• 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.

• Animal cells and systems
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• v.3 no.1
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• pp.53-58
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• 1999

The immunocytochemical method was used to identify the existence of voltage-dependent $Ca^{2＋}$-channels in mouse follicular oocytes. Three types of voltage-dependent $Ca^{2＋}$-channels were shown to exist in the follicular oocytes for the first time, the P/Q-type $Ca^{2＋}$-channel, the N-type $Ca^{2＋}$-channel, and the L-type $Ca^{2＋}$-channel. Among proven $Ca^{2＋}$-channels distributions of the P/Q-type $Ca^{2＋}$-channel and L-type $Ca^{2＋}$-channel showed localized staining (clustered pattern) on the oolemma. The distribution of the P/Q-type $Ca^{2＋}$-channel showed all localized staining, and the range of localized staining was from 1 to 8 in staining intensity. As the staining intensity increased from 1 to 8, the number of localized staining decreased. The L-type $Ca^{2＋}$-channel are homogeneously stained (29.4%-54.2%), while some of them (around 28.7%-44.1%) showed localized staining on the oolemma. However, the rest of them showed no staining at all (17.1%- 26.5%). On the contrary, the N-type $Ca^{2＋}$-channel showed mostly homogeneous staining, while nonstaining oocytes were around 33.8%. The rest showed localized staining (10%). However, staining intensity was much weaker than those of the P/Q-type and L-type $Ca^{2＋}$-channel. In fact, the N-type $Ca^{2＋}$-channel has been known to exist only in neurons (from ectoderm origin), but it is unknown how the N-type $Ca^{2＋}$-channel exists in the follicular oocytes (from mesoderm origin). Further studies are needed to examine the expression of $Ca^{2＋}$-channels during the developmental stages of the oocytes.

• Development and Reproduction
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• v.24 no.4
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• pp.297-306
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• 2020

Repetitive changes in the intracellular calcium concentration ([Ca²⁺]i) triggers egg activation, including cortical granule exocytosis, resumption of second meiosis, block to polyspermy, and initiating embryonic development. [Ca²⁺]i oscillations that continue for several hours, are required for the early events of egg activation and possibly connected to further development to the blastocyst stage. The sources of Ca²⁺ ion elevation during [Ca²⁺]i oscillations are Ca²⁺ release from endoplasmic reticulum through inositol 1,4,5 tri-phosphate receptor and Ca²⁺ ion influx through Ca²⁺ channel on the plasma membrane. Ca²⁺ channels have been characterized into voltage-dependent Ca²⁺ channels (VDCCs), ligand-gated Ca²⁺ channel, and leak-channel. VDCCs expressed on muscle cell or neuron is specified into L, T, N, P, Q, and R type VDCs by their activation threshold or their sensitivity to peptide toxins isolated from cone snails and spiders. The present study was aimed to investigate the localization pattern of N and P/Q type voltage-dependent calcium channels in mouse eggs and the role in fertilization. [Ca²⁺]i oscillation was observed in a Ca²⁺ contained medium with sperm factor or adenophostin A injection but disappeared in Ca²⁺ free medium. Ca²⁺ influx was decreased by Lat A. N-VDCC specific inhibitor, ω-Conotoxin CVIIA induced abnormal [Ca²⁺]i oscillation profiles in SrCl₂ treatment. N or P/Q type VDC were distributed on the plasma membrane in cortical cluster form, not in the cytoplasm. Ca²⁺ influx is essential for [Ca²⁺]i oscillation during mammalian fertilization. This Ca²⁺ influx might be controlled through the N or P/Q type VDCCs. Abnormal VDCCs expression of eggs could be tested in fertilization failure or low fertilization eggs in subfertility women.

• Clinical and Experimental Reproductive Medicine
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• v.28 no.1
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• pp.13-24
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• 2001

Objective: In muscle and neuronal cells, calcium channels have been classified by electrophysiological and pharmacological properties into (1) voltage-dependent $Ca^{2+}$-channel (1) P/Q-type $Ca^{2+}$-channel (2) N-type $Ca^{2+}$-channel (3) L-type $Ca^{2+}$-channel (4) T-type $Ca^{2+}$-channel (5) R-type $Ca^{2+}$-channel. The present study was done in order to investigate whether there is any difference in $Ca^{2+}$-channel distribution between activated and normally fertilized embryos. Methods: The immunocytochemical method was used to identify the existence of voltage-dependent $Ca^{2+}$-channels in parthenogenetically activated 2-cell embryos by ethanol and $SrCl_2$ treatment. These 2-cell embryos were obtained by exposure to 6% ethanol for 6 min and to 10 mM $SrCl_2$ for 2h. Results: P/Q-type $Ca^{2+}$-channels and L-type $Ca^{2+}$-channels have been identified. Whereas, three type of $Ca^{2+}$-channel P/Q-type, N-type, L-type have been identified in 2-cell embryos fertilized in vivo. Conclusion: Activation by ethanol was faster than those by $SrCl_2$. However, there was difference in DAB staining of the embryos between ethanol and $SrCl_2$ treatment (87.7% and 54.1 %). Intensity of staining was also different between ethanol- and $SrCl_2$-treated group. However, it has not been known why there was some difference in DAB staining and staining intensity in the present study.

• Progress in Medical Physics
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• v.8 no.1
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• pp.3-15
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• 1997

Adrenal chromaffin cells secrete catecholamine in response to acetylcholine. The secretory response has absolute requirement for extracellular calcium, indication that $Ca^{2+}$ influx through voltage dependent $Ca^{2+}$ channel (VDCC) is the primary trigger of the secretion cascade. Although the existence of various types of $Ca^{2+}$ channels has been explored using patch clamp technique in adrenal chromaffin cells, the contribution of different types of $Ca^{2+}$ channels to catecholamine secretion remains to be established. To investigate the quantative contribution of different types of $Ca^{2+}$ channels to cate-cholamine secretion, $Ca^{2+}$ current($I_{Ca}$) and the resultant membrane capacitance increment($\Delta{C}_{m}$) were simultaneoulsy measured. Software based phasor detector technique was used to monitor $\Delta{C}_{m}$. After blockade of L type VDCC with nicardipine (1$\mu$M), $I_{ca}$ was blocked to 43.85$\pm$6.72%(mean$\pm$SEM) of control and the resultant ㅿC$_{m}$ was reduced ot 30.10$\pm$16.44% of control. In the presence of nicardipine and $\omega$-conotoxin in GVIA(l$\mu$M), an N type VDCC antagonist, $I_{ca}$ was blocked to 11.62$\pm$2.96% of control and the resultant $\Delta{C}_{m}$ was reduced to 26.13$\pm$8.25% of control. Finally, in the presence of L, N, and P type $Ca^{2\pm}$ channel antagonists(nicardipine, $\omega$-Conotoxin GVIA, and $\omega$-agatoxin IVA, respectively), $I_{ca}$ and resultant $\Delta{C}_{m}$ were almost completely blocked. From the observation of parallel effects of $Ca^{2+}$ channel antagonists on $I_{ca}$ and $\Delta{C}_{m}$, it was concluded that L, N, and also P type $Ca^{2+}$ channels served and $Ca^{2+}$ source for exocytosis and no difference was observed in their efficiency to evoke exocytosis amost L, N, and P type $Ca^{2+}$ channels.

• Molecules and Cells
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• v.37 no.11
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• pp.804-811
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• 2014

The protease-activated receptor (PAR)-2 is highly expressed in endothelial cells and vascular smooth muscle cells. It plays a crucial role in regulating blood pressure via the modulation of peripheral vascular tone. Although several mechanisms have been suggested to explain PAR-2-induced hypotension, the precise mechanism remains to be elucidated. To investigate this possibility, we investigated the effects of PAR-2 activation on N-type $Ca^{2+}$ currents ($I_{Ca-N}$) in isolated neurons of the celiac ganglion (CG), which is involved in the sympathetic regulation of mesenteric artery vascular tone. PAR-2 agonists irreversibly diminished voltage-gated $Ca^{2+}$ currents ($I_{Ca}$), measured using the patch-clamp method, in rat CG neurons, whereas thrombin had little effect on $I_{Ca}$. This PAR-2-induced inhibition was almost completely prevented by ${\omega}$-CgTx, a potent N-type $Ca^{2+}$ channel blocker, suggesting the involvement of N-type $Ca^{2+}$ channels in PAR-2-induced inhibition. In addition, PAR-2 agonists inhibited $I_{Ca-N}$ in a voltage-independent manner in rat CG neurons. Moreover, PAR-2 agonists reduced action potential (AP) firing frequency as measured using the current-clamp method in rat CG neurons. This inhibition of AP firing induced by PAR-2 agonists was almost completely prevented by ${\omega}$-CgTx, indicating that PAR-2 activation may regulate the membrane excitability of peripheral sympathetic neurons through modulation of N-type $Ca^{2+}$ channels. In conclusion, the present findings demonstrate that the activation of PAR-2 suppresses peripheral sympathetic outflow by modulating N-type $Ca^{2+}$ channel activity, which appears to be involved in PAR-2-induced hypotension, in peripheral sympathetic nerve terminals.

• Progress in Medical Physics
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• v.12 no.1
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• pp.59-70
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• 2001

Adrenal chromaffin cells secrete catecholamine in response to acetylcholine. The secretory response has absolute requirement for extracellular calcium, indicating that $Ca^{2+}$ influx through voltage operated $Ca^{2+}$ channels is the primary trigger of the secretion cascade. Although the existence of various types of $Ca^{2+}$ channels has been explored using patch clamp technique in adrenal chromaffin cells, there is still disagreement with the types of $Ca^{2+}$ channels existed in different species. Therefore, we have tried to identify several distinct types of $Ca^{2+}$ channels in rat chromaffin cells. By using nicardipine(L type channel blocker), $\omega$-CgTx GVIA(N type channel blocker), and $\omega$-AgaTx VIA(P type channel blocker), it was identified that L, N, and P type $Ca^{2+}$ channel exist in rat adrenal chromaffin cells and the order of contribution of each channel type to whole cell $Ca^{2+}$ current was L type> N type> P type. type> P type.

• Journal of Ginseng Research
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• v.24 no.1
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• pp.18-22
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• 2000

In previous reports we have shown that ginsenosides inhibit high threshold voltage-dependent $Ca^{2+}$ channels in neuronal cells. However, these studies did not show whether ginsenosides-induced inhibition of $Ca^{2+}$ currents discriminates among the various $Ca^{2+}$ channel subtypes, although it is known that there are at least five different $Ca^{2+}$ channel subtypes in neuronal cells. In this study we investigated the effect of ginsenosides on high threshold voltage-dependent $Ca^{2+}$ channel subtypes using their selective $Ca^{2+}$ channel blockers nimodipine (L-type), $\omega$-conotoxin GVIA (N-type), or $\omega$-agatoxin IVA (P-type) in bovine chromaffin cells. We could observe that ginsenosides inhibited high threshold voltage-dependent $Ca^{2+}$ currents in a dose-dependent manner. The $IC_{50}$/ was about 120 $\mu$g/ml. Nimodipine had no effect on ginsenosides response. However, the effect of ginsenosides on $Ca^{2+}$ currents was reduced by $\omega$-conotoxin GVIA, $\omega$-agatoxin IVA, and mixture of nimodipine, $\omega$-contoxin GVIA, and $\omega$-agatoxin IVA. These data suggest that ginsenosides are negatively coupled to three types of calcium channels in bovine chromaffin cell, including an $\omega$-conotoxin GVIA-sensitive (N-type) channel, an $\omega$-agatoxin IVA-sensitive (P-type) channel and nimodipine/$\omega$-conotoxin GVIA/$\omega$-agatoxin IVA-resistant (presumptive Q-type) channel.Q-type) channel.

• The Korean Journal of Physiology and Pharmacology
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• v.8 no.5
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• pp.265-272
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• 2004

The present study was attempted to investigate the effect of cilnidipine (FRC-8635), which is a newly synthesised novel dihydropyridine (DHP) type of organic $Ca^{2+}$ channel blockers, on secretion of catecholamines (CA) evoked by acetylcholine (ACh), high $K^+$, DMPP and McN-A-343 from the isolated perfused rat adrenal gland. Cilnidipine $(1{\sim}10{\mu}M)$ perfused into an adrenal vein for 60 min produced relatively dose- and time-dependent inhibition in CA secretory responses evoked by ACh $(5.32{\times}10^{-3}M),\;DMPP\;(10^{-4}M\;for\;2\;min)$ and McN-A-343 $(10^{-4}M\;for\;2\;min)$. However, lower dose of cilnidipine did not affect CA secretion by high $K^+\;(5.6{\times}10^{-2}\;M)$, higher dose of it reduced greatly CA secretion of high $K^{+}$. Cilnidipine itself did fail to affect basal catecholamine output. In the presence of cilnidipine $(10{\mu}M)$, the CA secretory responses evoked by Bay-K-8644 $(10{\mu}M)$, an activator of L-type $Ca^{2+}$ channels and cyclopiazonic acid $(10{\mu}M)$, an inhibitor of cytoplasmic $Ca^{2+}$-ATPase were also inhibited. Moreover, ${\omega}-conotoxin\;GVIA\;(1{\mu}M)$, a selective blocker of the N-type $Ca^{2+}$ channels, given into the adrenal gland for 60 min, also inhibited time-dependently CA secretory responses evoked by Ach, high $K^+$, DMPP, McN-A-343, Bay-K-8644 and cyclopiazonic acid. Taken together, these results demostrate that cilnidipine inhibits CA secretion evoked by stimulation of cholinergic (both nicotinic and muscarinic) receptors from the isolated perfused rat adrenal gland without affecting the basal release. However, at lower dose, cilnidipine did not affect CA release by membrane depolarization while at larger dose inhibited that. It seems likely that this inhibitory effect of cilnidipine is exerted by blocking both L- and N-type voltage-dependent $Ca^{2+}$ channels (VDCCs) on the rat adrenomedullary chromaffin cells, which is relevant to inhibition of both the $Ca^{2+}$ influx into the adrenal chromaffin cells and intracellular $Ca^{2+}$ release from the cytoplasmic store. It is thought that N-type VDCCs may play an important role in regulation of CA release from the rat adrenal medulla.

• Asian-Australasian Journal of Animal Sciences
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• v.15 no.7
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• pp.949-956
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• 2002

There are several physiological and pharmacological evidences indicating that opening of voltage dependent $Ca^{2+}$ channels play a critical role in induction of acrosome reaction in mammalian sperm. We determined the intracellular free $Ca^{2+}$ concentration in ejaculated goat sperm using a fluorescent, $Ca^{2+}$-specific probe, Fura2/AM, after the suspension of sperm in KRB medium, capable of sustaining capacitation and the acrosome reaction. We used nifedipine, D-600 and diltiazem, the $Ca^{2+}$ channel antagonists belonging to the classes of dihydropyridines, phenylalkylamines and benzothiazepines, to investigate the possibility that L-type voltage gated $Ca^{2+}$ channels play a role in the progesterone-stimulated exocytotic response. Progesterone promoted a rise in intracellular $Ca^{2+}$ in goat sperm and addition of nifedipine (100 nM) just prior to progesterone induction, significantly inhibited both intracellular $Ca^{2+}$ rise and exocytosis suggesting that $Ca^{2+}$ channels are involved in the process. However, the intracellular $Ca^{2+}$ increase during the process of capacitation was not affected with the addition of nifedipine suggesting a role of focal channel for $Ca^{2+}$ during capacitation. Studies using monensin and nigericin, two monovalent cation ionophores showed that an influx of $Na^+$ also may play a role in the opening of $Ca^{2+}$ channels. These results strongly suggests that the entry of $Ca^{2+}$ channels with characteristics similar to those of L-type, voltage-sensitive $Ca^{2+}$ channels found in cardiac and skeletal muscle, is a crucial step in the sequence of events leading to progesterone induced acrosome reaction in goat sperm.