臺灣博碩士論文加值系統

內皮素-1 (endothelin-1，ET-1)為一強力血管收縮物質。在高血壓之動物模式已證實其血液中ET-1濃度有上升的現象，臨床研究也指出患有高血壓之病患，其血液中ET-1濃度較正常人高。ET-1必須先與細胞表面的內皮素受體結合才能產生多種生物功能，內皮素受體主要有兩種：A型(ETAR)及B型(ETBR)，ETAR主要分部於血管平滑肌細胞，受刺激時會造成血管收縮導致血壓上升；ETBR主要存在於內皮細胞，受刺激時會產生一氧化氮及前列腺素，使血管放鬆血壓下降。由於某些疾病(如高血壓、心血管疾病、敗血症、惡性腫瘤等)發生及惡化與血液中ET-1濃度升高有關，因此臨床上認為內皮素受體拮抗劑可作為高血壓等疾病之治療藥物。中草藥P1於本草綱目記載有降血壓功效，而我們實驗室之前與阮琪昌博士合作，發現P1含rETAR之拮抗劑，若給予高果糖飲食所引起高血壓之大鼠飲用P1水萃液，可降低大鼠之血壓，故在此想探討P1可否作為人類ETAR之拮抗劑。我們利用外送可表現重組質體到CHO-K1細胞以分別表現hETAR及hETBR，並選殖穩定之轉殖株(稱為CHO-ETAR及CHO-ETBR)，利用受體結合實驗可得到ETAR之Kd為1533pM，Bmax為233.78 fmol/2×105 cells；ETBR之Kd為482pM，Bmax為122.93 fmol/2×105 cells，顯示ET-1對hETBR之親合力大於hETAR。而ETAR拮抗劑(BQ610)以及ETBR拮抗劑(BQ788)可分別抑制ET-1和ETAR及ETBR結合。以ET-1刺激可使CHO-ETAR細胞內鈣離子濃度上升，並使CHO-ETBR細胞一氧化氮合成酶磷酸化，得知表現於CHO-K1細胞上之內皮素受體是具有功能的。以這兩株細胞為材料，發現P1可直接作用於受體上並抑制ET-1對內皮素受體之結合，P1對ETAR有67%之抑制效果，對ETBR有90%之抑制效果，且可連帶影響由ET-1刺激所引起之CHO-ETAR細胞內鈣離子濃度上升。經由不同比例酒精沉澱濃縮P1中之有效成分，發現F3.3.5x(在3.5倍酒精中不沉澱)對ETAR抑制效果較佳(抑制83%)，但對ETBR效果較弱(抑制37%)；而F2 2x(在2倍酒精中沉澱)對ETBR抑制效果較佳(抑制92%)，但對ETAR效果較弱(抑制48%)，由此可知P1中含有兩種不同之有效成分。以人類平滑肌及內皮細胞做實驗，P1及F3 3.5x分別對ETAR有67%及＞96%之抑制效果；而P1及F2 2x對ETBR有79%及87%之抑制效果。另外我們發現P1可以抑制由LPS刺激內皮細胞所引起之一氧化氮合成酶磷酸化現象。

Endothelin-1 (ET-1) is a powerful vasoconstrictor that contributes to blood pressure elevation. In different animal models and patients with hypertension, ET-1 is overexpressed in the plasma. The biological effects of ETs are mediated by two receptors：endothelin receptor type A (ETAR) and type B (ETBR). In the vasculature, ETAR is expressed predominantly on the cell membrane of vascular smooth muscle cells and mediates vasoconstrictor effects of ET-1. ETBR is expressed on endothelial cells. Its activation by ET-1 results in vasodilatation through generation of prostacyclin and nitric oxide (NO). Elevation of ET-1 level was found in patients of hypertension, cardiovascular disease, sepsis, and tumor. The endothelin receptor antagonist might be useful for therapeutic purpose. According to the codex P1, a Chinese herbal medicine, has been reported to treat hypertension and reduce the blood pressure. In the previous study, our lab and Dr. Juan found that P1 is rETAR antagonist and reduces the blood pressure in high fructose diet-induced hypertension in rats, and therefore I investigated whether P1 can act as a human ETAR antagonist too. cDNAs of hETAR and hETBR were cloned and inserted into pIRESneo vector. CHO-K1 cells were transfected with the recombinant vectors. The stable clones of CHO-K1 expressing the hETAR or hETBR were designated CHO-ETAR or CHO-ETBR, respectively. The [125I]-ET-1 binding assay was conducted Kd is 1533pM and and Bmax is 233.78 fmol/2×105 cells for CHO-ETAR. Kd is 482pM and Bmax is 122.93 fmol/2×105 cells for CHO-ETBR. The affinity of ET-1 for ETBR seems stronger than ETAR. [125I]-ET-1 binding was inhibited by ETAR specific antagonist BQ610 and the ETBR antagonist BQ788, respectively. ET-1 stimulates intracellular Ca2+ mobilization in CHO-ETAR and eNOS phosphorylation in CHO-ETBR. Binding assay and postreceptor signaling revealed that they are functionally active. We found that [125I]-ET-1 binding was inhibited by P1 water extract. Effective components of P1 directly interact with endothelin receptors and cause 67% inhibition for CHO-ETAR and 90% inhibition for CHO-ETBR. ET-1 stimulated intracellular Ca2+ mobilization was also inhibited by P1 in CHO-ETAR. I enriched the effective components in P1 by precipitating with different volumes of ethanol. We found that there are two effective components in P1. The component enriched in F3 3.5x (the supernatant portion with 3.5V ethanol) is more effective for ETAR (83% inhibition), but less effective for ETBR (37% inhibition). The another component enriched in F2 2x (the precipitate portion with 2V ethanol) is more effective for ETBR (92% inhibition), but less effective for ETAR (48% inhibition). On the other hand, a 67% and a >96% inhibition were achieved by P1 and F3 3.5x respectively when human primary smooth muscle cells were tested. P1 and F2 2x caused 79% and 87% inhibition for human primary endothelial cells. Moreover, we found that LPS stimulated eNOS phosphorylation in endothelial cell can inhibited by P1.

論文電子檔著作權授權書......................i
論文審定同意書..............................ii
誌謝........................................iii
中文摘要....................................iv
英文摘要....................................v
目錄........................................vi
圖目錄......................................vii
表目錄......................................viii
緒論 ...................................1
材料與方法..................................6
結果 ...................................18
討論 ...................................24
圖 ...................................28
表 ...................................43
參考文獻 ...................................49
附錄 ...................................53

1 Yanagisawa, M. et al., A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332, 411-415 (1988).
2 Elshourbagy, N.A. et al., Molecular characterization and regulation of the human endothelin receptors. J. Biol. Chem. 268, 3873-3879 (1993).
3 Schiffrin, E.L., Vascular endothelin in hypertension. Vascul. Pharmacol. 43, 19-29 (2005).
4 Shreenivas, S. & Oparil, S., The role of endothelin-1 in human hypertension. Clin. Hemorheol. Microcirc. 37, 157-178 (2007).
5 Gasic, S., Wagner, O.F., Vierhapper, H., Nowotny, P., & Waldhausl, W., Regional hemodynamic effects and clearance of endothelin-1 in humans: renal and peripheral tissues may contribute to the overall disposal of the peptide. J. Cardiovasc. Pharmacol. 19, 176-180 (1992).
6 Davenport, A.P. et al., Human endothelin receptors characterized using reverse transcriptase-polymerase chain reaction, in situ hybridization, and subtype-selective ligands BQ123 and BQ3020: evidence for expression of ETB receptors in human vascular smooth muscle. J. Cardiovasc. Pharmacol. 22 Suppl 8, S22-25 (1993).
7 Dhaun, N., Goddard, J., & Webb, D.J., The endothelin system and its antagonism in chronic kidney disease. J. Am. Soc. Nephrol. 17, 943-955 (2006).
8 Dhaun, N., Pollock, D.M., Goddard, J., & Webb, D.J., Selective and mixed endothelin receptor antagonism in cardiovascular disease. Trends Pharmacol. Sci. 28, 573-579 (2007).
9 Inoue, A. et al., The human endothelin family: three structurally and pharmacologically distinct isopeptides predicted by three separate genes. Proc. Natl. Acad. Sci. USA 86, 2863-2867 (1989).
10 Kimura, S. et al., Conversion of big endothelin-1 to 21-residue endothelin-1 is essential for expression of full vasoconstrictor activity: structure-activity relationships of big endothelin-1. J. Cardiovasc. Pharmacol. 13 Suppl 5, S5-7; discussion S18 (1989).
11 Arai, H., Hori, S., Aramori, I., Ohkubo, H., & Nakanishi, S., Cloning and expression of a cDNA encoding an endothelin receptor. Nature 348, 730-732 (1990).
12 Sakurai, T. et al., Cloning of a cDNA encoding a non-isopeptide-selective subtype of the endothelin receptor. Nature 348, 732-735 (1990).
13 Karne, S., Jayawickreme, C.K., & Lerner, M.R., Cloning and characterization of an endothelin-3 specific receptor (ETC receptor) from Xenopus laevis dermal melanophores. J. Biol. Chem. 268, 19126-19133 (1993).
14 Davenport, A.P., Kuc, R.E., Maguire, J.J., & Harland, S.P., ETA receptors predominate in the human vasculature and mediate constriction. J. Cardiovasc. Pharmacol. 26 Suppl 3, S265-267 (1995).
15 D'Orleans-Juste, P. et al., Function of the endothelin(B) receptor in cardiovascular physiology and pathophysiology. Pharmacol. Ther. 95, 221-238 (2002).
16 Pollock, D.M., Keith, T.L., & Highsmith, R.F., Endothelin receptors and calcium signaling. FASEB J. 9, 1196-1204 (1995).
17 Tsukahara, H., Ende, H., Magazine, H.I., Bahou, W.F., & Goligorsky, M.S., Molecular and functional characterization of the non-isopeptide-selective ETB receptor in endothelial cells. Receptor coupling to nitric oxide synthase. J. Biol. Chem. 269, 21778-21785 (1994).
18 de Nucci, G. et al., Pressor effects of circulating endothelin are limited by its removal in the pulmonary circulation and by the release of prostacyclin and endothelium-derived relaxing factor. Proc. Natl Acad. Sci. USA 85, 9797-9800 (1988).
19 Burke, S.E. et al., Evidence for vasoconstriction mediated by the endothelin-B receptor in domestic swine. J. Cardiovasc. Pharmacol. 35, 838-844 (2000).
20 Sokolovsky, M., Ambar, I., & Galron, R., A novel subtype of endothelin receptors. J. Biol. Chem. 267, 20551-20554 (1992).
21 Warner, T.D., Allcock, G.H., Corder, R., & Vane, J.R., Use of the endothelin antagonists BQ-123 and PD 142893 to reveal three endothelin receptors mediating smooth muscle contraction and the release of EDRF. Br. J. Pharmacol. 110, 777-782 (1993).
22 Schiffrin, E.L., Role of endothelin-1 in hypertension and vascular disease. Am. J. Hypertens. 14, 83S-89S (2001).
23 Battistini, B., D'Orleans-Juste, P., & Sirois, P., Endothelins: circulating plasma levels and presence in other biologic fluids. Lab. Invest. 68, 600-628 (1993).
24 Iglarz, M. & Schiffrin, E.L., Role of endothelin-1 in hypertension. Curr. Hypertens. Rep. 5, 144-148 (2003).
25 Rossi, G.P. et al., Endothelin-1 and its mRNA in the wall layers of human arteries ex vivo. Circulation 99, 1147-1155 (1999).
26 Hiroe, M. et al., Plasma endothelin-1 levels in idiopathic dilated cardiomyopathy. Am. J. Cardiol. 68, 1114-1115 (1991).
27 Ihling, C. et al., Coexpression of endothelin-converting enzyme-1 and endothelin-1 in different stages of human atherosclerosis. Circulation 104, 864-869 (2001).
28 Kowala, M.C. et al., Selective blockade of the endothelin subtype A receptor decreases early atherosclerosis in hamsters fed cholesterol. Am. J. Pathol. 146, 819-826 (1995).
29 Dupuis, J., Stewart, D.J., Cernacek, P., & Gosselin, G., Human pulmonary circulation is an important site for both clearance and production of endothelin-1. Circulation 94, 1578-1584 (1996).
30 Iskit, A.B. & Guc, O., Effects of endothelin and nitric oxide on organ injury, mesenteric ischemia, and survival in experimental models of septic shock. Acta Pharmacol. Sin. 24, 953-957 (2003).
31 Nelson, J., Bagnato, A., Battistini, B., & Nisen, P., The endothelin axis: emerging role in cancer. Nat. Rev. Cancer 3, 110-116 (2003).
32 Lahav, R., Heffner, G., & Patterson, P.H., An endothelin receptor B antagonist inhibits growth and induces cell death in human melanoma cells in vitro and in vivo. Proc. Natl. Acad. Sci. USA 96, 11496-11500 (1999).
33 Bazil, M.K., Lappe, R.W., & Webb, R.L., Pharmacologic characterization of an endothelinA (ETA) receptor antagonist in conscious rats. J. Cardiovasc. Pharmacol. 20, 940-948 (1992).
34 Verma, S., Bhanot, S., & McNeill, J.H., Effect of chronic endothelin blockade in hyperinsulinemic hypertensive rats. Am. J. Physiol. 269, H2017-2021 (1995).
35 Krum, H., Viskoper, R.J., Lacourciere, Y., Budde, M., & Charlon, V., The effect of an endothelin-receptor antagonist, bosentan, on blood pressure in patients with essential hypertension. Bosentan Hypertension Investigators. N. Engl. J. Med. 338, 784-790 (1998).
36 Attina, T., Camidge, R., Newby, D.E., & Webb, D.J., Endothelin antagonism in pulmonary hypertension, heart failure, and beyond. Heart 91, 825-831 (2005).
37 Haynes, W.G. et al., Systemic endothelin receptor blockade decreases peripheral vascular resistance and blood pressure in humans. Circulation 93, 1860-1870 (1996).
38 Weber, C. et al., Pharmacokinetics and pharmacodynamics of the endothelin-receptor antagonist bosentan in healthy human subjects. Clin. Pharmacol. Ther. 60, 124-137 (1996).
39 Goddard, J. et al., Endothelin-A receptor antagonism reduces blood pressure and increases renal blood flow in hypertensive patients with chronic renal failure: a comparison of selective and combined endothelin receptor blockade. Circulation 109, 1186-1193 (2004).
40 Nakov, R., Pfarr, E., & Eberle, S., Darusentan: an effective endothelinA receptor antagonist for treatment of hypertension. Am. J. Hypertens. 15, 583-589 (2002).
41 Nayler, W.G., Liu, J.J., Panagiotopoulos, S., & Casley, D.J., Streptozotocin-induced diabetes reduces the density of [125I]-endothelin-binding sites in rat cardiac membranes. Br. J. Pharmacol. 97, 993-995 (1989).
42 Juan, C.C. et al., Exogenous hyperinsulinemia causes insulin resistance, hyperendothelinemia, and subsequent hypertension in rats. Metabolism 48, 465-471 (1999).
43 Yang, D.M. et al., Tracking of secretory vesicles of PC12 cells by total internal reflection fluorescence microscopy. J. Microsc. 209, 223-227 (2003).
44 Juan, C.C., Au, L.C., Yang, F.Y., Yang, D.M., & Ho, L.T., An endothelin type A receptor-expressing cell to characterize endothelin-1 binding and screen antagonist. Anal. Biochem. 379, 27-31 (2008).
45 Duan, H. et al., Effect of anemonin on NO, ET-1 and ICAM-1 production in rat intestinal microvascular endothelial cells. J Ethnopharmacol 104, 362-366 (2006).
46 Rittirsch, D., Hoesel, L.M., & Ward, P.A., The disconnect between animal models of sepsis and human sepsis. J. Leukoc. Biol. 81, 137-143 (2007).
47 Buras, J.A., Holzmann, B., & Sitkovsky, M., Animal models of sepsis: setting the stage. Nat Rev Drug Discov 4, 854-865 (2005).
48 Iskit, A.B., Senel, I., Sokmensuer, C., & Guc, M.O., Endothelin receptor antagonist bosentan improves survival in a murine caecal ligation and puncture model of septic shock. Eur. J. Pharmacol. 506, 83-88 (2004).
49 Aramori, I. & Nakanishi, S., Coupling of two endothelin receptor subtypes to differing signal transduction in transfected Chinese hamster ovary cells. J. Biol. Chem. 267, 12468-12474 (1992).