• The Korean Journal of Physiology
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• v.27 no.1
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• pp.107-114
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• 1993

Most of voltage operated $Ca^{2+}$ channels can be divided into three types (T-, N-, and L-type), according to the electrical and pharmacological properties. Their distribution is closely related to cell specific functions. Properties of the voltage activated $Ca^{2+}$ current in mouse eggs were examined to classify channel types and to deduce the function by using whole cell voltage clamp technique. $Ca^{2+}$ currents appeared below -40 mV and reached a maximum at -15 mV (half maximum was -31 mV), then decayed rapidly (inactivation time constant ${\tau}=28.2{\pm}9.59$ ms at -10 mV within 50 ms after the onset of step depolarization. Activation and inactivation of the $Ca^{2+}$ channel was steeply dependent on voltage, in a relatively low range of $-70\;mV{\sim}-10 mV,$ half maximum of activation was -31 mV and that of inactivation was -39 mV, respectively. This current was not decreased significantly by nifedipine, a specific dihydropyridine $Ca^{2+}$ channel blocker in the range of $1\;{\mu}M\;to\;100{\mu}M.$ The inhibitory effect of $Ni^{2+}\;on\;Ca^{2+}$ current was greater than that of $Cd^{2+}.$ The conductance of $Ba^{2+}$ through the channel was equal to or lower than that of $Ca^{2+}$ These results implied that $Ca^{2+}$ current activated at a lower voltage in the mouse egg is carried via a $Ca^{2+}$ channel with similar properties that of the T-type channel.

• The Korean Journal of Physiology
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• v.28 no.2
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• pp.151-157
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• 1994

Intracellular free $Ca^{2+}$ contributes to regulation of various events occurring in vascular smooth muscle cells. One of these events is modulating the membrane iou currents. Single smooth muscle cells were isolated from rabbit mesenteric artery. Three kinds of $Ca^{2+}-activated\;current$ were studied with the patch clamp method. $Ca^{2+}-activated\;K^+\;current$ with a large oscillation was recorded in the depolarized potential range. The single channel conductance of this current was about 250 pS. It was abolished by replacing intracellular $K^+\;with\;Cs^+$. A $Ca^{2+}-activated$ nonselective cation current was observed in both the depolarized and hyperpolarized potential ranges. And it was blocked by replacement of extracellular $Na^+$ with N-methylglucamine (NMG) or extracellular application of $Cd^{2+}$. $Ca^{2+}-activated\;Cl^-\;current$ was revealed in the whole voltage range and was blocked by niflumic acid. These results indicate that at least three kinds of $Ca^{2+}-activated$ ionic currents exist in smooth muscle cells from rabbit superior mesenteric artery.

• The Korean Journal of Physiology and Pharmacology
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• v.8 no.2
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• pp.101-110
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• 2004

Voltage-sensitive release mechanism was pharmacologically dissected from the $Ca^{2+}-induced\;Ca^{2+}\;release$ in the SR $Ca^{2+}$ release in the rat ventricular myocytes patch-clamped in a whole-cell mode. SR $Ca^{2+}$ release process was monitored by using forward-mode $Na^+-Ca^{2+}$ exchange after restriction of the interactions between $Ca^{2+}$ from SR and $Na^+-Ca^{2+}$ exchange within micro-domains with heavy cytosolic $Ca^{2+}$ buffering with 10 mM BAPTA. During stimulation every 10 s with a pulse roughly mimicking action potential, the initial outward current gradually turned into a huge inward current of $-12.9{\pm}0.5\;pA/pF$. From the inward current, two different inward $I_{NCX}s$ were identified. One was $10\;{\mu}M$ ryanodine-sensitive, constituting $14.2{\pm}2.3%$. It was completely blocked by $CdCl_2$ (0.1 mM and 0.5 mM) and by $Na^+-depletion$. The other was identified by 5 mM $NiCl_2$ after suppression of $I_{CaL}$ and ryanodine receptor, constituting $14.8{\pm}1.6%$. This latter was blocked by either 10 mM caffeine-induced SR $Ca^{2+}-depletion$ or 1 mM tetracaine. IV-relationships illustrated that the latter was activated until the peak in $30{\sim}35\;mV$ lower voltages than the former. Overall, it was concluded that the SR $Ca^{2+}$ release process in the rat ventricular myocytes is mediated by the voltage-sensitive release mechanism in addition to the $Ca^{2+}-induced-Ca^{2+}\;release$.

• Korean Journal of Veterinary Research
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• v.34 no.1
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• pp.25-36
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• 1994

The inward tail current after a short depolarizing pulse has been known as Na-Ca exchange current activated by intracellular calcium which forms late plateau of the action potential in rabbit atrial myocytes. Chloride conductance which is also dependent upon calcium concentration has been reported as a possible tail current in many other excitable tissues. Thus, in order to investigate the exsitance of the calcium activated chloride current and its contribution to tail current, whole cell voltage clamp measurement has been made in single atrial cells of the rabbit. The current was recorded during repolarization following a brief 2 ms depolarizing pulse to +40mV from a holding potential of -70mV. When voltage-sensitive transient outward current was blocked by 2 mM 4-aminopyridine or replacement potassium with cesium, the tail current were abolished by ryanodine$(1{\mu}M)$ or diltiazem$(10{\mu}M)$ and turned out to be calcium dependent. The magnitudes of the tail currents were increased when intracellular chloride concentration was increased to 131 mM from 21 mM. The current was decreased by extracellular sodium reduction when intracellular chloride concentration was low(21 mM), but it was little affected by extracellular sodium reduction when intracellual chloride concentration was high(131 mM). The current-voltage relationship of the difference current before and after extracellular sodium reduction, shows an exponential voltage dependence with the largest magnitude of the current occurring at negative potentials, with is similar to current-voltage relationship at negative potentials, which is similar to current-voltage relationship of Na-Ca exchange current. The current was also decreased by $10{\mu}M$ niflumic acid and 1 mM bumetanide, which is well known anion channel blockers. The reversal potentials shifted according to changes in chloride concentration. The current-voltage relationships of the niflumic acid-sensitive currents in high and low concentration of chloride were well fitted to those predicted as chloride current. From the above results, it is concluded that calcium activated chloride component exists in the tail current with Na-Ca exchange current and it shows the reversal of tail current. Therefore it is thought that in the physiologic condition it leads to rapid end of action potential which inhibits calcium influx and it contributes to maintain the low intracellular calcium concentration with Na-Ca exchange mechanism.

• The Korean Journal of Physiology and Pharmacology
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• v.9 no.5
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• pp.269-273
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• 2005

In horizontal cells (HCs) that were freshly dissociated from goldfish retina, two types of voltagedependent calcium currents ($I_{Ca}$) were recorded using a patch-clamping configuration: a transient type current and a sustained type current. The cell was held at -40 mV, and the prepulse step of -90 mV was applied before command pulse between -65 and +55 mV. The transient $Ca^{2+}$ current was activated by depolarization to around -50 mV from a prepulse voltage of -90 mV lasting at least 400 ms and reached a maximal value near -25 mV. On the other hand, the sustained $Ca^{2+}$ current was induced by pre-inactivation for less than 10 ms duration. Its activation started near -10 mV and peaked at +20 mV. $Co^{2+}$ (2 mM) suppressed both of these two components, but nifedipine ($20{\mu}M$), L-type $Ca^{2+}$ channel antagonist, blocked only the sustained current. Based on the activation voltage and the pharmacolog$I_{Ca}$l specificity, the sustained current appears to be similar to L-type $I_{Ca}$ and the transient type to T-type $I_{Ca}$. This study is the first to confirm that transient type $I_{Ca}$ together with the sustained one is present in HCs dissociated from goldfish retina.

• The Korean Journal of Physiology and Pharmacology
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• v.4 no.5
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• pp.403-408
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• 2000

The outward currents elicited in hamster eggs by depolarizing pulses were studied. The currents appeared to comprise at least two components, a transient outward component $(I_{to})$ and a steady-state outward component $(I_{\infty}).\;I_{to}$ was transiently followed by the cessation of inward $Ca^{2+}$ current $(I_{Ca}),$ and its current-voltage (I-V) relation was a mirror image of that of $(I_{Ca}).$ Either blockade of $(I_{Ca})$ by $Co^{2+}$ or replacement of $Ca^{2+}$ with $Sr^{2+}$ abolished $I_{to}$ without change in $I_{\infty}.$ Intracellular EGTA (10 mM) inhibited $I_{to}$ but not $I_{\infty}.$ suggesting strongly that generation of $I_{to}$ requires intracellular $Ca^{2+}.$ Apamin (1 nM) abolished selectively $I_{to},$ indicatingthat $I_{to}$ is $Ca^{2+}-dependent\;K^+$ current. On the other hand, $I_{\infty}$ was $Ca^{2+}-independent.$ Both $I_{to}$ and $I_{\infty}$ were completely inhibited by internal $Cs^+$ and external TEA. The estimated reversal potential of $I_{to}$ was close to the theoretical $E_K.$ Taken together, both outward currents were carried by $K^+$ channels. From these results, $I_{to}$ is likely to be a current responsible for the hyperpolarizing responses seen in hamster eggs at fertilization.

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

• Proceedings of the Korean Biophysical Society Conference
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• 1999.06a
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• pp.61-62
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• 1999

;The mechanisms inducing hypertension are actively investigated and are still challenging topics. Basically hypertension must be caused by the disorder of $Ca^{2+}$ metabolism in vascular smooth muscle, such as the increase of $Ca^{2+}$ influx, the decrease of ci+ efflux, or the change of sensitivity of contractile protein etc. The one of cause of the increase of ci+ influx may be the change of ci+ channel activity. Even though the relationships of ci+ channel activity and hypertension were studied using various hypertension models, still it is not clear how much change of $Ca^{2+}$ channel activity in diabetes mellitus (DM) induced hypertension is occurred. We induced DM hypertension in SD rat and compared the $Ca^{2+}$ channel activity with age-matched normotensive SD rat. For inducing DM hypertension, left kidney was removed with 200 gm rat and, after 1 month, 60 mg/kg of streptozotocin was injected into peritoneal space to induce diabetes mellitus. Usually after 4-6 weeks, hypertension was fully induced. For isolating vascular smooth muscle cells (VSMC), we used mesenteric arteriole (3rd - 4th branch of mesenteric artery) of which diameter is below 150 urn. VSMCs were isolated enzymatically. $Ca^{2+}$ current was measured using whole cell patch clamp technique. All experiments were performed at $37^{\circ}C$. The cell membrane area of VSMC of DM hypertensive rat is larger than that of control VSMC($36.6{\pm}3.64{\;}pF{\;}vs{\;}22.4{\pm}1.29{\;}pF, {\;}mean{\pm}S.E.$) When we compared the current amplitude, the $Ca^{2+}$ current amplitude in VSMC of DM hypertensive rat is much larger than that in VSMC of normotensive age-matched rat. After $Ca^{2+}$ current amplitude was normalized by cell membrane area, the current amplitude in DM hypertension is increased to $249.1{\pm}15.9{\;}%{\;}(mean{\pm}S.E.M)$, which means the ;absolute current amplitude is about 4 times larger in DM hypertension. When we compared the steady state activation and inactivation. there were no noticeable differences. From these results. one of cause of the DM hypertension is due to the increase of $Ca^{2+}$ current amplitude. But it need further study why the $Ca^{2+}$ current is so large in VSMC of DM hypertension and how much $Ca^{2+}$ influx through $Ca^{2+}$ channel contribute to the increase of intracellular $Ca^{2+}$ and eventually contribute to development of hypertension.ypertension.

• The Korean Journal of Physiology and Pharmacology
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• v.3 no.6
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• pp.615-621
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• 1999

In spite many evidences has supported the cardioprotective effect of bradykinin, its direct effects at the cell level are still under question. We investigated the both effects of bradykinin (BK) on $Ca^{2+}-related$ ionic currents using whole cell voltage clamp technique in rabbit cardiomyocytes and on the intracellular $Ca^{2+}$ transient using calcium sensitive fluorescence dye, indo-1AM. Simultaneously with recording intracellular $Ca^{2+}$ transients, cell contractility was estimated from the changes in length of the electrical stimulated rat cardiac myocytes. L-type $Ca^{2+}$ current decreased by bradykinin at the entire voltage range. Inward tail current increased initially up to its maximum about 4 min after exposing myocytes to BK, and then gradually decreased again by further exposure to BK. This tail current decreased remarkably at washing BK off but slowly recovered ca. 20 min later. The change in cell contractility was similar to that in tail current showing initial increase followed by gradual decrease. Removal of BK brought remarkable decrease in contractility, which was recovered $15{\sim}20$ min after cessation of electrical stimulation. Bradykinin increased $Ca^{2+}$ transient initially but after some time $Ca^{2+}$ transient also decreased coincidentally with contractility. From these results, it is suggested that bradykinin exerts directly its cardioprotective effect on the single myocytes by decreasing the intracellular $Ca^{2+}$ level followed by an initial increase in $Ca^{2+}$ transient.

• 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.