• Korean Journal of Veterinary Research
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• v.42 no.1
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• pp.35-43
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• 2002

Cryopreservation is commonly used for an efficient utilization of semen, oocytes and embryos but has disadvantage in the survival, development of the post-thawed eggs. The high risk in the survival, development of eggs after thawing is thought to be caused by inappropriate internal regulation of $Ca^{2+}$ and/or formation of intracellular ice crystals. In this experiment, we tested whether the $Ca^{2+}$ current (iCa), a decisive factor to $Ca^{2+}$ entry, was altered in post-thawed oocytes by using whole cell voltage clamp technique. The quality and survival rates of the oocytes derived from both fresh and frozen groups were examined by morphology and FDA-test. Vitrified oocytes (VOs) were incubated for 4 hr after thawing and then donated to this experiment. Ethyleneglycol-ficoll-galactose (EFG) was used as a cryoprotectant for vitrification. The membrane potential was held at -80 mV and step depolarizations of 250 ms were applied from -50 mV to 50 mV in 10 mV increments. The survival rates showed a higher in VOs vitrified with EFG containing $Ca^{2+}$ than in VOs vitrified with EFG under the $Ca^{2+}$-free condition (82.0% vs 14%). In group with/without $Ca^{2+}$, the survival rates were significantly (P<0.01) difference. In the fresh metaphase II oocytes (FOs), current-voltage (I-V) relationship showed that iCa began to activate at -40 mV and reached its maximum at -10 mV. With same voltage pulses, inward currents were elicited in VOs. I-V relationships observed in VOs were similar to those in FOs. Time constants of activation and inactivation of the inward current shown in VOs were not different to those in FOs. This accordance in I-V relations and time constants in FOs with those in VOs indicates that the inward currents in FOs are unaltered by vitrification and thawing. Therefore, vitrification with EFG does not play as a factor to deteriorate $Ca^{2+}$ entry across the membrane of the oocytes.

• 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.

• The Korean Journal of Physiology and Pharmacology
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• v.7 no.1
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• pp.47-52
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• 2003

The prostate gland contains numerous neuroendocrine cells that are believed to influence the function of the prostate gland. Our recent study demonstrated the expression of both ${\alpha}1$- and ${\alpha}2$-ARs, signaling the release of stored $Ca^{2+}$ and the inhibition of N-type $Ca^{2+}$ channels, respectively, in rat prostate neuroendocrine cells (RPNECs). In this study, the effects of NA on the resting membrane potential (RMP) of RPNECs were investigated using a whole-cell patch clamp method. Fresh RPNECs were dissociated from the ventral lobe of rat prostate and identified from its characteristic shape; round or oval shape with dark cytoplasm. Under zero-current clamp conditions with KCl pipette solution, the resting membrane potential (RMP) of RPNECs was between -35 mV and -85 mV. In those RPNECs with relatively hyperpolarized RMP (＜-60 mV), the application of noradrenaline (NA, $1{\mu}M$) depolarized the membrane to around -40 mV. In contrast, the RPNECs with relatively depolarized RMP (＞-45 mV) showed a transient hyperpolarization and subsequent fluctuation at around -40 mV on application of NA. Under voltage clamp conditions (holding voltage, -40 mV) with CsCl pipette solution, NA evoked a slight inward current (＜-20 pA). NA induced a sharp increase of cytosolic $Ca^{2+}$ concentration ($[Ca^{2+}]_c$), measured by the fura-2 fluorescence, and the voltage clamp study showed the presence of charybdotoxin-sensitive $Ca^{2+}$-activated $K^+$ currents. In summary, adrenergic stimulation induced either depolarization or hyperpolarization of RPNECs, depending on the initial level of RMP. The inward current evoked by NA and the $Ca^{2+}$-activated $K^+$ current might partly explain the depolarization and hyperpolarization, respectively.

• The Korean Journal of Physiology and Pharmacology
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• v.17 no.3
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• pp.181-187
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• 2013

Reactive oxygen species (ROS) are generated in various cells, including vascular smooth muscle and endothelial cells, and regulate ion channel functions. $K_{Ca}3.1$ plays an important role in endothelial functions. However, the effects of superoxide and hydrogen peroxide radicals on the expression of this ion channel in the endothelium remain unclear. In this study, we examined the effects of ROS donors on $K_{Ca}3.1$ expression and the $K^+$ current in primary cultured human umbilical vein endothelial cells (HUVECs). The hydrogen peroxide donor, tert-butyl hydroperoxide (TBHP), upregulated $K_{Ca}3.1$ expression, while the superoxide donors, xanthine/xanthine oxidase mixture (X/XO) and lysophosphatidylcholine (LPC), downregulated its expression, in a concentration-dependent manner. These ROS donor effects were prevented by antioxidants or superoxide dismustase. Phosphorylated extracellular signal-regulated kinase (pERK) was upregulated by TBHP and downregulated by X/XO. In addition, repressor element-1-silencing transcription factor (REST) was downregulated by TBHP, and upregulated by X/XO. Furthermore, $K_{Ca}3.1$ current, which was activated by clamping cells with 1 ${\mu}M$ $Ca^{2+}$ and applying the $K_{Ca}3.1$ activator 1-ethyl-2-benzimidazolinone, was further augmented by TBHP, and inhibited by X/XO. These effects were prevented by antioxidants. The results suggest that hydrogen peroxide increases $K_{Ca}3.1$ expression by upregulating pERK and downregulating REST, and augments the $K^+$ current. On the other hand, superoxide reduces $K_{Ca}3.1$ expression by downregulating pERK and upregulating REST, and inhibits the $K^+$ current. ROS thereby play a key role in both physiological and pathological processes in endothelial cells by regulating $K_{Ca}3.1$ and endothelial function.

• Proceedings of the Korean Biophysical Society Conference
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• 2003.06a
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• pp.46-46
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• 2003

Salviae Miltiorrhizae Radix has been used for treatment of cardiovascular diseases in oriental medicine. To investigate the possible involvement of cardiac ion channel in this effect, we examined electrophysiological effects of the extract of Salviae Miltiorrhizae Radix on action potentials and ionic currents in rat ventricular myocytes. The extracts of Salviae Miltiorrhizae Radix were fractionated into nine fractions, and the effect of each fraction on action potential was tested. The fraction containing monomethyl lithospermic acid-A (LSA-A) induced a significant prolongation of action potential duration (APD). LSA-B which is a major component of Salviae Miltiorrhizae Radix, however, did not cause a significant effect. In voltage clamp experiments, the effects of LSA-A on K currents, Ca currents and Na currents were tested. Neither K currents nor L-type Ca currents were affected by LSA-A. On the contrary, LSA-A significantly slowed down the inactivation kinetics of the Na current with no effect on the fast component of the inactivation process. The amplitude of the peak current and the voltage-dependence of activation were not changed by LSA-A. The effect of LSA-A on Na current was abolished when high concentration of $Ca^{2+}$ buffer (10 mM BAPTA) was included in the pipette solution or when Ca2+ current was blocked by nicardipine (1 $\mu$M) in the bath solution.n.

• Journal of Microbiology and Biotechnology
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• v.25 no.8
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• pp.1371-1379
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• 2015

The Ca_v1.2 Ca²⁺ channel is essential for cardiac and smooth muscle contractility and many physiological functions. We mutated single, double, and quadruple sites of the four potential Asn (N)-glycosylation sites in the rabbit Ca_v1.2 into Gln (Q) to explore the effects of Nglycosylation. When a single mutant (N124Q, N299Q, N1359Q, or N1410Q) or Ca_v1.2/WT was expressed in Xenopus oocytes, the biophysical properties of single mutants were not significantly different from Ca_v1.2/WT. In comparison, the double mutant N124,299Q showed a positive shift in voltage-dependent gating. Furthermore, the quadruple mutant (QM; N124,299,1359,1410Q) showed a positive shift in voltage-dependent gating as well as a reduction of current. We tagged EGFP to the QM, double mutants, and Ca_v1.2/WT to chase the mechanisms underlying the reduced currents of QM. The surface fluorescence intensity of QM was weaker than that of Ca_v1.2/WT, suggesting that the reduced current of QM arises from its lower surface expression than Ca_v1.2/WT. Tunicamycin treatment of oocytes expressing Ca_v1.2/WT mimicked the effects of the quadruple mutations. These findings suggest that Nglycosylation contributes to the surface expression and voltage-dependent gating of Ca_v1.2.

• The Korean Journal of Physiology and Pharmacology
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• v.2 no.6
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• pp.733-742
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• 1998

Background: We have previously reported that not only cGMP but also 8-Br-cGMP or 8-pCPT-cGMP, specific and potent stimulators of cGMP-dependent protein kinase (cGMP-PK), increased basal L-type calcium current $(I_{Ca})$ in rabbit ventricular myocytes. Our findings in rabbit ventricular myocytes were entirely different from the earlier findings in different species, suggesting that the activation of cGMP-PK is involved in the facilitation of $I_{Ca}}$ by cGMP. However, there is no direct evidence that cGMP-PK can stimulate $I_{Ca}}$ in rabbit ventricular myocytes. In this report, we focused on the direct effect of cGMP-PK on $I_{Ca}}$ in rabbit ventricular myocytes. Methods and Results: We isolated single ventricular myocytes of rabbit hearts by using enzymatic dissociation. Regulation of $I_{Ca}}$ by cGMP-PK was investigated in rabbit ventricular myocytes using whole-cell voltage clamp method. $I_{Ca}}$ was elicited by a depolarizing pulse to +10 mV from a holding potential of -40 mV. Extracellular 8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphate (8-pCPT-cGMP), potent stimulator of cGMP-dependent protein kinase (cGMP-PK), increased basal $I_{Ca}}$. cGMP-PK also increased basal $I_{Ca}}$. The stimulation of basal $I_{Ca}}$ by cGMP-PK required both 8-Br-cGMP in low concentration and intracellular ATP to be present. The stimulation of basal $I_{Ca}}$ by cGMP-PK was blocked by heat inactivation of the cGMP-PK and by bath application of 8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate, Rp-isomer (Rp-pCPT-cGMP), a phosphodiesterase-resistant cGMP-PK inhibitor. When $I_{Ca}}$ was increased by internal application of cGMP-PK, IBMX resulted in an additional stimulation of $I_{Ca}}$. In the presence of cGMP-PK, already increased $I_{Ca}}$ was potentiated by bath application of isoprenaline or forskolin or intracellular application of cAMP. Conclusions: We present evidence that cGMP-PK stimulated basal $I_{Ca}}$ by a direct phosphorylation of L-type calcium channel or associated regulatory protein in rabbit ventricular myocytes.

• Progress in Medical Physics
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• v.11 no.1
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• pp.29-38
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• 2000

The signal transduction pathway for most neurotransmitter induced inhibition of $Ca^{2+}$ channels in sympathetic neurons involves a G-protein mediated, membrane-delimited mechanism without the participation of any known protein kinase. However, activation of protein kinase C (PKC) has been proposed as one of the intracellular mechanisms mediating some neurotransmitter induced $Ca^{2+}$ channel inhibition. In the present study, we investigated the effects of phorbol-12, 13-dibutyrate (PDBu) on $Ca^{2+}$ channel currents of acutely dispersed neurons from adult rat superior cervical ganglion (SCG) neurons using whole cell variant of the patch clamp technique. PDBu (500 nM), the activator of PKC, increased $Ca^{2+}$ channel currents and retarded the deactivation of tail currents. The effects of PDBu were voltage dependent and the maximal increase in the current amplitudes was observed between -10 to 10 mV (n=4). PDBu attenuated $Ca^{2+}$ current inhibition induced by norepinephrine (NE), which modulates $Ca^{2+}$ channels via a pertussis toxin (PTX)-sensitive pathway. Inhibition of PDBu by staurosporine (1 $\mu$M) blocked the effects of PDBu on current amplitudes and NE-induced G-protein mediated inhibition of $Ca^{2+}$ currents. Further experiment should be done to know if G-protein or $Ca^{2+}$ channel itself is the target of PKC phosphorvlation.phosphorvlation.

• Proceedings of the PSK Conference
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• 2003.10a
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• pp.94-95
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• 2003

Local cytosolic rises of $Ca^{2+}$ appears to be critical in the regulation of many cellular activities, including muscle contraction, neurotransmitter secretion, and cell death. Cardiac $Ca^{2+}$ signaling similarly begins with discrete and localized rises of $Ca^{2+}$($Ca^{2+}$ sparks) triggered by $Ca^{2+}$ current ($I_{Ca}$). The large local releases of $Ca^{2+}$ in turn modulate L-type $Ca_{v}$1.2( ${\alpha}_{1C}$ $Ca^{2+}$ channels, suggesting that discrete $Ca^{2+}$ cross-signaling may occur in the micro-domains of ${\alpha}_{1C}$/ryanodine receptors (RyRs). (omitted)

• Proceedings of the Korean Institute of Electrical and Electronic Material Engineers Conference
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• 2000.04b
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• pp.88-91
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• 2000

The $(Sr_{0.85}Ca_{0.15})TiO_3$(SCT) thin films are deposited on Pt-coated electrode(Pt/TiN/$SiO_2$/Si) using RF sputtering method. The crystallinity of SCT thin films is increased with increase of substrate temperature in the temperature range of 200~500$[^{\circ}C]$. V-I characteristics of SCT thin films show the increasing leakage current with the increases of deposition temperature. The conduction mechanism of the SCT thin films observed in the temperature range of 25~100$[^{\circ}C]$ can be divided into four characteristic regions with different mechanism by the increasing current.