• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.18-18
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• 2001

Despite of importance of integrated events of nucleus and microtubule remodeling in nuclear transferred embryos with somatic cells, little information is available on this subject. In this study we configured chromatin and microtubule organization following somatic cell nuclear transfer in pre- and non-activated bovine oocytes in order to clearify nuclear remodeling process and to demonstrate centrosome inheritance during nuclear transfer. The cumulus-oocyte complexes were collected from slaughterhouse and were matured in vitro for 20 h in TCM 199 supplemented hormone. Matured bovine oocytes were enucleated by aspirating the frist polar body and metaphase chromatin using a beveled pipette. Bovine fibroblast cells were fused into enucleated oocyte by electrical stimulation. Reconstructed oocytes were activated with ionomycine and 6-dimethylaminopurin, and then cultured in CRlaa medium. The organization of nuclear and microtubules were observed using laser-scanning confocal microscopy. At 1 hour after fusion, microtubule aster was seen near the transferred nucleus in most oocytes regardless activation condition. While most of fibroblast nuclei remodeled to premature chromosome condensation (PCC) and to the two masses of chromosome in non-activated oocytes, a few number of fibloblasts went to PCC and multiple pronuclear like structures in activated oocytes. Microtubular spindle was seen around condensed chromosome. Gamma-tubulin was detected in the vicinity of condensed chromosome, suggesting this is a transient spindle. The spindle seperated nucleus into two masses of chromatin which developed to the pronuclear like structures. Two pronuclear like structures were than apposed by microtubular aster and formed one syngamy like nuclear structure at 15 h following nuclear transfer. At 17 to 18 h after fusion, two centrosomes were seen near the nucleus, which nucleates micrtubules for two cell cleavage. While 31% of reconstructed oocytes in non-activated condition developed to morulae and blastocysts, a few reconstructed oocytes in pre-activated condition developed to the blastocyst. These results suggested introduction of foreign centrosome during nuclear transfer, which appeared to give an important role for somatic cell nuclear reprogramming.

• The Korean Journal of Physiology and Pharmacology
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• v.9 no.2
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• pp.95-101
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• 2005

In the heart, $Na^{+}-Ca^{2+}$ exchange (NCX) is the major $Ca^{2+}$ extrusion mechanism. NCX has been considered as a relaxation mechanism, as it reduces global $[Ca^{2+}]_i$ raised during activation. However, if NCX locates in the close proximity to the ryanodine receptor, then NCX would curtail $Ca^{2+}$ before its diffusion to global $Ca^{2+}_i$ This will result in a global $[Ca^{2+}]_i$ decrease especially during its ascending phase rather than descending phase. Therefore, NCX would decrease the myocardial contractility rather than inducing relaxation in the heart. This possibility was examined in this study by comparing NCX-induced extrusion of $Ca^{2+}$ after its release from SR in the presence and absence of global $Ca^{2+}_i$ transient in the isolated single rat ventricular myocytes by using patch-clamp technique in a whole-cell configuration. Global $Ca^{2+}_i$ transient was controlled by an internal dialysis with different concentrations of BAPTA added in the pipette. During stimulation with a ramp pulse from +100 mV to -100 mV for 200 ms, global $Ca^{2+}_i$ transient was suppressed only mildly, and completely at 1 mmol/L, and 10 mmol/L BAPTA, respectively. In these situations, ryanodine-sensitive inward NCX current was compared using $100{\mu}mol/L$ ryanodine, $Na^+$ depletion, 5 mmol/L $NaCl_2$ and $1{\mu}mol/L$ nifedipine. Surprisingly, the result showed that the ryanodine-sensitive inward NCX current was well preserved after 10 mmol/L BAPTA to 91 % of that obtained after 1 mmol/L BAPTA. From this result, it is concluded that most of the NCX-induced $Ca^{2+}$ extrusion occurs before the $Ca^{2+}$ diffuses to global $Ca^{2+})i$ in the rat ventricular myocyte.

• The Korean Journal of Physiology and Pharmacology
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• v.16 no.1
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• pp.25-30
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• 2012

Under some pathological conditions as bile flow obstruction or liver diseases with the enterohepatic circulation being disrupted, regurgitation of bile acids into the systemic circulation occurs and the plasma level of bile acids increases. Bile acids in circulation may affect the nervous system. We examined this possibility by studying the effects of bile acids on gating of neuronal (N)-type $Ca^{2+}$ channel that is essential for neurotransmitter release at synapses of the peripheral and central nervous system. N-type $Ca^{2+}$ channel currents were recorded from bullfrog sympathetic neuron under a cell-attached mode using 100 mM $Ba^{2+}$ as a charge carrier. Cholic acid (CA, $10^{-6}M$) that is relatively hydrophilic thus less cytotoxic was included in the pipette solution. CA suppressed the open probability of N-type $Ca^{2+}$ channel, which appeared to be due to an increase in (no activity) sweeps. For example, the proportion of sweep in the presence of CA was ~40% at +40 mV as compared with ~8% in the control recorded without CA. Other single channel properties including slope conductance, single channel current amplitude, open and shut times were not significantly affected by CA being present. The results suggest that CA could modulate N-type $Ca^{2+}$ channel gating at a concentration as low as $10^{-6}M$. Bile acids have been shown to activate nonselective cation conductance and depolarize the cell membrane. Under pathological conditions with increased circulating bile acids, CA suppression of N-type $Ca^{2+}$ channel function may be beneficial against overexcitation of the synapses.

• The Korean Journal of Physiology and Pharmacology
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• v.4 no.5
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• pp.385-391
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• 2000

Using the patch-clamp technique, we investigated the alteration of 4-aminopyridine(4-AP)-sensitive, voltage-dependent $K^+$ channel (Kv) in the mesenteric arterial smooth muscle cell (MASMC) of renovascular hypertensive model, one-kidney one-clip Goldblatt hypertensive rat (GBH). To isolate $K_V$ current, internal pipette solution contained 5 mM ATP and 10 mM EGTA. Under these condition, MASMC was depolarized by 4-AP, but charybdotoxin did not affect membrane potential. Membrane potential of hypertensive cell $(-40.3{\pm}3.2\;mV)$ was reduced when compared to that of normotensive cell $(-59.5{\pm}2.8\;mV).$ Outward $K^+$ current of hypertensive cell was significantly reduced when compared to normotensive cell. At 60 mV, the outward currents were $19.10{\pm}1.91$ and $14.06{\pm}1.05$ pA/pF in normotensive cell and hypertensive cell respectively. 4-AP-sensitive $K^+$ current was also smaller in hypertensive cell $(4.28{\pm}0.38\;pA/pF)$ than in normotensive cell $(7.65{\pm}0.52\;pA/pF).$ The values of half activation voltage $(V_{1/2})$ and slope factor (k1) as well as the values of half inactivation voltage $(V_{1/2})$ and slope factor (k1) were virtually similar between GBH and NTR. These results suggest that the decrease of 4-AP-sensitive $K^+$ current contributes to a depolarization of membrane potential, which leads to development of vascular tone in GBH.

• The Korean Journal of Physiology and Pharmacology
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• v.7 no.6
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• pp.341-348
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• 2003

Mechanical stimuli to the cardiac myocytes initiate many biochemical and physiological events. Stretch-activated cation channels have been suggested to mediate these events. In this study, cell-attached and inside-out excised-patch clamp methods were used to identify stretch-activated cation channels in adult rat atrial myocytes. Channel openings were increased in cell-attached configuration when negative pressure was applied to the pipette, and also in inside-out excised patches by negative pressure. The channel was not permeable to $Cl^-$, $Na^+$ and $Cs^+$, but selectively permeable to $K^+$, and the degree of activation was dependent on the magnitude of negative pressure (full activation at ${\sim} -50 mmHg). In symmetrical 140 mM KCl, the slope conductance was $51.2{\pm}3$ pS between the potentials of -80 and 0 mV and $55{\pm}6$ pS between 0 and +80 mV (n=5). Glibenclamide ($100{mu}M$) or ATP (2 mM) failed to block the channel openings, indicating that it is not ATP-sensitive $K^+$ channel. Arachidonic acid ($30{mu}M$), which has been shown to activate a $K^+$ channel cooperatively with membrane stretch, did not affect the channel activity. $GdCl_3$ ($100{mu}M$) also did not alter the activity. These results demonstrate that the mechanical stretch in rat atrial myocytes activates a novel $K^+$-selective cation channel, which is not associated with other $K^+$ channels such as ATP-sensitive and arachidonic acid-activated $K^+$ channel.

• The Korean Journal of Physiology and Pharmacology
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• v.9 no.1
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• pp.63-68
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• 2005

Hydrogen peroxide ($H_2O_2$) causes oxidative stress and is considered as an inducer of cell death in various tissues. Two-pore domain $K^+$ ($K_{2p}$) channels may mediate $K^+$ efflux during apoptotic volume decreases (AVD) in zygotes and in mouse embryos. In the present study, we sought to elucidate linkage between $K_{2p}$ channels and cell death by $H_2O_2$. Thus $K_{2p}$ channels (TASK-1, TASK-3, TREK-1, TREK-2) were stably transfected in HEK-293 cells, and cytotoxicity assay was preformed using cell counting kit-8 (CCK-8). Cell survival rates were calculated using the cytotoxicity assay data and dose-response curve was fitted to the $H_2O_2$ concentration. Ionic currents were recorded in cell-attached mode. The bath solution was the normal Ringer solution and the pipette solution was high $K^+$ solution. In HEK-293 cells expressing TREK-1, TREK-2, TASK-3, $H_2O_2$ induced cell death did not change in comparison to non-transfected HEK-293. In HEK-293 cells expressing TASK-1, however, dose-response curve was significantly shifted to the left. It means that $H_2O_2$ induced cell death was increased. In cell attached-mode recording, application of $H_2O_2$ (300μM) increased activity of all $K_{2p}$ channels. However, a low concentration of $H_2O_2$ ($50{\mu}M$) increased only TASK-1 channel activity. These results indicate that TASK-1 might participate in $K^+$ efflux by $H_2O_2$ at low concentration, thereby inducing AVD.

• The Korean Journal of Physiology and Pharmacology
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• v.7 no.2
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• pp.53-57
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• 2003

The glutamate receptors (GluRs) are key receptors for modulatory synaptic events in the central nervous system. It has been reported that glutamate increases the intracellular $Ca^{2+}$ concentration ($[Ca^{2+}]_i$) and induces cytotoxicity. In the present study, we investigated whether the glutamate-induced $[Ca^{2+}]_i$ increase was associated with the activation of ionotropic (iGluR) and metabotropic GluRs (mGluR) in substantia gelatinosa neurons, using spinal cord slice of juvenile rats (10${\sim}21 day). $[Ca^{2+}]_i$ was measured using conventional imaging techniques, which was combined with whole-cell patch clamp recording by incorporating fura-2 in the patch pipette. At physiological concentration of extracellular $Ca^{2+}$, the inward current and $[Ca^{2+}]_i$ increase were induced by membrane depolarization and application of glutamate. Dose-response relationship with glutamate was observed in both $Ca^{2+}$ signal and inward current. The glutamate-induced $[Ca^{2+}]_i$ increase at holding potential of -70 mV was blocked by CNQX, an AMPA receptor blocker, but not by AP-5, a NMDA receptor blocker. The glutamate-induced $[Ca^{2+}]_i$ increase in $Ca^{2+}$ free condition was not affected by iGluR blockers. A selective mGluR (group I) agonist, RS-3,5-dihydroxyphenylglycine (DHPG), induced $[Ca^{2+}]_i$ increase at holding potential of -70 mV in SG neurons. These findings suggest that the glutamate-induced $[Ca^{2+}]_i$ increase is associated with AMPA-sensitive iGluR and group I mGluR in SG neurons of rats.

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

• International Journal of Oral Biology
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• v.39 no.4
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• pp.229-236
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• 2014

Reactive oxygen species (ROS) and nitrogen species (RNS) are implicated in cellular signaling processes and as a cause of oxidative stress. Recent studies indicate that ROS and RNS are important signaling molecules involved in nociceptive transmission. Xanthine oxidase (XO) system is a well-known system for superoxide anions ($O{_2}^{{\cdot}_-}$) generation, and sodium nitroprusside (SNP) is a representative nitric oxide (NO) donor. Patch clamp recording in spinal slices was used to investigate the role of $O{_2}^{{\cdot}_-}$ and NO on substantia gelatinosa (SG) neuronal excitability. Application of xanthine and xanthine oxidase (X/XO) compound induced membrane depolarization. Low concentration SNP ($10{\mu}M$) induced depolarization of the membrane, whereas high concentration SNP (1 mM) evoked membrane hyperpolarization. These responses were significantly decreased by pretreatment with phenyl N-tert-butylnitrone (PBN; nonspecific ROS and RNS scavenger). Addition of thapsigargin to an external calcium free solution for blocking synaptic transmission, led to significantly decreased X/XO-induced responses. Additionally, X/XO and SNP-induced responses were unchanged in the presence of intracellular applied PBN, indicative of the involvement of presynaptic action. Inclusion of GDP-${\beta}$-S or suramin (G protein inhibitors) in the patch pipette decreased SNP-induced responses, whereas it failed to decrease X/XO-induced responses. Pretreatment with n-ethylmaleimide (NEM; thiol-alkylating agent) decreased the effects of SNP, suggesting that these responses were mediated by direct oxidation of channel protein, whereas X/XO-induced responses were unchanged. These data suggested that ROS and RNS play distinct roles in the regulation of the membrane excitability of SG neurons related to the pain transmission.

• The Korean Journal of Physiology and Pharmacology
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• v.10 no.1
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• pp.19-24
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• 2006

The intermediate conductance $Ca^{2+}-activated$ $K^+$ channels (SK4, IKCa1) are present in lymphocytes, and their membrane expression is upregulated by various immunological stimuli. In this study, the activity of SK4 was compared between Bal-17 and WEHI-231 cell lines which represent mature and immature stages of murine B lymphocytes, respectively. The whole-cell patch clamp with high-$Ca^{2+}$ ($0.8{\mu}M$) KCl pipette solution revealed a voltage-independent $K^+$ current that was blocked by clotrimazole (1 mM), an SK4 blocker. The expression of mRNAs for SK4 was confirmed in both Bal-17 and WEHI-231 cells. The density of clotrimazole-sensitive SK4 current was significantly larger in Bal-17 than WEHI-231 cells ($-11.4{\pm}3.1$ Vs. $-5.7{\pm}1.15$ pA/pF). Also, the chronic stimulation of B cell receptors (BCR) by BCR-ligation (anti-IgM Ab, $3{\mu}g$/ml, 8∼12 h) significantly upregulated the amplitude of clotrimazolesensitive current from $-11.4{\pm}3.1$ to $-53.1{\pm}8.6$ pA/pF in Bal-17 cells. In WEHI-231 cells, the effect of BCR-ligation was significantly small ($-5.7{\pm}1.15$ to $-9.0{\pm}1.00$ pA/pF). The differential expression and regulation by BCR-ligation might reflect functional changes in the maturation of B lymphocytes.