• The Korean Journal of Physiology
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• 제18권1호
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• pp.37-50
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• 1984

The effects of $Ca^{++}$ and its antagonists (verapamil and $La^{+++}$) upon the spontaneous contraction and the contracture induced by 60 mM K-Tyrode solution were studied in the isolated uterine muscle. Longitudinal muscle strips were prepared from the rat uteri at estrous stage. All experiments were performed in tris-buffered Tyrode solution which was aerated with 100% $O_2$ and kept at 35^{\circ}$. The results obtained were as follows: 1) In the uterine strips contracting spontaneously, both the amplitude of peak tension and the area of contraction curve increased dose-dependently in the range of $0.5${\sim}8$ mM $Ca^{++}$. The frequency of contraction increased as the concentration of $Ca^{++}$ increased up to 2 mM, but above this concentration the frequency decreased. In $Ca^{++}-free$ media, however, contraction did not develop. In the contracture induced by 60 mM K-Tyrode solution, the developed tension increased dose-dependently as the concentration of external $Ca^{++}$ increased to 8 mM. In the absence of external $Ca^{++}$ K-contracture appeared, but it was not sustained. 2) The spontaneous contraction of rat uterus was suppressed by verapamil in proportion to an increase of its concentration and totally abolished at the concentration of $3{\times}10^{-4}\;g/l$, but the spontaneous contraction re-appeared by addition of $Ca^{++}$. The amplitude of peak tension recovered completely but the recovery of frequency was incomplete. K-contracture decreased in a dose-dependent manner after the treatment with verapamil and totally disappeared at its concentration of $3{\times}10^{-4}\;g/l$. Even in this case contracture developed again by extra $Ca^{++}$. 3) The spontaneous contractile activity was inhibited by $La^{+++}$. At the concentration of $10^{-4}$M $La^{+++}$, fibrillation appeared. In the strip inhibited by $10^{-5}M\;La^{+++}$, contractility recovered completely by extra $Ca^{++}$ while in the $10^{-4}M\;La^{+++}$ treated preparation, the rhythmic spontaneous contraction did not develop even at the concentration of 16 mM $Ca^{++}$. After the initial transient depression of contracture tension by $10^{-3}M$ of $La^{+++}$, the strip stowed considerably large size of contracture, hardly influenced by external $Ca^{++}$ or verapamil. The results obtained in this experiment suggest that in the rat uterine muscle there would be some competitive actions between $Ca^{++}$ and its antagonists. It is speculated that $Ca^{++}$ plays an important role in the conduction of excitation, and $La^{+++}$ influences upon cellular $Ca^{++}$ mobilization and re-uptake process as well as transmembrane $Ca^{++}$ transport in a K-depolarized state.

• Clinical and Experimental Reproductive Medicine
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• 제29권2호
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• pp.139-147
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• 2002

Objective : This study was to establish a reproducible differentiation system from the parthenogenetic mouse embryonic stem (P-mES02) cells into functional cardiomyocytes like as in vitro fertilization mouse embryonic stem (mES01) cells. Materials and Methods: To induce differentiation, P-mES02 cells were dissociated and aggregated in suspension culture environment for embryoid body (EB) formation. For differentiation into cardiomyocytes, day 4 EBs were treated with 0.75% dimethyl sulfoxide (DMSO) for another 4 days (4-/4+) and then were plated onto gelatin-coated dish. Cultured cells were observed daily using an inverted light microscope to determine the day of contraction onset and total duration of continuous contractile activity for each contracting focus. This frequency was compared with the results of DMSO not treated P-mES02 group (4-/4-) and mES01 groups (4-/4+ or 4-/4-). For confirm the generation of cardiomyocytes, beating cell masses were treated with trypsin-EDTA, dispersed cells were plated onto glass coverslips and incubated for 48 h. Attached cells were fixed using 4% paraformaldehyde and incubated with specific antibodies (Abs) to detect cardiomyocytes (anti-sarcomeric ? -actinin Ab, 1 : 100; anti-cardiac troponin I Ab, 1 : 2000) for 1 h. And the cells were finally treated with FITC or TRITC labelled 2nd Abs, respectively, then they were examined under fluorescence microscopy. Results: Rhythmically contracting areas in mES01 or P-mES02 cells were firstly appeared at 9 or 10 days after EBs plating, respectively. The highest cumulative frequency of beating EBs was not different in both treatment groups (mES01 and P-mES02, 4-/4+) with the results of 61.3 % at 13 days and 69.8% at 15 days, respectively. Also, the contracting duration of individual beating EBs was different from minimal 7 days to maximal 53 days. However, DMSO not treated groups (mES01 and P-mES02, 4-/4-) also had contracting characteristics although their frequency was a few compared to those of DMSO treated groups (6.0% and 4.0%). Cells recovered from the spontaneously contracting areas within EBs in both treated groups were stained positively with muscle specific anti-sarcomeric ? -actinin Ab and cardiac specific anti-cardiac troponin I Ab. Conclusion: This study demonstrated that the P-mES02 cell-derived cardiomyocytes displayed similarly structural properties to mES01 cell-derived cardiomyocytes and that the DMSO treatment enhanced the cardiomyocytes differentiation in vitro.

• The Korean Journal of Physiology
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• 제18권2호
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• pp.139-150
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• 1984

Vanadate의 회장 평활근에 대한 수축작용이 Na-K-ATPase를 억제하기 때문인지를 구명하기 위하여 Na-K-ATPase를 억제하는 ouabain과의 작용의 차이를 관찰한 결과 다음과 같은 결론을 얻었다. 1) Ouabain에 의해 나타나는 수축은 2중 peak를 나타내었으나 vanadate 의해서는 단일 Peak만을 보였다. 2) Ouabain에 의한 수축은 atropine$(2{\times]10^{-6}M)$에 의해 강력하게 억제되었으나 vanadate의 작용은 영향을 받지 않았다. 3) Ouabain에 의한 수축은 vanadate에 비해 외부의 $Ca^{++}4농도 및 Ca-길항제에 대해 민감하게 영향을 받았다. 4) 용액내 $Na^+$이 없을때 혹은 고농도의 $K^+$존재하에서 ouabain에 의한 수축반응은 거의 나타나지 않았으나 vanadate에 의한 수축은 영향을 받지 않았다. 5) Vanadate에 의한 수축은 ouabain 존재시에 더욱 증가되었다. 6) 3시간동안 incubation한 결과 vanadate는 ouabain과 달리 세포내 $Na^+$의 농도에 영향을 미치지 못하였다. 이상의 결과로 보아 ouabain과 vanadate는 서로 다른 기전에 의해 회장 평활근에서 수축반응을 유발시키는 것으로 추측된다.

• 대한수의학회지
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• 제46권4호
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• pp.323-326
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• 2006

Suaeda (S.) asparagoides $M_{IQ}$, one of the halophyte groups, has been used as a folk remedy for digestive disturbances in Korea. However, its pharmacological activity on gastrointestinal motility has not been reported yet. In this study, the effects of this halophyte extracts with various solvent fractions (ethanol, hexane, chloroform, ethyl acetate, butanol, and water) on mice ileal spontaneous motility was examined. All solvent fractions at the concentration of $100{\mu}g/ml$ showed inhibitory actions on spontaneous motility of ileum with the potency order of water > 70% ethanol > hexane ${\gg}$ chloroform ${\geq}$ butanol ${\geq}$ ethyl acetate, respectively. In addition, the water fraction of extracts from S. asparagoides $M_{IQ}$ (WFSA) dose-dependently ($1-100{\mu}g/ml$) inhibited the amplitude of spontaneous phasic contraction and area under the contractile curve (AUC). The inhibitory effect of water fraction at the concentration of $10{\mu}g/ml$ was not affected by tetrodotoxin (TTX), $Na^+$ channel blocker ($1{\mu}M$), and $N^w$-nitro-L-arginine Methyl Ester (L-NAME), nitric oxide synthase inhibitor ($100{\mu}M$). However, cyclopiazonic acid (CPA, $10{\mu}M$), inhibitor of sarcoplasmic reticulum $Ca^{2+}$-ATPase, almost blocked the inhibitory effects of WFSA ($10{\mu}g/ml$) on the spontaneous phasic contraction of mouse ileum. But, CPA did not inhibit the lowering basal tone effects of WFSA. The result of this study showed that various extracts of S. asparagoides $M_{IQ}$ induce inhibitory effects on spontaneous contraction of mice ileal segments. More over, the polar solvent fractions were shown to be more potent than non-polar solvent fractions. The effects of S. asparagoides $M_{IQ}$ extracts are not mediated by nerve or nitric oxide. The inhibitory effects of WFSA at least partially mediated by sarcoplasmic reticulum $Ca^{2+}$-ATPase. However, further study is required to determine the exact pharmacological mechanisms of this halophyte on its gastrointestinal motility inhibitory effects.

• Archives of Pharmacal Research
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• 제29권11호
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• pp.1024-1031
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• 2006

The role of mitogen-activated protein kinase (MAPK) in the decreased contractile response to phorbol ester in aortic smooth muscle strips from deoxycorticosterone acetate (DOCA)-salt hypertensive rats was examined. Norepinephrine (NE) evoked greater contractility in aortic strips from DOCA rats than in those of sham-operated rats. 12-Deoxyphorbol 13-isobutyrate (DPB) induced contraction in $Ca^{2+}-free$ medium, which was diminished in strips from DOCA rats compared to sham-operated rats. Vasoconstrictions induced by these stimulants were inhibited by SB203580 and PD098059, inhibitors of p38 MAPK and extracellular signal-regulated kinase (ERK) 1/2, respectively, in both strips. The phosphorylation of p38 MAPK and ERK1/2 induced by NE was greater in strips from DOCA rats compared to those from sham-operated rats, and this phosphorylation was inhibited by the kinase inhibitors. DPB increased the phosphorylation of p38 MAPK and ERK1/2 in strips from both animals, and the increment of p38 MAPK phosphorylation by the stimulant was diminished in strips from DOCA rats compared to sham-operated rats. These findings suggest that the $Ca^{2+}-independent$ contraction evoked by DPB results from the activation of MAPKs in rat aortic smooth muscle and that the attenuated contractility by DPB in DOCA rat appears to be associated with diminished p38 MAPK activity.

• BMB Reports
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• 제55권11호
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• pp.565-570
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• 2022

Pulmonary arterial hypertension (PAH) is a progressive and devastating disease whose pathogenesis is associated with a phenotypic switch of pulmonary arterial vascular smooth muscle cells (PASMCs). Bone morphogenetic protein (BMP) signaling and potassium two pore domain channel subfamily K member 3 (KCNK3) play crucial roles in PAH pathogenesis. However, the relationship between BMP signaling and KCNK3 expression in the PASMC phenotypic switching process has not been studied. In this study, we explored the effect of BMPs on KCNK3 expression and the role of KCNK3 in the BMP-mediated PASMC phenotypic switch. Expression levels of BMP receptor 2 (BMPR2) and KCNK3 were downregulated in PASMCs of rats with PAH compared to those in normal controls, implying a possible association between BMP/BMPR2 signaling and KCNK3 expression in the pulmonary vasculature. Treatment with BMP2, BMP4, and BMP7 significantly increased KCNK3 expression in primary human PASMCs (HPASMCs). BMPR2 knockdown and treatment with Smad1/5 signaling inhibitor substantially abrogated the BMP-induced increase in KCNK3 expression, suggesting that KCNK3 expression in HPASMCs is regulated by the canonical BMP-BMPR2-Smad1/5 signaling pathway. Furthermore, KCNK3 knockdown and treatment with a KCNK3 channel blocker completely blocked BMP-mediated anti-proliferation and expression of contractile marker genes in HPAMSCs, suggesting that the expression and functional activity of KCNK3 are required for BMP-mediated acquisition of the quiescent PASMC phenotype. Overall, our findings show a crosstalk between BMP signaling and KCNK3 in regulating the PASMC phenotype, wherein BMPs upregulate KCNK3 expression and KCNK3 then mediates BMP-induced phenotypic switching of PASMCs. Our results indicate that the dysfunction and/or downregulation of BMPR2 and KCNK3 observed in PAH work together to induce aberrant changes in the PASMC phenotype, providing insights into the complex molecular pathogenesis of PAH.

• The Korean Journal of Physiology
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• 제21권2호
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• pp.259-272
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• 1987

This was study carried out to investigate the effect of calcium on spontaneous contraction and electrical activity induced by ethanol in gastric smooth muscle. After peeling off the mucous membrane from the isolated whole stomach of 102 cats, two kinds of small muscle preparations $(2.0{\times}0.2\;cm)$, one longitudinal and the other circular, were excised from the fundus, the corpus and the antrum portion of each whole stomach specimen. The isometric contraction of the small muscle preparation was measured in a cylinder-shaped chamber filled with Krebs-Ringer-dextrose solution (pH 7.4, temperature $36{\pm}0.5^{\circ}C$) bubbling with 5% $CO_2$ in $O_2$. A large muscle preparation $(5.0{\times}1.2\;cm)$ was excised from the anterior wall of the corpus-antrum portion of the same specimen in 72 of 102 cats. The gastric electrical activity (slow wave and spike potential) was monopolarly recorded by four capillary electrodes (Ag-AgCl), of which two were placed on the corpus and two on the antrum, in a muscle chamber filled with the same solution as described above. Changes in the amplitude of the contraction, frequency of the gastric slow wave and the production of the spike potential were observed after adding ethanol and/or under the treatments with verapamil, $CaCl_2$ and Ca-free Krebs-Ringer-dextrose solution. The results were as follows: 1) After adding ethanol, the spontaneous phasic contraction of the corpus was reduced dose-dependently (0.125-2.0%), which was totally abolished by higher concentrations (2.0-8.0%) of ethanol. 2) The corporal phasic contraction was also completely abolished by verapamil $(3{\times}10^{-5}\;M)$ or Ca-free Krebs-Ringer-dextrose solution. The contraction was increased by $CaCl_2\;(1.8{\times}10^{-3}\;M)$, but the inhibitory effect of ethanol on the contraction persisted even under the treatment with $CaCl_2$. 3) At higher concentrations, ethanol caused tonic contraction of both preparations from the fundus, the corpus and the antrum in a dose-dependent manner. The tonic contraction of the fundus produced by ethanol was not influenced by $CaCl_2$ or verapamil, whereas the tonic contraction was not produced by ethanol in tile Ca-free solution. 4) Frequency of gastric slow wave was decreased dose-dependently by the addition of ethanol (0.25-1.0%), and tile slow wave was not produced by higher concentration of ethanol (2.0%). 5) The frequency of slow wave was significantly reduced by verapamil only and the inhibitory influence of ethanol on the slow wave frequency was reinforced by verapamil. 6) The treatment of $CaCl_2$ increased significantly the slow wave frequency, and attenuated the inhibitory effect of ethanol on the frequency. It is therefore suggested that ethanol regulates the phasic contraction and the production of slow wave by interfering with the transport of calcium in the stomach muscle of the cat.

• 생명과학회지
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• 제22권4호
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• pp.499-507
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• 2012

군소인 Aplysia kurodai의 중추신경절로부터 발견된 myomodulin A (MMA, PMSMLRLamide)와 myomodulin E (MME, GLQMLRLamide)는 $Mytilus$ $edulis$의 anterior byssus retractor muscle (ABRM)을 활성측정시스템으로 사용하여 정제되었다. 정제된 MMA와 MME는 연체동물에서 발견된 myomodulin 계열의 펩타이드와 동일한 일차구조를 지닌다. MME의 구조와 활성간의 상관관계를 알아보기 위해서 MME, 유도체 및 다른 신경성 펩타이드들을 합성하였다. MME의 유도체인 Des[$Gly^1$]-MME, Des[$Gly^1,Leu^2$]-MME 및 Des[$Gly^1,Leu^2,Gln^3$]-MME의 일차구조는 각각 LQMLRLamide, QMLRLamide 및 MLRLamide이다. 합성 물질들을 사용하여 ABRM에 대한 phasic contraction을 측정하였다. MME는 $1{\times}10^{-9}$ M 또는 더 높은 농도에서 ABRM의 phasic contraction을 저해하였다. 또한 MME는 $1{\times}10^{-8}$ M에서 catch-tension에 대해 이완활성을 나타내었다. 합성 펩타이드들을 사용하여 Africa giant snail, $Achatina$ $fulica$의 소낭과 penial retractor muscle에 대해서도 활성을 측정하였다. MME와 유도체들은 소낭에 대해서는 수축반응을 보였지만, penial retractor muscle에 대해서는 이완 활성을 나타내었다. 이러한 결과들은 MME와 그 유도체들은 연체동물의 다양한 조직에 대해 조절 효과를 가지고 있다는 것을 의미한다. 본 연구는 생체 내에서 발생하는 신경 및 circuit의 변화를 조절하는 작용 연구에 대한 기본적인 자료가 될 것이다.

• 한국어병학회지
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• 제14권1호
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• pp.46-53
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• 2001

본 연구에서는 경골어류인 나일틸라피아(Oreochromis niloticus)와 이스라엘잉어(Cyprinus carpio)의 장관에 대한 neurokinin-1(NK-1)수용체 효능제인 substance p(SP)와 neurokinin-2(NK-2)수용체 효능제인 neurokinin A(NKA)의 수축활성과 작용기전을 비교 분석하였다. SP와 NKA는 모두 나일틸라피아와 이스라엘잉어의 장관 평활근에 대하여 농도 의존적인 수축반응을 나타내었다. 그러나 이들에 의한 수축반응의 경향은 어종에 따라 다르게 나타났다. 즉, 나일틸라피아 장관 평활근에 대하여서는 SP가 NKA에 비하여 높은 친화력과 효능을 나타내었으나, 이스라엘잉어의 장관 평활근에 대하여서는 SP와 NKA의 수축반응은 유의한 차이가 없었다. 나일틸라피아 및 이스라엘잉어의 장관 평활근에 대한 SP와 NKA의 수축반응들은 모두 NK-1 수용체 차단제인 L-732,138에 의해서는 비경쟁적으로 길항되었으나, NK-2 수용체 차단제인 MDL 29913에 의해서는 영향을 받지 않았다. 한편, 선택적 무스카린성 수용체 길항제인 atropine($5{\times}10^{-7}$M)및 신경절 차단제인 tetrodotoxin($2{\times}10^{-7}$M)은 나일틸라피아 및 이스라엘잉어의 장관 평활근에 있어서 SP에 의해 유발된 수축반응들은 모두 유의성 있게 억제시켰으나, NKA에 의해 유발된 수축반응들에 대하여서는 유의성 있는 영향을 나타내지 않았다. 이상의 결과들을 종합하여 보면, SP및 NKA는 모두 어류의 장관 평활근에 대하여 강력한 수축효과를 나타내나 그 작용기전은 다르다고 추정된다. 즉, SP및 NKA의 장관 수축작용은 주로 장관 평활근에 존재하는 NK-1 수용체를 매개한 직접적인 작용에 기인하는 것으로 사료되나 SP의 경우에는 tachykinin 수용체를 통한 직접작용 이외에도 콜린성 신경말단(cholinergic nerve terminal)을 자극하는 간접적인 경로도 관여하는 것으로 추정된다.

• Journal of Yeungnam Medical Science
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• 제11권2호
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• pp.293-302
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• 1994

유뇨증의 치료제로서 널리 사용되고 있는 imipramine의 작용기전에 관한 학설이 여러가지가 있으나 그 중 콜린성수용체 봉쇄작용을 관찰하기 위한 방법으로 평활근 세포를 분리배양하여 단일세포에 대한 acetylcholine의 수축작용과 이에 대한 imipramine의 길항작용을 atropine의 그것과 비교해 보기로 하였다. 개의 방광을 적출하여 $0{\sim}4^{\circ}C$의 K-H 용액내에서 $2{\times}2mm$크기의 평활근 절편을 얻어 $36^{\circ}C$의 collagenase 용액에 넣고 95%/5% $O_2/CO_2$, 혼합산소 공급하에서 17~20분동안 배양하여 분리된 부유세포군을 5 ml test tubes에 나누어 담고 acetylcholine을 $10^{-14}M{\sim}10^{-9}M$의 농도로 첨가하였다. Acrolein 1%를 가하여 수축한 세포를 고정시킨 후, 위상차 현미경에 장착한 CCTV camera로 채취한 영상을 microscaler로 전송하고 monitor상에서 세포의 길이를 측정하였다. 분리된 세포들은 acetylcholine에 의해 5초 이내에 최대 수축을 보였으며 이후 120초까지 수축상태를 지속하였다. Atropine은 acetylcholine 유발 수축을 atropine $10^{-7}M$ 에서부터 농도의존적으로 억제하였으며, imipramine도 acetylcholine 유발수축을 atropine과 같은 경향으로 농도의존적으로 억제하였는데, imipramine $10^{-9}M$의 저농도에서도 유의한 억제를 보였다. 이상의 결과를 종합하면 본 실험에 사용된 조건하에서의 collagense를 이용한 세포 분리법은 단일세포를 대상으로 하는 실험을 위하여 가용한 방법이며, imipramine은 개의 방광평활근 단일세포에서 atropine보다 더 강력한 콜린성 수용체 봉쇄작용을 나타낸다고 생각되었다.