평지방막에 융합된 골격근의 single ATP-sensitive K⁺ channel의 이온투과성에 대한 연구

류판동,Ryu, Pan-dong 대한수의학회 1992 大韓獸醫學會誌 Vol.32 No.4

Properties of unitary ATP-sensitive $K^+$ channels were studied using planar lipid bilayer technique. Vesicles were prepared from bullfrog (Rana catesbeiana) skeletal muscle. ATP-sensitive $K^+$ (K (ATP)) channels were identified by their unitary conductance and sensitivity to ATP. In the symmetrical solution containing 200mM KCI, 10mM Hepes, 1mM EGTA and pH 7.2, single K (ATP) channels showed a linear current-voltage relations with slight inward rectification. Slope conductance at reversal potential was $60.1{\pm}0.43$ pS(n=3)). Micromolar ATP reversibly inhibited the channel activity when applied to the cytoplasmic side. In the range of -50~+50 mV, the channel activity was not voltage-dependent, but the channel gating within a burst was more frequent at negative voltage range. Varying the concentrations of external/internal KCl(mM) to 40/200, 200/200, 200/100 and 200/40 shifted reversal potentials to $-30.8{\pm}2.9$(n=3), $-1.1{\pm}2.7$(n=3), 10.5 and 30.6(mV), respecrivety. These reversal potentials were close to the expected values by the Nernst equation, indicating nearly ideal selectivity for $K^+$ over $Cl^-$. Under bi-ionic conditions of 200mM external test ions and 200mM internal $K^+$, the reversal potentials for each test ion/K pair were measured. The measured reversal potentials were used for the calculation of the releative permeability of alkali cations to $K^+$ ions using the Goldman-Hodgkin-Katz equation. The permeability sequence of 5 cations relative to $K^+$ was $K^+$(1), $Rb^+$(0.49), $Cs^+$(0.27), $Na^+$(0.027) and $Li^+$(0.021). This sequence was recognized as Eisenman's selectivity sequence IV. In addition, modelling the permeation of $K^+$ ion through ATP-sensitive $K^+$ channel revealed that a 3-barrier 2-site multiple occupancy model can reasonably predict the observed current-voltage relations.

랫드 척수후각 단일세포 분리 및 특성에 관한 연구

한성규,류판동,Han, Seong-Kyu,Ryu, Pan-Dong 대한약리학회 1996 대한약리학잡지 Vol.32 No.2

The spinal dorsal horn is the area where primary afferent fibers terminate and cutaneous sensory information is processed. A number of putative neurotransmitter substances, including excitatory and inhibitory amino acids and peptides, are present in this region. In this study, single neurons of the spinal dorsal horn were acutely isolated and the properties of whole cell current and responses to excitatory and inhibitory neurotransmitters were studied by patch clamp technique. Transverse slice ($(300{\mu}m$) of lumbar spinal cords from young rats$(7{\sim}14\;days)$ were sequentially treated with two pretenses(pronase 0.75 mg/ml and thermolysin 0.75 mg/ml), then single neurons were mechanically dissociated. These neurons showed near-intact morphology such as multipolar, ellipsoidal and bipolar, and pyramidal cells and we recorded the typical whole cell currents of $K^+$, $Ca^{2+}$ and ligand-operated channels from these neurons. Glutamate $(30{\mu}M)$ and N-methyl-D-aspartate(NMDA, $30{\mu}M)$ induced inward currents of $117{\pm}12.4$ pA(n=5) and $49{\pm}6.9$ pA(n=3), respectively. Glycine $(1{\mu}M)$ potentiated glutamate-induced currents $4{\sim}5$ times and NMDA-induced currents $8{\sim}10$ times. In addition, glycine $(30{\mu}M)$ induced Inward current ($31{\pm}6.1$ nA, n=2), which was rapidly desensitized after the peak to a new steady-state level. However, the inward currents induced by ${\gamma}-amino$ butyric acid(GABA, $1{\mu}M$) decreased continuously after the peak($226{\pm}41.6$ pA, n=3) under the similar experimental condition. The ionic currents and pharmacological responses of isolated neurons in this work were similar to those observed in vivo or in vitro spinal cord slice, indicating that acutely isolated neurons could be effectively used for further pharmacological studies.

K⁺ 통로 조절 약물이 마우스 골격근의 피로현상에 미치는 영향

이기호,류판동,이문한,이항,Lee, Ki-ho,Ryu, Pan-dong,Lee, Mun-han,Lee, Hang 대한수의학회 1995 大韓獸醫學會誌 Vol.35 No.2

The density of ATP-sensitive potassium($K_{APT}$) channels, that open as intracellular ATP concentration falls below a critical level, is very high in skeletal muscle surface membrane and those high density may imply that $K_{ATP}$ channels have very important physiological roles. To elucidate a role of $K_{ATP}$ in relation to fatigue, the modulating effects of potassium channel openers and blockers on the fatigue velocity(FV) of mouse extensor hallucis longus muscle(EHL) were investigated in vitro. Twitch contraction was induced by an electrical field stimulation (EFS: 24-48V, 20ms, 0.2-4Hz) and resulting contraction force was isometrically recorded. The twitch forces were gradually decreased to 25% of initial contraction force(ICF) in $37.52{\pm}1.55sec$($mean{\pm}s.e.m.$, n=135), indicating the fatigue phenomena. The mean velocity for development of the fatigue was measured during the period that twitch force decreased to half($FV_{0/0.5}$) and during the period from half to 25%($FV_{0.5/0.25}$) of ICF. The fatigue was induced once every one hour and the tissue response was stable for up to 4 hours. In control condition, ICF was $5.8{\pm}0.12g$ (n=144) and decreased to 50% of ICF with the mean fatigue velocity of $0.182{\pm}0.006g/sec$($FV_{0/0.5}$, n=135) and from 50% to 25% of ICF with $0.084{\pm}0.004g/sec$($FV_{0.5/0.25}$, n=135). Cromakalim($50{\mu}M$) significantly increased $FV_{0.5/0.25}$(n=4). Glibenclamide($IC_{50}>50{\mu}M$), $Ba^{2+}$($IC_{50}=10{\mu}M$), 4-aminopyridine($FV_{0/0.5}$, $IC_{50}=0.5mM$; $FV_{0.5/0.25}$, $IC_{50}=2mM$) decreased both $FV_{0/0.5}$ and $FV_{0.5/0.25}$ concentration-dependently up to 75%. $TEA^+$(30mM), E-4031($10{\mu}M$), tolbutamide(1mM) decreased $FV_{0.5/0.25}$, but apamin(300nM) and $TEA^+$(10mM) showed no significant effects. Our results suggest that activation of the $K_{ATP}$ channels may be major cause of $K^+$ outflux during development of the fatigue and the isolated EHL muscle could be an useful experimental preparation in studying the fatigue phenomena in skeletal muscle. In addition, the possibility of activation of delayed rectifier during the fatigue development remains to be studied further.

쥐자궁근의 운동성에 대한 $K^+$채널 개방제의 이완 작용

김희정,이문한,류판동,Kim, Hee-Jeong,Lee, Mun-Han,Ryu, Pan-Dong 대한약리학회 1994 대한약리학잡지 Vol.30 No.2

$K^+$ channel openers (KCOs) are known to have a wide range of effects by opening the $K^+$ channel in plasma membranes of various smooth muscles, cardiac muscle and pancreatic ${\beta}-cell$. In the present study, we investigated the effects of 5 types of KCOs, cromakalim, RP49356, pinacidil, nicorandil and diazoxide on the contractility of isolated rat uterus. All KCOs tested inhibited the uterine contraction induced by 0.2 nM oxytocin in a dose-dependent manner. Individual KCO and its $pD_2$ values were cromakalim 6.5, RP49356 6.3, pinacidil 5.92, nicorandil 4.43 and diazoxide 4.18. The relaxant effects of KCO were inhibited by glibenclamide (0.3, 1 and $10\;{\mu}M$) with $pA_2$ values of cromakalim 6.91, RP49356 6.59, pinacidil 6.55, nicorandil 5.97 and diazoxide 6.37. In addition, the relaxant effect of cromakalim or pinacidil was antagonised by TEA, a non-selective $K^+$ channel blocker, but not by apamin. Contractions induced by low concentration of KCI (< 40 mM) were inhibited by cromakalim $(100{\mu}M)$ and nicorandil $(300{\mu}M)$, but those evoked by higher concentration (> 40 mM) of KCI were little affected. In ovariectomized rat uterus, cromakalim dose-dependently inhibited oxytocin-induced contraction and glibenclamide $(10{\mu}M)$ inhibited the relaxant effect of cromakalim with $pD_2$ and $K_B$ values of 7.48 and $1.26{\times}10^{-7}M$, respectively. In estrogen-primed rat uterus, these values were 6.51 and $1.57{\times}10^{-7}M$, respectively, indicating that the cromakalim is less effective on the estrogen-treated uterine smooth muscle. Our results suggest that the KCO-sensitive $K^+$ channels participate in the motility of uterine smooth muscle and such channels are, at least in part, under the control of estrogen. In addition, our data Indicate that the type of $K^+$ channels activated by KCO is ATP-sensitive $K^+$ channels which is blocked by glibenclamide.

해마절편의 허혈성 $K^+$ 축적에 대한 $K^+$채널 조절 약물의 작용

최진규,전보권,류판동,Choi, Jin-Kyu,Chun, Boe-Gwun,Ryu, Pan-Dong 대한약리학회 1997 The Korean Journal of Physiology & Pharmacology Vol.1 No.6

Loss of synaptic transmission and accumulation of extracellular $K^+([K^+]_O)$ are the key features in ischemic brain damage. Here, we examined the effects of several $K^+$channel modulators on the early ischemic changes in population spike (PS) and $[K^+]_o$ in the CA1 pyramidal layer of the rat hippocampal slice using electrophysiological techniques. After onset of anoxic aglycemia (AA), orthodromic field potentials decreased and disappeared in $3.3{\pm}0.22\;min$ $(mean{\pm}SEM,\;n=40)$. The hypoxic injury potential (HIP), a transient recovery of PS appeared at $6.0{\pm}0.25\;min$ (n=40) in most slices during AA and lasted for $3.3{\pm}0.43\;min$. $[K^+]_o$ increased initially at a rate of 0.43 mM/min (Phase 1) and later at a much faster rate (12.45 mM/min, Phase 2). The beginning of Phase 2 was invariably coincided with the disappearance of HIP. Among $K^+$ channel modulators tested such as 4-aminopyridine (0.03, 0.3 mM), tetraethylammonium (0.1 mM), NS1619 $(0.3{\sim}10\;{\mu}M)$, niflumic acid (0.1 mM), glibenclamide $(40\;{\mu}M)$, tolbutamide $(300\;{\mu}M)$ and pinacidil $(100\;{\mu}M)$, only 4-aminopyridine (0.3 mM) induced slight increase of $[K^+]_o$ during Phase 1. However, none of the above agents modulated the pattern of Phase 2 in $[K^+]_o$ in response to AA. Taken together, the experimental data suggest that 4-aminopyridine-sensitive $K^+$channels, large conductance $Ca^{2+}-activated$ $K^+$ channels and ATP-sensitive $K^+$ channels may not be the major contributors to the sudden increase of $[K^+]_o$ during the early stage of brain ischemia, suggesting the presence of other routes of $K^+$ efflux during brain ischemia.

랫드 흑질 신경세포의 전기적 특성과 ATP-sensitive K⁺채널의 전류밀도

한성규,박진봉,류판동,Han, Seong-kyu,Park, Jin-bong,Ryu, Pan-dong 대한수의학회 2000 大韓獸醫學會誌 Vol.40 No.2

Substantia nigra is known to highly express glibenclamide binding site, a protein associated to ATP-sensitive $K^{+}$ ($K_{ATP}$) channel in the brain. However, the functional expression of $K_{ATP}$ channels in the area is not yet known. In this work, we attempted to estimate the functional expression of $K_{ATP}$ channels in neurons of the substantia nigra pars compacta (SNC) in young rats using slice patch clamp technique. Membrane properties and whole cell currents attributable to $K_{ATP}$ channel were examined by the current and voltage clamp method, respectively. In SNC, two sub-populations of neurons were identified. Type I (rhythmic) neurons had low frequency rebound action potentials ($4.5{\pm}0.25Hz$, n=75) with rhythmic pattern. Type II (phasic) neurons were characterized by faster firing ($22.7{\pm}3.16Hz$, n=12). Both time constants and membrane capacitance in rhythmic neurons ($34.0{\pm}1.27$ ms, $270.0{\pm}11.83$ pF) and phasic neurons ($23.7{\pm}4.16$ ms, $184{\pm}35.2$ pF) were also significantly different. The current density of $K_{ATP}$ channels was $6.1{\pm}1.47$ pA/pF (2.44~15.43 pA/pF, n=8) at rhythmic neurons of young rats. Our data show that in SNC there are two types of neurons with different electrical properties and the density of $K_{ATP}$, channel of rhythmic neuron is about 600 channels per neuron.

축산식품 중에 잔류하는 Gentamicin 검사를 위한 ELISA 개발에 관한 연구

김재명,이문한,이항,류판동,조명행,박종명,Kim, Jae-Myung,Lee, Mun-Han,Lee, Hang,Ryu, Pan-Dong,Cho, Myung-Haing,Park, Jong-Myung 한국식품위생안전성학회 1994 한국식품위생안전성학회지 Vol.9 No.3

An enzyme-linked immunosorbent assay(ELISA) was developed for the detection of residual gentamicin(GM) in edible animal products. The immunogen(GM-KLH conjugate) and coating antigen(GM-BSA conjugate) were prepared by coupling GM sulfate to keyhole limpet hemocyanin(KLH) and bovine serum albumin(BSA) in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, respectively. Polyclonal antibody to GM was produced in rabbits(New Zealand White, female) by using the immunogen and the antibody titer was measured by indirect ELISA. A competitive ELISA was developed using GM-bovine serum albumin conjugate as a coating antigen, GM(as standards or sample), polyclonal antibody to GM, secondary antibody conjugated with horseradish peroxidase as an enzyme, and H2O2 and o-phenylenediamine dihydrochloride as a substrate and a chromophore, respectively. The detection limit of GM was 10 ng/ml and the standard curve of GM(n=26) was linear up to 10 $\mu\textrm{g}$/ml in this competitive ELISA system. There were no cross-reactivities of the partially purified antibody between GM and the various antibiotice such as amikacin, benzyl-penicillin, chloramphenicol, erythromycin, furazlidone, kanamycin, neomycin, oleandomycin, streptomycin, sulfathiazole and thiamphenicol(CR50<0.05%)

Phenytoin의 태아 구개열 유발과 모체 혈청 Corticosterone 증가에 대한 Dimethyl Sulfoxide 억제 작용

이재권,이창업,이문한,류판동,이영재,성하정,Lee, Jae-Kwon,Lee, Chang-Eop,Lee, Mun-Han,Ryu, Pan-Dong,Lee, Young-Jae,Sung, Ha-Jung 한국독성학회 1992 Toxicological Research Vol.8 No.2

It is well known that phenytoin (PHT), a commonly prescribed anticonvulsant, has teratogenicity in experimental animals and human. The major malformation induced by PHT in mouse is cleft palate. The mechanisms of the embryotoxic effects of PHT are unknown. However, PHT and synthetic glucocorticoids share several features with respect to their teratogenicity, and it was known that PHT increased maternal corticosterone level. Therefore PHT-induced cleft palate may be mediated indirectly by elevated maternal corticosterone. Recently it was reported that secalonic acid Dinduced cleft palate and elevated endogenous corticosterone level, and that such effects were antagonized by DMSO. The purpose of this work was to investigate whether the elevated maternal corticosterone is associated with the teratogenicity of PHT in the ICR mouse fetuses by treatment with PHT or PHT plus DMSO. PHT (74mg/kg, BW) was daily administered intraperitoneally on day 10~12 of gestation with and without DMSO(2ml/kg, BW), and the fetal malformation was observed on day 18. Maternal serum corticosterone and fetal PHT levels were determined by HPLC. The results are summarized as follows. 1)The percentage of cleft palate incidense in fetuses following treatment with PHT on day 10~12 of gestation was 51.7%. 2)There was a significant decrement in the cleft palate incidence in fetuses to 30.8% in the group treated with PHT plus DMSO compared with 51.7% in that with PHT alone. 3) Maternal serum corticosterone levels following treatment with PHT on day 10~12 of gestation increased by 116~343% compared with that of vehicle control. Such effect was antagonized by DMSO. 4)PHT concentration in the fetuses was not affected by DMSO. These results suggest that PHT-induced cleft-palate in fetuses seems to be closely associated with the elevation of maternal corticosterone level.

Xylazine-ketamine 병용마취가 토끼의 혈중 ACTH, corticosterone 및 glucose 농도에 미치는 영향

박권무,이용화,한성규,류판동,Park, Kwon-moo,Li, Long-hua,Han, Seong-kyu,Ryu, Pan-dong 대한수의학회 1999 大韓獸醫學會誌 Vol.39 No.1

Anesthetic agents are useful in inducing the anesthesia for surgical operations and various biological experiments, but they can disturb the body homeostasis and cause the stress in animals. Much efforts have been directed on reducing such side effects of anesthesia. In this work, we measured the serum ACTH, corticosterone and glucose concentration in rabbits to compare the degree of stress induced by two commonly-used anesthetics, ketamine, xylazine, and the combination of xylazine and ketamine. 1. The anesthesia was induced in about 10 min in the rabbits treated with xyalzine, ketamine and xylazine-ketamine. The duration of complete loss of righting reflex were 12, 13 and 115 min in the groups treated with xylazine, ketamine and xylazine-ketamine, respectively. 2. Serum ACTH concentrations in all treatment groups were higher than those in control group. At 30 min after the administration of the drugs, serum ACTH levels in ketamine-treated group were significantly higher than those in control, xylazine- and xylazine-ketamine-treated groups. However, at 1, 2, 5 and 9 hours after the drug administration, serum ACTH levels in xylazine-treated-group were higher than those in control. 3. Serum corticosterone levels in xylazine- and xylazine-ketamine-treated groups were lower than those in control or ketamine-treated groups at 0.5 and 1 hour after the administration. However, at 5 and 9 hours after the administration, serum corticosterone levels in xylazine- and xylazine-ketamine-treated groups were significantly higher than those in ketamine-treated group or control. 4. Serum glucose levels transiently increased to 3 times of the pre-injection levels at 0.5 and 1 hours after the administration in xylazine or xylazine-ketamine-treated groin, but were not changed in control and ketamine-treated group. These results indicate that xylazine-induced stress lasts longer than ketamine-induced, suggesting that the difference in stress-related hormone levels during anesthesia could be due to the differences in modes of actions of individual drugs used and the depth of anesthesia.

Pharmacokinetics and tissue levels of a sustained-release recombinant porcine somatotropin in pigs

한성규,박상균,장병선,심석권,류판동,Han, Seong-kyu,Park, Sang-kyoon,Chang, Byoung-sun,Shim, Seog-kwon,Ryu, Pan-dong The Korean Society of Veterinary Science 1998 大韓獸醫學會誌 Vol.38 No.1

서방형 돼지성장호르몬(sustained-release formulation of porcine somatotropin, PST-SR)을 1주 간격으로 6차례 피하 및 근육주사하고 혈액과 조직중의 돼지 성장호르몬(PST)과 insulin-like growth factor 1(IGF-1)의 농도를 측정하여 다음과 같은 결과를 얻었다. 대조군의 혈중 PST와 IGF-1의 농도는 각각 2.41과 95.2 ng/ml 이었다. 1. PST-SR을 투여한 후 PST의 혈중농도는 8시간만에 최대에 도달하여(30 ng/ml) 곧 감소하였다. 혈중농도 반감기(decay half life)는 91~227시간이었다. IGF-1의 혈중농도는 투여후 12시간에 최대에 도달하였으며(165 ng/ml), 이후 서서히 감소되었고 반감기는 77~99시간이었다. 2. 혈중 PST농도-시간의 자료는 제재에서 PST가 유리되는 과정에는 두단계 즉, 투여후 24시간까지의 유리속도가 빠른 단계와 그 이후의 유리속도가 느린 단계가 있음을 보여주었다. 3. 여섯번의 반복투여기간에는 PST의 혈중농도는 투여직후 증가하여 24시간 이후 다음 투여전까지 지속적으로 감소되는 패턴이 반복되었고, 최종투여후 1주일경에는 정상수준으로 회복되었다. 반면에 투여가 반복됨에 따라 매 투여직후의 PST의 혈중 최고치는 다소 증가되는 경향을 보였다(20~40 ng/ml). IGF-1의 혈중농도는 투여가 반복됨에 따라 누적적인 증가현상이 뚜렷하였으며, 이후 2주일후 까지도 정상농도보다 높게 유지되었다(200ng/ml). 임상용량 투여군에서 PST 및 IGF-1의 혈중농도는 투여경로에 따른 차이는 나타나지 않았다. 4. 최종(6번째) 투여후 6, 8, 10, 14일에 조사한 간장, 신장, 소장, 근육, 지방 및 주사부위의 조직중의 PST 농도는 6일째에 이미 대조군 수준으로 회복되었다. IGF-1의 경우 최종투여후 6일에는 간장, 신장, 소장, 지방조직에서 정상보다 높은 농도로 잔류하나 이후 14일까지 모두 대조군 수준으로 감소되었다. 5. 이상의 결과는 본 실험에서 사용된 서방형 PST제제는 최소 1주간 유효성이 유지되며, 동시에 PST는 투여 6일째에, IGF-1은 투여후 14일에 정상수준으로 회복됨을 보여주고 있다.