• 동의생리병리학회지
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• 제31권4호
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• pp.226-232
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• 2017

In this study, ethanol extract of Epimedium koreanum Nakai(EEKN) enhanced melanogenesis by inducing expression of tyrosinase and tyrosinase-related protein-1 (TRP-1). But EEKN did not increase the protein expression of tyrosinase-related protein 2 (TRP-2). Moreover, EEKN enhanced tyrosinase activity and melanin contents of B16F10 cells. EEKN raised the expression of CREB phosphorylation and microphthalmia-associated transcription factor (MITF) as a key transcription factor for tyrosinase expression regulating melanogenesis. And PKC inhibitor H89 supressed that EEKN induced tyrosinase activity, melanin contents, and expression of tyrosinase, TRP-1. These results suggest that melanogenesis-promoting effect of EEKN was correlated with regulation of tyrosinase and TRP-1 protein through cAMP/PKC pathway.

• Molecules and Cells
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• 제23권2호
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• pp.138-144
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• 2007

Previously we showed that the human GM3 synthase gene was expressed during the induction of megakaryocytic differentiation in human leukemia K562 cells by phorbol 12-myristate 13-acetate (PMA). In this study we found that treatment of PMA-induced K562 cells with $G{\ddot{o}}6976$, a specific inhibitor of PKC, and U0126, an inhibitor of the extracellular signal-regulated kinase (ERK) reduced expression of GM3 synthase, whereas wortmannin, an inhibitor of phosphoinositide 3-kinase (PI3K) did not. Moreover, activation of ERK and cAMP response element binding protein (CREB) was prevented by pretreatment with $G{\ddot{o}}6976$ and U0126. PMA stimulated the promoter activity of the 5'-flanking region from -177 to -83 region of the GM3 synthase gene, and mutation or deletion of a CREB site located around -143 of the promoter reduced PMA-stimulated promoter activity, as did the inhibitors $G{\ddot{o}}6976$ and U0126. Our results demonstrate that induction of GM3 synthase during megakaryocytic differentiation in PMA-stimulated human leukemia K562 cells depends upon the PKC/ERK/CREB pathway.

• 치위생과학회지
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• 제6권1호
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• pp.1-9
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• 2006

기계적 자극에 의한 골조직의 개조에서 압박측은 일차적으로 파골세포에 의하여 골기질의 흡수를 위한 유전자 발현에 의하여 기시된다. 그러나 기질을 이루는 유기 단백질의 흡수에 관여하는 단백용해효소의 세포 내 작용 기전은 여전히 완전히 이해되지 않고 있다. 이 연구는 파골세포에서 용해용소체효소인 cathepsin k에 주목하여, cAMP 길항제와 PKA 억제제 및 adenylate cyclase 촉진제에 의한 cathepsin k 생성의 촉진 또는 억제 효과의 기전을 해명하는데 그 목적을 두었다. cAMP 길항제인 Rp-cAMP와 PKA 억제제인 KT5720과 H89는 cathepsin K의 세포 내성숙을 차단하였으며, 대조적으로 adenylate cyclase 촉진제 forskolin은 파골세포에서의 cathepsin K의 생성과 성숙을 유인하는 것으로 나타났다. 특히 cathepsin K의 생성과 성숙에 관여하는 신호전달이 protein kinase C(PKC)와 관련성을 검정하기 위하여 백서의 골세포를 PKC의 선택적 억제제인 calphostin C로 처리하였을 때 아무런 영향이 없는 것으로 나타남으로써 calphostin C는 파골 세포에 의해 매개된 cathepsin K의 생성과 성숙과는 무관한 것으로 밝혀졌다. 이는 파골세포에서의 cathepsin K의 성숙은 cAmp-PKA 신호전달 경로에 의해 조절됨을 의미한다. 분비된 전구효소는 M6P 수용체를 통하여 세포 내로 다시 진입할 수 있는 잠재성을 가지고 있기 때문에 이러한 가능성을 차단하기 위하여 M6P가 존재 또는 결여된 상태에서 cAMP 길항제인 Rp-cAMP와 PKA 억제제인 KT5720 및 H89를 시험하였다. 그 결과 Rp-cAMP, KT5720 또는 H89에 의한 cathepsin K의 M6P용량 비례적 생성 억제가 관찰 되었다. 또한 M6P를 주었을 때 Rp-cAMP, KT5720와 H89의 작용이 증가된을 보였다. 이상에서와 같이 Rp-cAMP, KT5720와 H89의 cathepsin K 생성 방해를 통한 골흡수 억제는 골다공증 또는 관절염의 치료와 같은 골흡수의 억제를 필요로 하는 분야에서의 임상적응용 가능성을 시사한다.

• 대한의생명과학회지
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• 제10권4호
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• pp.375-383
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• 2004

It has been reported that osteoporosis induced by the deficiency of estrogens in menopause is associated with the unbalance of Ca/sup 2+/ and Pi levels. Proximal tubule is very important organ to regualte Ca/sup 2+/ and Pi level in the body. However, the effect of estrogens on Ca/sup 2+/ and Pi regulation was not elucidated. Thus, we examined the effect of 17-β estradiol (E₂) on Ca/sup 2+/ and Pi uptake in the primary cultured rabbit renal proxiaml tubule cells. In the present study, E₂(> 10/sup -9/M) decreases Ca/sup 2+/uptake and stimulates Pi uptake over 3 days. E₂-induced decrease of Ca/sup 2+/ uptake and stimulation of Pi uptake were blocked by actinomycin D (a gene transcription inhibitor), cycloheximide (a protein synthesis inhibitor). tamoxifen, and progesterone (estrogen receptor antagonists). E₂-induced decrease of Ca/sup 2+/ uptake and stimulation of Pi uptake were blocked by SQ22536 (an adenylate cyclase inhibitor), Rp-cAMP (a cAMP antagonist), and PKI (a protein kinase A inhibitor). Indeed, E₂ increased cAMP formation. In addition, E₂-induced decrease of Ca/sup 2+/ uptake and stimulation of Pi uptake were blocked by staurosporine, H-7, and bisindolylmaleimide I (protein kinase C inhibitors) and E₂ translocated PKC from cytoslic fraction to membrane fraction. In conclusion, E₂ decreased Ca/sup 2+/ uptake and stimulated Pi uptake via cAMP and PKC pathway in the PTCs.

• The Korean Journal of Physiology and Pharmacology
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• 제1권4호
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• pp.413-423
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• 1997

The importance of the kidney in the development of hypertension was first demonstrated by Goldblatt and his colleagues more than fifty years ago. Many hormones and other regulatory factors have been proposed to play a major role in the development of hypertension. Among these factors angiotensia II (ANG II) is closely involved in renal hypertension development since it directly regulates $Na^+$ reabsorption in the renal proximal tubule. Thus the aim of the present study was to examine signaling pathways of low dose of ANC II on the $Na^+$ uptake of primary cultured rabbit renal proximal tubule cells (PTCs) in hormonally defined seum-free medium. The results were as follows: 1) $10^{-11}$ M ANG II has a significant stimulatory effect on growth as compared with control. Alkaline phosphatase exhibited significantly increased activity. However, leucine aminopeptidase and ${\gamma}-glutamyl$ transpeptidase activity were not significant as compared with control. In contrast to $10^{-11}$ M ANG II stimulated $Na^+$ uptake $(108.03{\pm}2.16% of that of control)$, $10^{-9}$ M ANG II inhibited ($92.42{\mu}2.23%$ of that of control). The stimulatory effect of ANG II on $Na^+$ uptake was amiloride-sensitive and inhibited by losartan (ANG II receptor subtype 1 antagonist) and not by PD123319 (ANG II receptor subtype 2 antagonist). 2) Pertussis toxin (PTX) alone inhibited $Na^+$ uptake by $85.52{\pm}3.52%$ of that of control. In addition, PTX pretreatment prevented the AMG II-induced stimulation of $Na^+$ uptake. 8-Bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP), forskolin, and isobutylmethylxanthine (IBMX) alone inhibited $Na^+$ uptake by $88.79{\pm}2.56,\;80.63{\pm}4.38,\;and\;84.47{\pm}4.74%$ of that of control, respectively, and prevented the ANG II-induced stimulation of $Na^+$ uptake. However, $10^{-11}$ M ANG II did not stimulate cAMP production. 3) The addition of 12-O-te-tradecanoylphorbol-13-acetate (TPA, 0.01 ng/ml) to the PTCs produced significant increase in $Na^+$ uptake ($114.43{\pm}4.05%$ of that of control). When ANG II and TPA were added together to the PTCs, there was no additive effect on $Na^+$ uptake. Staurosporine alone had no effect on $Na^+$ uptake, but led to a complete inhibition of ANG II- or TPA-induced stimulation of Na'uptake. ANG II treatment resulted in a $111.83{\mu}4.51%$ increase in total protein kinase C (PKC) activity. In conclusion, the PTX-sensitive PKC pathway is the main signaling cascade involved in the stimulatory effects of ANG II on $Na^+$ uptake in the PTCs.

• 대한수의학회지
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• 제41권3호
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• pp.319-325
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• 2001

Several recent studies demonstrate that cAMP accumulation evokes marked changes in magnesium ($Mg^{2+}$) homeostasis. The goal of this study was to investigate the effect of melatonin, the principal hormone of the vertebral pineal gland, on $Mg^{2+}$ regulation in perfused guinea pig hearts. We hypothesized that melationin would regulate $Mg^{2+}$ efflux induced by adrenergic drugs and cAMP analogues because melatonin inhibites adneylate cyclase (AC) and phospholipase C(PLC) in the hearts. The $Mg^{2+}$ content in the perfusate was significantly higher in the presence than in the absence of melatonin. The addition of forskolin, isoproterenol or dimaprit to perfused hearts induced a marked $Mg^{2+}$ efflux. These effluxes were not inhibited by melatonin. The $Mg^{2+}$ efflux could also be induced by phenylephrine, a ${\alpha}_1$-adrenoceptor agonist. This phenylephrine-induced $Mg^{2+}$ efflux was inhibited by melatonin. In addition, the phenylephrine-induced $Mg^{2+}$ efflux was potentiated by PMA, a protein kinase C(PKC) activator. This $Mg^{2+}$ efflux was inhibited by melatonin. In conclusion, these data suggest that melatonin regulates $Mg^{2+}$ homeostasis and the inhibitory effect of melatonin on ${\alpha}_1$-adrenoceptor-stimulated $Mg^{2+}$ efflux may occur through an inhibition of PLC pathway in perfused guinea pig hearts.

• Molecular & Cellular Toxicology
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• 제4권3호
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• pp.224-229
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• 2008

Eucommia ulmoides (Duchung) is commonly used for treatment of diabetes in Korean traditional medicine. However, the exact mechanism of its anti-diabetic effect has not yet been fully elucidated. In this study, the effect of E. ulmoides extract on glucose uptake was investigated in L6 rat skeletal muscle cells. E. ulmoides extract stimulated the activity of phosphatidylinositol (PI) 3-kinase that is a major regulatory molecule in glucose uptake pathway. Protein kinase B (PKB) and protein kinase C-${\xi}$ (PKC-${\xi}$), downstream mediators of PI 3-kinase, were also activated by E. ulmoides extract. We assessed the activity of AMP-activated protein kinase (AMPK), another regulatory molecule in glucose uptake pathway. Phosphorylation level of AMPK did not change with treatment of E. ulmoides extract. Phosphorylations of p38 mitogen activated protein kinase (p38 MAPK) and acetyl-CoA carboxylase (ACC), downstream mediators of AMPK, were not significantly different. Taken together, our results suggest that E. ulmoides may stimulate glucose uptake through PI 3-kinase but not AMPK in L6 skeletal muscle cells.

• 대한수의학회지
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• 제36권4호
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• pp.783-794
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• 1996

이온운반계는 생체의 각기 다른 세포의 성장을 조절하는 성장조절인자들의 효과를 매개하는데 깊은 관련이 있는 것으로 보고되고 있다. 신장 근위세뇨관에서 솔변 연 $Na^+/H^+$ 상호운반계는 사구체에서 여과된 나트륨의 재흡수와 수소이온의 분비를 조절하는 중요한 기능을 수행한다. 이 연구는 초대배양된 신장 근위세뇨관세포의 나트륨 운반을 Insulin-like Growth Factor-I(IGF-I)이 어떤 경로를 통하여 조절하는지를 알아보고자 실시하였다. 결과는 아래와 같다. 1. 초대배양된 신장 근위세뇨관세포에서 $Na^+$ uptake는 시간의존적으로 증가되었으며, 30분동안 $Na^+$ uptake를 실시한 결과 세포외 NaCl 농도의존적으로 $Na^+$ uptake를 유의성있게 감소시켰다(대조군; $40.11{\pm}1.76$, 140mM군; $17.82{\pm}0.94pmole\;Na^+/mg\;protein/min$). 2. $Na^+$ uptake는 iodoacetic acid(IAA, $1{\times}10^{-4}M$) 또는 valinomycin($5{\times}10^{-6}M$)처리시 대조군에 비해 각각 $50.51{\pm}4.4%$와 $57.65{\pm}2.27%$ 억제되었으며, ouabain($5{\times}10^{-5}M$)을 처리한 경우는 $140.23{\pm}3.37%$ 증가되었다. IGF-I($1{\times}10^{-5}M$)으로 배양한 세포를 actinomycin D($1{\times}10^{-7}M$)와 cycloheximide($4{\times}10^{-5}M$)로 처리시 $Na^+$ uptake는 대조군에 비해 각각 $90.21{\pm}2.39%$와 $89.64{\pm}3.69%$로 감소되었다. 3. IGF-I으로 배양한 세포에서 세포외 cAMP는 농도의존적($10^{-8}-10^{-4}M$)으로 $Na^+$ uptake를 유의성있게 감소시켰고, 3-isobutyl-1-methyl-xanthine(IBMX, $5{\times}10^{-5}M$)도 억제시켰다. Pertussis toxin(PTX, 50pg/ml)이나 cholera toxin(CTX, $1{\mu}g/ml$)의 처리시에도 $Na^+$ uptake는 억제되었다. 세포외 phorbol 12-myristate 13 acetate(PMA) 또한 농도의존적(1-100ng/ml)으로 $Na^+$ uptake를 감소시켰다. 그러나 staurosporine($1{\times}10^{-7}M$)은 $Na^+$ uptake에 영향을 미치지 않았으며 PMA와 stauiosporine을 동시에 처리했을 때도 $Na^+$ uptake는 억제되지 않았다. 결론적으로 초대배양된 토끼 신장 근위세뇨관세포에서 $Na^+$ uptake는 막전위와 세포내 에너지 의존적이며 IGF-I은 부분적으로 단백질 및 RNA 합성을 통해서 그리고 세포내 cAMP나 PKC 경로를 통해서 $Na^+$ uptake를 조절하는 것으로 생각된다.

• KSBB Journal
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• 제21권6호
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• pp.466-473
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• 2006

본 연구에서는 췌장 베타세포의 인슐린 분비 촉진 물질로 선별된 방선균 배양액에서 유래한 YHB-2017 (genistein)의 인슐린 분비 촉진 활성의 특성을 조사하고 그 작용 기전을 밝히고자 하였다. YHB-2017는 췌장소도에서 glucose 농도가 16 mM일 때 농도 의존적으로 인슐린 분비를 대조군에 비해 2배 이상 촉진시켰으며, 5.5 mM 이하의 glucose 농도에서는 인슐린 분비 촉진 활성이 거의 없는 것으로 나타났다. MIN6 세포를 이용한 YHB-2017의 인슐린 분비 촉진 활성 특성을 분석한 결과, PKA inhibitor (H89)에 의해서 활성이 저해되었으며, 세포막의 $K_{ATP}$ channel를 배제하고 단순히 칼슘이온을 최대로 세포내로 유입시킨 조건인 diazoxide ($200\;{mu}M$)와 KCI (35 mM)를 첨가한 경우에 YHB-2017는 인슐린 분비 촉진 활성을 나타내 $K_{ATP}$ channel-independent pathway를 통한 인슐린 분비 촉진 기전을 추정할 수 있었다. 베타세포의 단백질 인산화에 대한 영향을 조사한 결과 YHB-2017는 고농도 glucose 조건에서만 PKA 기질과 cAMP response element-binding protein (CREB)의 인산화를 증가시키는 것으로 나타났고, PKC 기질의 인산화에는 영향이 없었다. 또한, YHB-2017를 18시간동안 베타세포에 처리하였으나 인슐린 유전자 발현에는 영향을 주지 않았다. 이상의 결과를 종합하여 볼 때 YHB-2017는 기존의 sulphonylurea 계열 약물과는 다른 작용 기전에 의해 췌장 베타세포에서 인슐린 분비를 촉진시키며, 그 기전은 PKA경로를 통해 amplify 신호를 활성화시키는데 관여하는 것으로 추정된다.