摘要
囊性纤维化跨膜电导调节因子(cystic fibrosis transmembrane conductance regulator, CFTR)是一种CAMP/PKA依赖的上皮氯离子通道,同时又是某些膜转运蛋白的调节因子。属于ATP结合盒(ATP binding cassette, ABC)转运蛋白超家族的成员。CFTR在哺乳动物中所有与分泌和吸收有关的上皮组织(例如小肠、气道、胰腺、汗腺、输精管细胞、心肌细胞、血管平滑肌细胞)中广泛表达。CFTR的主要功能是介导跨上皮细胞Cl-转运、控制氨敏感的上皮细胞Na+通道(epithelial Na+ channel, ENaC)、促进HCO3-分泌及调节K+通道等,因此CFTR与多种生理和病理状态密切相关。CFTR功能障碍会引起致死性遗传疾病囊性纤维化病(cystic fibrosis, CF)、特发性慢性胰腺炎(idiopathic chronic pancreatiti, ICP)、干眼病(keratoconjunctivitis sicca, KCS)、习惯性便秘(habitual constipation)等;而霍乱毒素和耐热性致病性大肠杆菌内毒素导致的分泌型腹泻(secretary diarrhea)、多囊肾病(polycystic kidney)等则是CFTR活动过强引起的。因此寻找能够调节野生型和突变型CFTR功能的小分子调节剂的工作近年来受到了广泛重视并取得了较大进展,目前仍是CFTR有关研究的重要目标之一。
我们利用含?F508-CFTR(或wt-CFTR)的表达质粒与一种对卤族元素碘离子高度敏感的荧光绿蛋白突变体EYFP-H148Q的表达质粒共转染Fischer大鼠甲状腺上皮细胞,得到稳定表达该蛋白的两种细胞系(FRT/?F508-CFTR /EYFP-H148Q和FRT/wt-CFTR /EYFP-H148Q-I152L)。以此为筛选模型对49种生物碱类化合物和20种挥发油类化合物进行筛选,目的是为了筛选出wt-CFTR氯离子通道激活剂和?F508-CFTR氯离子通道激活剂及蛋白质质膜转运障碍纠正剂,并对其分子药理学特征进行系统研究。我们得到了8种生物碱类化合物和6种挥发油类化合物对wt-CFTR均具有明显的激活作用。由于前期本实验室已对生物碱中有明显活性的菏叶碱和白鲜碱有系统的研究,因此我们对其余六种生物碱类化合物进行进一步的研究分析,同时我们也分析了挥发油类化合物中活性最强的对羟基苯甲酸丁酯。结果表明,六种生物碱类化合物和对羟基苯甲酸丁酯对野生型CFTR Cl-通道具有明显的激活作用,其中盐酸罂粟碱、尼莫地平、对羟基苯甲酸丁酯对?F508突变型通道开放障碍也有明显的激活作用,同时胡椒碱也表现出了一定的活性。此外,六种生物碱类化合物和对羟基苯甲酸丁酯对G551D突变型CFTR Cl-通道都无激活作用,也不能纠正?F508-CFTR蛋白胞内转运的障碍。上述七种单体化合物对野生型和突变型CFTR Cl-通道的激活作用具有作用迅速、可逆、剂量依赖的特点,初步机制分析结果表明它们可能是通过与CFTR直接结合而发挥作用的。
本研究中七种单体化合物对CFTR Cl-通道具有激活作用不仅为阐明CFTR的结构与功能之间的关系,以及作为先导化合物开发探索治疗与CFTR突变有关疾病等方面具有重要用途,而且为进一步明确这七种化合物其它方面的药理机制奠定了基础。
The CAMP-activated cystic fibrosis transmembrane conductance regulator (CFTR) belongs to a family of proteins called traffic ATPases or ABC transporters. Its mutation will cause the most prevalence lethal hereditary disease-cystic fibrosis (CF). It is most prominently expressed in the apical membrane of various epithelia in mammal involved secretion and absorption (such as intestine, airways, pancreas, sweat ducts), which works as Cl- channel and also a regulator of other anion channels. Therefore, CFTR is closely related to many physiological and pathological conditions. Impaired function of CFTR Cl- channels in CF disrupts transepithelial ion transport, thus leads to the wide-ranging manifestations of the disease such as idiopathic chronic pancreatitis (ICP), keratoconjunctivitis sicca (KCS), habitual constipation. Evidence also showed that over active of CFTR is associated with such diseases as secretory diarrhea and polycystic kidney disease. Identification of small-molecule modulators to regulate the function of wild-type and mutant CFTR have been attracted many attention in recent years and have obtained substantial progress, which is still one of most important fields of CFTR study.
A stably transfected Fischer thyroid epithelial (FRT) cell lines co-expressing ?F508-CFTR or wt-CFTR and a green fluorescent protein mutant with ultra-high halide sensitivity (EYFP-H148Q) was generated as assay for the drug screening. Using this cell model, 8 alkaloid compounds and 6 volatile compounds were screened for CFTR-mediated halide transport. Previously, we have a systematic study to Holland and Dictamnine which have significantly activition, so we make further research and analysis on the remaining six compounds of alkaloid compounds, at the same time we also analyzed
Butyl-phydroxybenzoate (Bpb) which has strongest activity in the volatile oil compounds. As a result, the six alkaloid compounds and Bpb can attivate wild-type CFTR chloride channel gating. Papaverine hydrochloride, Nimodipine, Bpb and Piperine can stimulated ?F508-CFTR mediate anion transport, but had no effect on ?F508-CFTR misprocessing defect or G551D-CFTR channel gating defect. Pharmacological properties of the seven compounds were characterized systematically in terms of the dose-dependent, reversibility, activition time-course and so on. The study provided clues that they activate CFTR chloride channel through a direct binding mechanism.
This studies indicated that we regard the seven compounds not only as important tools in better understanding the structure and function of CFTR but aslo as a lead compound to develop pharmacological therapy of CFTR-related disease. On the other hand, it lays a foundation for them at further exploring other aspects pharmacology mechanism.
引文
[1] Gadsby DC, Vergani P, Csanady L. The ABC protein turned chloride channel whose failure causes cystic fibrosis [J]. Nature, 2006, 440(7083):477~83.
[2] Stigers KD, Soth MJ., Nowick JS. Designed molecule that fold tomimic protein secondary structures[J]. Current opinion in chemical Biology, 1999, 6:714~723.
[3] Livnah O, Stura EA., Johnson DL., et al. Functional mimicry of a protein hormone by a peptide agonist: the EPO receptor complex at 2.8?[J]. Science,1996;273:464~471.
[4] Bunnett NW, Bouvier M., De Blasi A. Peptide G-protein-coupled receptors meet at Erice[J]. Trends in Pharmacological. Sciences,1998,19:343~346.
[5] Hondeghem LM, Katzung BG. Time-and voltage-dependent interactions of antiarrhythmic drugs with cardiac sodium channels[J]. Biochem Biophys Acta ,1977,472(3-4) : 373~98.
[6] Kopito,R.R. Biosynthesis and degradation of CFTR. Physiol. Rev. 1999.79 (Suppl.): S167~S173.
[7] Berger,H.A.,M.P.anderson. Identification and regulation of the cystic fibrosis transmembrane conductance regulator-generated chloride channel[J]. J.Clin .Invest, 1991,88:1422~1431.
[8] Robert R, Savineau JP, Norez C, et al. Expression and function of cystic fibrosis transmembrane conductance regulator in rat intrapulmonary arteries[J]. Eur Respir J, 2007,30(5):857~64.
[9] Weiner SA, Caputo C, Bruscia E, et al. Rectal potential difference and the functional expression of CFTR in the gastrointestinal epithelia in cystic fibrosis mouse models [J]. Pediatr Res, 2008, 63(1):73~8.
[10] Pan P, Guo Y, Gu J. Expression of cystic fibrosis transmembrane conductance regulator in ganglion cells of the hearts [J]. Neurosci Lett, 2008, 441(1):35~8.
[11] Robert R, Thoreau V, Norez C, et al. Regulation of the cystic fibrosis transmembrance conductance regulator channel by beta-adrenergic agonists and vasoactive intestinal petide in rat smooth muscle cells and its role in vasorelaxation[J]. J Biol Chem, 2004, 279(20):21160~21168.
[12] Robert R, Norez C, Becq F. Disruption of CFTR chloride channel alters mechanical properties and CAMP-dependent Cl- transport of mouse aortic smooth muscle cells[J]. J Physiol, 2005, 568(Pt 2):483~495.
[13] Gao Z, Sun HY, Lau CP, et al. Evidence for cystic transmembrance conductance regulator chloride current in swine ventricular myocytes[J]. J Mol Cell Cardiol, 2007, 42(1):98~105.
[14] Xu WM, Shi QX, Chen WY, et al. Cystic fibrosis transmembrance conductance regulator is vital to sperm fertilizing capacity and male fertility[J]. Proc Natl Acad Sci, 2007, 104(23):9816~9821.
[15] Hernandez-Gonzalez E O, Trevino C L, Castellano L E, et al. Involvement of CFTR in Mouse Sperm Capacitation[J]. J Biol Chem, 2007, 282(33):24397~24406.
[16] DorwartM, Thibodeau P, Thomas P. Cystic fibrosis recent structural insights[J]. J Cyst Fibros, 2004, (3 Supp 12):91~94.
[17] Scott Olenych Investigation of CFTR intermolecular structure and protein-protein interavtion[J]. Diss Abstr int, 2002, 63-08, Section B:3593.
[18] Chappe V, Irvine T, Liao J, et al. Phosphorylation of CFTR by PKA promotes binding of the regulatory domain[J].EMBO J, 2005, 24(15):2730~2740.
[19] Sheppard, D. N., and Welsh, M. J. Physiol. Rev, 1999, 79, S23~S45.
[20] Horisberger J D. ENaC-CFTR interactions: the role of electrical coupling of ion fluxes explored in an epithelial cell model. Pflugers Arch, 2003,445(4):522~528.
[21] Chambers LA, Rollins BM, Tarran R. Liquid movement across the surface epithelium of large airways [J]. Respir Physiol Neurobiol., 2007,159(3):256~70.
[22] Li J, Allen KT, Sun XC, et al. Dependence of cAMP meditated increases in Cl- and HCO3- permeability on CFTR in bovine corneal endothelial cells. Exp Eye Res, 2008,86(4):684~90.
[23] Kim JK, Yoo HY, Kim SJ, et al. Effects of sevoflurane on the CAMP-induced short-circuit current in mouse tracheal epithelium and recombinant Cl- (CFTR) and K+ (KCNQ1) channels [J]. Br J Anaesth, 2007,99(2):245~51.
[24] Bachhuber T, Konig J, Voelcker T, et al. Cl- interference with the epithelial Na+ Channel ENaC[J]. J Biol Chem, 2005, 280(36):31587~31594.
[25] Burghardt B, ElkaermL, Kwon TH, et al. Distribution of aquaporin water channels AQP1 and AQP5 in the ductal system of the human pancreas[J]. Gut, 2003, 52 (7): 1008~16.
[26] Guggino WB, Banks2Schlegel SP. Macromolecular interactions and ion transport in cystic fibrosis[J]. Am J RespirCrit CareM ed, 2004,170 (7):815~20.
[27] Welsh M.J., and Smith A.E. Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis[J]. Cell, 1993, 73:1251~1254.
[28] Nousia-A rvan itak is S Cystic fibrosis and the pancreas: recent scientific advances[J]. J Clim Gastroenterol, 1999, 29(2):138~142
[29] Hillesheim J, Tuo B, Jorna H, Houtsmuller AB, Shenolikar S, Weinman EJ, Donowitz M, Seidler U, de Jonge HR, Hogema BM.Cystic fibrosis transmembrane conductance regulator activation is reduced in the small intestine of Na+/H+ exchanger 3 regulatory factor 1 (NHERF-1)- but Not NHERF-2-deficient mice.J Biol Chem. 20078;282(52):37575~84.
[30] Sellers ZM, Mann E, Smith A, Hyun Ko K, Giannella R, Cohen MB, Barrett KE, Dong H.Heat-stable enterotoxin of Escherichia coli (STa) can stimulate duodenal HCO3- secretion via a novel GC-C- and CFTR-independent pathway.FASEB J. 2008 Jan 4.
[31] Canale-Zambrano JC, Poffenberger MC, Cory SM, et al. Intestinal phenotype of variable-weight cystic fibrosis knockout mice.Am J Physiol Gastrointest Liver Physiol.2007 Jul, 293(1):G222~9.
[32] Eggermont E, De Boeck K.Related Articles, Small-intestinal abnormalities in cystic fibrosis patients.Eur J Pediatr.1991 Oct;150(12):824~8.
[33] Thiagarajah JR, Verkman AS.CFTR pharmacology and its role in intestinal fluid secretion.Curr Opin Pharmacol. 2003c;3(6):594~9.
[34] Sonawane ND, Hu J, Muanprasat C, Verkman AS.Luminally active, nonabsorbable CFTR inhibitors as potential therapy to reduce intestinal fluid loss in cholera.FASEB J. 2006 Jan;20(1):130~2.
[35] Goodman BE, Percy WH.CFTR in cystic fibrosis and cholera: from membrane transport to clinical practice.Adv Physiol Educ. 2005 Jun, 29(2):75~82.
[36] Thiagarajah JR, Verkman AS.New drug targets for cholera therapy.Trends Pharmacol Sci. 2005 Apr;26(4):172-5.
[37] Park R W, Grand RJ. Related Artieles, Gastrointestinal manifestations of eystic fibrosis:a review. Gastroenterology.1981 Dec;81(6):1143~61.
[38] Knowles, M.R., M. J. Stutts, A.Spock, et al. Abnormal ion permeation through cystic fibrosis respiratory epithelium.Seience,1983, 221:1067~1070.
[39] Quinton PM. Chloride impermeability in cystic fibrosis.Nature. 1983 Feb 3;301(5899):421~2.
[40] Mall MA.Role of Cilia, Mucus, and Airway Surface Liquid in Mucociliary Dysfunction: Lessons from Mouse Models.J Aerosol Med. 2008 Jan 16.
[41] Lamireau T, Monnereau S, Martin S,et al. Epidemiology of liver disease in cystic fibrosis: a longitudinal study[J]. J Hepatol, 2004, 41(6):920~5.
[42] Efrati O, Barak A, Modan-Moses D, et al. Liver cirrhosis and portal hypertension in cystic fibrosis.Eur J Gastroenterol Hepatol. 2003, 15(10):1073~8.
[43] Chillon M, Casals T, Mercier B, Bassas L, Lissens W, Silber S, Romey MC, Ruiz Romero J, Verlingue C, Claustres M, Nunes V, Ferec C, Estivill X. Mutations in the cystic fibrosis gene in patients with congenital absence of the vas deferens. N Engl J Med 1995, 332: 1475~1480.
[44] Knowles M, Gatzy J, Boucher R. Increased bioelectric potential difference across respiratory epithelia in cystic fibrosis[J]. N Engl J Med. 1981 Dec 17;305(25):1489~95.
[45] Knowles M, Gatzy J, Boucher R. Relative ion permeability of normal and cystic fibrosis nasal epithelium. J Clin Invest. 1983 May;71(5):1410~7.
[46] Widdicombe, J. H. Cystic fibrosis and beta-adrenergic response of airway epithelial cell cultures. Am. J. Physiol. 1986, 251 (Regulatory Intigrative Comp. Physiol. 20): R818~R822.
[47] Khan T Z, Wagener J S, Bost T, et al. Early pulmonary inflammation in infants with cystic fibrosis[J]. Am J Respir Crit Care Med,1995,151:1075~1082.
[48] Yang Y, Janich S, Cohn J A, et al. The common variant of cystic fibrosis transmembrane conductance regulator is recognized by hsp70 and degraded in a pre-Golgi nonlysosomalcompartment[J]. Proc. Natl. Acad. Sci,1993, 90:9480~9484.
[49] Loo M A, Jensen T J, Cui L, et al. Perturbation of Hsp90 interaction with nascent CFTR prevents its maturation and accelerates its degradation by the proteasome[J]. EMBO J,1998,17(23):6879~6887.
[50] Farinha CM, Nogueira P, Mendes F, et al. The human DnaJ homologue (Hdj)-1/heat-shock protein (Hsp) 40 co-chaperone is required for the in vivo stabilization of the cystic fibrosis transmembrane conductance regulator by Hsp70[J]. Biochem J,2002, 366(3):797~806.
[51] Torres VE, Rossetti S, Harris PC.Update on autosomal dominant polycystic kidney disease[J]. Minerva Med. 2007, 98(6):669~91. [52] Chang MY, Ong AC. Autosomal dominant polycystic kidney disease: recent advances in pathogenesis and treatment.Nephron Physiol[J]. 2008, 108(1):p1~7.
[53] Rapoport J.Autosomal dominant polycystic kidney disease: pathophysiology and treatment[J]. QJM. 2007 Jan, 100(1):1-9.
[54] McLigeyo SO.Autosomal dominant polycystic kidney disease - a systemic disorder.Afr J Health Sci. 1998 Jul-Dec;5(3-4):114~7.
[55]梅长林,戴兵等。多囊肾病慢性进展的分子发病机制及治疗对策[J]。中国实用内科杂志,2007,27(1):53~55.
[56] Li H, Findlay IA, Sheppard DN. The relationship between cell proliferation, Cl- secretion, and renal cyst growth: a study using CFTR inhibitors[J]. Kidney Int, 2004, 66(5):1926~38.
[57] Wilson PD.Cystic fibrosis transmembrane conductance regulator in the kidney: clues to its role?Exp Nephrol. 1999, 7(4):284~9.
[58] Sullivan LP, Wallace DP, Grantham JJ. Chloride and fluid secretion in polycystic kidney disease[J]. J Am Soc Nephrol. 1998 May;9(5):903~16.
[59] KOICHI NAKANISHI, WILLIAM E. SWEENEY, JR. Role of CFTR in Autosomal Recessive Polycystic Kidney Disease[J]. J Am Soc Nephrol, 2001,12:719~725.
[60] Heaton ND, Pryor JP. Vasa aplasia and cystic fibrosis.Br J Urol. 1990 Nov;66(5):538~40.
[61] Anguiano A, Oates RD, Amos JA, et al. Congential bilateral absence of the vas deferens. A primarily genital form of cystic fibrosis[J]. JAMA 1992, 267: 1794~1797.
[62] Chillon M, Casals T, Mercier B, et al. Mutations in the cystic fibrosis gene in patients with congenital absence of the vas deferens[J]. N Engl J Med 1995, 332: 1475~1480.
[63] Chan HC, Shi QX, Zhou CX, et al. Critical role of CFTR in uterine bicarbonate secretion and the fertilizing capacity of sperm [J] .MolCell Endocrinol, 2006, 250(1-2): 106~113.
[64] Hodges CA, Palmert MR, Drumm ML. Infertility in Females with Cystic Fibrosis is Multifactorial: Evidence from Mouse Models.Endocrinology. 2008 Mar 6; [Epub ahead of print]
[65] Rowlands DK, Tsang LL, Cui YG, et al. Upregulation of cystic fibrosis transmembraneconductance regulator expression by oestrogen and Bak Foong Pill in mouse uteri.Cell Biol Int. 2001;25(10):1033~5.
[66] Hernández-González EO, Trevi?o CL, Castellano LE, et al. Involvement of cystic fibrosis transmembrane conductance regulator in mouse sperm capacitation[J]. J Biol Chem. 2007, 282(33):24397~406.
[67] Salleh N, Baines DL, Naftalin RJ, Milligan SR.The hormonal control of uterine luminal fluid secretion and absorption.J Membr Biol[J]. 2005, 206(1):17~28.
[68] Jarzabek K, Zbucka M, Pepiński W, Szamatowicz J, Domitrz J, Janica J, Wo?czyński S, Szamatowicz M.Cystic fibrosis as a cause of infertility. Reprod Biol[J]. 2004, 4(2):119~29.
[69] Kopelman H, Durie P, Gaskin K, et al. Pancreatic fluid secretion and protein hyperconcentration in cystic fibrosis[J].N Engl J Med,1985,312(6):329~334.
[70]姚新生。天然药物化学[M]。第2版.北京人民卫生出版社,1997, 483。
[71]川芎嗪有效成分的研究—川芎嗪的药理研究[J]。中华医学杂志,1997, 57:464。
[72]唐小卿,曹建国。甲基莲心碱的药理作用[J]。中国药理学通报,2004,20(1):8-10。
[73]周际安,丁耀贤,曾繁典等。蝙蝠葛碱对心血管患者血小板聚集性的影响[J]。中国药理学通报,1994,10(1):33-36。
[74]李晓天,王广基。关附甲素代谢产物关附胺醇在大鼠体内的药代动力学研究[J]。中国药科大学学报,2005,36(2):142。
[75]杨宇杰,王春民。野婴粟生物碱化学成分和药理作用研究进展[J]。中草药,2005, 36(10):1595~1597。
[76]周贤春,何春霞,苏力坦·阿巴白克力。生物碱的研究进展[J]。生物技术通讯,2006, 17(3):476~479。
[77]李丹,王平权。苦参碱类的研究进展及临床应用[J]。中草药,1996, 27(5):696。
[78]吕梅香,曾和平,王晓娟等。农药用生物碱的研究进展[J]。农药,2004, 143(16):249-253。
[79]马兴铭,李红玉,尹少甫等.藏咬砂生槐子生物碱抗炎艺菌活性的研究[J]。中医药学报,2004, 32(5):23~25。
[80]余建强,蒋袁絮。槐定碱,氧化槐定碱的药理学研究进展[J]。宁夏医学院学报,2005, 27(1):78~80。
[81]茅益民,曾民德,陆伦根等。氧化苦参碱胶囊治疗慢性病毒性肝炎的随机双盲安慰剂对照多中心临床研究[J],2002, 7(4):222~224。
[82] Agarwa US, Rastogi RP. Chemical examination of water 2 soluble fraction of Mappiafoetia Miers[J]. Indian J Chem, 1975, 13(7):758.
[83]秦竹丽,江元汝。超临界萃取技术在生物碱提取中的应用进展[J]。化工时刊,2006, 20(7):60~63。
[84]天津轻工业学院食品工业教学研究室.食品添加剂(修订本)[M]。北京轻工业出版社,1985,20~27。
[85]陈业高。植物化学成分[M]。北京化学工业出版社,2005, 158~162。
[86]滑艳,邓雁如,汪汉卿。各种挥发油的药理活性及在医学方面的应用[J]。天然产物研究与开发,2003, 15(5):467~470。
[87] Ma T, Thiagarajah JR, Yang H, et al. Thiazolidinone CFTR inhibitor identified by high-throughput screening blocks cholera toxin-induced intestinal fluid secretion[J]. J Clin Invest, 2002,110(11):1651~1658.
[88] Caci E, Caputo A, Hinzpeter A, et al. Evidence for direct CFTR inhibition by CFTR(inh)-172 based on Arg347 mutagenesis [J]. Biochem J, 2008, 413(1):135~42.
[89] Galietta LV, Jayaraman S, Verkman AS. Cell-based assay for high-throughput quantitative screening of CFTR chloride transport agonists[J]. Am J Physiol Cell Physiol, 2001, 281(5):C1734~1742.
[90] Ma T, Vetrivel L, Yang H, et al. High-affinity activators of cystic fibrosis transmembrane conductance regulator (CFTR) chloride conductance identified by high-throughput screening [J]. J Biol Chem, 2002, 277(40):37235~37241.
[91] Akiyama T, Ishida J, Nakagawa S, et al. Genistein, a specific inhibitor of tyrosine-specific protein kinases [J]. J Biol Chem, 1987,262(12):5592~5595.
[92]林森,侯曙光,金伶伶等。荷叶碱对野生型和突变CFTR氯离子通道的激活作用。中国临床药理学与治疗学,2008,13(2):138 ~143。
[93] Liu J, Liu L, Wang S, Xu L, Yu B, Lin, S, Hou S, Zhou N, Jin L, Yang H*. Dictamine stimulates cystic fibrosis transmembrane conductance regulator Cl- transport. Chem. Res. Chinese U . 2007, 23(5): 554~557.
[94] Zsuzsa Bebok, James F Collawn, John Wakefield, at all. Failure of CAMP agonists to activate rescued ?F508 CFTR in CFBE41o- airway epithelial monolayers [J]. J Physiol, 2005,569(Pt2):601~615.
[95] Yoo CL, Yu GJ, Yang B, et al. 4'-Methyl-4,5'-bithiazole-based correctors of defective delta F508-CFTR cellular processing [J]. Bioorg Med Chem Lett, 2008, 18(8):2610~4.
[96] Bompadre SG, Li M, Hwang TC. Mechanism of G551D-CFTR (cystic fibrosis transmembrane conductance regulator) potentiation by a high affinity ATP analog [J]. J Biol Chem, 2008, 283(9):5364~9.
[97] Al-Nakkash L, Springsteel MF, Kurth MJ, et al. Activation of CFTR by UCCF-029 and genistein [J]. Bioorg Med Chem Lett, 2008,18(14):3874~7.
[98] Schmidt A, Hughes LK, Cai Z, et al. Prolonged treatment of cells with genistein modulates the expression and function of the cystic fibrosis transmembrane conductance regulator [J].Br J Pharmacol, 2008,153(6):1311~23.
[99]佟继铭,李兰芳,宋成军。野婴粟碱对甲醛致痛反应的镇痛作用及其机制。中草药,2004, 35(9):1029~1030。
[100] JOHN E C, YUJI Numaguchi, GREGG H, et al. Intra arterial papaverine infusion for cerebral vasopasm after subarachnoid hemorrhage[J]. Am J Neuroradial, 1995, 6:27.
[101] KAKU Y, YONE K Y, TSUKAHARA T, et al. Superselective intraarterial infusion of papaverine for the treatment of cerebral vasospasm after subarachiid hemorrhage[J]. J Neurosury, 1992, 77:842.
[102] ASANO J, TANISHIMO T, SASAKIJ, et al. European study group on nimodipine in severe head injury: A multicenter trial of the efficacy of nimodipine on outcome after severe head injury[J]. J Neurosury, 1994, 80:797.
[103] Lim S , Tomita K, Carramori G, et al . Low2dose t heophylline reduces eosinophilic inflammation but not exhaled nit ric oxide in mildast hma[J ] . AmJ Respir Crit Care Med, 2001, 164: 273~276.
[104] Culpit t SV, de Matos C, Russell RE, et al. Effect of t heophylline on induced sputum inflammatory indices and neut rophil chemotaxis in COPD[J] . Am J Respir Crit Care Med, 2002, 165: 1371~1376.
[105] Pauwels RA, Buist AS, Calverley PM, Jenkins CR, Hurd SS. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: NHLBI/WHO Global Initiative for Chronic Obstructive Lung Disease (GOLD) Workshop summary. Am.J Respir Crit Care Med 2001;163:1256~1276.
[106] Rennard SI.Treatment of stable chronic obstructive pulmonary disease. Lancet 2004;364:791~802.
[107] Howell RE, Muehsam WT, Kinnier WJ. Mechanism for the emetic side effect of xanthine bronchodilators. Life Sci 1990, 46:563~568.
[108] Nishmura, J.&Van Breemen,C.(1989). Diect regulation of smooth muscle contractile elements by second messengers. Biochem. Biophys. Res. Commun., 163, 929~935.
[109]聂乾忠,夏延斌,曾晓楠等。辣椒素的代谢途径及生理功能的研究进展.食品与机械。2008, 24(2):141~145。
[110] KENTARO K, MIKIN. Mechanism of potent antieroxidative effect of capsaicin[J]. Biochimicaet Biophysicaacta, 2002, 1573:84~92.
[111] Wu CC, Lin J P, Yang J S, et al. Capsaicin induced cell cycle arrest and apopotosis in human esophagus epidermoid carcinoma CE81T/VGH cells through the elevation of intracellular reactive oxygen species and Ca2+ producrions and caspase-3 activation[J]. Mutant Res, 2006, 60 1(1-2):71.
[112] Vijayan KK, Thampuran A. Phamacology, toxicology and clinical applications of black pepper[J]. Harwood Academic, 2000, 55~66.
[113] D’Hooge R, Pei Y, Raes A, et al. Anticonvulsant activity of piperine on seizures induced by excitatory amino acid receptor agonist[J]. Arzneimittel Forschung Drug Reseach, 1996(46):557~560.
[114] Renaud Robert, Caroline Norez and Frederic Becq.Disruption of CFTR chloridechannel alters mechanical properties and Camp-dependent Cl- transport of mouse aortic smooth muscle cells[J]. J hysiol, 2005, 568: 483~95.
[115] Hwang TC, Sheppard DN. Molecular pharmacology of the CFTR Cl- channel[J]. Trends Pharmacol Sci, 1999, 20(11):448~453.
[116] Gadsby DC, Nairn AC. Regulation of CFTR Cl- ion channels by phosphorylation and dephorylation[J]. Adv Second Messenger Phosphoprotein Res, 1999, 33:79~106.
[117] Becq F, Jensen TJ, Chang XB, et al. Phosphatase inhibitors activate normal and defective CFTR chloride channels[J]. Proc Natl Acad Sci USA, 1994, 91(19):9160~9164.
