• BMB Reports
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• v.34 no.2
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• pp.182-187
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• 2001

Phospholiapse D (PLD), and phosphatidic acid generated by it, have been implicated in receptor-mediated intracellular signaling. Carbachol (CCh) is known to activate PLD1, and protein kinase C (PKC) is known to mediate in this signaling pathway In recent reports (Kim et al., 1999b; Kim et al., 2000), we published our observations of the direct phosphorylation of PLD1 by PKC and we described the phosphorylation-dependent regulation of PLD1 activity. In this study, we investigated the phasphorylation and compartmentalization of PLD1 in terms of CCh signaling in M3 muscarinic receptor (M3R)-expressing COS-7 cells. CCh treatment of COS-7 cells transiently coexpressing PLD1 and M3R stimulated PLD1 activity and induced direct phosphorylation of PLD1 by PKC. The CCh-induced activation and phosphorylation of PLD1 was completely blocked upon pretreatment of the cells with PKC-specific inhibitors. We looked at the localization of the PLD1 phosphorylation by PKC and found that PLD1 was mainly located in the caveolin-enriched membrane (CEM) fraction. Based on these results, we conclude that CCh induces the activation and phosphorylation of PLD1 via PKC and that the phosphorylation of PLD1 occurs in caveolae.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1994.04a
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• pp.274-274
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• 1994

본 연구에서는 토끼신장 근위세뇨관 일차배양세포에서 브라디키닌의 생리작용이 phospholipase D (PLD)에 의해 매개되는지를 살펴 보기위해 PLD 효소반응의 특이한 성질인 transphosphatidylation 반응의 생성물인 phosphatidylethanol (PEth) 의 세포내 양을 측정함으로 PLD 효소의 관련성을 규명할 수 있었다. 시간경과에 따른 phosphatidic acid (PA) 및 diacylglycerol (DAG) 의 생성을 살펴본 결과 PA가 DAG보다 먼저 생성되어 최고치 (30초)에 도달하였고 DAG는 1분이후부터 5분까지 서서히 생성되는 양상을 나타내었다. 또한 0.5에서 5％까지의 에탄올 존재하에 PA 및 PE소 생성량을 비교해본 결과 에탄올량이 증가함에 따라 PA는 감소하는 반민 PEth 의 생성은 계속 증가하였다. 한편 브라디키닌 농도 변화 실험에서는 브라디키닌농도가 증가함에 따라 PA 및 PEth 둘다 생성이 증가되었다. 이러한 결과로부터 토끼신장 근위세뇨관 세포막에 존재하는 브라디키닌수용체는 브라디키닌에 의해 activation 시 PLD를 직접적으로 활성화시켜 그들의 작용을 세포내로 전달한다는 사실을 알 수 있었다. 또한 PLD 효소활성의 activator로 수용체효능 제외에 칼슘이온, protein kinase C (PKC) 등이 몇몇 다른 실험에 의해 밝혀져 있고, G protein 역시 PLD 효소 활성을 조절하는 역할이 있음이 알려졌다. calcium ionophore 및 칼슘채널길항제인 verapamil을 이용한 실험에서 우리는 브라디키닌의 PLD 활성화는 칼슘이온에 의존적인 경로 및 비의존적인 경로가 같이 존재함을 알수 있었다. 또한 브라디키닌의 PLD 활성화기전이 PKC 의존적인지를 살펴보기위해 PKC activator(PMA) 및 inhibitor (staurosporine)를 이용한 실험에서 브라디키닌은 신장세포에서 PKC를 통하여 PLD를 활성화시킴으로 신호전달을 하는 것으로 추측되었다. 마지막으로 가수분해안되는 G protein 유도체인 GTPrS 및 G protein 활성물질 NaF, 백일해독소등을 이용한 실험에서 G protein 의 PLD 조절활성을 확인할 수 있었다.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1996.04a
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• pp.207-207
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• 1996

Through molecular cloning, five muscarinic receptors have been identified. The muscarinic receptors can be generally grouped according to their coupling to either stimulation of phospholipase C (m1, m3, and m5) or the inhibition of adenylate cyclase (m2 and m4). Each m1, m3, and m5 receptors has the additional potential to couple to the activation of phospholipase A$_2$, C, and D, tyrosine kinase, and the mobilization of Ca$\^$2+/. However, the differences in coupling efficiencies to different second messenger systems between these receptors have not been studied well. Ectopic expression of each of these receptors in mammalian cells has provided the opportunity to evaluate the signal transduction of each in some detail. In this work we compared the coupling efficiencies of the m1, m3 and m5 muscarinic receptors expressed in chinese hamster ovary (CHO) cells to the Ca$\^$2+/ mobilization and the stimulation of neuronal nitric oxide synthase (nNOS). Because G protein/PLC/PI turnover/[(Ca$\^$2+/])i/NOS pathway was supposed as a main pathway for the production of nitric oxide via muscarinic receptors, we studied on ml, m3 and m5 receptors. Stimulation of guanylate cyclase activity in detector neuroblastoma cells was used as an index of generation nitric oxide (NO) in CHO cells. The agonist carbachol increased the cGMP formation and the intracellular [Ca$\^$2+/] in concentration dependent manner in three types of receptors and the increased cGMP formation was significantly attenuated by scavenger of NO or inhibitor of NOS. m5 receptors was most efficiently coupled to stimulation of nNOS, And, the coupling efficiencies to the stimulation of neuronal nitric oxide synthase in three types of receptors were parallel with them to the Ca$\^$2+/ mobilization.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1996.04a
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• pp.187-187
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• 1996

세포질에 존재하는 100 kDa Phospholipase $A_2$(cPLA$_2$)는 인지질의 sn-2 위치의 에스테르결합을 가수분해함으로서 Prostaglandin과 Leukotriene등 Eicosanoids 생합성의 전구체인 아라키돈산과 Platelet activating factor(PAF)를 생합성하는 전구체를 동시에 생성시키는 효소로 염증과 세포손상등에 중요한 역할이 기대된다. 본 효소의 활성화 기전을 규명하고자 하는 최근의 활발한 연구에도 불구하고 불명확한 점이 많은 것이 현실이다. 특히 세포를 자극하였을 때 유리되는 아라키돈산의 증가율과 세포를 파괴한 후 조제한 가용성분획에서 측정한 활성의 증가율과는 많은 차이를 나타냈다. 이러한 결과부터 cPLA$_2$ 효소 자체를 활성화시키는 어떤 인자를 가정하였다. 최근, PLA$_2$의 또다른 형태인 14 kDa의 분비성 PLA$_2$의 in vitro 활성을 증가시키는 인자가 동정되어 그 생화학적 특성이 규명되고 있으나 이 인자는 cPLA$_2$의 활성에는 아무런 증가효과를 나타내지 않았다. 본 연구자들은 소의 뇌조직에서 cPLA$_2$의 활성을 증가시키는 인자를 발견하고 그의 생화학적인 특성을 규명하였다. 돼지 비장에서 정제한 cPLA$_2$를 사용하였으며 소의 뇌 조직의 가용성 분획으로부터 본 활성화 인자를 동정하였으며 그 활성분획을 양이온 크로마토그라피로서 Mono S EPLC와 Superose 12 Sepharose gel filtration 크로마토그라피를 이용하여 더욱 분리한 결과 약 70 kDa과 25 kDa에서 각각 용출되었다. 이렇게 부분정제한 활성은 췌장에서 분리한 group I과 흰주의 group I과 흰주의 혈소판에서 분리한 group II PLA$_2$에 대해서는 아무런 증가효과를 나타내지 않는 반면, cPLA$_2$의 활성만을 약 5배 증가시켰다. 본 활성은 cPLA$_2$ 효소량의 증가에 따라 활성의 증가효과가 정차 감소하므로 화학량적인 반응(Stoichiometric reaction)일 것으로 예상되었다.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1996.04a
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• pp.206-206
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• 1996

The C-terminus ends of the second putative transmembrane domains of both m1 and m2 muscarinic receptors contain a triplet of amino acid residues consisting of leucine (L), tyrosine (Y) and threonine (T), This triplet is repeated as LYT-LYT in m2 receptors at the interface between the second transmembrane domain and the first extracellular loop. Interestingly, however, it is repeated in a transposed fashion (LYT-TYL) in the sequence of m1 receptors. In this work we employed site-directed mutagenesis to investigate the possible significance of this unique sequence diversity for determining the distinct differential drug-receptor interaction and cellular function at m1 muscarinic receptor. Mutation of the LYTTYL sequence of m1 receptors to the corresponding m2 receptor LYTLYT sequence, however, did not result in a significant change in the binding affinity of the agonist carbachol or in the affinity of the majority of a series of receptor antagonists which are able to discriminate between wild-type m1 and m2 receptors. Surprisingly, the LYTLYT ml receptor mutant demonstrated markedly enhanced coupling to activation of phospholipase C without a change in its coupling to increased cyclic AMP formation. There was also an enhanced receptor sensitivity in transducing elevation of intracellular Ca$\^$2+/. These changes were not due to alterations in the rate of receptor. desensitization or sequestration, On the other hand, the reverse LYTLYT-LYTTYL mutation in the m2 receptor did not alter its coupling to inhibition of adenylate cyclase, but slightly enhanced its coupling to stimulation of PI hydrolysis, Our data suggest that the LYTTYL/LYTLYT sequence difference between ml and n12 muscarinic receptors is not involved in determining receptor pharmacology. On the other hand, while these differences might play a role in the modulation of muscarinic receptor coupling to PI hydrolysis, they are not important for specifying coupling of various subtypes of muscarinic receptors to different cellular signaling pathways.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1996.04a
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• pp.219-219
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• 1996

Mucin release from hamster tracheal surface epithelial(HTSE) cells can be stimulated by extracellular ATP via activation of P$_2$ purinoceptors located on the cell surface which appears to be coupled to phospholipase C via G proteins. However, our preliminary data indicate that the ATP-induced mucin release involves, in part, activation of PKC, but not an increase in the intracellular Ca++ level, suggesting the presence of another pathway which is separate from the PLC-PKC pathway, In this study, we intended to confirm the previous observation and subsequently identify an additional mechanism. Confluent HTSE cells were metabolically labeled with either $^3$H-glucosamine or $^3$H-arachidonic acid(AA), and release of either $^3$H-mucin or $^3$H-AA was quantified following various treatments. $^3$H-mucin was assayed using the sepharose CL-4B gel-filtration method, whereas $^3$H-AA liberation was measured by counting $^3$H-radioactivity in the chase medium. We found that: (1)Desensitization of PKC by pretreatment with PMA completely abolished the mucin releasing effect of PMA but partially inhibited the ATP-induced mucin release; (2) ATP increases release of $^3$H-AA in a dose-dependent fashion; (3) mepacrine, an inhibitor of PLA$_2$, attenuates ATP-induced mucin release in a dose-dependent fashion. These results confirm our previous notion that the PLC-PKC pathway is responsible, in part, for ATP-induced mucin release. Furthermore, activation of PLA$_2$ appears to be an additional pathway which is involved in ATP-induced mucin release.

• Biomedical Science Letters
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• v.22 no.4
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• pp.127-139
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• 2016

A species of the fungal genus Cordyceps has been used as an ingredient of traditional Chinese medicine. In this study, we prepared cordycepin-enriched WIB-801CE, an ethanol extract from culture solution of Cordyceps militaris-hypha, and evaluated its antiplatelet effects on human platelet aggregation. WIB-801CE dose-dependently inhibited ADP-, collagen-, and thrombin-induced platelet aggregation. These antiplatelet effects by WIB-801CE were associated with the attenuation of thromboxane $A_2$ ($TXA_2$) production and serotonin release by ADP, collagen, and thrombin. The inhibition of $TXA_2$ production by WIB-801CE was due to the inhibition of cyclooxygenase-1, $TXA_2$ synthase, and cytosolic phospholipase $A_2$ activity. Therefore, these data suggest that WIB-801CE may be a beneficial component against protection from platelet aggregation-mediated thrombotic disease.

• BMB Reports
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• v.44 no.9
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• pp.572-577
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• 2011

Elevated phospholipase D (PLD) expression prevents cell cycle arrest and apoptosis. However, the roles of PLD isoforms in cell proliferation and apoptosis are incompletely understood. Here, we investigated the physiological significance of the interaction between PLD2 and protein kinase CKII (CKII) in HCT116 human colorectal carcinoma cells. PLD2 interacted with the CKII${\beta}$ subunit in HCT116 cells. The C-terminal domain (residues 578-933) of PLD2 and the N-terminal domain of CKII${\beta}$ were necessary for interaction between the two proteins. PLD2 relocalized CKII${\beta}$ to the plasma membrane area. Overexpression of PLD2 reduced CKII${\beta}$ protein level, whereas knockdown of PLD2 led to an increase in CKII${\beta}$ expression. PLD2-induced CKII${\beta}$ reduction was mediated by ubiquitin-dependent degradation. The C-terminal domain of PLD2 was sufficient for CKII${\beta}$ degradation as the catalytic activity of PLD2 was not required. Taken together, the results indicate that the C-terminal domain of PLD2 can regulate CKII by accelerating CKII${\beta}$ degradation in HCT116 cells.

• BMB Reports
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• v.44 no.9
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• pp.566-571
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• 2011

Although the phospholipase C (PLC)${\beta}$-1 isoform is associated with spontaneous seizure and distinctively expressed in the telencephalon, the distribution of PLC${\beta}$-1 expression in the epileptic gerbil hippocampus remains controversial. Therefore, we determined whether PLC${\beta}$-1 is associated with spontaneous seizure in an animal model of genetic epilepsy. In the present study, PLC${\beta}$-1 immunoreactivity was down-regulated in seizure-sensitive (SS) gerbils more than in seizure-resistant (SR) gerbils. The expression of PLC${\beta}$-1 within calretinin (CR)-positive neurons was rarely detected within the dentate hilar region of SS gerbils. PLC${\beta}$-1 immunoreactivity in the hippocampus was significantly elevated as compared to that in pre-seizure SS gerbil 3 h post-ictal. These findings suggest that alterations in PLC${\beta}$-1 immunoreactivity in the SS gerbil hippocampus may be closely related to the epileptic state of the gerbil brain and transiently elevated PLC${\beta}$-1 protein levels following seizure episodes. Such alterations may be compensatory responses in the SS gerbil hippocampus.

• Development and Reproduction
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• v.9 no.2
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• pp.115-121
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• 2005

The present report aimed at evaluating the effect of bisphenol A(BPA) and diethylstilbestrol(DES) on Leydig or Sertoli cell-lines. To identify the differences in the susceptibility to BPA upon different cell-types, assay of the cell viability was done on TM3(Leydig cells) and TM4(Sertoli cells) cell-lines. The result indicates that Sertoli cells are more sensitive to low dose of BPA than Leydig cells. Also, the BPA- or DES-treated Sertoli cells showed a reduction of phospholipase D(PLD) activity identically. According to the confirmation of the mRNA expression of fas receptor and fas ligand in the BPA-treated cells, fas/fasL system activated by BPA will deliver the apoptosis signal onto Sertoli TM4 cells. However, Fas/FasL system was not activated in the DES-treated cells unlike the BPA-treated cells.