Effects of Extracellular $Ca^{2+}$ and $Ca^{2+}$-Antagonists on Endothelium-Dependent Relaxation in Rabbit Aorta

서석효,구용숙,박춘옥,황상익,김기환,Suh, Suk-Hyo,Goo, Yong-Sook,Park, Choon-Ok,Hwang, Sang-Ik,Kim, Ki-Whan The Korean Physiological Society 1990 대한생리학회지 Vol.24 No.1

토끼 흉부 대동맥을 이용하여 내피세포 의존성 혈관이완에 대한 세포외 $Ca^{2+}$과 여러가지 $Ca^{2+}$ 길항제의 효과를 분석하여 EDRF의 작용기전을 밝혀 보고자 하였다. 대동맥 횡단 절편의 등장성 수축은 $10^{-7}\;M$ 노에피네프린으로 유발시켰으며, $10^{-6}\;M$ 사세틸콜린으로 내피세포 의존성 혈관이완을 일으켰다. 내피세포는 작은 솜뭉치로 부드럽게 문질러서 제거하였으며, hemolysate를 사용하여 EDRF에 대한 헤모글로빈의 효과를 관찰하였다. 결과를 종합하면 다음과 같다. 1) 아세틸콜린에 의한 내피세포 의존성 혈관이완은 두 시기, 즉 초기급속이완기와 후기완만이완기로 나타났다. 2) 세포외 $Ca^{2+}$을 낮추면, 아세틸콜린에 의한 내피세포 의존성 혈관이완이 감소하였으며, 특히 후기완만이완기가 감소하였다. 3) Verapamil, nifedipine, $Mn^{2+}$ 및 $Cd^{2+}$은 내피세포 의존성 혈관이완에 영향이 없었던 반면 $La^{3+}$와 $Co^{2+}$는 억제시켰다. 4) 헤모글로빈을 투여하면 내피세포가 없는 절편에서는 기초긴장도의 변화가 없었으나 내피세포가 있는 절편에서는 기초긴장도가 증가하였고 아세틸콜린에 의한 내피세포 의존성 혈관이완도 완전히 억제되었다. 이상의 결과로부터 세포외 $Ca^{2+}$은 주로 후기완만이완기에 작용하며 이때 사용되는 $Ca^{2+}$ 유입 통로는 $Ca^{2+}$ 길항제로 억제되지 않는 것으로 결론지을 수 있다. The effects of extracellular $Ca^{2+}$ and various $Ca^{2+}$ antagonists on endothelium-dependent relaxation to acetylcholine were studied in the isolated rabbit thoracic aorta in order to elucidate the control mechanism of endothelium derived relaxing factor (EDRF) release. Endothelium was removed from aortic strips by gentle rubbing with cotton ball. The effect of hemoglobin on basal tension was also observed with hemolysate. The results obtained were as follows: 1) Endothelium-dependent relaxation (EDR) to acetylcholine (ACh) showed biphasic pattern; the initial rapid relaxation phase and the late slow relaxation phase. 2) With the depletion of the extracellular $Ca^{2+}$, EDR was gradually suppressed, especially the late slow relaxation. 3) Verapamil, nifedipine, $Mn^{2+}$ and $Cd^{2+}$ had not any effect on EDR, while $La^{3+}$ and $Co^{2+}$ suppressed EDR completely. 4) The resting tension of the strips with rubbed endothelium was not altered by the addition of hemoglobin. That of the strips with intact endothelium, however, was enhanced and EDR to ACh was completely blocked From these results, we suggest that extracellular $Ca^{2+}$ is necessary for ACh-induced slow relaxation while $Ca^{2+}$ antagonists have not any effect on EDR.

세포외 Ca₂+ 와 Ca₂+ 길항제들이 내피세포성 이완인자의 분비에 미치는 영향

서석효 東國大學校醫學硏究所 1994 東國醫學 Vol.2 No.-

세포외 Ca2+와 Ca2+ 길항제들이 혈관 내피세포에 의한 혈관평활근 세포내 cyclic GMP 농도 상승과 내피세포 의존성 이완에 미치는 영향을 알아보고자 하였다. 2.0 kg 정도의 토끼를 pentobarbital sodium (40mg/kg)를 사용하여 마취하였으며 총경동맥을 절단 실혈시켜 즉사시켰으며, 흉부 및 복부 대동맥과 총경동맥을 절취하였다. 절취한 총경동맥과 대동맥에서 organic bath 실험방법, bioassay 실험방법, 그리고 cyclic GMP 농도 측정 방법으로 각각 세포외 Ca2+와 Ca2+ 길항제들이 내피세포 의존성 이완에 미치는 영향, 내피세포성 이완인자의 분비에 미치는 영향, 그리고 혈관평활근 세포내 cyclic GMP 농도 상승에 미치는 영향을 관찰하였다. 실험 결과는 다음과 같다. 1. 아세틸콜린에 의한 내피세포 의존성 이완은 세포외 Ca2+ 농도를 낮추었을 때 감소하였다. 2. 내피세포성 이완인자 분비 혈관을 관류하는 용액에서 Ca2+을 제거하고 관류 후 Ca2+을 주입 시험절편을 관류할 때에는 정상 Ca2+ 농도로 조정했을 때 시험절편의 이완이 감소하였다. 3. 내피세포 의존성 이완은 organic Ca2+ 길항제와 inorganic Ca2+ 길항제인 Mn2+에 의해서는 억제되지 않았다. 그러나 inorganic Ca2+ 길항제 중 La3+와 Co2+에 의해서는 억제되었다. 4. 아세틸콜린에 의한 내피세포 의존성 cyclic GMP 농도 증가는 Co2+와 Ca2+ 제거 용액에 의해서는 유의하게 억제되었으나, verapamil, nifedipine, Mn2+에 의해서는 억제되지 않았다. 이상의 실험 결과로 미루어 내피세포성 이완인자의 분비에 세포외 Ca2+이 필요하나 Ca2+ 길항제들은 내피세포성 이완인자의 분비에 영향이 없는 것으로 결론 내릴 수 있었다. The effects of extracellular Ca2+ and Ca2+ antagonosts on endothelium-dependent cyclic GMP increase in the vascular smooth muscle cells by acetylcholine and endothelium-dependent relaxation to acetylcholime were studied in rabbit arteries, using organic bath, bioassay experiment, and cyclic GMP measurement. The magnitudes of endothelium-dependent relaxations by acetylcholine were decreased by the treatment with low Ca2+ solution. EDRF release was attenuated with Ca2+ free solution. Endothelium-dependent relaxations by acetylcholine were not attenuated with the treatment of Mn2+ or organic Ca2+-antagonists. Co2+ and La3+ inhibited endothelium-dependent relaxation. Cyclic GMP levels of the strips treated with Co2+ and Ca2+ -free solution were significantly decreased compared with that of the strips with intact endothelium. Whereas, cyclic Gmp levels of the strips treated with verapamil, nifedipine, and Ca2+ were not decreased, compared with that of the strips with intact endothelium. Therefore, it could be concluded that extracellular Ca2+ is necessary for EDRF release but Ca2+-antagonists have no effect no the release.

토끼 대동맥 평활근의 내피세포 의존성 이완에 미치는 Ca²⁺ 및 Ca²⁺ 길항제의 효과

서석효(Suh, Suk-Hyo),구용숙(Goo, Yong-Sook),박춘옥(Park, Choon-Ok),황상익(Hwang, Sang-Ik),김기환(Kim, Ki-Whan) 대한생리학회 1990 대한생리학회지 Vol.24 No.1

토끼 흉부 대동맥을 이용하여 내피세포 의존성 혈관이완에 대한 세포외 Ca²⁺과 여러가지 Ca²⁺ 길항제의 효과를 분석하여 EDRF의 작용기전을 밝혀 보고자 하였다. 대동맥 횡단 절편의 등장성 수축은 10^-7 M 노에피네프린으로 유발시켰으며, 10^-6 M 사세틸콜린으로 내피세포 의존성 혈관이완을 일으켰다. 내피세포는 작은 솜뭉치로 부드럽게 문질러서 제거하였으며, hemolysate를 사용하여 EDRF에 대한 헤모글로빈의 효과를 관찰하였다. 결과를 종합하면 다음과 같다. 1) 아세틸콜린에 의한 내피세포 의존성 혈관이완은 두 시기, 즉 초기급속이완기와 후기완만이완기로 나타났다. 2) 세포외 Ca²⁺을 낮추면, 아세틸콜린에 의한 내피세포 의존성 혈관이완이 감소하였으며, 특히 후기완만이완기가 감소하였다. 3) Verapamil, nifedipine, Mn²⁺ 및 Cd²⁺은 내피세포 의존성 혈관이완에 영향이 없었던 반면 La³⁺와 Co²⁺는 억제시켰다. 4) 헤모글로빈을 투여하면 내피세포가 없는 절편에서는 기초긴장도의 변화가 없었으나 내피세포가 있는 절편에서는 기초긴장도가 증가하였고 아세틸콜린에 의한 내피세포 의존성 혈관이완도 완전히 억제되었다. 이상의 결과로부터 세포외 Ca²⁺은 주로 후기완만이완기에 작용하며 이때 사용되는 Ca²⁺ 유입 통로는 Ca²⁺ 길항제로 억제되지 않는 것으로 결론지을 수 있다. The effects of extracellular Ca²⁺ and various Ca²⁺ antagonists on endothelium-dependent relaxation to acetylcholine were studied in the isolated rabbit thoracic aorta in order to elucidate the control mechanism of endothelium derived relaxing factor (EDRF) release. Endothelium was removed from aortic strips by gentle rubbing with cotton ball. The effect of hemoglobin on basal tension was also observed with hemolysate. The results obtained were as follows: 1) Endothelium-dependent relaxation (EDR) to acetylcholine (ACh) showed biphasic pattern; the initial rapid relaxation phase and the late slow relaxation phase. 2) With the depletion of the extracellular Ca²⁺, EDR was gradually suppressed, especially the late slow relaxation. 3) Verapamil, nifedipine, Mn²⁺ and Cd²⁺ had not any effect on EDR, while La³⁺ and Co²⁺ suppressed EDR completely. 4) The resting tension of the strips with rubbed endothelium was not altered by the addition of hemoglobin. That of the strips with intact endothelium, however, was enhanced and EDR to ACh was completely blocked From these results, we suggest that extracellular Ca²⁺ is necessary for ACh-induced slow relaxation while Ca²⁺ antagonists have not any effect on EDR.

Effects of Electrolytes and Drugs on the Inhibitory Junction Potentials Recorded from the Antrum of Guinea-pig Stomach

구용숙,서석효,이석호,황상익,김기환,Goo, Yong-Sook,Suh, Suk-Hyo,Lee, Suk-Ho,Hwang, Sang-Ik,Kim, Ki-Whan The Korean Physiological Society 1990 대한생리학회지 Vol.24 No.1

The effects of electrolytes, adenosine, ATP, 5-hydroxytryptamine (5-HT, serotonin) and ketanserin on the inhibitory junction potentials (IJPs) were investigated to clarify the interactions of these drugs with the neurotransmitters released from non-adrenergic, non-cholinergic nerves in the antrum of guinea-pig stomach. Electrical responses of antral circular muscle cells were recorded intracellularly using glass capillary microelectrode filled with 3 M KCI. All experiments were performed in Tris-buffered Tyrode soluition which was aerated with 100% $O_{2}$ and kept at $35^{\circ}C$. The results obtained were as follows: 1) Inhibitory junction potential (IJP) was recorded in antral strip, while excitatory junction potential (EJP) was recorded in fundic strip. 2) IJP recorded in antral strip was not influenced by atropine $(10^{-6}\;M)$ and guanethidine $(5{\times}10^{-6})$. 3) The amplitude of IJP increased in high $Ca^{2+}$ solution, while that of IJP decreased in high $Mg^{2+}$ solution or by $Ca^{2+}$ antagonist (verapamil). Apamin, $Ca^{2+}$-activated $K^{+}$ channel blocker blocked IJP completely. 4) ATP and adenosine decreased the amplitude of IJP. 5) 5-HT decreased the amplitude of IJP with no change of the amplitude of slow waves, while ketanserin (5-HT type 2 blocker) decreased the amplitude of slow waves markedly with no change in that of IJP. From the above results, the following conclusions could be made. 1) IJP recorded in antral strip is resulted from neurotransmitters released from non-adrenergic, non-cholinergic nerves. 2) An increase in the concentration of external $Ca^{2+}$ enhances the release of neurotransmitters from non-adrenergic, non-cholinergic nerves which activate the $Ca^{2+}$-dependent $K^{+}$ channel. 기니피그 유문동 부위를 절제한 뒤 점막층을 박리하고 윤상근 주행방향으로 길이 10 mm, 너비 2 mm 되는 조직 절편을 만들어 수평형 실험용기에 넣어 핀으로 고정하였다. 유리미세전극을 세포내에 삽입하여 서파를 기록하면서 조직양편에 설치한 백금자극전극(직경 0.5 mm)에 강도 $10{\sim}50V$, 기간 $50{\sim}100\;{\mu}s$ 되는 자극파를 주어 신경-근 부위의 접합부 전압을 기록하여 다음과 같은 결과를 얻었다. 1) 위저부에서는 흥분성 접합부 전압이, 유문동에서는 억제성 접합부 전압이 기록되었고 유문동의 억제성 접합부 전압은 atropine($10^{-6}\;M)$과 guanethidine$(5{\times}10^{-6}\;M)$을 동시 처치했을 때 영향을 받지 않았다. 2) 세포외 $Ca^{2+}$ 농도를 높였을 때(7 mM)는 억제성 접합부 전압의 크기가 증가하고 세포외 $Mg^{2+}$ 농도를 높였을 때(5 mM)와 verapamil($10^{-5}\;M$)을 주었을 때는 억제성 접합부 전압의 크기가 감소하였다. 3) 아데노신을 투여하였을 때와 ATP를 투여했을 때는 모두 억제성 접합부 전압의 크기가 감소하였다. 4) 5-HT$(10^{-6}\;M)$을 투여했을 때는 서파크기에는 변화없이 억제성 접합부 전압의 크기만 감소하였고 5-HT type 2 길항제인 ketanserin$(5{\times}10^{-6}\;M)$을 투여했을 때는 서파크기는 현저히 감소한 반면 억제성 접합부 전압크기는 변화가 없었다. 이상의 결과로부터 유문동에서 기록되는 억제성 접합부 전압은 비아드레날린, 비콜린 동작성 신경에 의해 유발되며 $Ca^{2+}$은 비아드레날린 비콜린 동작성 신경에서 신경흥분전달물질의 유리를 촉진시키고 분비된 신경흥분전달물질로 인해 $Ca^{2+}$ 의존성 $K^{+}$ 통로가 활성화되어 억제성 접합부 전압의 크기를 증가시킨다고 사료된다.

세포 외 K+에 의한 혈관 수축성 조절 기전: 혈관평활근 수축성과 내피세포 의존성 이완에 미치는 영향

유지영,설근희,서석효,안재호 대한흉부외과학회 2004 Journal of Chest Surgery (J Chest Surg) Vol.37 No.3

Nitric Oxide에 의한 혈관내피세포 내 Ca2+ 조절 기전:Cyclic GMP 의존적 및 비의존적 기전.

전성희,설근희,서석효,박성훈 대한심장학회 2004 Korean Circulation Journal Vol.34 No.6

Background and Objectives:Nitric oxide (NO) reduces the intracellular Ca2+ concentration ([Ca2+]i) in smoothmuscle cells, whereas the effect of NO on [Ca2+]i in endothelial cells is still controversial. Therefore, theeffect of NO on the [Ca2+]i, and its mechanism in mouse aortic endothelial cells (MAEC) and human umbilicalvein endothelial cells (HUVEC) were examined. Materials and Methods:In primary cultured MAEC andHUVEC, cells were loaded with fura 2-AM and [Ca2+]i and measured using a microfluorometer. Results:The NOdonor, sodium nitroprusside (SNP), reduced the [Ca2+]i in 72% of the cells tested (n=100). In the remainingcells, the effect of SNP was biphasic, or the [Ca2+]i was increased. In addition, the membrane-permeablecGMP, 8-bromo cGMP, decreased the [Ca2+]i. The effects of SNP and 8-bromo cGMP were inhibited by thesoluble guanylate cyclase inhibitor, 1H-[1,2,4] oxadiazole[4,3-a]quinoxalin-1-one (ODQ), and the cGMP-dependentprotein kinase inhibitor, KT5823, respectively. In contrast, in the presence of 8-bromo cGMP or ODQ,SNP increased the [Ca2+]i. Conclusion:These results suggest that NO inhibits the [Ca2+]i through a cGMPdependentmechanism and increases the [Ca2+]i through a cGMP-independent mechanism. (Korean Circulation J200434(6):600-609) 배경 및 목적:혈관내피세포에서 분비되는 nitric oxide(NO)는 혈관평활근세포 Ca2+ 농도([Ca2+]i)를 감소시키지만 혈관Korean Circulation J 2004;34(6):600-609 608내피세포 [Ca2+]i에 미치는 영향에 대해서는 다양한 보고가 있으며 그 기전도 명확하지 않다. 이에 본 연구에서는 혈관내피세포에서 NO가 [Ca2+]i에 미치는 영향과그 기전을 밝히고자 하였다.방 법:일차 배양 기법을 이용하여 분리한 쥐의 대동맥 혈관내피세포(mouse aortic endothelial cell, MAEC)와 인간제대정맥 내피세포(human umbilical vein endothelialcell, HUVEC)에서 NO에 의한 [Ca2+]i 변화를 측정하였다. 결 과: SNP는 혈관내피세포 [Ca2+]i에 대하여 다양한 반응 을 보였다. 농도 의존적으로 [Ca2+]i가 감소한 경우는 72%의 세포(총 100세포)에서 관찰되었으며 나머지 세 포에서는 오히려 증가하는 경우, 일시적 감소 후 증가하 는 경우, 저농도에서 감소하다가 고농도에서 증가하는 경우 등 다양한 반응을 보였다. NO 생성 억제제인 LNAME도 혈관내피세포 [Ca2+]i에 대하여 세포에 따라 감소 혹은 증가시키는 서로 상반된 효과를 보였다. 혈 관내피세포 [Ca2+]i는 세포막을 통과하는 cGMP인 8- bromo cGMP에 의하여 감소되었으며, 이러한 8-bromo cGMP의 효과는 cGMP 억제제인 KT5823에 의하여 억제되었다. 혈관내피세포 [Ca2+]i를 감소시키는 SNP 의 효과는 NO dependent soluble guanylate cyclase 억제제인 ODQ에 의하여 억제되었다. 혈관내피세포를 8-bromo cGMP로 전처치하거나 또는 ODQ로 전처치 하고 SNP를 투여하는 경우 저농도에서도 [Ca2+]i가 증 가하였다. 결 론: 이상의 연구결과로 NO는 혈관내피세포 [Ca2+]i를 cGMP 의존적인 기전으로 감소시키며 비의존적 기전으 로는 증가시킬 수 있음을 알 수 있었다. 그러므로 혈관 내피세포에서 NO는 cGMP 의존적 기전과 비의존적 기 전의 상대적인 활성도 정도에 따라서 [Ca2+]i에 영향을 미치며 NO 분비에 영향을 줄 것으로 추정된다.

Oxidized Low-density Lipoprotein- and Lysophosphatidylcholine-induced Ca2+ Mobilization in Human Endothelial Cells

김문영,최수승,서석효,Guo Hua Liang,김지애,최신구 대한약리학회 2009 The Korean Journal of Physiology & Pharmacology Vol.13 No.1

The effects of oxidized low-density lipoprotein (OxLDL) and its major lipid constituent lysophosphatidylcholine (LPC) on Ca2+ entry were investigated in cultured human umbilical endothelial cells (HUVECs) using fura-2 fluorescence and patch-clamp methods. OxLDL or LPC increased intracellular Ca2+ concentration ([Ca2+]i), and the increase of [Ca2+]i by OxLDL or by LPC was inhibited by La3+ or heparin. LPC failed to increase [Ca2+]i in the presence of an antioxidant tempol. In addition, store-operated Ca2+ entry (SOC), which was evoked by intracellular Ca2+ store depletion in Ca2+-free solution using the sarcoplasmic reticulum Ca2+ pump blocker, 2, 5-di-t-butyl-1, 4-benzohydroquinone (BHQ), was further enhanced by OxLDL or by LPC. Increased SOC by OxLDL or by LPC was inhibited by U73122. In voltage-clamped cells, OxLDL or LPC increased [Ca2+]i and simultaneously activated non-selective cation (NSC) currents. LPC-induced NSC currents were inhibited by 2-APB, La3+ or U73122, and NSC currents were not activated by LPC in the presence of tempol. Furthermore, in voltage-clamped HUVECs, OxLDL enhanced SOC and evoked outward currents simultaneously. Clamping intracellular Ca2+ to 1 μM activated large-conductance Ca2+-activated K+ (BKCa) current spontaneously, and this activated BKCa current was further enhanced by OxLDL or by LPC. From these results, we concluded that OxLDL or its main component LPC activates Ca2+-permeable Ca2+-activated NSC current and BKCa current simultaneously, thereby increasing SOC.

Isolation and In Vitro Culture of Vascular Endothelial Cells from Mice

최신규,김지예,김관창,서석효 대한약리학회 2015 The Korean Journal of Physiology & Pharmacology Vol.19 No.1

In cardiovascular disorders, understanding of endothelial cell (EC) function is essential to elucidatethe disease mechanism. Although the mouse model has many advantages for in vivo and in vitroresearch, efficient procedures for the isolation and propagation of primary mouse EC have beenproblematic. We describe a high yield process for isolation and in vitro culture of primary EC frommouse arteries (aorta, braches of superior mesenteric artery, and cerebral arteries from the circle ofWillis). Mouse arteries were carefully dissected without damage under a light microscope, and smallpieces of the vessels were transferred on/in a Matrigel matrix enriched with endothelial growthsupplement. Primary cells that proliferated in Matrigel were propagated in advanced DMEM with fetalcalf serum or platelet-derived serum, EC growth supplement, and heparin. To improve the purity ofthe cell culture, we applied shearing stress and anti-fibroblast antibody. EC were characterized bya monolayer cobble stone appearance, positive staining with acetylated low density lipoprotein labeledwith 1,1’-dioctadecyl-3,3,3’,3’-tetramethyl-indocarbocyanine perchlorate, RT-PCR using primers forvon-Willebrand factor, and determination of the protein level endothelial nitric oxide synthase. Oursimple, efficient method would facilitate in vitro functional investigations of EC from mouse vessels.

혈관내피세포 기능 이상과 에스트로겐, 프로게스테론 관계에 대한 연구

김미경,이수윤,김영주,서석효,박미해 대한산부인과학회 2006 대한산부인과학회 학술대회 Vol.92 No.-

Contradictory Effects of Superoxide and Hydrogen Peroxide on KCa3.1 in Human Endothelial Cells

최신규,나혜영,김지애,조성은,서석효 대한약리학회 2013 The Korean Journal of Physiology & Pharmacology Vol.17 No.3

Reactive oxygen species (ROS) are generated in various cells, including vascular smooth muscle and endothelial cells, and regulate ion channel functions. KCa3.1 plays an important role in endothelial functions. However, the effects of superoxide and hydrogen peroxide radicals on the expression of this ion channel in the endothelium remain unclear. In this study, we examined the effects of ROS donors on KCa3.1 expression and the K+ current in primary cultured human umbilical vein endothelial cells (HUVECs). The hydrogen peroxide donor, tert-butyl hydroperoxide (TBHP), upregulated KCa3.1 expression,while the superoxide donors, xanthine/xanthine oxidase mixture (X/XO) and lysophosphatidylcholine (LPC), downregulated its expression, in a concentration-dependent manner. These ROS donor effects were prevented by antioxidants or superoxide dismustase. Phosphorylated extracellular signal-regulated kinase (pERK) was upregulated by TBHP and downregulated by X/XO. In addition, repressor element-1-silencing transcription factor (REST) was downregulated by TBHP, and upregulated by X/XO. Furthermore, KCa3.1 current, which was activated by clamping cells with 1 μM Ca2+ and applying the KCa3.1 activator 1-ethyl-2-benzimidazolinone, was further augmented by TBHP,and inhibited by X/XO. These effects were prevented by antioxidants. The results suggest that hydrogen peroxide increases KCa3.1 expression by upregulating pERK and downregulating REST, and augments the K+ current. On the other hand, superoxide reduces KCa3.1 expression by downregulating pERK and upregulating REST, and inhibits the K+ current. ROS thereby play a key role in both physiological and pathological processes in endothelial cells by regulating KCa3.1 and endothelial function.