• Proceedings of the Korean Biophysical Society Conference
• /
• 2003.06a
• /
• pp.37-37
• /
• 2003

We have shown the $Ca^{2+}$-activated chloride current is present in cardiac myocyte in rabbit pulmonary vein (Kim et al., 2002). This current amplitude was increased as [N $a^{+}$]$_{i}$ was increased and we suggested this chloride current may be involve in the spontaneous action potential frequency change. Since this current is activated by the increase of intracellular $Ca^{2+}$, we would like to test what is the inducer of the increase of [C $a^{2+}$]$_{i}$ between a L-type $Ca^{2+}$-current or a reverse mode of N $a^{+}$-C $a^{2+}$ exchange current. White rabbit (1.5 kg) was used and anesthetized with Ketamin (100 mg/kg). Pulmonary vein (PV) was isolated and sleeve area between left atrium and PV was dissected. Using collagenase (Worthington 0.7 mg/cc), single cardiac myocytes were isolated. In the presence of 15 mM of N $a^{＋}$, three steps of voltage pulses were applied (holding potential : -40 ㎷, -80 ㎷ for 50 msec, 30 ㎷ for 5 msec, 10 ㎷ steps from -70 ㎷ to 60 ㎷). The inward and outward tail current was activated after brief 5 msec prepulse. The outward tail current was blocked by the removal of extracellular chloride substituted by glucuronic acid or by a chloride channel blocker, 5 mM 9-AC. But the inward tail current was still remained even though the amplitude was decreased. The reversal potentials were changed to the direction of the change of chloride equilibrium potential ( $E_{Cl}$ ) but the shift of equilibrium potential was not enough to match to the theoretical equilibrium potential shift. In the presence of L-type $Ca^{2+}$ channel blocker, nifedipine 1 uM, inward tail currents were greatly reduced but the outward current tail currents were still remained. In the presence of N $a^{+}$-C $a^{2+}$ exchange current blocker, 10 uM KB-R7943, the inward and outward tail currents were blocked almost completely. We tried to test the $Ca^{2+}$sensitivity of the chloride current with various [C $a^{2+}$]$_{i}$ in pipette solution from 100 nM to 1 uM but we failed to activate $Ca^{2＋}$-activated chloride currents even though the cell became contracted in the presence of 1 uM $Ca^{2+}$. From these results, we could conclude that the increase of [C $a^{2+}$]$_{i}$ to activate the outward $Ca^{2+}$-activated chloride current was mainly induced by the activation of the reverse mode of N $a^{+}$-C $a^{2+}$ exchanger, But for the increase of [C $a^{2+}$]$_{i}$ to activate the inward tail current, L-type $Ca^{2+}$ current may be the major provoking current. Since the cytosolic increase of [C $a^{2+}$]$_{i}$ through pipette solution have failed to activate $Ca^{2+}$-activated chloride current, this chloride current may have very low $Ca^{2+}$ sensitivity or a comparmental increase $Ca^{2+}$ such as in subsarcolemmal space may activate the chloride current. Since there are several reports and models that the increase of $Ca^{2+}$ in subsarcolemmal space would be over several to tens of uM, both possibility may be valid together.uM, both possibility may be valid together.

• Molecules and Cells
• /
• v.27 no.5
• /
• pp.525-531
• /
• 2009

We studied the effect of carbachol on pacemaker currents in cultured interstitial cells of Cajal (ICC) from the mouse small intestine by muscarinic stimulation using a whole cell patch clamp technique and $Ca^{2+}$-imaging. ICC generated periodic pacemaker potentials in the current-clamp mode and generated spontaneous inward pacemaker currents at a holding potential of -70 mV. Exposure to carbachol depolarized the membrane and produced tonic inward pacemaker currents with a decrease in the frequency and amplitude of the pacemaker currents. The effects of carbachol were blocked by 1-dimethyl-4-diphenylacetoxypiperidinium, a muscarinic $M_3$ receptor antagonist, but not by methotramine, a muscarinic $M_2$ receptor antagonist. Intracellular $GDP-{\beta}-S$ suppressed the carbachol-induced effects. Carbachol-induced effects were blocked by external $Na^+$-free solution and by flufenamic acid, a non-selective cation channel blocker, and in the presence of thapsigargin, a $Ca^{2+}$-ATPase inhibitor in the endoplasmic reticulum. However, carbachol still produced tonic inward pacemaker currents with the removal of external $Ca^{2+}$. In recording of intracellular $Ca^{2+}$ concentrations using fluo 3-AM dye, carbachol increased intracellular $Ca^{2+}$ concentrations with increasing of $Ca^{2+}$ oscillations. These results suggest that carbachol modulates the pacemaker activity of ICC through the activation of non-selective cation channels via muscarinic $M_3$ receptors by a G-protein dependent intracellular $Ca^{2+}$ release mechanism.

• Korean Journal of Environmental Biology
• /
• v.18 no.2
• /
• pp.255-262
• /
• 2000

This study was carried out to investigate the physiological changes induced acutely with the low doses of lead acetate in the synaptosomes from the cerebrum and brain stem of the rat. The general uptake patterns of [$^3$H]-serotonin were observed in synaptosomes, as a model of presynaptic nerve terminal, from the cerebrum and brain stem. And the effects of the low doses of lead acetate on the uptake process were investigated id vitro and in vivo. The Km value of the uptake of the [$^3$H]-serotonin by the synaptosomes was 0.5 $\mu$M in the cerebrum and 0.1 $\mu$M in the brain stem. These low values reveal that the synaptosomes from the cerebrum and the brain stem have a high affinity to [$^3$H]-serotonin, especially in brain stem. The uptake of $\mu$M-serotonin was dependant on the sodium and potassium ions. When being treated with ouabain, the $Na^+$ $-K^+$ ATPase inhibitor, the uptake of [$^3$H]-serotonin was reduced. This supports strongly that the uptake of [$^3$H]-serotonin was sensitive to the changes of the concentrations of the sodium and potassium ions. When the calcium channel blocker, verapamil, was treated, the uptake of [$^3$H]-serotonin was changed only in synaptosomes from the brain stem. The uptake of [$^3$H]-serotonin was reduced by the lead treatment in the synaptosomes from the cerebrum and brain stem in vitro and in vivo. [lead acetate, synaptosomes, $^3$H-serotonin, rat]

• The Korean Journal of Physiology and Pharmacology
• /
• v.16 no.5
• /
• pp.327-332
• /
• 2012

Sertraline is a commonly used antidepressant of the selective serotonin reuptake inhibitors (SSRIs) class. In these experiments, we have used the whole cell patch clamp technique to examine the effects of sertraline on the major cardiac ion channels expressed in HEK293 cells and the native voltage-gated $Ca^{2+}$ channels in rat ventricular myocytes. According to the results, sertraline is a potent blocker of cardiac $K^+$ channels, such as hERG, $I_{Ks}$ and $I_{K1}$. The rank order of inhibitory potency was hERG > $I_{K1}$ > $I_{Ks}$ with $IC_{50}$ values of 0.7, 10.5, and 15.2 ${\mu}M$, respectively. In addition to $K^+$ channels, sertraline also inhibited $I_{Na}$ and $I_{Ca}$, and the $IC_{50}$ values are 6.1 and 2.6 ${\mu}M$, respectively. Modification of these ion channels by sertraline could induce changes of the cardiac action potential duration and QT interval, and might result in cardiac arrhythmia.

• Natural Product Sciences
• /
• v.19 no.4
• /
• pp.290-296
• /
• 2013

In preliminary tests, we examined the effect of several fractions isolated from fermented pine needle extract on pacemaker potentials in cultured interstitial cells of Cajal (ICCs) from the mouse colon using a whole cell patch clamp technique. Among these fractions, Fraction 3 (F3) elicited the most powerful depolarization of membrane. Therefore, the aim of the present study was to investigate the effect of F3 obtained from fermented extract of Pinus densiflora needle on pacemaker potentials in ICCs and to establish its mechanism of action. Colonic ICCs generated spontaneous periodic pacemaker potentials in the current-clamp mode. F3 depolarized the membrane and decreased the frequency and amplitude of pacemaker potentials in a dose-dependent fashion. The F3-induced effects on pacemaker potentials were blocked by methoctramine, a muscarinic $M_2$ receptor antagonist, and by glycopyrrolate, a muscarinic $M_3$ receptor antagonist. The F3-induced effects on pacemaker potentials were blocked by external $Na^+$-free solution and by flufenamic acid, a non-selective cation channel blocker, as well as by the removal of external $Ca^{2+}$ and in the presence of thapsigargin, a $Ca^{2+}$-ATPase inhibitor in the endoplasmic reticulum. Taken together, these results suggest that F3 of pine needle extract modulates the pacemaker activity of colonic ICCs by the activation of non-selective cation channels via muscarinic $M_2$ and $M_3$ receptors. And external $Ca^{2+}$ influx and intracellular $Ca^{2+}$ release are involved in F3 actions on ICCs.

• International Journal of Oral Biology
• /
• v.33 no.4
• /
• pp.217-221
• /
• 2008

Bicuculline is one of the most commonly used $GABA_A$ receptor antagonists in electrophysiological research. Because of its poor water solubility, bicuculline quaternary ammonium salts such as bicuculline methiodide (BMI) and bicuculline methbromide are preferred. However, a number of studies have shown that BMI has non-$GABA_A$ receptor-mediated effects. The substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) is implicated in the processing of nociceptive signaling. In this study, we investigated whether BMI has non-GABA receptor-mediated activity in Vc SG neurons using a whole cell patch clamp technique. SG neurons were depolarized by application of BMI ($20{\mu}M$) using a high $Cl^-$ pipette solution. GABA ($30-100{\mu}M$) also induced membrane depolarization of SG neuron. Although BMI is known to be a $GABA_A$ receptor antagonist, GABA-induced membrane depolarization was enhanced by co-application with BMI. However, free base bicuculline (fBIC) and picrotoxin (PTX), a $GABA_A$ and $GABA_C$ receptor antagonist, blocked the GABA-induced response. Furthermore, BMI-induced membrane depolarization persisted in the presence of PTX or an antagonist cocktail consisting of tetrodotoxin ($Na^+$ channel blocker), AP-5 (NMDA receptor antagonist), CNQX (non-NMDA receptor antagonist), and strychnine (glycine receptor antagonist). Thus BMI induces membrane depolarization by directly acting on postsynaptic Vc SG neurons in a manner which is independent of $GABA_A$ receptors. These results suggest that other unknown mechanisms may be involved in BMI-induced membrane depolarization.

• Journal of Ginseng Research
• /
• v.36 no.1
• /
• 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.

• Journal of Ginseng Research
• /
• v.35 no.2
• /
• pp.219-225
• /
• 2011

Korean red ginseng (KRG) is a valuable and important traditional medicine in East Asian countries and is currently used extensively for botanical products in the world. KRG has both stimulatory and inhibitory effects on the central nervous system (CNS) suggesting its complicated action mechanisms. The substantia gelatinosa (SG) neurons of the trigeminal subnucleus caudalis (Vc) are involved in orofacial nociceptive processing. Some studies reported that KRG has antinociceptive effects, but there are few reports of the functional studies of KRG on the SG neurons of the Vc. In this study, a whole cell patch clamp study was performed to examine the action mechanism of a KRG extract on the SG neurons of the Vc from juvenile mice. KRG induced short-lived and repeatable inward currents on all the SG neurons tested in the high chloride pipette solution. The KRG-induced inward currents were concentration dependent and were maintained in the presence of tetrodotoxin, a voltage gated $Na^+$ channel blocker. The KRG-induced inward currents were suppressed by 6-cyano-7-nitroquinoxaline-2,3-dione, a non-N-methyl-D-aspartate (NMDA) glutamate receptor antagonist and/or picrotoxin, a gamma-aminobutyric acid $(GABA)_A$ receptor antagonist. However, the inward currents were not suppressed by d,l-2-amino-5-phosphonopentanoic acid, an NMDA receptor antagonist. These results show that KRG has excitatory effects on the SG neurons of the Vc via the activation of non-NMDA glutamate receptor as well as an inhibitory effect by activation of the $GABA_A$ receptor, indicating the KRG has both stimulatory and inhibitory effects on the CNS. In addition, KRG may be a potential target for modulating orofacial pain processing.

• The Korean Journal of Physiology and Pharmacology
• /
• v.15 no.5
• /
• pp.285-289
• /
• 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.

• The Korean Journal of Physiology and Pharmacology
• /
• v.24 no.5
• /
• pp.433-440
• /
• 2020

The substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) is the first relay site for the orofacial nociceptive inputs via the thin myelinated Aδ and unmyelinated C primary afferent fibers. Borneol, one of the valuable time-honored herbal ingredients in traditional Chinese medicine, is a popular treatment for anxiety, anesthesia, and antinociception. However, to date, little is known as to how borneol acts on the SG neurons of the Vc. To close this gap, the whole-cell patch-clamp technique was applied to elucidate the antinociceptive mechanism responding for the actions of borneol on the SG neurons of the Vc in mice. In the voltage-clamp mode, holding at -60 mV, the borneol-induced non-desensitizing inward currents were not affected by tetrodotoxin, a voltage-gated Na⁺ channel blocker, 6-cyano-7-nitro-quinoxaline-2,3-dione, a non-N-methyl-ᴅ-aspartate (NMDA) glutamate receptor antagonist and DL-2-amino-5-phosphonopentanoic acid, an NMDA receptor antagonist. However, borneol-induced inward currents were partially decreased in the presence of picrotoxin, a γ-aminobutyric acid (GABA)_A receptor antagonist, or strychnine, a glycine receptor antagonist, and was almost suppressed in the presence of picrotoxin and strychnine. Though borneol did not show any effect on the glycine-induced inward currents, borneol enhanced GABA-mediated responses. Beside, borneol enhanced the GABA-induced hyperpolarization under the current-clamp mode. Altogether, we suggest that borneol contributes in part toward mediating the inhibitory GABA and glycine transmission on the SG neurons of the Vc and may serve as an herbal therapeutic for orofacial pain ailments.