• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1998년도 Proceedings of UNESCO-internetwork Cooperative Regional Seminar and Workshop on Bioassay Guided Isolation of Bioactive Substances from Natural Products and Microbial Products
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• pp.148-149
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• 1998

It has been known that potassium channel openers are a new class of molecules that have attracted general interest because of their potent antihypertensive activity in vivo and vasorelaxant activity in vitro (Hamilton and Weston, 1989). In the present study, it was attempted to examine the effect of the potassium channel opener on catecholamine (CA) secretion evoked by cholinergic stimulation, membrane depolarization and calcium mobilization from the isolated perfused rat adrenal gland. The perfusion of pinacidil (30-300 uM) into an adrenal vein for 20 min produced relatively dose-dependent inhibition in CA secretion evoked by ACh (5.32 mM), high $K^{+}$ (56 mM), DMPP (100 uM for 2 min), McN-A-343 (100 uM for 2 min), cyclopiazonic acid (10 uM for 4 min) and Bay-K-8644 (10 uM for 4 min). Also, under the presence of minoxidil (100 uM), which is also known to be a potassium channel activator, CA secretory responses evoked by ACh, high potassium, DMPP, McN-A-343, Bay-K-8644 and cyclopiazonic acid were also significantly depressed. However, in adrenal glands preloaded with pinacidil (100 uM) under the presence of glibenclamide (1 uM), an antidiabetic sulfonylurea that has been shown to be a specific blocker of ATP-regulated potassium channels (for 20 min), CA secretory responses evoked by ACh, high potassium, DMPP, McN-A-343, Bay-K-8644 and cyclopiazonic acid were considerably recovered to a considerable extent of the normal release as compared to that of pinacidil only. These results, taken together, suggest that pinacidil cause the marked inhibition of CA secretion evoked by stimulation of cholinergic (both nicotinic and muscarinic) receptors as well as by membrane depolarization, indicating strongly that this effect may be mediated by inhibiting influx of extracellular calcium and release in intracellular calcium in the rat adrenomedullary chromaffin cells. Furthermore, these findings suggest strongly that these potassium channel openers-sensitive membrane potassium channels also play an important role in regulating CA secretion.

• 대한약리학회지
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• 제32권1호
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• pp.57-66
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• 1996

파충류 골격근의 근소포체에서 칼슘유리 채널의 존재를 밝히고저 뱀 골격근에서 근소포체를 분리하여 SDS-PAGE 전기영동, RyR의 정제, $[^3H]ryanodine$ 결합실험 및 $^{45}Ca$ 유리 실험으로 아래와 같은 결과를 얻었다. 1) 뱀골격근 소포체도 단일 band의 high molecular weight 단백을 가지고 있고, 그 mobility는 포유류 골격근의 것과 유사했다. 2) RyR의 정제과정에서 얻어진 $[^3H]ryanodine$의 peak 결합 분획에서 high molecular weight의 단백분획이 발견되었다. 3) 뱀 골격근 SR vesicles에 대한 $[^3H]ryanodine$의 maximum binding site와 Kd값은 각각 6.36 pmole/mg protein과 17.62nM이었으며, $[^3H]ryanodine$의 특이성 결합은 칼슘과 AMP에 의해 유의성있게 증가되었고 (P<0.005), tetracaine에 의해 억제되지 않았으나 ruthenium red와 $MgCl_2$에 의해 일부만 억제되었다. 4) 근 소포체로부터 $^{45}Ca$ 유리는 낮은 농도의 칼슘 $(1{\sim}10{\mu}M)$과 AMP에 의해 증가되었고 (P<0.05), 고농도의 칼슘 $(300{\mu}M)$, tetracaine, ruthenium red 또는 $MgCl_2$에 의해 억제되었다 (P<0.05). 이상의 실험성적으로 파충류 (뱀)의 골격근에도 칼슘유리 채별이 있어 근 수축시 세포내 칼슘 농도 조절에 관여할 수 있을 것으로 여겨지며, 채널의 기능적 특징 일부가 포유류의 것과 유사한 것으로 사료된다.

• 한국발생생물학회지:발생과생식
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• 제24권4호
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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.

• The Korean Journal of Physiology and Pharmacology
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• 제3권2호
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• pp.199-205
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• 1999

Vibrio vulnificus cytolysin caused platelet cytolysis and increased intracellular calcium concentration $([Ca^{2+}]_i)$ of rat platelets in a concentration-dependent manner. In the presence of V. vulnificus cytolysin (3 HU/ml), lactate dehydrogenase (LDH) activity was increased from $1.3{\pm}0.4%$ of control to $64.3{\pm}3.4%$ in platelet suspension buffer. In $Ca^{2+}-free$ platelet suspension buffer, however, V. vulnificus cytolysin did not induce $[Ca^{2+}]_i$ increase and LDH release. Addition of EGTA (2 mM) to suspension buffer after the initial $Ca^{2+}$ influx reversed $[Ca^{2+}]_i$ to the control level. However, a $Ca^{2+}$ channel blocker verapamil $(20\;{\mu}M)$ or mefenamic acid $(20\;{\mu}M)$ did not inhibit V. vulnificus cytolysin-induced $[Ca^{2+}]_i$ increase and LDH release. Divalent cations such as $Co^{2+},\;Cd^{2+}\;or\;Mn^{2+}$ (2 mM each) also did not alter V. vulnificus cytolysin-induced $[Ca^{2+}]_i$ increase and LDH release. V. vulnificus cytolysin (3 HU/ml)-induced calcium influx was completely blocked by lanthanum (2 mM). Lanthanum (2 mM) also completely blocked V. vulnificus cytolysin (3 HU/ml)-induced LDH release. Osmotic protectants such as, raffinose, sucrose or PEG600 (50 mM each) did not inhibit the lytic activity of V. vulnificus cytolysin. In conclusion, lanthanum sensitive $Ca^{2+}$ influx plays a significant role in Vibrio vulnificus cytolysin-induced platelet cytolysis and thrombocytopenia in V. vulnificus infection.

• 한국어병학회지
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• 제9권1호
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• pp.41-51
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• 1996

경골어류의 혈관평활근에 대한 adrenaline성 조절기작을 규명의 일환으로 틸라피아의 배대동맥을 사용하여 Adrenergic agonist의 효과와 그 매개에 관여하는 수용체의 subtype에 대한 연구를 하였으며 그 결과는 다음과 같다. 1. Epinephrine, norepinephrine, phenylephrine, clonidine 및 methoxamine은 tilapia의 배대동맥에 대하여 농도의존적인 혈관수축효과만을 나타내었으며, 효력은 epinephrine, norepinephrine, phenylnephrine, clonidine, methoxamine의 순이었으며, 이들 수축반응은 혈관내피세포의 존재유무에 영향을 받지 않았다. 2. Epinephrine, norepinephrine, phenylephrine 및 clonidine 의 농도의존적인 혈관수축반응곡선은 선택적인 $\alpha_2$-adrenergic 수용체 길항제인 yohimbine의 농도가 증가함에 따라 오른쪽으로 평행이동 되었으며, epinephrine과 norepinephrine은 선택적인 $\alpha_1$-수용체 길항제인 prazosin의 농도가 증가함에 따라 오른쪽으로 평행이동되었다. 3. Epinephrine과 norepinephrine의 혈관수축반응은 calcium제거 생리적 완충용액에서는 각각 약 41%, 51% 소실되었며, calcium 유입차단제인 verapamil에 의해서도 거의 유사한 경향을 보였다. 이상의 실험결과들을 종합하면 Catecholamine류는 수축효과만을 나타내었으며 혈관내피세포 존재유무와는 무관하였다. 이러한 수축작용은 $\alpha_1$- 및 $\alpha_2$-adrenergic receptor가 모두 매개하였으며 voltage dependent $Ca^{2+}$ channel을 통하여 유입된 세포외액의 $Ca^{2+}$과 세포내 $Ca^{2+}$의해 일어난다고 사료된다.

• 한국발생생물학회지:발생과생식
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• 제7권2호
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• pp.95-103
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• 2003

세포내 칼슘은 다양한 세포에서 중요한 생리적 반응을 일으키며, ruthenium red와 ryanodine은 중요한 칼슘 조절자로 작용한다. Ruthenium red는 세포내 칼슘 저장고에서의 calcium induced calcium release(CICR)를 저해한다. Ryanodine은 ryanodine 통로를 통한 칼슘 방출을 촉진한다. 본 실험은 두 조절자가 생쥐 난자와 초기배아의 세포내 칼슘이온 농도에 영향을 미치는지 여부와 그 유효농도를 알아보고자 수행하였다 난자 및 초기배아내 칼슘이온 함량 변화는 Fluo-3/AM을 이용하여 공초점 레이저주사 현미경을 사용하여 실시간으로 측정하였다. Ruthenium red는 고농도(30$\mu$M, 300$\mu$M)에서 난자와 초기배아의 세포내 칼슘이온 농도를 저하시켰고, ryandoine은 저농도(0.01$\mu$M)에서 세포내 칼슘이온 농도를 증가시켰지만 고농도(10$\mu$M)에서는 세포내 칼슘이온 농도를 감소시켰다. 본 실험결과를 보면, ruthenium red와 ryanodine은 생쥐의 난자 및 초기배아에서도 세포내 칼슘이온 농도에 영향을 미쳤고, 그 유효농도는 근세포를 포함한 체세포와는 차이가 있었다.

• 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.

• International Journal of Oral Biology
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• 제40권4호
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• pp.211-216
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• 2015

Nitric Oxide (NO) is an important signaling molecule in the nociceptive process. Our previous study suggested that high concentrations of sodium nitroprusside (SNP), a NO donor, induce a membrane hyperpolarization and outward current through large conductances calcium-activated potassium ($BK_{ca}$) channels in substantia gelatinosa (SG) neurons. In this study, patch clamp recording in spinal slices was used to investigate the sources of $Ca^{2+}$ that induces $Ca^{2+}$-activated potassium currents. Application of SNP induced a membrane hyperpolarization, which was significantly inhibited by hemoglobin and 2-(4-carboxyphenyl) -4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (c-PTIO), NO scavengers. SNP-induced hyperpolarization was decreased in the presence of charybdotoxin, a selective $BK_{Ca}$ channel blocker. In addition, SNP-induced response was significantly blocked by pretreatment of thapsigargin which can remove $Ca^{2+}$ in endoplasmic reticulum, and decreased by pretreatment of dentrolene, a ryanodine receptors (RyR) blocker. These data suggested that NO induces a membrane hyperpolarization through $BK_{ca}$ channels, which are activated by intracellular $Ca^{2+}$ increase via activation of RyR of $Ca^{2+}$ stores.

• 대한약리학회지
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• 제31권1호
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• pp.11-15
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• 1995

연수의 세로토닌 신경계는 내재성 하행성 동통 억제계 (endogenous descending pain inhibitory system) 에 있어서 중추적인 역할을 하고 있다. 연수의 세로토닌 신경세포에 대한 cholecystokinin (CCK) 및 second messenger systems에 작용하는 약물들의 작용을 알아보기 위하여, 쥐의 태자 (태생 14일) 로부터 연수를 분리하여 10동안 배양한 후 5-hydroxytryptamine (5-HT)의 분비에 대한 cholecystokinin (CCK) 및 second messenger systems에 작용하는 약물의 영향을 연구하였다. 배양 10일된 세포에 여러 neuropeptide들을 $10{\mu}M$ 농도로 48 시간동안 자극한 결과, CCK 와 substance P에 의하여 5-HT의 분비가 증가됨을 관찰하였다. Somatostatin, proctolin, thyrotropin releasing hormone, 및 interleukin-6 은 5-HT의 분비에 있어서 아무런 영향이 없었다. 어떠한 second messenger가 CCK에 의한 5-HT 분비에 연관되어 있나를 알아보기 위하여 calcium channel 봉쇄제인 nimodipine, 그리고 calmodulin 길항제인 calmidazolium의 영향을 살펴본 결과 nimodipine ($1{\mu}M$)은 거의 완전하게, 그리고 calmidazolium ($1{\mu}M$)은 부분적으로 유의하게 CCK에 의한 5-HT의 분비를 억제하였다. 또한 adenyl cyclase의 활성도를 높이는 forskolin ($5{\mu}M$)은 5-HT의 분비를 증가시켰지만 protein kinase C(PKC)를 활성화시키는 phorbol myristate acetate (PMA)는 $2{\mu}M$ 농도에서 5-HT의 분비에 아무런 영향을 미치지 아니하였다. 이상의 연구결과, calcium channel을 통한 calcium influx와 세포내 calmodulin이 CCK에 의한 5-HT분비 증가에 있어서 중요한 역할을 함을 제시한다. 또한, 5-HT의 분비에 있어서 cyclic AMP system이 중요한 역할을 하나, PKC system은 5-HT의 분비에 연관이 없음을 제시하고 있다.

• The Korean Journal of Physiology and Pharmacology
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• 제2권2호
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• pp.173-184
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• 1998

The present study was undertaken to examine the influence of glucocorticoids on the secretory responses of catecholamines (CA) evoked by acetylcholine (ACh), DMPP, McN-A-343, excess K^+$ and Bay-K-8644 from the isolated perfused rat adrenal gland and to clarify the mechanism of its action. The perfusion of the synthetic glucocorticoid dexamethasone (10-100\;{\mu}M$) into an adrenal vein for 20 min produced a dose-dependent inhibition in CA secretion evoked by ACh (5.32 mM), excess K^+$ (a membrane-depolarizor 56 mM), DMPP (a selective nicotinic receptor agonist, 100\;{\mu}M$ for 2 min), McN-A-343 (a muscarinic receptor agonist, 100\;{\mu}M$ for 4 min), Bay-K-8644 (a calcium channel activator, 10\;{\mu}M$ for 4 min) and cyclopiazonic acid (a releaser of intracellular $Ca^{2+}$, 10\;{\mu}M$ for 4 min). Similarly, the preperfusion of hydrocortisone (30\;{\mu}M$) for 20 min also attenuated significantly the secretory responses of CA evoked by nicotinic and muscarinic receptor stimulation as well as membrane-depolarization, $Ca^{2+}$ channel activation and the release of intracellular $Ca^{2+}$. Furthermore, even in the presence of betamethasone (30{\mu}M$), CA secretion evoked by ACh, excess K^+$, DMPP and McN-A-343 was also markedly inhibited. Taken together, the present results suggest that glucocorticoids cause the marked inhibition of CA secretion evoked by both cholinergic nicotinic and muscarinic receptor stimulation from the isolated perfused rat adrenal gland, indicating strongly that this inhibitory effect may be mediated by inhibiting influx of extracellular calcium as well as the release of intracellular calcium in the rat adrenomedullary chromaffin cells.