Application of HPLC with Electrochemical Detection to Assaying Tyrosine Hydroxylase Activity and Dopamine Content in Dissociated Cultures of Fetal Rat Brainstem

송동근,위명복,박찬웅,김영희,Song, Dong-Keun,Wie, Myung-Bok,Park, Chan-Woong,Kim, Yung-Hi The Korean Society of Pharmacology 1991 대한약리학잡지 Vol.27 No.1

쥐(태령 14일) 뇌간 세포배양에서 발달과정에 따른 Tyrosine hydroxylase(TH) 활성 및 dopamine의 양적 증가를 전기화학 HPLC를 이용하여 측정하였다. TH 활성 및 dopamine의 양은 배양 7일까지 점차 증가하였다. 배양 7일째에 여러 약물들의 dopamine 대사에 대한 영향을 조사하였다. TH 억제제인 ${\alpha}-methyl-p-tyrosine$ 및 aromatic amiono acid decarboxylase 억제제인 NSD-1015는 효과적으로 dopamine을 고갈시켰다. Dopamine은 reserpine에 의해 고갈되었고, parglyine에 의해 증가되었다. 일주일 배양한 세포의 배양액에 tetrodotoxin$(0.1\;{\mu}M$)을 7일간 투여하였을 때 TH 활성은 현저히 감소하였다. 이상의 결과들은 배양세포의 dopamine 대사가 뇌 dopamine 대사를 충실히 반영함을 나타낸다. 뇌간 세포의 배양에서 HPLC-전기화학검출을 이용한 TH 활성 및 dopamine의 측정은 중추 dopamine계의 약리 및 독성 연구에 유용하리라 사료된다. We measured the developmental increase of tyrosine hydroxylase(TH) activity and dopamine content with high performance liquid chromatography with electrochemical detection(HPLC-EC) in dissociated cultures of fetal rat brainstem(E14). TH activity and dopamine content increased progressively upto 7 days in vitro, when the effects of various drugs on the dopamine contents were studied. ${\alpha}-Methyl-p-tyrosine$, a TH inhibitor and NSD-1015, an inhibitor of aromatic amiono acid decarboxylase effectively depleted dopamine contents. Dopamine contents were depleted by reserpine and increased by pargyline. When cultures grown for 1 week in control medium were then exposed to tetrodotoxin$(0.1\;{\mu}M$) for 7 days, exposure to tetrodotoxin markedly decreased TH activity. All the above results indicate that dopamine metabolism in the cultered cells reflect reliably the property of brain dopamine metabolism. We suggest that measuring TH activity and dopamine content in brainstem culture with HPLC-EC can be useful tool in the study of pharmacology as well as toxicology of the central dopaminergic system.

Effect of Cholecystokinin on Serotonin Release from Cultured Neurons of Fetal Rat Medulla Oblongata

송동근,조현미,이태희,서홍원,김영희,Song Dong-Keun,Cho Hyun-Mi,Lee Tae-Hee,Suh Hong-Won,Kim Yung-Hi The Korean Society of Pharmacology 1995 대한약리학잡지 Vol.31 No.1

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. Serotonergic neurons in medulla oblongata play an important role in the endogenous descending pain inhibitory system. To illucidate the factors involved in the regulation of medullary serotonergic neurons, we studied the effects of cholecystokinin (CCK) and agents acting on various second messenger systems on 5-hydroxytryptamine (5-HT) release from cultured neurons of rat fetal (gestational age 14th day) medulla oblongata. Cultured cells maintained for 10 days in vitro were stimulated for 48 hours with CCK or other neuropeptides at 10 micromolar concentration. CCK ($10{\mu}M$) and substance P ($10{\mu}M$) significantly increased. 5-HT release. However, somatostatin, proctolin, thyrotropin releasing hormone, and interleukin-6 did not have any effects on 5-HT release. Nimodipine ($1{\mu}M$), a calcium channel blocker, almost completely, and calmidazolium ($1{\mu}M$), a calmodulin antagonist, significantly inhibited the CCK-induced 5-HT release. The total 5-HT content (intracellular 5-HT plus released 5-HT) was significantly increased by CCK. However, the intracellular 5-HT content was not significantly changed by CCK. Forskolin ($5{\mu}M$), an adenylate cyclase activiator, but not $2{\mu}M$ phorbol myristate acetate (PMA), a protein kinase C activator, significantly enhanced 5-HT release. The total 5-HT content (intracellular 5-HT plus released 5-HT) was significantly increased by forskolin. However, the intracellular 5-HT content was not significantly changed by forskolin. PMA had no effect on intracellular 5-HT levels. These results suggest that CCK regulates serotonergic neurons in the medulla oblongata by enhancing 5-HT secretion through calcium influx and caimodulin, and that cyclic AMP system but not protein kinase C system is involved in 5-HT release.

액적 표면에서 증발 및 응축을 고려한 곡면 형상 액적 제거장치의 제거 효율에 대한 수치 해석

송동근,Song, Dong Keun 한국입자에어로졸학회 2016 Particle and Aerosol Research Vol.12 No.4

Removal of water contents in a gas is needed in industrial field of gas processing related on energy production/conversion, and environmental treatment. Inertial separators are economic devices for separating droplets from the gas stream. For accurate understanding of removal process in a curved vane mist eliminator, a numerical model including turbulent dispersion, evaporation and condensation of water vapor at surface of droplets is required. A two-stage curved vane mist eliminator has been modeled, and fluid flow of mixture of air and water vapor and droplet trajectories were solved simultaneously with taking into account two-way coupling. Removal efficiency of droplets with various inlet condition of relative humidities (RH, 40%, 90%, and 100%) were compared. As RH increased, the effect of evaporation decreased and inertial separation efficiencies of droplets obtained increased especially for droplets of diameter below 10 micrometers.

A Study on Serum Dopamine-${\beta}$-Hydroxylase Activity and Thermostability in a Randomly Selected Population of Adults

송동근,서유헌,우종인,신상구,박찬웅,Song Dong-Keun,Suh Yoo-Hun,Woo Jong-Inn,Shin Sang-Goo,Park Chan-Woong The Korean Society of Pharmacology 1986 대한약리학잡지 Vol.22 No.1

Distributions of serum dopamine-${\beta}$-hydroxylase(DBH) activity and thermostability in a randomly selected population of Korean adults were studied to investigate the genetic factor which could exert an influence on the serum DBH activity and thermostability. The results were followings: 1. The mean serum DBH activity in a randomly selected population of adults was $18.3{\pm}4.5$ umol/min/l($mean{\pm}SD;n=327$) which showed no age or sex variation. 2. The frequency distribution showed no isolated subgroup with very low serum DBH activity. 3. When the ratio of enzyme activity after heating $55^{\circ}C$ for 20 minutes to that before heating (heated-to-control or H/C ratio). was used as a measure of thermostability, the mean serum DBH H/C ratio in a randomly selecfld population, of adults was $0.90{\pm}0.17(mean{\pm}SD; n=327)$ which showed no age or sex variation. 4. Serum DBH H/C ratio showed unimodal, homogeneous distribution. 5. There was significant negative correlation between serum DBH activity and H/C ratio(r=-0.39. p<0.01). 6. Subjects with H/C ratio less than 0.1 had significantly higher basal enzyme activity $(22.2{\pm}4.5)$ $(mean{\pm}SD;n=33)$ umo1/min/1 than those with H/C ratio more than 1.09 $(15.5{\pm}3.3)$ $(mean{\pm}SD;n=32)$ umo1/min/1. This study shows that the distribution patterns of serum DBH activity and thermostability of Korean population are considerably different from those of Caucasian and it might be a line of evidence for the different inheritance pattern of plasma DBH enzyme between these racial groups. 한국인에 있어서 혈청 dopamine-${\beta}$-hydroxylase(DBH)에 대한 유전적 양상을 알아 보기 위해 정상성인 327명에서 혈청 DBH활성도 및 thermostability를 측정한 결과 다음과 같은 결과를 얻었다. 1. 정상성인 327명의 평균혈청 DBH 활성도는 $18.3{\pm}4.5(SD)umol/min/l$였으며 연령 및 성별에 따른 차이는 없었다. 2. 혈청 DBH활성도의 분포는 unimodol한 분포를 보였으며 매우 낮은 효소 활성도는 관찰할 수 없었다. 3. 정상성인 327명의 평균 thermostability index(H/C : heated-to-control)는 $0.90{\pm}0.17(SD)$ 이었으며 연령 및 성별에 따른 차이는 없었다. 4. H/C 비의 분포는 unimodal한 homogenous분포를 보였다. 5. 혈청 DBH 활성도와 thermosta bility 사이에는 유의한 역상관 관계가 있었다. (r=-0.39, p<0.01). 6. H/C 0.7 미만인 군의 평균 혈청 DBH 활성도는 $22.2{\pm}4.5(SD)umol/min/l$ 였으며 H/C 1.09이상인 군의 평균 혈청 DBH 활성도는 $15.5{\pm}33(SD)umol/min/l$로서 유의한 차이가 있었다(P<0.01). 이상의 결과에서 정상 한국인에 있어서 혈청 DBH 활성도 및 thermostability의 분포 양상이 Caucasian과는 상당히 다른 점을 알 수 있었다.

흰쥐 태 뇌간의 세포배양에서 HPLC-전기화학검출을 이용한 Tyrosine Hydroxylase 활성 및 Dopamine의 정량

송동근(Dong-Keun Song),위명복(Myung-Bok Wie),박찬웅(Chan-Woong Park),김영희(Yung-Hi Kim) 대한약리학회 1991 대한약리학잡지 Vol.27 No.1

We measured the developmental increase of tyrosine hydroxylase(TH) activity and dopamine content with high performance liquid chromatography with electrochemical detection(HPLC-EC) in dissociated cultures of fetal rat brainstem(E14). TH activity and dopamine content increased progressively upto 7 days in vitro, when the effects of various drugs on the dopamine contents were studied. α-Methyl-p-tyrosine, a TH inhibitor and NSD-1015, an inhibitor of aromatic amiono acid decarboxylase effectively depleted dopamine contents. Dopamine contents were depleted by reserpine and increased by pargyline. When cultures grown for 1 week in control medium were then exposed to tetrodotoxin(0.1μM) for 7 days, exposure to tetrodotoxin markedly decreased TH activity. All the above results indicate that dopamine metabolism in the cultered cells reflect reliably the property of brain dopamine metabolism. We suggest that measuring TH activity and dopamine content in brainstem culture with HPLC-EC can be useful tool in the study of pharmacology as well as toxicology of the central dopaminergic system. 쥐(태령 14일) 뇌간 세포배양에서 발달과정에 따른 Tyrosine hydroxylase(TH) 활성 및 dopamine의 양적 증가를 전기화학 HPLC를 이용하여 측정하였다. TH 활성 및 dopamine의 양은 배양 7일까지 점차 증가하였다. 배양 7일째에 여러 약물들의 dopamine 대사에 대한 영향을 조사하였다. TH 억제제인 α-methyl-p-tyrosine 및 aromatic amiono acid decarboxylase 억제제인 NSD-1015는 효과적으로 dopamine을 고갈시켰다. Dopamine은 reserpine에 의해 고갈되었고, parglyine에 의해 증가되었다. 일주일 배양한 세포의 배양액에 tetrodotoxin(0.1μM)을 7일간 투여하였을 때 TH 활성은 현저히 감소하였다. 이상의 결과들은 배양세포의 dopamine 대사가 뇌 dopamine 대사를 충실히 반영함을 나타낸다. 뇌간 세포의 배양에서 HPLC-전기화학검출을 이용한 TH 활성 및 dopamine의 측정은 중추 dopamine계의 약리 및 독성 연구에 유용하리라 사료된다.

수냉식 화격자 내부 유로 형상에 따른 냉각 성능 해석 및 비교

송동근(Dong-Keun Song),김상복(Sangbok Kim) 대한기계학회 2017 대한기계학회 춘추학술대회 Vol.2017 No.5

ACM을 이용한 가스 투과막 특성 해석 모델 개발

송동근(Dong-keun Song),유상석(Sangseok Yu) 대한기계학회 2015 대한기계학회 춘추학술대회 Vol.2015 No.11

A membrane is used to separate pure gas from gas mixture. Many different types of membranes are developed for separation of gases. In this study, the process simulation of gas separation is carried out to understand the physics of gas transport through the membrane. Aspen custom modeler is a tool to develop the simulation model of membrane gas transport that is easily extended to process simulation environment. Gas transport through the membrane depends on geometric parameters. In particular, membrane length is a core parameter to transport gases. In this study, three different membrane modeling approach are investigated those are lumped model, multi-cell model and discretization model. Even though multi-cell model results in very similar accuracy to discretization model, multi-cell model is strongly depended on number of cells. Finally, the discretization model is turned out to be most comprehensive and useful model for parametric study.

흡입구 위치에 따른 Venturi 폭기 성능 해석

송동근(Dong-Keun Song) 대한기계학회 2016 대한기계학회 춘추학술대회 Vol.2016 No.4

해마추상체 신경세포에서 칼슘에 의한 신경섬유 성장억제에 대한 칼파인 억제제의 영향

송동근(Dong-Keun Song),Stanley B. Kater,Donald L. Mykles 대한약리학회 1993 대한약리학잡지 Vol.29 No.2

칼슘이온은 신경섬유 성장의 중요한 조절인자이나 그 정확한 작용기전은 불명확하다. 세포골격 단백은 in vivo 및 in vitro에서 칼슘의존성 단백분해효소(칼파인)에 의해 신속히 분해되므로, 칼슘이온에 의한 신경섬유의 퇴행에 있어서 칼파인의 관련성을 추구하기위하여, 배양된 해마신경세포에서 칼슘이온 ionophore인 A23187에 의한 신경섬유의 성장억제가 칼파인의 억제제인 EST 및 MDL 28170에 의해 차단되는지를 조사하였다. A23187은 100nM의 농도에서 축삭에는 영향이 없이 수상돌기의 퇴행을 유발하였으나, 300 nM의 농도에서는 축삭의 성장을 억제하였다. EST(5 혹은 20 uM) 및 MDL 28170(20 uM)은 100 nM A23187의 수상돌기에 대한 작용과 300 nM A23187의 축삭에 대한 작용을 효과적으로 차단하였다. EST는 A23187에의한 세포내 칼슘이온의 증가를 차단하지 못하였다. 이상의 결과는 해마추상체세포에서 칼슘에 의한 신경섬유의 퇴행이 칼파인에 의해 매개됨을 시사한다. Ca²⁺ is an important regulator of neurite elongation and growth cone movements but the mechanism(s) mediating these Ca²⁺-dependent effects is unclear. Since cytoskeletal proteins are rapidly degraded by Ca²⁺-dependent proteinases (calpains) in vitro and in vivo, we investigated whether Ca²⁺-induced pruning or regression of neuronal processes is mediated by calpains. Isolated hippocampal pyramidal-like neurons were cultured and the ability of the membrane-permeable calpain inhibitors EST (etyl (+)-(2S,3S)-3-[(S)-methyl-1-(3-methlbutylcarbamoyl)-butylcarbamoyl]2-oxiranecarboxylate) and MDL28170 (carbobenzoxyl-Val-Phe-H) to block the Ca²⁺ ionophore A23187-induced suppression in neurite outgrowth was investigated. Addition of 100 nM A23187 to the culture medium resulted in a retraction of dendrites without altering axonal elongation. The addition of 300 nM A23187 to the culture medium resulted in a signiciant decrease in the rate of axonal elongation as well as a retraction of dendritic processes. Administration of EST (5 or 20μM) to the culture medium completely blocked the pruning effect of 100 nM A23187 on dendrites and of 300 nM A23187 on axons, while EST alone did not significantly affect neurite outgrowth rate. MDL 28170 (20μM) showed the same effect as EST in preventing ionophore-induced pruning of dendrites and axons at 100 nM and 300 nM concentrations, respectively, of A23187. EST (20μM) did not block the A23187-induced rise of [Ca²⁺]_i as measured with fura-2. These results show that Ca²⁺-induced pruning of neurites in isolated hippocampal pyramidal neurons is mediated by calpains.

미세기포 발생 기술과 환경 플랜트 분야 응용

송동근(Dong-Keun Song),W.S. Hong,W.H. Shin,S.B. Kim 대한기계학회 2014 대한기계학회 춘추학술대회 Vol.2014 No.05