Transferrin Receptors in the Liver Cell Membrane of Carcinogen (3-methyl-4-dimethyl-arninoazobenzene) Treated Rat

이재흔,노의선,허강민,이충식,석정호,Lee, Jae-Heun,Ro, Eu-Sun,Hur, Gang-Min,Lee, Choong-Sik,Seok, Jeong-Ho The Korean Society of Pharmacology 1993 대한약리학잡지 Vol.29 No.1

화학물질에 의한 간암 유발과정에서 transferrin receptor (TfR)의 변동을 밝히기 위해 간을 부분절제한 정상백서의 재생간과 발암물질로 3-Me-DAB를 8주간 투여한 백서 또는 약물 투여 후 부분 간절제 수술을 행하여 세포분열을 유도시킨 백서 간조직으로부터 parenchymal cell (PC)과 nonparenchymal cell (NPC)를 분리하고 각각의 세포막을 제조하여 $^{125}I-transferrin$ 결합실험을 실시한 바 다음과 같은 성적을 얻었다. 1. 3-Me-DAB 투여에 의하여 간조직에서 oval cell의 증식, 재생성 변화, 결절형성, 담관의 증식 및 담관세포암 등의 현저한 조직학적 변화가 동반되었다. 그러나 간세포증식을 더욱 촉진시키기 위하여 부분간절제 수술을 하였을 때 수술 후 경과에 따른 형태학적 변동은 큰 차이가 없었다. 2. 정상 재생간의 PC 및 NPC homogenate에서 transferrin 결합량은 부분간 절제 수술 후 1일 및 3일에 증가되었으며 수술 후 7일에 정상으로 회복되었다. 3-Me-DAB 투여에 의해 두세포군에서 모두 정상세포보다 높게 나타났으며 재생기간에 따라 계속 증가되었다. 3. 정상간의 NPC 세포막에서 transferrin 최대 결합량 (Bmax)은 PC 세포막에서 보다 많이 분포되어 있었으며, Kd는 양세포막에서 5.05 또는 6.3nM로 비슷하였다. 4. 재생간의 NPC 및 PC 세포막에서 transferrin 결합량은 부분 간절제 수술 후 1일 및 3일에 $40{\sim}50%$ 증가되었고 수술 후 7일에 정상치로 회복되었다. 5. 3-Me-DAB 처치에 의하여 NPC 및 PC 세포막의 transferrin 결합량은 정상 간세포막에서 보다 약 3배 증가되었고, 3-Me-DAB 투여후 재생간의 NPC 세포막에서는 부분 간절제 수술 후 3일까지 증가된 후 감소되는 양상인데 반해 PC 세포막에서는 수술 후 7일까지 계속 증가되었다. 6. 3-Me-DAB 투여 후 NPC 및 PC 세포막 transferrin binding site에서 Kd치가 $3.1{\sim}4.1\;nM$과 $25.4{\sim}54.1\;nM$인 두 종류가 존재하는 것으로 나타났다. 이상의 실험성적으로 TfR는 1) 간조직의 PC 및 NPC 세포에 모두 분포되어 있으며, 2) 정상 재생간 및 3-Me-DAB의 처리 후 간세포에서의 세포막 TfR의 증가는 세포내 합성량의 증가에 의하여 일어나며, 3) 정상 재생간의 세포막 TfR는 한 종류의 high affinity site $(Kd,\;<3.1{\sim}7.5\;nM)$에 의하여 증가되나, 3-Me-DAB 처리 후 간세포막에서는 정상에서와 같은 high affinity형 이외에 affinty가 낮은 다른 형태의 TfR $(Kd,\;25.4{\sim}54.1\;nM)$가 세포막으로 출현됨으로써 크게 증가되는 것으로 사료된다. To investigate the alteration of transferrin receptor (TfR) in the proliferating or transformed liver cells, $^{125}I-transferrin$ binding experiment was carried out in the isolated parenchymal cells (PC) or nonparenchymal cells (NPC) from normal regenerated rat liver after partial hepatectomy and from the liver of 3-methyl-4-dimethyl-aminoazobenzene (3-Me-DAB) treated rat. With the administration of 3-Me-DAB for 8 weeks, the liver tissue showed marked morphologic changes of oval cell proliferation, regenerations of hepatocytes, and atypical proliferations of bile ducts, but these changes were little affected by partial hepatectomy. Transferrin binding values in PC or NPC homogenate from the regenerated liver of normal rat, were increased by 3rd day and diminished to control level at 7th day after partial hepatectomy. With the treatement of 3-Me-DAB for 8 weeks, transferrin binding sites in homogenates were higher than those of normal rat liver and increased by 7th day after partial hepatectomy. Transferrin binding sites (Bmax) in the cell membrane of NPC were higher than those of PC of normal rat liver, but there was no significant difference in Kd values between both groups (5.05, 6.3 nM). In the normal resenerated rat liver, transferrin binding sites in the PC or NPC plasma membrane, were increased by 3rd day and diminished to control level at 7th day after partial hepatectomy. With 3-Me-DAB tratment, transferrin binding sites in both liver NPC and PC plasma membrane were increased about 3 folds, compared to those in each plasma membrane of normal rat liver. And after partial hepatectomy of 3-Me-DAB trated rat, transferrin binding sites were increased by the 3rd day in the NPC plasma membrane but increased by the 7th day in the PC plasma membrane. In the transferrin binding sites of the PC or NPC plasma membrane of 3-Me-DAB treated liver, two kinds of Kd values $(3.1{\sim}4.7\;nM,\;25.4{\sim}54.1\;nM)$ were detected. The present results suggest that 1) TfRs are distributed in the liver PC as well as NPC; 2) Increased TfRs in PC or NPC plasma membrane of normal regenerated liver after partial hepatectomy and 3-Me-DAB treated rat liver, may be due to increased intracellular synthesis; 3) Increased TfRs in normal regenerated liver after partial hepatectomy might be related to the expression of a single type of high affinity site $(Kd,\;3.1{\sim}7.5\;nM)$, but in 3-Me-DAB treated rat liver might be related to the expression of high and low affinity types of receptors $(Kd,\;25.4{\sim}54.1\;nm)$.

돼지 소장 평활근 세포막에서의 Calcium 이동에 미치는 Calcium entry blockers 의 영향

석정호(Jeong-Ho Seok),임종호(Jong-Ho Lim),이재흔(Jae-Heun Lee) Jeong-Ho Seok, Jong-Ho Lim and Jae-Heun Lee 대한약리학회 1986 대한약리학잡지 Vol.22 No.2

최근 심근세포 또는 신경세포에서 발표된 여러 종류의 calcium channel중 calcium antagonist로 차단되는 channel 또는 차단되지 않는 channel 등이 있는지 알아보기 위해 실험을 시행하였다. 돼지 소장 평활근으로부터 고농도의 KCl(150mM)로 부하된 세포 포막낭을 만들어 고농도의K⁺ 또는 전기자극으로 ⁴⁵Ca의 이동을 유발시켜 다음과 같은 성적을 얻었다. 저농도의 K⁺용액에서의 ⁴⁵Ca이동보다 고농도의 K⁺-용액에서의 ⁴⁵Ca이동이 유의하게 증가되었으며(p<0. 05) 이때 유입되는 ⁴⁵Ca의 양은 시간에 따라 서서히 감소되었다. 전기자극(3V, 15Hz, 25msec)을 하였을때 유입되는 ⁴⁵Ca의 양은 전기자극을 하지 않은 대조군에 비하여 현저하게 증가되었고, 자극시간에 따른 ⁴⁵Ca의 유입량은 2분 동안 계속 증가되었다. Diltiazem 또는 nifedipine을 처치하였을때, 고농도의 K⁺-용액에 의한 ⁴⁵Ca의 유입은 억제되지 않았으나 전기자극에 의해 유도되는 ⁴⁵Ca의 유입은 유의하게 억제되었다(p<0.005). 상기의 실험성적으로 돼지 소장 평활근으로부터 분리한 세포막에서의 calcium이동 중 전기자극에 의해 이루어지는 것은 calcium antagonist로 차단되는 calcium channel을 통하여 이루어지는 것으로 사료된다. There are some evidence for the presence of more than one type of calcium channels. To investigate whether organic calcium antagonist sensitive calcium channels exist in the isolated sarcolemmal membrane, we prepared high KCl-loaded sarcolemmal vesicle from the procine small instine, and induced calcium transport by high K⁺ concentration or by electrical stimulation after preincubation of KCl-loaded vesicle in the low potassium solution. Calcium transport induced by high K⁺ concentration (84.7mM) was significantly increased (p<0.05), compared with that by low K⁺ concentration (2.08 mM), and not inhibited by diltiazem (10^-6 M). Calcium transport was inactivated with time. By continuous electrical stimulation (3V, 15Hz, 25m see), calcium transport was markedly increased, and inhibited significantly by dilltiazem (10^-6 M) and nifedipine (10^-6 M) (p<0.005), compared with the value of control without electrical stimulation. Calcium transport by electrical stimulation was not inactivated with time for at least 2 min. From these results, it was concluded that there was organic calcium antagonist sensitive channel in the isolated intestinal sarcolemma membrane, which was activated by electrical stimulation.

뱀장어 근육내 Ryanodine Receptor의 기능 및 면역학적 성질

석정호(Jeong Ho Seok),이연수Yeon Soo Lee),남장현(Jang Hyeon Nam),최숙정(Suk Jeong Choi),홍장희(Jang Hee Hong),이재흔(Jae Heun Lee) 대한약리학회 1995 대한약리학잡지 Vol.31 No.2

To investigate the functional and immunological properties of the Ca-release channel in the sarcoplasmic reticulum(SR) of the eel skeletal muscle, [³H]ryanodine binding, SDS gel electrophoresis, ⁴⁵Ca release studies, and immunoblot assay were carried out in the SR of the eel skeletal muscle. Maximal binding sites(Bmax) and K_D values of [³H]ryanodine for Ca-release channel of the SR of the eel skeletal muscle were 19.44 ± 1.40 pmole/mg protein and 15.55 ± 1.69 nM, respectively. [³H]Ryanodine binding to RyR was increased by calcium and AMP. The SR of the eel skeletal muscle has two high molecular weight bands on the SDS PAGE. The mobility of upper band was more slower than the single band of the rabbit skeletal muscle, and that of the lower band was similar with the single band of canine cardiac muscle. Vesicular ⁴⁵Ca-release was activated by calcium. Ca-induced ⁴⁵Ca-release was significantly inhibited by MgCl₂(2 mM), ruthenium red(10 μM) or tetracaine(1 mM), but not by high concentration of calcium itself. AMP-induced ⁴⁵Ca-release was slightly occurred only in the absence of calcium, it was not inhibited by MgCl₂ or ruthenium red. Caffeine also increased ⁴⁵Ca-release from the SR vesicles, but it was not affected by MgCl₂ or ruthenium red. Polyclonal Ab against rat skeletal muscle RyR is reacted with that of rabbit, but not reacted with that of the eel skeletal muscle. These results suggested that ryanodine receptor of the SR of the eel skeletal muscle is showing some similar properties with that of mammalian skeletal muscle, but might be an another isotype channel having two bands which is less sensitive to AMP, not cross-reacted with antisera against rat RyR, and not inhibited by high concentration of calcium.

Effect of Calcium Entry Blockers on the Calcium Transport in the Isolated Sarcolemmal membrane from the Porcine Small Intestine

석정호,임종호,이재흔,Seok, Jeong-Ho,Lim, Jong-Ho,Lee, Jae-Heun The Korean Society of Pharmacology 1986 대한약리학잡지 Vol.22 No.2

There are some evidence for the presence of more than one type of calcium channels. To investigate whether organic calcium antagonist sensitive calcium channels exist in the isolated sarcolemmal membrane, we prepared high KCl-loaded sarcolemmal vesicle from the procine small instine, and induced calcium transport by high $K^+$ concentration or by electrical stimulation after preincubation of KCl-loaded vesicle in the low potassium solution. Calcium transport induced by high $K^+$ concentration (84.7mM) was significantly increased (p<0.05), compared with that by low $K^+$ concentration (2.08 mM), and not inhibited by diltiazem $(10^{-6}\;M)$. Calcium transport was inactivated with time. By continuous electrical stimulation (3V, 15Hz, 25m see), calcium transport was markedly increased, and inhibited significantly by dilltiazem $(10^{-6}\;M)$ and nifedipine $(10^{-6}\;M)$ (p<0.005), compared with the value of control without electrical stimulation. Calcium transport by electrical stimulation was not inactivated with time for at least 2 min. From these results, it was concluded that there was organic calcium antagonist sensitive channel in the isolated intestinal sarcolemma membrane, which was activated by electrical stimulation. 최근 심근세포 또는 신경세포에서 발표된 여러 종류의 calcium channel중 calcium antagonist로 차단되는 channel 또는 차단되지 않는 channel 등이 있는지 알아보기 위해 실험을 시행하였다. 돼지 소장 평활근으로부터 고농도의 KCl(150mM)로 부하된 세포 포막낭을 만들어 고농도의$K^+$ 또는 전기자극으로 $^{45}Ca$의 이동을 유발시켜 다음과 같은 성적을 얻었다. 저농도의 $K^+$용액에서의 $^{45}Ca$이동보다 고농도의 $K^+$-용액에서의 $^{45}Ca$이동이 유의하게 증가되었으며(p<0. 05) 이때 유입되는 $^{45}Ca$의 양은 시간에 따라 서서히 감소되었다. 전기자극(3V, 15Hz, 25msec)을 하였을때 유입되는 $^{45}Ca$의 양은 전기자극을 하지 않은 대조군에 비하여 현저하게 증가되었고, 자극시간에 따른 $^{45}Ca$의 유입량은 2분 동안 계속 증가되었다. Diltiazem 또는 nifedipine을 처치하였을때, 고농도의 $K^+$-용액에 의한 $^{45}Ca$의 유입은 억제되지 않았으나 전기자극에 의해 유도되는 $^{45}Ca$의 유입은 유의하게 억제되었다(p<0.005). 상기의 실험성적으로 돼지 소장 평활근으로부터 분리한 세포막에서의 calcium이동 중 전기자극에 의해 이루어지는 것은 calcium antagonist로 차단되는 calcium channel을 통하여 이루어지는 것으로 사료된다.

기관(氣官) 상피세포 생리 및 약리 실험모델로서의 공기-액체 접면 일차배양법 연구

이충재 ( Choong Jae Lee ),이재흔 ( Jae Heun Lee ),석정호 ( Jeong Ho Seok ),허강민 ( Gang Min Hur ) 한국응용약물학회 2002 Biomolecules & Therapeutics(구 응용약물학회지) Vol.10 No.4

Characterization of Calcium Release Channel (Ryanodine Receptor) in Sarcoplasmic Reticulum of Crustacean Skeletal Muscle

석정호,정정구,허강민,이재흔,Seok, Jeong-Ho,Jung, Jung-Koo,Hur, Gang-Min,Lee, Jae-Heun The Korean Society of Pharmacology 1994 대한약리학잡지 Vol.30 No.1

갑각류 골격근의 SR에서 칼슘유리 channel protein complex의 성격을 규명하기 위해 민물가재 및/또는 바다가재의 SR vesicles을 분리하여 $^{45}Ca$ 유리, $[^3H]ryanodine$결합, 및 immunoblot 실험을 실시하여 다음과 같은 결과를 얻었다. 1.민물가재 SR의 $[^3H]ryanodine$결합 실험에서 민물가재 SR의 maximal binding site및 affinity모두 바다가재에서 보다 낮았으나, high affinity binding site이었다. Extravesicles 칼슘농도를 증가시켰을 때 $[^3H]ryanodine$결합은 약간 증가되었으나, AMP나 AMP와 caffeine을 동시에 첨가하였을 때는 현저히 증가되었다(p<0.05). 이런 증가 현상은 $MgCl_2$나 tetracaine으로 유의성 있게 억제되었으나(p<0.001), ruthenium red에 의해서는 약간 억제되었다. 2.민물가재 SR을 전기영동하였을 때 바다가재의 ryanodine receptor band (HMWBr)와 비슷하나 포유류의 것(HMWBS) 보다는 약간 빠른 mobility를 나타낸다. 3.바다가재 HMWBr에 대한 polyclonal Ab를 이용한 민물가재, 바다가재 및 토끼 골격근의 칼슘유리 channel간의 면역학적 교차반응에서 민물가재와 바다가재의 칼슘유리 channel 간에는 교차반응이 있었으나, 포유류의 것과는 아무런 반응이 없었다. 4.민물가재 SR에서 $^{45}Ca$유리는 extravesicles의 칼슘농도 증가에 따라서 증가되었고, 낮은 외부 칼슘 농도에서 바다가재 보다 빠르게 일어났으나, AMP와 caffeine에 의해 영향을 받지 않았고, $MgCl_2$와 tetracaine으로 약간($3{\sim}8%$) 그리고 고농도의 ruthenium red로 중등도(23%) 억제되었다. 이상의 실험성적으로 갑각류 칼슘유리 channel protein은 포유류의 것과는 기능적으로나 면역학적으로 매우 다른 특징을 가지고 있고, 민물가재와 바다가재 칼슘유리 channel은 서로 유사한 특징을 갖지만, 민물가재의 칼슘유리 channel이 바다가재의 것보다 외부칼슘에 예민한 기능을 갖는 것으로 사료된다. To characterize the SR Ca-release channel protein complex of crustacean, $^{45}Ca-release,\;[^3H]ryanodine$ binding, and immunoblot studies were carried out in the crayfish and/or lobster skeletal sarcoplasmic reticulum. Bmax and affinity of crayfish SR to ryanodine were lower than those of lobster SR. AMP (5mM) increased $[^3H]ryanodine$ binding significantly in both vesicles (P<0.05). $Mg^{2+}$(5mM) or tetracaine(1mM) inhibited $[^3H]ryanodine$ binding significantly in both vesicles (P<0.001), but ruthenium red $(10\;{\mu}M)$ inhibited it moderately. In SDS polyacrylamide gel electrophoretic analysis of crayfish SR vesicles, there was a high molecular weight band that showed similar mobility with Ca-release channel protein of lobster skeletal SR, but more rapid mobility (HMWBr) than that of rabbit skeletal SR (HMWBS). Immunoblot analysis showed that polyclonal Ab to lobster skeletal SR Ca-release channel protein was react with HMWBr of crayfish skeletal SR, but not with that of HMWBs of rabbit skeletal SR. ^{45}Ca-release from crayfish skeletal SR vesicles was increased by the increase of extravesicular calcium from $1{\mu}M$ to 1mM. This Ca-release phenomenon was similar, but more sensitive in the low concentration of $Ca^{2+}$, compared to that from lobster SR vesicles. AMP (5mM) or caffeine (10mM) did not affect to $^{45}Ca-release.\;^{45}Ca-release$ was inhibited slightly ($3{\sim}8%\;by\;Mg^{2+}$) (5mM) or tetracaine (1mM), and moderately (23%) by high concentration of ruthenium red $(300\;{\mu}M)$. From the above results, it is suggested that SR Ca-release channel protein of crustacean has different properties from that of the rabbit, and similar properties between crayfish and lobster in functional and immunological aspects, but Ca-release via crayfish channel may be more sensitive to calcium.