this study, single neurons were acutely isolated and the properties of whole cell current and responses to excitatory and inhibitory neurotransmitters were studied by the patch clamp method. Young rats (7-14 days) were anesthetized with diethyl-ether, and the lumbar spinal cord was excised and cut transversely at a thickness of 30$\mu\textrm{m}$ by Vibroslicer. The treatment of spinal slices with low concentration of proteases (pronase and thermolysin 0.75 mg/$m\ell$) and mechanical dissociation yielded isolated neurons with near intact morphology. Multipolar, ellipsoidal and bipolar, and pyramidal cells were shown. By applying step voltage pulses to neurons held at -70 mV, two types of inward currents and one outward currents observed. The fast activating and inactivating inward current was the Na$\^$+/ current because of its fast kinetics and blocking by 0.5${\mu}$M TTX, a specific blocker of Na$\^$+/ channel. The second type of inward currents were sustained. Based on their kinetics and current-voltage relations, it was likely that the second type of inward current was the voltage-dependent Ca$\^$2+/ current. In the presence of TTX, the steady-state currents mainly represented outward K$\^$+/ current which looked like the delayed rectifier K$\^$+/ current. In addition, the membrane currents produced by agonist of excitatory amino acid (EAA) receptor and the endogenous transmitter candidate L-glutamate were recorded in isolated whole-cell voltage clamped neurons as well as responses to inhibitory amino acids (${\gamma}$-amino butyric acid, glycine). Drugs were applied by a method that allows complete exchange of the solution within 1 sec; an infinite number of solutions can be applied to a single cell.

• The Korean Journal of Physiology and Pharmacology
• /
• 제9권6호
• /
• pp.347-352
• /
• 2005

The effects of nitric oxide (NO) on inhibitory neurotransmitter receptors and some types of inhibitory receptors in dissociated rod bipolar cell (RBC) were investigated. In the whole cell voltage-clamping mode, the gamma-aminobutyric acid (GABA) activated current showed both sustained and transient components. GABA activated transient current was fully blocked by bicuculine, a $GABA_A$ receptor antagonist. The cis-4-aminocrotonic acid (CACA), a $GABA_C$ receptor agonist, evoked the sustained current that was not blocked by bicuculline (BIC). Glycine activated the transient current. These results indicate that the RBCs possess $GABA_A$, $GABA_C$, and glycine inhibitory receptors. Sodium nitroprusside (SNP), a NO analogue, reduced the currents activated by $GABA_A$ receptor only, however, did not reduce the currents activated by either $GABA_C$ or glycine receptors. This study signifies further that only NO depresses the fast inhibitory response activated by $GABA_A$ receptor in RBC. We, therefore, postulate that NO might depress the light-on/off transient inhibitory responses in RBCs in the rat retina.

• The Korean Journal of Physiology and Pharmacology
• /
• 제1권5호
• /
• pp.477-484
• /
• 1997

Intracellular recordings of oscillatory firing (bursting activity) were obtained from Purkinje cells (PCs) in rat cerebellar slices. Apamin inhibited post-burst hyperpolarizations (PBHs) progressively and finally terminated oscillatory firing activity of PCs. Apamin did not affect the amplitude or duration of the after-hyperpolarization (AHP) between spikes within the burst. In the voltage clamp mode, apamin shifted the whole-cell, quasi-steady state I/V relationship in an inward direction and abolished the zero slope resistance (ZSR) region by blocking outward current. Nickel ($Ni^{2+}$) terminated oscillatory activity and also abolished the ZSR region. However, $Ni^{2+}$ did not have progressive blocking action on the post-burst hyperpolarization before it blocked oscillatory activity. $Ni^{2+}$ blocked an inward current at potentials positive to approximately -65 mV, which was responsible for the ZSR region and outward current at more negative potentials. These data indicated that oscillatory activity of PCs is sustained by a balance between a slow $Ni^{2+}$-sensitive inward current and an apamin-sensitive outward current in the region of ZSR of the whole-cell I/V curve.

• The Korean Journal of Physiology
• /
• 제28권2호
• /
• pp.215-224
• /
• 1994

The presence of a calcium current $(i_{Ca^{2+}})$ passed via a specific channel was examined in the unfertilized hamster egg using the whole-cell voltage clamp technique. Pure inward current was isolated using a $Ca^{2+}-rich$ pipette solution containing 10 mM TEA. This current was independent of external $Na^+$ and was highly sensitive to the $Ca^{2+}$ concentration in the bathing solution, indicating that the inward current is carried by $Ca^{2+}$. The maximal amplitude was $-4.12{\pm}0.58nA\;(n=12)$ with 10mM $Ca^{2+}$ at -3OmV from a holding potential of -8OmV. This current reached its maximum within 20ms beyond -3OmV and decayed rapidly with an inactivation time constant $({\tau})$ of 15ms. Activation and inactivation of this $i_{Ca^{2+}}$ was steeply dependent on the membrane potential. The $i_{Ca^{2+}}$ began to activate at the lower voltage of -55 mV and reached its peak at -35 mV, being completely inactivated at potentials more positive than -40 mV. These result suggest that $i_{Ca^{2+}}$ in hamster eggs passes through channels with electrical properties similar to low voltage-activated T-type channels. Other results from the present study support this suggestion; First, the inhibitory effect of $Ni^{2+}\;(IC_{50}=13.7\;{\mu}M)$ was more potent than $Cd^{2+}\;(IC_{50}=123\;{\mu}M)$. Second, $Ba^{2+}$ conductance was equal to or below that of $Ca^{2+}$. Third, $i_{Ca^{2+}}$ in hamster eggs was relatively insensitive to nifedipine $(IC_{50}=96.6\;{\mu}M)$, known to be a specific t-type blocker. The physiological role of $i_{Ca^{2+}}$ in the unfertilized hamster eggs remains unclear. Analysis from steady-state inactivation activation curves reveals that only a small amount of this current will pass in the voltage range $(-70{\sim}-30\;mV)$ which partially overlaps with the resting membrane potential. This current has the property that it can be easily activated by a weak depolarization, thus it may trigger a certain kind of a intracellular event following fertilization which may cause oscillations in the membrane potential.

• The Korean Journal of Physiology and Pharmacology
• /
• 제20권2호
• /
• pp.193-200
• /
• 2016

Sertraline, a selective serotonin reuptake inhibitor (SSRI), has been reported to lead to cardiac toxicity even at therapeutic doses including sudden cardiac death and ventricular arrhythmia. And in a SSRI-independent manner, sertraline has been known to inhibit various voltage-dependent channels, which play an important role in regulation of cardiovascular system. In the present study, we investigated the action of sertraline on Kv1.5, which is one of cardiac ion channels. The effect of sertraline on the cloned neuronal rat Kv1.5 channels stably expressed in Chinese hamster ovary cells was investigated using the whole-cell patch-clamp technique. Sertraline reduced Kv1.5 whole-cell currents in a reversible concentration-dependent manner, with an $IC_{50}$ value and a Hill coefficient of $0.71{\mu}M$ and 1.29, respectively. Sertraline accelerated the decay rate of inactivation of Kv1.5 currents without modifying the kinetics of current activation. The inhibition increased steeply between -20 and 0 mV, which corresponded with the voltage range for channel opening. In the voltage range positive to +10 mV, inhibition displayed a weak voltage dependence, consistent with an electrical distance ${\delta}$ of 0.16. Sertraline slowed the deactivation time course, resulting in a tail crossover phenomenon when the tail currents, recorded in the presence and absence of sertraline, were superimposed. Inhibition of Kv1.5 by sertraline was use-dependent. The present results suggest that sertraline acts on Kv1.5 currents as an open-channel blocker.

• The Korean Journal of Physiology and Pharmacology
• /
• 제10권2호
• /
• pp.71-77
• /
• 2006

The goal of this study was to analyze the effects of genistein, a widely used tyrosine kinase inhibitor, on cloned Shaw-type $K^+$ currents, Kv3.1 which were stably expressed in Chinese hamster ovary (CHO) cells, using the whole-cell configuration of patch-clamp techniques. In whole-cell recordings, genistein at external concentrations from 10 to $100{\mu}M$ accelerated the rate of inactivation of Kv3.1 currents, thereby concentration-dependently reducing the current at the end of depolarizing pulse with an $IC_{50}$ value of $15.71{\pm}0.67{\mu}M$ and a Hill coefficient of $3.28{\pm}0.35$ (n=5). The time constant of activation at a 300 ms depolarizing test pulses from -80 mV to +40 mV was $1.01{\pm}0.04$ ms and $0.90{\pm}0.05$ ms (n=9) under control conditions and in the presence of $20{\mu}M$ genistein, respectively, indicating that the activation kinetics was not significantly modified by genistein. Genistein $(20{\mu}M)$ slowed the deactivation of the tail current elicited upon repolarization to -40 mV, thus inducing a crossover phenomenon. These results suggest that drug unbinding is required before Kv3.1 channels can close. Genistein-induced block was voltage-dependent, increasing in the voltage range $(-20\'mV{\sim}0\'mV)$ for channel opening, suggesting an open channel interaction. Genistein $(20{\mu}M)$ produced use-dependent block of Kv3.1 at a stimulation frequency of 1 Hz. The voltage dependence of steady-state inactivation of Kv3.1 was not changed by $20{\mu}M$ genistein. Our results indicate that genistein blocks directly Kv3.1 currents in concentration-, voltage-, time-dependent manners and the action of genistein on Kv3.1 is independent of tyrosine kinase inhibition.

• 한국의학물리학회지:의학물리
• /
• 제14권1호
• /
• pp.54-67
• /
• 2003

N형 칼슘전류의 비활성화 vs 전압곡선은 U형을 보인다 - 즉 칼슘 내향전류의 크기와 비활성화 정도가 어느 정도 일치한다. 이러한 U형 비활성화는 순수한 전압의존성 기전으로 설명되어져 왔으나 칼슘의존성 비활성화 기전 또한 보고되었다. 이 연구에서는 흰쥐 상행 경동맥 결절뉴론을 단일 세포로 얻은 후, whole cell patch clamp technique를 사용하여 N형 칼슘전류를 기록하고, 세포외액의 charge carrier 로서 바륨과 칼슘을 사용하면서, 칼슘이 N형 칼슘통로의 비활성화에 미치는 역할을 알아보았다. charge carrier 로 칼슘을 사용하였을 경우에 바륨을 사용하였을 때에 비하여 비활성화 정도가 증가하였으며 이러한 증가는 세포속 $Ca^{2＋}$ Chelator가 11 mM EGTA 로부터 20 mM BAPTA 로 치환되어도 계속 관찰되었다. 비활성화 vs 전압 곡선은 바륨과 칼슘 모두에서 U형이었다. charge carrier 를 칼슘으로 치환시 추가로 유도되는 비활성화 정도는 바륨사용시의 비활성화 정도와 역비례관계를 보여 두 이온에서 같은 기전으로 비활성화가 일어날 가능성을 시사하였다. 이러한 가능성을 지원해 주는 결과로 5초의 긴 저분극 자극시 바륨과 칼슘을 써서 얻은 전류기록은 2중 지수함수로 잘 그려낼 수 있었고, 그 결과 빠른 성분(시정수: -150 ms) 과 느린 성분(시정수 -2500 ms) 를 얻었다. 칼슘이 각각의 성분에 미치는 효과는 각기 달라서 빠른 성분의 amplitude는 증가하였고 느린 성분의 시정수는 빨라졌다. 칼슘에 의해 빠른 성분의 amplitude는 증가하였으므로 이는 더 많은 채널이 빠른 경로로 비활성화되었음을 시사한다. 빠른 성분의 시정수는 변화하지 않았으므로, 이는 비촬성화의 빠른 경로는 칼슘과 바륨에서 같음을 시사하며 즉 비활성화 기전이 칼슘의존성이 아님을 보여주는 증거이다. 그러나 비활성화의 느린 성분은 칼슘에 의해 그 시정수가 빨라졌으므로 칼슘의존성일 가능성이 있다.

• The Korean Journal of Physiology and Pharmacology
• /
• 제13권3호
• /
• pp.209-214
• /
• 2009

The striatum receives glutamatergic afferents from the cortex and thalamus, and these synaptic transmissions are mediated by ${\alpha}$-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and N-methyl D-aspartate (NMDA) receptors. The purpose of this study was to characterize glutamate receptors by analyzing NMDA/AMPA ratio and rectification of AMPA and NMDA excitatory postsynaptic currents (EPSCs) using a whole-cell voltage-clamp method in the dorsal striatum. Receptor antagonists were used to isolate receptor or subunit specific EPSC, such as (DL)-2-amino-5-phosphonovaleric acid (APV), an NMDA receptor antagonist, ifenprodil, an NR2B antagonist, CNQX, an AMPA receptor antagonist and IEM-1460, a GluR2-lacking AMPA receptor blocker. AMPA and NMDA EPSCs were recorded at - 70 and + 40 mV, respectively. Rectification index was calculated by current ratio of EPSCs between + 50 and - 50 mV. NMDA/AMPA ratio was 0.20${\pm}$0.05, AMPA receptor ratio of GluR2-lacking/GluR2-containing subunit was 0.26${\pm}$0.05 and NMDA receptor ratio of NR2B/NR2A subunit was 0.32${\pm}$0.03. The rectification index (control 2.39${\pm}$0.27) was decreased in the presence of both APV and combination of APV and IEM-1460 (1.02${\pm}$0.11 and 0.93${\pm}$0.09, respectively). These results suggest that the major components of the striatal glutamate receptors are GluR2-containing AMPA receptors and NR2A-containing NMDA receptors. Our results may provide useful information for corticostriatal synaptic transmission and plasticity studies.

• The Korean Journal of Physiology
• /
• 제26권1호
• /
• pp.15-25
• /
• 1992

In order to elucidate the properties of the background current whole cell patch clamp studies were performed in rabbit ventricular cells. Ramp pulses of ${\pm}80\;mV$ from holding potential of 40 mV(or 20 mV) at the speed of 0.8 V/sec were given every 30 sec(or 10 sec) and current-voltage diagrams(I-V curve) were obtained. For the activation of the background current isoprenaline, adenosine 3',5'-cyclic monophosphate(dBcAMP), guanosine 3',5'-cyclic monophosphate(cGMP), and $N^6$-2'-o-dibutyryladenosine 3',5'-cyclic monophosphate(dBcAMP) were applied after all known current systems were blocked with 2mM Ba, 1 mM Cd ,5 mM Ni, 10 ${\mu}M$ diltiazem, 10 ${\mu}m$ ouabain, and 20 mM tetraethylammonium(TEA). The conductance of background current in control was $0.65{\pm}0.69$ nS at 0 mV, its I-V curves was almost linear and reversed near 50 mV. When there was no taurine in pipette solution, isoprenaline hardly activated the background current but when taurine existed in pipette solution, isoprenaline activated the larger background current. Cyclic AMP or cyclic GMP alone had little effect on the activation of the background current, while cGMP potentiated cGMP effect. When the background current was activated with cGMP and cAMP, isoprenaline could not further increased the background current. The background current activated by isoprenaline depended on extracellular $Cl^-$ concentration and its reversal potential was shifted according to chloride equilibrium potential. The change of extracellular $Na+$ concentration had little effect on reversal potential of the background current activated by isoprenaline.

• Journal of Ginseng Research
• /
• 제36권1호
• /
• pp.47-54
• /
• 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.