• 대한약리학회지
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• 제31권1호
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• pp.63-74
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• 1995

저산소 상태에서는 부신수질로부터 카테콜아민 (CA) 유리작용이 활성화되지만 반면에 소의 배양 chromaffin cell에서는 고통도의 $K^+$에 의한 CA 분비작용이 억제된다고 알려져 있다. 본 연구에서는 적출 흰쥐 관류부신에서 콜린성 자극과 막탈분극에 의한 CA 분비작용에 대한 저산소증의 영향을 검색하고 그 작용기전을 규명코자 하였다. 본 연구목적을 위하여, 적출 흰쥐 관류부신을 이용, 저산소증이 니코틴($N_1$), 무스카린($M_1$) 수용체 흥분약, 막탈분극 약물, 칼슘채널 활성화 약물, 세포내 칼슘유리 약물 및 ACh에 의한 CA 분비에 미치는 영향을 연구하였으며, 저산소증은 95% 질소 및 5% 이산화탄소 혼합가스를 Krebs액에 주입하여 유발시켰으며, $3{\sim}4$시간동안 유지하였다. 저산소증 유발시, DMPP ($100{\mu}M$), McN-A-343 ($100{\mu}M$), ACh (5.32 mM), Bay-K-8644 ($10{\mu}M$) 및 high $K^+$ (56 mM)에 의한 CA 분비작용을 시간의존적으로 점차 유의성인 감소를 나타내었다. 그러나, cyclopiazonic acid ($10{\mu}M$)에 의한 CA 분비반응에는 하등의 영향을 일으키지 못하였다. 또한 저산소증 자체가 CA의 기초분비 작용에는 영향을 미치지 않았다. 이와같은 실험결과로 보아, 저산소증시 콜린성 자극 및 막탈분극에 의한 CA 분비 작용이 억제되며, 이러한 억제작용은 chromaffin cell내로 $Ca^{++}$ 유입을 직접적으로 억제시키는 결과에 기인되며, 세포내 칼슘저장고로부터 칼슘유리작용과는 관계없는 것으로 사료된다.

• Biomolecules & Therapeutics
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• 제3권1호
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• pp.21-27
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• 1995

The only compounds with antagonistic activity via AT$_1$receptor, one of two subtypes of angiotensin II (AII) receptor, have been demonstrated to block the vasoconstriction effects of AII and thereby provide therapeutic potential. This initiated the search for compounds with high specific affinity to AT$_1$receptor and their effective screening methods. The radioligand binding assay for the AII receptor is regarded as the primary method for the evaluation of AT$_1$receptor antagonists for their activity. In this paper, we characterized the liver AT$_1$receptor and describe the efficient method of the radioligand binding assay using rat liver as a source of AT$_1$receptor. Equilibrium binding studies with rat adrenal cortex, adrenal medulla, liver and bovine adrenal showed that the specific bindings of [$^3$H] AII were saturable in all tissues and the Scatchard plots of those data were linear, suggesting a single population of binding sites. Hill slopes were very near to the unity in all tissues. Kinetic studies of [$^3$H) AII binding in rat liver homogenates yielded two association rate constants, 4.10$\times$10$^{7}$ M$^{-1}$ min$^{-1}$ and 4.02$\times$10$^{9}$ M$^{-1}$ min$^{-1}$ , with a single dissociation rate constant, 7.07$\times$10$^{-3}$ min-$^{-1}$ , possibly due to the partial dissociation phenomenon. The rank order of inhibition potencies of [$^3$H] AII binding in rat liver was AII>Sarile>Losartan>PD 123177. Rat liver homogenates revealed to have very high density of homogeneous population of the AT$_1$receptor subtype, as the specifically bound [$^3$H] AII was not inhibited by PD 123177, the nonpeptide antagonist of AT$_2$. The results of this study demonstrated that the liver homogenates from rats could be the best receptor preparation for the AT$_1$receptor binding assay and provide an efficient system for the screening of newly synthesized candidate compounds of AT$_1$receptor antagonist.

• 대한약리학회지
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• 제24권1호
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• pp.1-10
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• 1988

카테콜아민 생합성에 관여하는 마지막 효소인 phenylethanolamine N-methyltransferase는 Norepinephrine을 epinephrine으로 전환시키는 중요한 효소이다. PNMT효소의 발현은 epinephrine 신경세포의 발현에 필수적이다. 따라서 PNMT유전자를 크로닝하여 그 구조를 결정하고, 유전자 발현연구를 하는 것은 상당히 중요한 일이다. 그러나 최근에 저자가 bovine cDNA를 처음으로 분리하여 그 구조를 보고한 것 외에는 아직까지 인간 PNMT cDNA나, 전체 genomic DNA의 분리 보고는 없다. 이에 저자들은 인간 PNMT유전자의 전체구조와 여러 종(species) 사이의 진화적인 관계를 규명하기 위해서 human genomic library(Charon 4A)를 만들고, 이 library 이용하여 bovine cDNA를 probe로 13.1 Kb길이의 genomic clone을 분리 크로닝하는데 성공하였다. 이 유전자는 두개의 EcoRI site가 포함되어 있어서, EcoRI제한효소에 의해서 7.5 Kb, 5.0 Kb,0.6 Kb로 분리되었으며, Southern과 dot blot 실험 에서 보면 5.0 Kb와 0.6 Kb에 exon이 흩어져 존재하고 있으며, 7.5 Kb는 flanking sequence로 판명되었다.

• The Korean Journal of Physiology
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• 제26권2호
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• pp.113-122
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• 1992

It is well known that chromaffin cells of adrenal medulla secrete catecholamine in response to sympathetic nerve activation and the influx of $Ca^{2+}$ through the voltage dependent $Ca^{2+}$ channels (VDCC) in the cell membrane do a major role in this secretory process. In this study, we explored the effect of divalent cations on VDCC of rat chromaffin cells. Rat (Sprague-Dawley rat, 150-250 gm) chromaffin cells were isolated and cultured. Standard giga seal, whole cell recording techniques were employed to study $Ca^{2+}$ current with external and internal solutions that could effectively isolate VDCC currents $(NMG\;in\;external\;and\;TEA\;and\;Cs^{2+}\;in\;internal\;solution)$. The voltage dependence and the inactivation time course of VDCC in our cells were identical to those of bovine chromaffin cells. A persistent inward current was first activated by depolarizing step pulse from the holding potential (H.P.) of -80 mV to -40 mV, increased to maximum amplitude at around +10 mV, and became smaller with progressively higher depolarizing pulses to reverse at around +60 mV. The inactivation time constant $(\tau)$, fitted from the long duration test potential (2 sec) was $1295.2{\pm}126.8$ msec $(n=20,\;1\;day\;of\;culture,\;mean\;{\pm}S.E.M.)$ and the kinetic parameters were not altered along the culture duration. Nicardipine $(10\;{\mu}M)$ blocked the current almost completely. Among treated divalent cations such as $Cd^{2+},\;Co^{2+},\;Ni^{2+},\;Zn^{2+}\;and\;,Mn^{2+},\;Cd^{2+}$ was the most potent blocker on VDCC. When the depolarizing step pulse from -80 mV to 10 mV was applied, the equilibrium dissociation constant $(K_d)$ of $Cd^{2+}\;was\;39\;{\mu}M,\;K_d\;of\;Co^{2+}\;was\;100\;{\mu}M\;and\;K_d\;of\;Ni^{2+}];was];780{\mu}M.$ The principal findings of this study are as follows. First, the majority of $Ca^{2+}$ channels in rat chromaffin cells are well classified to L-type $Ca^{2+}$ channel in the view of kinetics and pharmacology. Second, all divalent cations tested could block the $Ca^{2+}$ current and the most potent blocker among the tested was $Cd^{2+}$.

• 한국가축번식학회지
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• 제19권4호
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• pp.307-322
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• 1996

The corpus luteum (CL) is formed by the action of a surge of luteinizing hormone (LH) on the pre-ovulatory follicle. Luteal cells derived from granulosa and theca interna cells continue to secrete progesterone for about two weeks. LH in domestic animals is essential for the normal secretion of progesterone at all stages of the luteal phase. For this process in the rodents, 20$\alpha$-hydroxysteroid dehydrogenase (20$\alpha$-HSD) is indispensable. 20$\alpha$-HSD is an enzyme to be a biologically inactive steroid. This enzyme plays a critical role in the regulation of the rat luteal function and reported to be present in steroid-producing tissues such as the testis and adrenal gland. We have purified 20$\alpha$-HSD and found two distinct 20$\alpha$-HSD molecules (HSD-1 and HSD-2). Their molecular weights are both estimated to be 33kd.The amino acid compositions of HSD-1 and HSD-2 are mostly similar, but there is a slight difference in the content of lysine. We demonstrated that 1) CL of previous generations contribute more to whole ovarian 20$\alpha$-HSD activity, 2) newly formed corpora lutea contain only 20$\alpha$-HSD-1 activity, and 3) old CL express activities of each HSD isozyme as shown in the luteal tissue of cycling rats on the day of diestrus where only degenerating old CL exist. The increase in 20$\alpha$-HSD activity identified seems to be related to the increase in the numbers of 20$\alpha$-HSD-positive cells. Interestingly, 20$\alpha$-HSD-1 activities were strongly found in the follicle fluids and theca interna cells by immunohistochemical study. Thus, the activity of 20$\alpha$-HSD may be related to a survival mechanism of those luteal cells and follicles remaining in the ovaries. Luteal cells arise from two sources. The small luteal cells are all of theca cell origin, while the large luteal cells are mainly of granulosa cell origin. CL of Korean Native Cattle, as those of other animal species, contains two morphologycally and functionally distinct luteal cell populations, such as small and large luteal cells as well as nonluteal cells. In all reproductive states except in the late luteal phase, the bovine CL also contained more small luteal cells than large luteal cells. Luteal tissue secretes a variety of growth factors (proteins) and the pattern of secretion changes during all stages of the luteal phase. These growth factors could be important in regulating the function of the bovine corpus luteum and may act in a potential endocrine autocrine and paracrine mechanisms. Therefore, further work has to be done to elucidate the role of growth factors in the ovary, especially in the corpus luterum. Interest should be focussed on interaction of these growth factors in the regulation of luteal cell and the localization of cytokine synthesis in differnet luteal cells.

• Journal of Yeungnam Medical Science
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• 제9권2호
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• pp.288-301
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• 1992

소의 중추신경계의 신경전달인자에 의한 세포막에서의 정보전달 과정에 관여하는 PLC 활성화에 G-단백질의 관여 여부를 관찰하기 위하여 소의 뇌조직의 PLC ${\beta}$, ${\gamma}$ 및 ${\delta}$를 얻어 각 isozyme의 특성을 관찰하였다. 기질용액에 phosphatidyl choline(PC)을 첨가시 PLC 각 isozyme 마다 정도의 차이는 있으나 증가 양상을 보였으며 PLC ${\delta}$는 $100{\mu}M$ $Ca^{2+}$ 농도에서 높은 활성도 증가를 보였다. 세포막 소포체를 형성하기 위하여 $PIP_2$기질과 PC에 detergent로 cholate와 deoxycholate 농도에 따른 PLC 효과 관찰에서 cholate 농도 0.2%에서 1%까지 증가할 때 효소 활성도의 지속적인 상승이 관찰되었고, deoxycholate는 농도가 0.2%에서 높았다가 0.4%에서 낮아졌고 1%까지 증가함에 따라 PLC 효소 활성도는 약간 증가하였다. 기질액에 뇌추출액을 첨가하여 cholic acid 농도에 따른 PLC의 효과를 관찰한 결과 cholic acid 농도 0.2%에서 보다 1%에서 각 isozyme 모두에서 PLC활성도가 증가하였다. 소의 여러 장기에서 PLC isozyme의 분포정도를 방사면역측정방법으로 관찰하였을 때 뇌조직에 가장 많이 분포하고 있으며 특히 PLC ${\beta}$, ${\gamma}$가 많았고, PLC ${\delta}$는 부신에서 가장 많이 분포하였다. 다음으로 PLC ${\beta}$는 부신과 위, PLC${\gamma}$는 부신과 폐순이었다. PLC 효소가 활성화될 때 G-단백질의 관여 여부에 관하여 cholate 0.2%와 0.1%에서 G-단백질과 GTPrS 및 PLC의 결합정도의 관찰은 조직분쇄시료를 소의 뇌 및 부신조직을 이용하여 $^{35}S$-GTPrS 첨가시와 단세포군 항체를 이용한 경우 모두에서 1.49% 이하의 낮은 결합 정도를 관찰하였다. 그래서 정제된 PLC isozyme과 G-단백질 $Go{\alpha}$, $G{\beta}{\gamma}$, Gmix, $Gi{\alpha}$ 및 $Gt{\alpha}$ 각각에 대한 효과 관찰에 서 $Go{\alpha}$와 $G{\beta}{\gamma}$는 PLC ${\beta}$와 ${\delta}$의 활성도를 증가시켰고, PLC ${\gamma}$는 별 영향이 없었으며 Gmix에서는 세효소 모두 증가시켰다. $Gi{\alpha}$는 PLC ${\beta}$와 ${\gamma}$에서만 증가하였다. $Gt{\alpha}$는 PLC ${\beta}$ 및 ${\gamma}$에서 억제하였고 PLC ${\delta}$에서는 증가 양상을 보였다. 그러므로 PLC 활성화에 G-단백질의 관여가 인지되며 PLC isozyme과 G-단백질의 종류에 따라 대개의 경우 증가하는 경향이나 일부는 억제 내지는 별 영향이 없는 것으로 나타났다.

• 고려인삼학회:학술대회논문집
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• 고려인삼학회 1998년도 Advances in Ginseng Research - Proceedings of the 7th International Symposium on Ginseng -
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• pp.40-46
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• 1998

We investigated the influence of the root of Panax ginseng C. A. Meyer on the secretion of catecholamines from bovine adrenal chromaffin cells, which are used as a model of nervous systems. In two major parts extracted from the ginseng root, the crude saponin fraction, but not the non-saponin fraction, reduced the secretion from the cells, stimulated by acetylcholine (ACh). Ginseng saponins (ginsenosides) are classified into three groups, the panaxadiol, the panaxatriol and the oleanolic acid groups, on the basis of the chemical structures of their saponins. Both the panaxadiol and the panaxatriol saponins, excluding only one oleanolic acid saponin ginsenoside-Ro, generally reduced the ACh-evoked secretion. The inhibitory effects of the panaxatriol were much stronger than those of the panaxadiol. However, ginsenoside-Rg, and -Rh3 in the panaxadiol saponins were the potent inhibitors comparable to the panaxatriol saponins. Ginsenoside-Rg2 in the panaxatriol was the most effective. It is probable that the ginsenoside inhibition of the catecholamine secretion is due to the suppression of the function of the nicotinic ACh receptor-cation channels. On the other hand, ginsenoside-Rg2 did not affect the angiotensin II-, the bradykinin-, the histamine- and the neurotensin- induced catecholamine secretions from the chromaffin cells and the muscarine- and the histamine- induced contraction of the ileum in guinea-pigs. Ginsenoside-Rbl, a panaxadiol saponin, and ginsenoside-Ro had no or only a slight effect on them. On the contrary, ginsenoside-Rg3 not only competitively inhibited the muscarine-induced ileum contraction but also reduced the angiotensin R -, the bradykinin-, the histamine- and the neurotensin-induced catecholamine secretions. Thus, the ginseng root contains active ingredients, namely some ginsensides, which suppress the responses induced by receptor stimulation. The inhibitory effects of ginseng saponins may be one of the action mechanisms for the pharmacological effects of the Panax ginseng root.