토끼 경동맥 평활근에서 Isoproterenol의 이완작용에 미치는 Phorbol Esters의 효과

김법영,이영우 대한신경외과학회 1995 Journal of Korean neurosurgical society Vol.24 No.7

토끼 경동맥에서 phorbol estersr가 isoporterenol에 의한 혈관 긴장도의 조절에 미치는 protein kinase C(PKC)의 작용을 밝히기 위하여 경동맥환에서 등척성 수축을 기록하여 phorbol esters인 phorbol 12,13-di-butyrate(PDBu)와 phorbol 12-myristate 13-acetate(PMA)의 효과를 관찰하였다. β-Adrenergic agonist인 isoproterenol은 phenylephrine에 의한 수축을 이완하였으나 phorbol esters에 의한 수축에는 영향을 미치지 아니하였다. Isoproterenol에 의한 혈관 이완작용은 내피세포의 제거나 methylene blue나 nitro-L-arginine의 전처치에 의하여 감소하였다. 수축을 야기하지 않는 농도는 phorbol esters의 전처치는 내피세포 의존성 및 비의존성으로 isoproterenol에 의해 야기된 혈관이완을 억제하였다. Isoproterenol에 의해 야기된 혈관 이완작용에 대한 PDBu의 억제작용은 수축반응에서 보인 양상과는 달리 PMA의 억제작용보다 낮은 효과를 보였으나 PDBu의 수축효과는 PMA보다 더 강하게 나타났다. PMA는 forskolin의 농도반응에 영향을 미치지 않았다. 그리고 protein kinase C inhibitor인 staurosporine은 isoprocerenol에 의해 야기된 이완과 수축반응에 대한 이들 약물의 작용을 억제하였다. 이상과 같은 결과는 isoproterenol에 의해 야기된 혈관 이완작용은 protein kinase C의 활성제인 phorbol esters에 의해 억제되며, 이는 수축반응에 각기 다른 isozyme이 관여하는 것으로 사료된다. The effects of phorbol esters were studied in rabbit carotid artery to evaluate the action of protein kinase C on the regulation of vascular tone by isoproterenol. The vascular rings. 2 mm in width, were myographied isometrically in an isolated organ bath and the effects of phorbol 12,13-dibutyrate(PDBu) and phorbol 12-myristate 13-acetate(PMA) were determined. Isoproterenol a beta adrenergic agonist, relaxed the vessel which was precontracted by phenylephrine, but not that by phorbol esters. The action of isoproterenol was attenuated by removal of endothelium or pretreatment with methylene blue or nitro-L-arginine. The pretreatment with phorbol esters at concentrations which did not induce contraction, decreased isoproterenol-induced relaxation of vascular rings with or without endothelium. The action of PDBu on isoproterenol-induced relaxation was less effective than that of PMA, unlike those observed in contractile response. but the contractile effect of the former was more potent than that of the latter. PMA did not affect relaxant effect of forskolin, an activator of adenyl cyclase. Staurosporine, a protein kinase C inhibitor, inhibited the action of these drugs on both isoproterenol-induced relaxation and the contractile response. These results suggest that the relaxation induced by isoproterenol was reduced by the activation of protein kinase C. which may be isozyme different from that involved in contractile response.

섬유아세포 증식에 대한 Phorbol myristate acetate의 효과

윤석주,박낭운,김인산,손건영,조준승 慶北大學校 醫科大學 1995 慶北醫大誌 Vol.36 No.1

목적 : 이 연구는 Phorbol myristate acetate (PMA)가 강력한 암촉진제로서 세포내 신호전달체인 protenin kinase C (PKC)의 활성화를 통하여 여러가지 작용을 나타낸다. 사람 두배수체인 섬유아세포와 불멸성 섬유아세포인 Swiss 3T3 세포의 증식 즉 DNA 합성에 대해 PMA의 미치는 효과와 이것의 PKC와의 관계를 조사하는데 있다. 재료 및 방법 : 사람 폐섬유아세포(HEL299) 및 Swiss 3T3세포를 밀생상태가 될때까지 배양한후 fetal vine serum이 없는 배지에서 48시간 두어 휴지기 상태로 만들어 필요한 시약과 [H^3]thymidine를 넣어 24시간 배양해서 세포내 DNA 합성에 이용된 [H^3]thymidine의 방사능을 측정하여 비교하였다. 결과 : 사람 및 Swiss 3T3 섬유아세포의 DNA 합성은 PMA단독투여 때는 약2배로 증가되었으나 colchicine 혹은 insulin을 함께 투여하면 그 양상은 달랐다. 즉 PMA는 Swiss 3T3 세포에서 insulin에 의한 DNA 합성 증가작용을 더욱 촉진시켰으나, 사람의 섬유아세포에서는 insulin이나 colchicine에 의한 DNA합성 증가를 오히려 억제하였다. 사람의 섬유아세포에 비활성 phorbol ester인 4α-phorbol-12, 13-didecanoate(4α-PDD) 단독 투여하니 PMA 무여군과는 달리 DNA 합성이 대조군과 별 차이가 없었으며, colchicine에 의한 DNA 합성증가에도 별로 영향을 미치지 않았다. Swiss 3T3 세포에 비교적 선택적으로 PKC 활성을 억제하는 staurosporine을 농도를 증가시키면서 투여하니 PMA에 의한 DNA합성 증가효과는 감소되었다. 결론 : PMA는 사람 섬유아세포나 Swiss 3T3 세포에 약간의 증식효과를 가지나 세포 자극제에 의한 DNA 합성 증가효과에 대해서는 세포의 종류 및 상태에 따라 다른 결과를 나타내었다. 또한 PMA의 효과는 PKC를 통하여 일어남을 시사하였다. Proliferation of fibroblasts around the damaged tissue plays a key role in the wound healing and the development of tissue fibrosis. Phorbol myristate acetate (PMA) is known to be a potent tumor promoter and its intracellular receptor, protein kinase (PKC) is known to play important roles in many signaling pathways, including those involved in cell proliferation. In cultured human diploid tang fibroblasts and Swiss 3T3 cells, the effects of PMA on the DNA synthesis and the involvement cf PKC in its effects were studied. PMA alone stimulated DNA syntheis by the both cells slightly. However, the treatment of the both cells with PMA resulted in an opposite effect on the DNA syntheis stimulated by colchicine and/or insulin. PMA inhibited DNA synthesis in human fibroblasts stimulated by insulin and colchicine, whereas PMA potentiated insulin-stimulated DNA synthesis in Swiss 3T3 cells. 4α-phorbol-12, 13-didecanoate(4α-PDD), known to be inactive for PKC, was without effect in DNA synthesis by human fibroblasts in the either absence or presence of colchicine. Staurosporine, a relatively specific inhibitor for PKC abolished the effect of PMA on DNA synthesis in Swiss 3T3 cells in the presence of insulin. These results suggest that PKC is involved in the regulation of DNA synthesis in fibroblasts but its outcome may be different according to the culture conditions and the cells used.

토끼 경동맥에서 Acetylcholine의 작용에 대한 Phorbol Ester의 조절 작용

노용래,이상호,이영우 대한신경외과학회 1994 Journal of Korean neurosurgical society Vol.23 No.12

Authors studied the regulatory mechanism of protein kinase C on the action of acetylcholine in rabbit carotid artery. The arterial rings were myobphied isometrically in an isolated organ bath. In this study, acetylcholine relaxed phenylephrine-induced contraction of rabbit carotid artery in the presence of endothelium. In the pretreatment of methylene blue or nitro-L-arginine, the action of acetylchioline was reduced. Pretreatment of phorbol 12-myristate IZacetate(PMA) attenuated the action of acetylcholine, but PMA did not attenuated it in the presence of staurosporine, suggesting that protein kinase C suppressed the action of acetylcholine. The potency of phorbol ester on the action of acetylcholine was PMA>phohol 1213dibutyrate(PDBu) >phorbol 12,13-diacetate(PDA), but the direct effect of phorbol on the contraction of arterial rings was PDBu>PMA>PDA. This implied that protein kinase C involved in the contraction of smooth muscle and the attenuation of the action of acetylcholine were different PMA did not affect on A23187- and sodium nitroprusside-induced vasorelaxation. Acetylcholine increased tissue cGMP contents, which was reduced by PMA, These results suggest that in rabbit carotid artery protein kinase C reduce acetylcholine-stimuated endothelium derived relaxing factor(EDRF) release by affecting membrane receptor, and do not affect on the function of EDRF and cGMP production in the smooth muscle.

방선균 분리주 No. 1882-5로부터 Phorbol ESter에 의해 유도되는 K562 Cell의 소포형성을 억제하는 물질의 분리와 동정

안종석,안순철,이현선,박문수,오원근,김보연,민태익 한국산업미생물학회 1992 한국미생물·생명공학회지 Vol.20 No.5

Phorbol ester에 의해 유도되는 K562 cell의 소포 형성을 저해하는 물질을 생산하는 방선균 분리주 No. 1882-5를 토양으로부터 분리하였다. 분리주 No. 1882-5로부터 용매추출, Amberlite XAD-4, silica, Lobar low pressure LC를 거쳐 항진균 활성물질 MT 1882-Ⅰ과 K562 cell의 소포형성을 억제하는 활성물질 MT 1882-Ⅱ를 분리하였다. 이 물질들에 대한 이화학적 성질의 조사와 UC, ^1H-NMR, ^13C-NMR, mass spectrum의 분석에 의하여 MT 1882-Ⅰ은 piericidin A_1(C_25H_370_4N, MW 415), MT 1882-Ⅱ는 glucopiericidin A(C_31H_470_9N, MW 577)로 동정하였다. We isolated Actinomycetes strain No. 1882-5, which produces the inhibitor on the bleb formation of K562 cell induced by phorbol ester, from soil sample. Through solvent extraction, Amberlite XAD-4, silica and Lobar low pressure LC, antifungal antibiotic MT 1882-I and bleb forming inhibitor MT 1882-Ⅱ were purified from strain No. 1882-5. MT 1882-Ⅰ was identified as piericidin A_1(C_25H_37O_4N, M.W.415) and MT 1882-Ⅱ as glucopiericidin A(C_31H_47O_9N, M.W.577) from the analysis of physico-chemical properties and UV, ^1H-NMR, ^13C-NMR, and mass spectra of these compounds.

Molecular Mechanisms of Protein Kinase C-induced Apoptosis in Prostate Cancer Cells

Gonzalez-Guerrico, Anatilde M.,Meshki, John,Xiao, Liqing,Benavides, Fernando,Conti, Claudio J.,Kazanietz, Marcelo G. Korean Society for Biochemistry and Molecular Biol 2005 Journal of biochemistry and molecular biology Vol.38 No.6

Protein kinase C (PKC) isozymes, a family of serine-threonine kinases, are important regulators of cell proliferation and malignant transformation. Phorbol esters, the prototype PKC activators, cause PKC translocation to the plasma membrane in prostate cancer cells, and trigger an apoptotic response. Studies in recent years have determined that each member of the PKC family exerts different effects on apoptotic or survival pathways. $PKC{\delta}$, one of the novel PKCs, is a key player of the apoptotic response via the activation of the p38 MAPK pathway. Studies using RNAi revealed that depletion of $PKC{\delta}$ totally abolishes the apoptotic effect of the phorbol ester PMA. Activation of the classical $PKC{\alpha}$ promotes the dephosphorylation and inactivation of the survival kinase Akt. Studies have assigned a pro-survival role to $PKC{\varepsilon}$, but the function of this PKC isozyme remains controversial. Recently, it has been determined that the PKC apoptotic effect in androgen-dependent prostate cancer cells is mediated by the autocrine secretion of death factors. $PKC{\delta}$ stimulates the release of $TNF{\alpha}$ from the plasma membrane, and blockade of $TNF{\alpha}$ secretion or $TNF{\alpha}$ receptors abrogates the apoptotic response of PMA. Molecular analysis indicates the requirement of the extrinsic apoptotic cascade via the activation of death receptors and caspase-8. Dissecting the pathways downstream of PKC isozymes represents a major challenge to understanding the molecular basis of phorbol ester-induced apoptosis.

Characterization of AJH-836, a diacylglycerol-lactone with selectivity for novel PKC isozymes

Cooke, Mariana,Zhou, Xiaoling,Casado-Medrano, Victoria,Lopez-Haber, Cynthia,Baker, Martin J.,Garg, Rachana,Ann, Jihyae,Lee, Jeewoo,Blumberg, Peter M.,Kazanietz, Marcelo G. American Society for Biochemistry and Molecular Bi 2018 The Journal of biological chemistry Vol.293 No.22

<P>Diacylglycerol (DAG) is a key lipid second messenger downstream of cellular receptors that binds to the C1 domain in many regulatory proteins. Protein kinase C (PKC) isoforms constitute the most prominent family of signaling proteins with DAG-responsive C1 domains, but six other families of proteins, including the chimaerins, Ras-guanyl nucleotide-releasing proteins (RasGRPs), and Munc13 isoforms, also play important roles. Their significant involvement in cancer, immunology, and neurobiology has driven intense interest in the C1 domain as a therapeutic target. As with other classes of targets, however, a key issue is the establishment of selectivity. Here, using [H-3]phorbol 12,13-dibutyrate ([H-3]PDBu) competition binding assays, we found that a synthetic DAG-lactone, AJH-836, preferentially binds to the novel PKC isoforms PKC and PKCE relative to classical PKC and PKCII. Assessment of intracellular translocation, a hallmark for PKC activation, revealed that AJH-836 treatment stimulated a striking preferential redistribution of PKCE to the plasma membrane relative to PKC. Moreover, unlike with the prototypical phorbol ester phorbol 12-myristate 13-acetate (PMA), prolonged exposure of cells to AJH-836 selectively down-regulated PKC and PKCE without affecting PKC expression levels. Biologically, AJH-836 induced major changes in cytoskeletal reorganization in lung cancer cells, as determined by the formation of membrane ruffles, via activation of novel PKCs. We conclude that AJH-836 represents a C1 domain ligand with PKC-activating properties distinct from those of natural DAGs and phorbol esters. Our study supports the feasibility of generating selective C1 domain ligands that promote novel biological response patterns.</P>

전처치된 지치 유래 naphthoquinone의 혈관 수축성 조절 효과

제현동 ( Hyun Dong Je ) 대구가톨릭대학교 자연과학연구소 2015 자연과학연구논문집 Vol.13 No.1

The present study was undertaken to investigate the influence of naphthoquinone (shikonin) on vascular smooth muscle contractility and to determine the mechanism involved. We hypothesized that naphthoquinone, the primary ingredient of Lithospermum erythrorhizon, plays a role in vascular relaxation through inhibition of Rho-kinase in rat aortae. Intact or denuded arterial rings from male Sprague-Dawley rats were used and isometric tensions were recorded using a computerized data acquisition system. Interestingly, naphthoquinone significantly inhibited fluoride, phorbol ester or thromboxane A2 mimetic-induced contraction in denuded muscles suggesting that additional pathways different from endothelial nitric oxide synthesis such as inhibition of Rho-kinase or MEK might be involved in the vasorelaxation. Furthermore, naphthoquinone nonspecifically inhibited fluoride- or phorbol ester-induced contraction suggesting the mechanism including inhibition of fluoride- or phorbol ester-induced increases in MYPT1 or ERK1/2 phosphorylation. This study provides evidence that naphthoquinone induces vascular relaxation through inhibition of Rho-kinase or MEK in rat aortae.

TGF β유도 Type 1 Collagen Promotor 활성에 미치는 Phorbor ester와 Okadaic acid의 영향

전은주,박낭운,김인산,이병헌,최제용,조준승 慶北大學校 醫科大學 1995 慶北醫大誌 Vol.36 No.2

목적 : 이 연구는 섬유아세포에서 collagen 유전자 발현을 조절하는 기전을 밝히려는 노력의 일환으로, HEL299 섬유아세포에서 transforming grow factor β(TGFβ)에 의해 증가된 collagen합성 및 promoter활성에, 세포내 단백질의 인산화에 중요한 역할을 하는 protein phosphatase의 길항제이며 non-phorbol ester tumor promoter인 okadaic acid (OKA)와 Protein kinase C (PKC) 활성제인 phorbol myristate acetate (PMA)가 어떤 영향을 미치는가를 알아보았다. 재료 및 방법 : 사람 폐 섬유아세포인 HEL299 세포를 일정한 상태로 배양한 후 PMA 또는 OKA의 일정량을 단독 혹은 TGFβ와 병합투여하여 24시간 작용시키면서 마지막 6시간 동안 [^3H]proline을 첨가하여 이것의 총단백질과 collagen에의 편입도를 측정·비교하였다. 한편 α_2(Ⅰ)procollagen promoter의 활성도 측정을 위해 HEL299세포에 10㎍의 pMS-3.5/CAT plasmid를 transfection시키고 24시간 배양한 다음 PMA 또는 OKA의 일정량을 단독 혹은 TGFβ와 병합 투여하여 48시간 작용시켜서 세포를 회수하고 CAT활성을 측정하여, 대조 plasmid인 pSV2/CAT를 transfection 시켰을 때의 CAT 활성에 대한 상대적인 값으로 표시하였다. 결과 : OKA와 PMA는 다같이 % collagen합성을 대조군에 비하여 각각 53.4%, 53.9% 억제하였으며, TGF에 의한 collagen 합성 증가도 효과적으로 억제하는 것으로 나타났다. α_2(Ⅰ)procollagen promoter의 3500bp 부위를 포함하는 pMS-3.5/CAT plasmid를 이용한 transfection 실험에서 OKA와 PMA는 그 자체만으로는 promoter 활성을 억제하지 않았지만, TGFβ에 의한 활성 증가에 대해서는 억제효과를 나타내었다. 결론 : 이 결과를 통해서 볼 때, TGFβ에 의한 collagen합성의 증가에 관여하는 인자는 세포내 단백질의 인산화 정도에 영향을 받는다고 할 수 있다. In order to elucidate the regulation mechanism of collagen gene expression in HEL299 fibroblast, the effects of two well-known chemicals affecting phosphorylation status of the intracellular proteins, okadaic acid (OKA), a protein phosphatase inhibitor, and phorbol myristate acetate (PMA), a protein kinase C activator, on TGFβ-induced type Ⅰ collagen synthesis and α_2(Ⅰ) collagen promoter activity were examined. Both OKA and PMA per se inhibited the collagen synthesis by 53.5% and 53.9%, respectively. In addition, the incresase of collagen synthesis induced by TGFβ was suppressed by both OKA and PMA. In transfection experiment using pMS-3.5/CAT composed of -3500∼+58 of α_2(Ⅰ) collagen promoter and CAT gene, OKA and PMA per se did not inhibit the promoter activity. However, they could suppress the effect of TGFβ on collagen promoter activity. These results support the view that factors mediating the effect of TGFβ on type Ⅰ collagen synthesis and α_1(Ⅰ) promoter activity might be regulated by the phosphorlylation status of the intracellular proteins.

Phorbol Ester-induced Contraction Through p38 Mitogen-activated Protein Kinase is Diminished in Aortas from DOCA-Salt Hypertensive Rats

Lee, Chang-Kwon,Kim, Jung-Kwan,Won, Kyung-Jong,Lee, Hwan-Myung,Kim, Hyo-Jin,Roh, Hui-Yul,Park, Hyo-Jun,Shin, Hwa-Sup,Park, Tae-Kyu,Kim, Bo-Kyung,Lee, Sang-Mok The Pharmaceutical Society of Korea 2006 Archives of Pharmacal Research Vol.29 No.11

The role of mitogen-activated protein kinase (MAPK) in the decreased contractile response to phorbol ester in aortic smooth muscle strips from deoxycorticosterone acetate (DOCA)-salt hypertensive rats was examined. Norepinephrine (NE) evoked greater contractility in aortic strips from DOCA rats than in those of sham-operated rats. 12-Deoxyphorbol 13-isobutyrate (DPB) induced contraction in $Ca^{2+}-free$ medium, which was diminished in strips from DOCA rats compared to sham-operated rats. Vasoconstrictions induced by these stimulants were inhibited by SB203580 and PD098059, inhibitors of p38 MAPK and extracellular signal-regulated kinase (ERK) 1/2, respectively, in both strips. The phosphorylation of p38 MAPK and ERK1/2 induced by NE was greater in strips from DOCA rats compared to those from sham-operated rats, and this phosphorylation was inhibited by the kinase inhibitors. DPB increased the phosphorylation of p38 MAPK and ERK1/2 in strips from both animals, and the increment of p38 MAPK phosphorylation by the stimulant was diminished in strips from DOCA rats compared to sham-operated rats. These findings suggest that the $Ca^{2+}-independent$ contraction evoked by DPB results from the activation of MAPKs in rat aortic smooth muscle and that the attenuated contractility by DPB in DOCA rat appears to be associated with diminished p38 MAPK activity.

Phorbol Ester-induced Contraction Through p38 Mitogen-activated Protein Kinase is Diminished in Aortas from DOCA-Salt Hypertensive Rats

Chang-Kwon Lee,Junghwan Kim,원경종,Hwan Myung Lee,Hyo Jin Kim,Hui Yul Roh,Hyo-Jun Park,신화섭,박태규,Bokyung Kim,이상목 대한약학회 2006 Archives of Pharmacal Research Vol.29 No.11

The role of mitogen-activated protein kinase (MAPK) in the decreased contractile response to phorbol ester in aortic smooth muscle strips from deoxycorticosterone acetate (DOCA)-salt hypertensive rats was examined. Norepinephrine (NE) evoked greater contractility in aortic strips from DOCA rats than in those of sham-operated rats. 12-Deoxyphorbol 13-isobutyrate (DPB) induced contraction in Ca2+-free medium, which was diminished in strips from DOCA rats compared to sham-operated rats. Vasoconstrictions induced by these stimulants were inhibited by SB203580 and PD098059, inhibitors of p38 MAPK and extracellular signalregulated kinase (ERK) 1/2, respectively, in both strips. The phosphorylation of p38 MAPK and ERK1/2 induced by NE was greater in strips from DOCA rats compared to those from shamoperated rats, and this phosphorylation was inhibited by the kinase inhibitors. DPB increased the phosphorylation of p38 MAPK and ERK1/2 in strips from both animals, and the increment of p38 MAPK phosphorylation by the stimulant was diminished in strips from DOCA rats compared to sham-operated rats. These findings suggest that the Ca2+-independent contraction evoked by DPB results from the activation of MAPKs in rat aortic smooth muscle and that the attenuated contractility by DPB in DOCA rat appears to be associated with diminished p38 MAPK activity.