• Journal of Ginseng Research
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• 제24권1호
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• pp.18-22
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• 2000

앞의 연구에서 우리는 진세노사이드가 신경세포에 존재하는 high-threshold voltage-dependent $Ca^{2+}$ channel을 억제한다는 것을 발표하였다. 그러나, 이러한 연구는 진세노사이드가 여러 칼슘 채널subtypes중 어느 특정 칼슘 채널만을 선택적으로 조절한다는 것을 보여주지는 않았다. 따라서 이 연구에서 우리는 여러 칼슘 채널subtypes에 선택적으로 작용하는 약물 혹은 toxins을 이용하여 진세노사이드가 어느 종류의 칼슘 채널 subtypes를 억제하는가를 bovine chromaffin cell을 이용하여 연구하였다. 사용한 물질은nimodipine(L-type 칼슘 채널 길항제), $\omega$-conotoxin GVIA (N-type $Ca^{2+}$ channel 길항제), $\omega$-agatoxin IVA(P-type 칼슘 채널 길항제)이었다. 연구 결과 진세노사이드는 bovine chromaffin 세포에 존재하는 high-threshold 칼슘 current을 투여 농도별로 억제하였다. $IC_{50}$/은 약 120 $\mu$g/ml인 것으로 나타났다. nimodipine은 진세노사이드에 의한 칼슘 currents억제 작용에 영향을 미치지 않은 것으로 나타났다. 그러나, $\omega$-conotoxin GVIA, $\omega$-agatoxin IVA 및 nimodipine+$\omega$-conotoxin GVIA+$\omega$-agatoxin IVA을 처리한 세포에서는 진세노사이드에 의한 칼슘 currents억제 작용이 현저하게 줄어 들었다. 이러한 연구 결과들은 진세노사이드가 L-type 칼슘 채널은 억제하지 않고, 주로 N-, p-, 및 Q-type칼슘 채널을 억제한다는 것을 보여주고 있다

• The Korean Journal of Physiology and Pharmacology
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• 제1권6호
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• pp.625-637
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• 1997

Calcium ions are implicated in a variety of physiological functions, including enzyme activity, membrane excitability, neurotransmitter release, and synaptic transmission, etc. Calcium antagonists have been known to be effective for the treatment of exertional angina and essential hypertension. Selective and nonselective voltage-dependent calcium channel blockers also have inhibitory action on the acute and tonic pain behaviors resulting from thermal stimulation, subcutaneous formalin injection and nerve injury. This study was undertaken to investigate the effects of iontophoretically applied $Ca^{++}$ and its antagonists on the responses of WDR (wide dynamic range) cells to sensory inputs. The responses of WDR cells to graded electrical stimulation of the afferent nerve and also to thermal stimulation of the receptive field were recorded before and after iontophoretical application of $Ca^{++}$, EGTA, $Mn^{++}$, verapamil, ${\omega}-conotoxin$ GVIA, ${\omega}-conotoxin$ MVIIC and ${\omega}-agatoxin$ IVA. Also studied were the effects of a few calcium antagonists on the C-fiber responses of WDR cells sensitized by subcutaneous injection of mustard oil (10%). Calcium ions and calcium channel antagonists ($Mn^{++}$, verapamil, ${\omega}-conotoxin$ GVIA & ${\omega}-agatoxin$ IVA) current-dependently suppressed the C-fiber responses of WDR cells without any significant effects on the A-fiber responses. But ${\omega}-conotoxin$ MVIIC did not have any inhibitory actions on the responses of WDR cell to A-fiber, C-fiber and thermal stimulation. Iontophoretically applied EGTA augmented the WDR cell responses to C-fiber and thermal stimulations while spinal application of EGTA for about $20{\sim}30\;min$ strongly inhibited the C-fiber responses. The augmenting and the inhibitory actions of EGTA were blocked by calcium ions. The WDR cell responses to thermal stimulation of the receptive field were reduced by iontophoretical application of $Ca^{++}$, verapamil, ${\omega}-agatoxin$ IVA, and ${\omega}-conotoxin$ GVIA but not by ${\omega}-conotoxin$ MVIIC. The responses of WDR cells to C-fiber stimulation were augmented after subcutaneous injection of mustard oil (10%, 0.15 ml) into the receptive field and these sensitized C-fiber responses were strongly suppressed by iontophoretically applied $Ca^{++}$, verapamil, ${\omega}-conotoxin$ GVIA and ${\omega}-agatoxin$ IVA. These experimental findings suggest that in the rat spinal cord, L-, N-, and P-type, but not Q-type, voltage-sensitive calcium channels are implicated in the calcium antagonist-induced inhibition of the normal and the sensitized responses of WDR cells to C-fiber and thermal stimulation, and that the suppressive effect of calcium and augmenting action of EGTA on WDR cell responses are due to changes in excitability of the cell.

• The Korean Journal of Physiology and Pharmacology
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• 제11권1호
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• pp.21-30
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• 2007

The present study was designed to establish comparatively the inhibitory effects of cilnidipine(CNP), nifedipine(NIF), and $\omega$-conotoxin GVIA(CTX) on the release of CA evoked by cholinergic stimulation and membrane depolarization from the isolated perfused model of the rat adrenal medulla. CNP(3 ${\mu}M$), NIF(3 ${\mu}M$), and CTX(3 ${\mu}M$) perfused into an adrenal vein for 60 min produced greatly inhibition in CA secretory responses evoked by ACh($5.32{\times}10^{-3}M$), DMPP($10^{-4}M$ for 2 min), McN-A-343($10^{-4}M$ for 2 min), high $K^+(5.6{\times}10^{-2}M)$, Bay-K-8644($10^{-5}M$), and cyclopiazonic acid($10^{-5}M$), respectively. For the CA release evoked by ACh and Bay-K-8644, the following rank order of potency was obtained: CNP>NIF>CTX. The rank order for the CA release evoked by McN-A-343 and cyclopiazonic acid was CNP>NIF>CTX. Also, the rank orders for high $K^+$ and for DMPP were NIF>CTX>CNP and NIF>CNP>CTX, respectively. Taken together, these results demonstrate that all voltage-dependent $Ca^{2+}$ channels(VDCCs) blockers of cilnidipine, nifedipine, and $\omega$-conotoxin GVIA inhibit greatly the CA release evoked by stimulation of cholinergic(both nicotinic and muscarinic) receptors and the membrane depolarization without affecting the basal release from the isolated perfused rat adrenal gland. It seems likely that the inhibitory effects of cilnidipine, nifedipine, and $\omega$-conotoxin GVIA are mediated by the blockade of both L- and N-type, L-type only, and N-type only VDCCs located on the rat adrenomedullary chromaffin cells, respectively, which are relevant to $Ca^{2+}$ mobilization. It is also suggested that N-type VDCCs play an important role in the rat adrenomedullary CA secretion, in addition to L-type VDCCs.

• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 2001년도 학술 발표회 진행표 및 논문초록
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• pp.47-47
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• 2001

Opening of $Ca^{2＋}$ -channels represents the final common pathway for insulin secretion in pancreatic beta-cells and related cell lines. We investigated voltage-sensitive calcium channels (VSCCs) and insulin secretion in RINm5F, an insulinoma cell line derived from rat pancreatic beta-cells. Several types of VSCCs were identified in RINm5f cells： dihydropyridine-sensitive L-type, $\omega$-conotoxin GVIA-sensitive N-type, $\omega$-agatoxin IVA-sensitive P-type channels, and $\omega$-conotoxin MVIIC sensitive Q-type channels.(omitted)

• The Korean Journal of Physiology and Pharmacology
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• 제10권5호
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• pp.255-261
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• 2006

Melittin-induced nociceptive responses are mediated by selective activation of capsaicin-sensitive primary afferent fibers and are modulated by excitatory amino acid receptor, cyclooxygenase, protein kinase C and serotonin receptor. The present study was undertaken to investigate the peripheral and spinal actions of voltage-gated calcium channel antagonists on melittin-induced nociceptive responses. Changes in mechanical threshold and number of flinchings were measured after intraplantar (i.pl.) injection of melittin $(30\;{\mu}g/paw)$ into mid-plantar area of hindpaw. L-type calcium channel antagonists, verapamil [intrathecal (i.t.), 6 or $12\;{\mu}g$; i.pl.,100 & $200\;{\mu}g$; i.p., 10 or 30 mg], N-type calcium channel blocker, ${\omega}-conotoxin$ GVIA (i.t., 0.1 or $0.5\;{\mu}g$; i.pl., $5\;{\mu}g$) and P-type calcium channel antagonist, ${\omega}-agatoxin$ IVA (i.t., $0.5\;{\mu}g$; i.pl., $5\;{\mu}g$) were administered 20 min before or 60 min after i.pl. injection of melittin. Intraplantar pre-treatment and i.t. pre- or post-treatment of verapamil and ${\omega}-conotoxin$ GVIA dose-dependently attenuated the reduction of mechanical threshold, and melittin-induced flinchings were inhibited by i.pl. or i.t. pre-treatment of both antagonists. P-type calcium channel blocker, ${\omega}-agatoxin$ IVA, had significant inhibitory action on flinching behaviors, but had a limited effect on melittin-induced decrease in mechanical threshold. These experimental findings suggest that verapamil and ${\omega}-conotoxin$ GVIA can inhibit the development and maintenance of melittin-induced nociceptive responses.

• The Korean Journal of Physiology and Pharmacology
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• 제6권2호
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• pp.87-91
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• 2002

The aim of this study was to investigate the role of $Ca^{2+}-channel$ blockers in norepinephrine (NE) release from rat hippocampus. Slices and synaptosomes were incubated with $[^3H]-NE$ and the releases of the labelled products were evoked by 25 mM KCl stimulation. Nifedipine, diltiazem, nicardipine, flunarizine and pimozide did not affect the evoked and basal release of NE in the slice. But, diltiazem, nicardipine and flunarizine decreased the evoked NE release with a dose-related manner without any change of the basal release from synaptosomes. Also, a large dose of pimozide produced modest decrement of NE release. ${\omega}-conotoxin$ (CTx) GVIA decreased the evoked NE release in a dose-dependent manner without changing the basal release. And ${\omega}-CTxMVIIC$ decreased the evoked NE release in the synaoptosomes without any effect in the slice, but the effect of decrement was far less than that of ${\omega}-CTxGVIA.$ In interaction experiments with ${\omega}-CTxGVIA,\;{\omega}-CTxMVIIC$ slightly potentiated the effect of ${\omega}-CTxGVIA$ on NE release in the slice and synaptosomal preparations. These results suggest that the NE release in the rat hippocampus is mediated mainly by N-type $Ca^{2+}-channels,$ and that other types such as L-, T- and/or P/Q-type $Ca^{2+}-channels$ could also be participate in this process.

• 한국의학물리학회지:의학물리
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• 제8권1호
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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.

• The Korean Journal of Physiology and Pharmacology
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• 제5권6호
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• pp.511-519
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• 2001

Nimopidine, one of dihydropyridine derivatives, has been widely used to pharmacologically identify L-type Ca currents. In this study, it was tested if nimodipine is a selective blocker for L-type Ca currents in sensory neurons and heterologous system. In mouse dorsal root ganglion neurons (DRG), low concentrations of nimodipine $(<10\;{\mu}M),$ mainly targeting L-type Ca currents, blocked high-voltage-activated calcium channel currents by ${\sim}38%.$ Interestingly, high concentrations of nimodipine $(>10\;{\mu}M)$ further reduced the 'residual' currents in DRG neurons from ${\alpha}_{1E}$ knock-out mice, after blocking L-, N- and P/Q-type Ca currents with $10\;{\mu}M$ nimodipine, $1\;{\mu}M\;{\omega}-conotoxin$ GVIA and 200 nM ${\omega-agatoxin$ IVA, indicating inhibitory effects of nimodipine on R-type Ca currents. Nimodipine $(>10\;{\mu}M)$ also produced the inhibition of both low-voltage-activated calcium channel currents in DRG neurons and ${\alpha}_{1B}\;and\;{\alpha}_{1E}$ subunit based Ca channel currents in heterologous system. These results suggest that higher nimodipine $(>10\;{\mu}M)$ is not necessarily selective for L-type Ca currents. While care should be taken in using nimodipine for pharmacologically defining L-type Ca currents from native macroscopic Ca currents, nimodipine $(>10\;{\mu}M)$ could be a useful pharmacological tool for characterizing R-type Ca currents when combined with toxins blocking other types of Ca channels.

• The Korean Journal of Physiology and Pharmacology
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• 제18권4호
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• pp.297-305
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• 2014

Flavonoids have an ability to suppress various ion channels. We determined whether one of flavonoids, cyanidin-3-glucoside, affects adenosine 5'-triphosphate (ATP)-induced calcium signaling using digital imaging methods for intracellular free $Ca^{2+}$ concentration ([$Ca^{2+}$]i), reactive oxygen species (ROS) and mitochondrial membrane potential in PC12 cells. Treatment with ATP ($100{\mu}M$) for 90 sec induced [$Ca^{2+}$]i increases in PC12 cells. Pretreatment with cyanidin-3-glucoside ($1{\mu}g/ml$ to $100{\mu}g/ml$) for 30 min inhibited the ATP-induced [$Ca^{2+}$]i increases in a concentration-dependent manner ($IC_{50}=15.3{\mu}g/ml$). Pretreatment with cyanidin-3-glucoside ($15{\mu}g/ml$) for 30 min significantly inhibited the ATP-induced [$Ca^{2+}$]i responses following removal of extracellular $Ca^{2+}$ or depletion of intracellular [$Ca^{2+}$]i stores. Cyanidin-3-glucoside also significantly inhibited the relatively specific P2X2 receptor agonist 2-MeSATP-induced [$Ca^{2+}$]i responses. Cyanidin-3-glucoside significantly inhibited the thapsigargin or ATP-induced store-operated calcium entry. Cyanidin-3-glucoside significantly inhibited the ATP-induced [$Ca^{2+}$]i responses in the presence of nimodipine and ${\omega}$-conotoxin. Cyanidin-3-glucoside also significantly inhibited KCl (50 mM)-induced [$Ca^{2+}$]i increases. Cyanidin-3-glucoside significantly inhibited ATP-induced mitochondrial depolarization. The intracellular $Ca^{2+}$ chelator BAPTA-AM or the mitochondrial $Ca^{2+}$ uniporter inhibitor RU360 blocked the ATP-induced mitochondrial depolarization in the presence of cyanidin-3-glucoside. Cyanidin-3-glucoside blocked ATP-induced formation of ROS. BAPTA-AM further decreased the formation of ROS in the presence of cyanidin-3-glucoside. All these results suggest that cyanidin-3-glucoside inhibits ATP-induced calcium signaling in PC12 cells by inhibiting multiple pathways which are the influx of extracellular $Ca^{2+}$ through the nimodipine and ${\omega}$-conotoxin-sensitive and -insensitive pathways and the release of $Ca^{2+}$ from intracellular stores. In addition, cyanidin-3-glucoside inhibits ATP-induced formation of ROS by inhibiting $Ca^{2+}$-induced mitochondrial depolarization.

• 한국의학물리학회지:의학물리
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• 제15권4호
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• pp.220-227
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• 2004

N형 칼슘통로의 비활성화기전에 관하여는 아직까지도 막전압의존성 기전과 칼슘의존성 기전간에 논란이 계속되고 있다. 2003년에 의학물리에 발표한 논문1)에서 본 연구자는 N형 칼슘통로의 비활성화 기전은 2가지 성분 -빠른 성분과 느린 성분을 가지고 있고 빠른 성분은 칼슘의존적이 아니며 오직 느린 성분만이 칼슘의존적일 가능성을 제시하였다. 본 논문에서는 막전압의존성 기전이 옳건 칼슘의존성 기전이 옳건 간에 세포 신호전달 체계로서 비활성화와 연계된 기전이 필요하므로 이러한 맥락에서 인산화 기전을 연구하였다. 흰쥐 경동맥 결절뉴론을 단일 세포로 얻은 후 whole cell patch clamp technique를 사용하여 N형 칼슘전류를 기록하고 대조 세포내액을 사용하였을 때와 phosphatase inhibitor인 okadaic acid를 포함한 세포내액을 사용하였을 때의 차이를 비교하였다. Okadaic acid에 의하여 비활성화정도가 증가되었고 이러한 okadaic acid 효과는 주로 N형 통로를 통하여 영향을 미침을 N형 칼슘통로 억제제인 $\omega$-conotoxin GVIA를 사용함으로써 확인하였다. Okadaic acid에 의한 비활성화 증가 효과는 protein kinase를 비특이적으로 억제하는 staurosporine에 의하여 억제되었고 또한 calmodulin dependent protein kinase의 특이적 억제제인 lavendustin C에 의하여 억제되었으므로 인산화과정이 N형 칼슘통로 비활성화와 관련되어 있고 특히 calmodulin을 통한 인산화과정이 주로 관여함을 확인하였다. 본 연구자가 발표한 선행논문1)에 의해 외부의 2가 양이온에 의해 빠른 비활성화가 진행되며, 본 논문에 의하여 인산화과정에 의해 빠른 비활성화가 촉진된다는 사실이 확인되었다. 그러나 본 연구결과만으로는 인산화과정이 비활성화 자체라고는 볼 수 없으며 단지 인산화과정에 의해 비활성화가 가속되었다고 해석할 수 밖에 없다. 인산화과정이 비활성화자 체인지 여부는 2가 양이온이 칼슘통로에 작용하는 결합부위에 관한 연구 및 인산화 부위가 칼슘통로인지 아니면 다른 조절 부위인지 여부를 확인할 수 있는 연구가 진행되어야 확실히 알 수 있을 것이다.