延腦鼻端腹外側核（rostral ventrolateral medulla）為交感神經血管運動的起源，當有機磷毒藥美文松（mevinphos）作用在大白鼠延腦鼻端腹外側核時，會誘發嚴重的心血管反應。
本論文研究欲探討大白鼠延腦鼻端腹外側核中酪氨酸激酶受體 tropomyosin-related kinase（Trk）與酪氨酸激酶 Src，於大白鼠延腦鼻端腹外側核美文松模式（mevinphos intoxication）所誘導之交感神經興奮（sympathoexcitatory）與交感神經抑制（sympathoinhibitory）這兩個不同心血管反應分期所扮演的角色。微量注射美文松（10 nmol）至大白鼠延腦鼻端腹外側核，產生兩個不同分期：美文松中毒第一期（交感神經興奮心血管反應作用）與第二期（交感神經抑制心血管反應作用）；以西方墨點浸漬法探討大白鼠延腦鼻端腹外側核蛋白質表現量發現，於美文松中毒第一期，酪氨酸激酶受體 Trk 活化態（p-Trk490）表現量提升，而酪氨酸激酶Src 活化態（p-Src416）表現量大增僅於美文松中毒第二期；有趣的是，於大白鼠延腦鼻端腹外側核前處理酪氨酸激酶受體 Trk 專一性抑制劑（K252a; 1 pmol）除了抑制美文松中毒第一期中酪氨酸激酶受體 Trk 活化態（p-Trk490）與未磷酸化的 Trk A 及 Trk B 蛋白質表現量，更能抑制一氧化氮合成酶第一型（nitric oxide synthase I）的蛋白質表現，進而減弱交感神經興奮心血管反應過程；而前處理酪氨酸激酶 Src 專一性抑制劑（SU6656; 100 pmol）除了抑制酪氨酸激酶 Src 活化態（p-Src416）蛋白質表現，也抑制一氧化氮合成酶第二型（nitric oxide synthase II）的蛋白質表現，因此抑制了美文松中毒第二期的交感神經抑制心血管反應。
本論文研究中，我們認為 Trk 或 Src 這兩個不同的酪氨酸激酶，於大白鼠延腦鼻端腹外側核美文松中毒模式中，分別參與心血管反應之第一期與第二期。酪氨酸激酶受體 Trk 參與大白鼠延腦鼻端腹外側核美文松中毒模式第一期，透過 Trk A 或 Trk B 活化 Y490 的位置後，影響一氧化氮合成酶第一型蛋白質產生，作用在交感神經興奮心血管反應過程中。酪氨酸激酶 Src 參與大白鼠延腦鼻端腹外側核美文松中毒模式第二期，透過活化態 p-Src416 蛋白質表現大量提升，影響一氧化氮合成酶第二型蛋白質生成，作用在交感神經抑制心血管反應過程中。

Mevinphos (Mev) is an organophosphate insecticide that acts on the rostral ventrolateral medulla (RVLM), the origin of sympathetic vasomotor tone, to induce cardiovascular responses. This study investigated the role of Trk (tropomyosin-related kinase) (receptor form) or Src (non-receptor form) tyrosine kinase at the RVLM in Mev-induced cardiovascular responses. Bilateral microinjection of Mev (10 nmol) into the RVLM elicited two distinct phases of cardiovascular responses, designated Phase I (sympathoexcitatory) and Phase II (sympathoinhibitory) Mev intoxication. Western blot assay showed that whereas p-Trk490 was increased during Phase I, p-Src416 was increased only during Phase II Mev intoxication. Interestingly, application of a Trk specific inhibitor (K252a; 1 pmol) or Src specific inhibitor (SU6656; 100 pmol) into the bilateral RVLM blunted the Mev-elicited sympathoexcitatory or sympathoinhibitory effect, respectively. Besides, K252a was limited to block NOS I protein expression in the RVLM during Mev intoxication, SU6656 only inhibited NOS II protein expression in the RVLM during Mev intoxication.
We conclude that Trk tyrosine kinase (p-Trk490) in the RVLM participates in the Phase I cardiovascular responses during Mev intoxication, Src tyrosine kinase (p-Src416) in the RVLM participates in the Phase II cardiovascular responses associated with Mev intoxication.

目錄


頁
中文摘要 3-5
Abstract 6-8
前言 9-25
實驗動機與目的 26-28
材料與方法 29-35
結果 36-46
討論 47-59
結論 60-61
未來展望 62-63
參考文獻 64-74
附圖 75-132

Abram CL and Courtneidge SA (2000) Src family tyrosine kinases and growth factor signaling. Exp Cell Res 254: 1-13.

Arevalo JC, Pereira DB, Yano H, Teng KK and Chao MV (2006) Identification of a switch in neurotrophin signaling by selective tyrosine phosphorylation. J Biol Chem 281: 1001-1007.

Barbacid M, Lamballe F, Pulido D and Klein R (1991) The trk family of tyrosine protein kinase receptors. Biochim Biophys Acta 1072: 115-127.

Bardin PG and Van Eeden SF (1990) Organophosphate poisoning: grading the severity and comparing treatment between atropine and glycopyrrolate. Crit Care Med 18: 956-960.

Benito-Gutierrez E, Garcia-Fernandez J and Comella JX (2006) Origin and evolution of the Trk family of neurotrophic receptors. Mol Cell Neurosci 31: 179-192.

Brown DL and Guyenet PG (1985) Electrophysiological study of cardiovascular neurons in the rostral ventrolateral medulla in rats. Circ Res 56: 359-369.

Brugge JS, Cotton PC, Queral AE, Barrett JN, Nonner D and Keane RW (1985) Neurones express high levels of a structurally modified, activated form of pp60c-src. Nature 316: 554-557.

Buday L and Downward J (1993) Epidermal growth factor regulates p21ras through the formation of a complex of receptor, Grb2 adapter protein, and Sos nucleotide exchange factor. Cell 73: 611-620.

Cerutti C, Barres C and Paultre C (1994) Baroreflex modulation of blood pressure and heart rate variabilities in rats: assessment by spectral analysis. Am J Physiol 266: H1993-H2000.

Chan JYH, Wang LL, Wu KL and Chan SHH (2001c) Reduced functional expression and molecular synthesis of inducible nitric oxide synthase in rostral ventrolateral medulla of spontaneously hypertensive rats. Circulation 104: 1676-1681.

Chan JYH, Wang SH and Chan SHH (2001a) Differential roles of iNOS and nNOS at rostral ventrolateral medulla during experimental endotoxemia in the rat. Shock 15: 65-72.

Chan JYH, Chan SHH and Chang AYW (2004) Differential contributions of NOS isoforms in the rostral ventrolateral medulla to cardiovascular responses associated with mevinphos intoxication in the rat. Neuropharmacology 46: 1184-1194.

Chan JYH, Chan SHH, Li FCH, Tsai CY, Cheng HL and Chang AYW (2005) Phasic cardiovascular responses to mevinphos are mediated through differential activation of cGMP/PKG cascade and peroxynitrite via nitric oxide generated in the rat rostral ventrolateral medulla by NOS I and II isoforms. Neuropharmacology 48: 161-172.

Chan SHH, Wang LL, Ou CC and Chan JYH (2002) Contribution of peroxynitrite to fatal cardiovascular depression induced by overproduction of nitric oxide in rostral ventrolateral medulla of the rat. Neuropharmacology 43: 889-898.

Chan SHH, Wang LL, Wang SH and Chan JYH (2001b) Differential cardiovascular responses to blockade of nNOS or iNOS in rostral ventrolateral medulla of the rat. Br J Pharmacol 133: 606-614.

Chan SHH, Wang LL and Chan JYH (2003) Differential engagements of glutamate and GABA receptors in cardiovascular actions of endogenous nNOS or iNOS at rostral ventrolateral medulla of rats. Br J Pharmacol 138: 584-593.

Chang AYW, Chan JYH and Chan SHH (2003) Differential distribution of nitric oxide synthase isoforms in the rostral ventrolateral medulla of the rat. J Biomed Sci 10: 285-291.

Chang AYW, Chan JYH, Kao FJ, Huang CM and Chan SHH (2001) Engagement of inducible nitric oxide synthase at the rostral ventrolateral medulla during mevinphos intoxication in the rat. J Biomed Sci 8: 475-483.

Chuang FR, Jang SW, Lin JL, Chern MS, Chen JB and Hsu KT (1996) QTc prolongation indicates a poor prognosis in patients with organophosphate poisoning. Am J Emerg Med 14: 451-453.

Cunningham ME, Stephens RM, Kaplan DR and Greene LA (1997) Autophosphorylation of activation loop tyrosines regulates signaling by the TRK nerve growth factor receptor. J Biol Chem 272: 10957-10967.

Dampney RA (1994) Functional organization of central pathways regulating the cardiovascular system. Physiol Rev 74: 323-364.

Dampney RA, Goodchild AK, Robertson LG and Montgomery W (1982) Role of ventrolateral medulla in vasomotor regulation: a correlative anatomical and physiological study. Brain Res 249: 223-235.

Feinstein DL, Galea E, Cermak J, Chugh P, Lyandvert L and Reis DJ (1994a) Nitric oxide synthase expression in glial cells: suppression by tyrosine kinase inhibitors. J Neurochem 62: 811-814.

Feinstein DL, Galea E and Reis DJ (1994b) Suppression of glial iNOS expression by tyrosine kinase inhibitors. Ann N Y Acad Sci 738: 325-328.

Forstermann U and Kleinert H (1995) Nitric oxide synthase: expression and expressional control of the three isoforms. Naunyn Schmiedebergs Arch Pharmacol 352: 351-364.

Forstermann U, Boissel JP and Kleinert H (1998) Expressional control of the 'constitutive' isoforms of nitric oxide synthase (NOS I and NOS III). FASEB J 12: 773-790.

Forstermann U, Kleinert H, Gath I, Schwarz P, Closs EI and Dun NJ (1995) Expression and expressional control of nitric oxide synthases in various cell types. Adv Pharmacol 34: 171-186.

Frame MC (2002) Src in cancer: deregulation and consequences for cell behaviour. Biochim Biophys Acta 1602: 114-130.

Guo W, Robbins MT, Wei F, Zou S, Dubner R and Ren K (2006) Supraspinal brain-derived neurotrophic factor signaling: a novel mechanism for descending pain facilitation. J Neurosci 26: 126-137.

Harder KW, Moller NP, Peacock JW and Jirik FR (1998) Protein-tyrosine phosphatase alpha regulates Src family kinases and alters cell-substratum adhesion. J Biol Chem 273: 31890-31900.

Hilaire G and Duron B (1999) Maturation of the mammalian respiratory system. Physiol Rev 79: 325-360.

Holtzman DM and Lowenstein DH (1995) Selective inhibition of axon outgrowth by antibodies to NGF in a model of temporal lobe epilepsy. J Neurosci 15: 7062-7070.

Holtzman DM, Lee S, Li Y, Chua-Couzens J, Xia H, Bredt DS and Mobley WC (1996) Expression of neuronal-NOS in developing basal forebrain cholinergic neurons: regulation by NGF. Neurochem Res 21: 861-868.

Hung SP, Hsu JR, Lo CP, Huang HJ, Wang JP and Chen ST (2005) Genistein-induced neuronal differentiation is associated with activation of extracellular signal-regulated kinases and upregulation of p21 and N-cadherin. J Cell Biochem 96: 1061-1070.

Ignarro LJ (1989) Endothelium-derived nitric oxide: actions and properties. FASEB J 3: 31-36.

Jin ZG, Ueba H, Tanimoto T, Lungu AO, Frame MD and Berk BC (2003) Ligand-independent activation of vascular endothelial growth factor receptor 2 by fluid shear stress regulates activation of endothelial nitric oxide synthase. Circ Res 93: 354-363.

Jonnala RR and Buccafusco JJ (2001) Inhibition of nerve growth factor signaling by peroxynitrite. J Neurosci Res 63: 27-34.

Kagiyama S, Tsuchihashi T, Abe I and Fujishima M (1997) Cardiovascular effects of nitric oxide in the rostral ventrolateral medulla of rats. Brain Res 757: 155-158.

Kaplan DR and Miller FD (1997) Signal transduction by the neurotrophin receptors. Curr Opin Cell Biol 9: 213-221.

Katz DM (2005) Regulation of respiratory neuron development by neurotrophic and transcriptional signaling mechanisms. Respir Physiol Neurobiol 149: 99-109.

Klocker N, Cellerino A and Bahr M (1998) Free radical scavenging and inhibition of nitric oxide synthase potentiates the neurotrophic effects of brain-derived neurotrophic factor on axotomized retinal ganglion cells In vivo. J Neurosci 18: 1038-1046.

Klocker N, Kermer P, Gleichmann M, Weller M and Bahr M (1999) Both the neuronal and inducible isoforms contribute to upregulation of retinal nitric oxide synthase activity by brain-derived neurotrophic factor. J Neurosci 19: 8517-8527.

Krause DS and Van Etten RA (2005) Tyrosine kinases as targets for cancer therapy. N Engl J Med 353: 172-187.

Krukoff TL (1999) Central actions of nitric oxide in regulation of autonomic functions. Brain Res Brain Res Rev 30: 52-65.

Kuo TBJ, Yang CCH and Chan SHH (1997) Selective activation of vasomotor component of SAP spectrum by nucleus reticularis ventrolateralis in rats. Am J Physiol 272: H485-H492.

Lee JY, Lowell CA, Lemay DG, Youn HS, Rhee SH, Sohn KH, Jang B, Ye J, Chung JH and Hwang DH (2005) The regulation of the expression of inducible nitric oxide synthase by Src-family tyrosine kinases mediated through MyD88-independent signaling pathways of Toll-like receptor 4. Biochem Pharmacol 70: 1231-1240.

Lin MT and Lin JH (2000) Involvement of tyrosine kinase in the pyrogenic fever exerted by NOS pathways in organum vasculosum laminae terminalis. Neuropharmacology 39: 347-352.

Lipski J, Kanjhan R, Kruszewska B and Rong W (1996) Properties of presympathetic neurones in the rostral ventrolateral medulla in the rat: an intracellular study “in vivo”. J Physiol 490: 729-744.

Maness PF (1986) pp60c-src encoded by the proto-oncogene c-src is a product of sensory neurons. J Neurosci Res 16: 127-139.

Martin-Zanca D, Mitra G, Long LK and Barbacid M (1986) Molecular characterization of the human trk oncogene. Cold Spring Harb Symp Quant Biol 51: 983-992.

Morrison SF, Milner TA and Reis DJ (1988) Reticulospinal vasomotor neurons of the rat rostral ventrolateral medulla: relationship to sympathetic nerve activity and the C1 adrenergic cell group. J Neurosci 8: 1286-1301.

Murphy SM, Bergman M and Morgan DO (1993) Suppression of c-Src activity by C-terminal Src kinase involves the c-Src SH2 and SH3 domains: analysis with Saccharomyces cerevisiae. Mol Cell Biol 13: 5290-5300.

Pereira SP, Medina SV and Araujo EG (2001) Cholinergic activity modulates the survival of retinal ganglion cells in culture: the role of M1 muscarinic receptors. Int J Dev Neurosci 19: 559-567.

Purchio AF, Erikson E, Brugge JS and Erikson RL (1978) Identification of a polypeptide encoded by the avian sarcoma virus src gene. Proc Natl Acad Sci U S A 75: 1567-1571.

Rider JA, Puletti EJ and Swader JI (1975) The minimal oral toxicity level for mevinphos in man. Toxicol Appl Pharmacol 32: 97-100.

Rodriguez J, Quignard JF, Fagni L, Lafon-Cazal M and Bockaert J (1994) Blockade of nitric oxide synthesis by tyrosine kinase inhibitors in neurones. Neuropharmacology 33: 1267-1274.

Ross CA, Ruggiero DA, Park DH, Joh TH, Sved AF, Fernandez-Pardal J, Saavedra JM and Reis DJ (1984) Tonic vasomotor control by the rostral ventrolateral medulla: effect of electrical or chemical stimulation of the area containing C1 adrenaline neurons on arterial pressure, heart rate, and plasma catecholamines and vasopressin. J Neurosci 4: 474-494.

Samdani AF, Newcamp C, Resink A, Facchinetti F, Hoffman BE, Dawson VL and Dawson TM (1997) Differential susceptibility to neurotoxicity mediated by neurotrophins and neuronal nitric oxide synthase. J Neurosci 17: 4633-4641.

Schlessinger J (2000) Cell signaling by receptor tyrosine kinases. Cell 103: 211-225.

Schlessinger J (2000) New roles for Src kinases in control of cell survival and angiogenesis. Cell 100: 293-296.

Segal RA, Bhattacharyya A, Rua LA, Alberta JA, Stephens RM, Kaplan DR and Stiles CD (1996) Differential utilization of Trk autophosphorylation sites. J Biol Chem 271: 20175-20181.

Sobreviela T, Jaffar S and Mufson EJ (1998) Tyrosine kinase A, galanin and nitric oxide synthase within basal forebrain neurons in the rat. Neuroscience 87: 447-461.

Spyer KM (1994) Central nervous mechanisms contributing to cardiovascular control. J Physiol 474: 1-19.

Sugrue MM, Brugge JS, Marshak DR, Greengard P and Gustafson EL (1990) Immunocytochemical localization of the neuron-specific form of the c-src gene product, pp60c-src(+), in rat brain. J Neurosci 10: 2513-2527.

Takahashi H, Kojima T, Ikeda T, Tsuda S and Shirasu Y (1991) Differences in the mode of lethality produced through intravenous and oral administration of organophosphorus insecticides in rats. Fundam Appl Toxicol 16: 459-468.

Tatosyan AG and Mizenina OA (2000) Kinases of the Src family: structure and functions. Biochemistry (Mosc) 65: 49-58.

Thoby-Brisson M, Cauli B, Champagnat J, Fortin G and Katz DM (2003) Expression of functional tyrosine kinase B receptors by rhythmically active respiratory neurons in the pre-Botzinger complex of neonatal mice. J Neurosci 23: 7685-7689.

Thomas ML and Brown EJ (1999) Positive and negative regulation of Src-family membrane kinases by CD45. Immunol Today 20: 406-411.

Triedman JK and Saul JP (1994) Blood pressure modulation by central venous pressure and respiration. Buffering effects of the heart rate reflexes. Circulation 89: 169-179.

Tzeng SF and Huang HY (2003) Downregulation of inducible nitric oxide synthetase by neurotrophin-3 in microglia. J Cell Biochem 90: 227-333.

Verberk MM (1977) Incipient cholinesterase inhibition in volunteers ingesting monocrotophos or mevinphos for one month. Toxicol Appl Pharmacol 42: 345-350.

Verberk MM and Salle HJ (1977) Effects on nervous function in volunteers ingesting mevinphos for one month. Toxicol Appl Pharmacol 42: 351-358.

Wakade AR and Wakade TD (1983) Mechanism of negative feed-back inhibition of norepinephrine release by alpha-adrenergic agonists. Neuroscience 9: 673-677.

Wan Q, Man HY, Braunton J, Wang W, Salter MW, Becker L and Wang YT (1997) Modulation of GABAA receptor function by tyrosine phosphorylation of beta subunits. J Neurosci 17: 5062-5069.

Wang H and Zhou XF (2002) Injection of brain-derived neurotrophic factor in the rostral ventrolateral medulla increases arterial blood pressure in anaesthetized rats. Neuroscience 112: 967-975.

Watcharasit P, Tucholski J and Jope RS (2001) Src family kinase involvement in muscarinic receptor-induced tyrosine phosphorylation in differentiated SH-SY5Y cells. Neurochem Res 26: 809-816.

Xiong H, Yamada K, Jourdi H, Kawamura M, Takei N, Han D, Nabeshima T and Nawa H (1999) Regulation of nerve growth factor release by nitric oxide through cyclic GMP pathway in cortical glial cells. Mol Pharmacol 56: 339-347.

Yang CH, Shyr MH, Kuo TBJ, Tan PP and Chan SHH (1995b) Effects of propofol on nociceptive response and power spectra of electroencephalographic and systemic arterial pressure signals in the rat: correlation with plasma concentration. J Pharmacol Exp Ther 275: 1568-1574.

Yang MW, Kuo TBJ, Lin SM, Chan KH and Chan SHH (1995a) Continuous, on-line, real-time spectral analysis of SAP signals during cardiopulmonary bypass. Am J Physiol 268: H2329-H2335.

Yen DH, Yen JC, Len WB, Wang LM, Lee CH and Chan SHH (2001) Spectral changes in systemic arterial pressure signals during acute mevinphos intoxication in the rat. Shock 15: 35-41.

Yen DH, Yien HW, Wang LM, Lee CH and Chan SHH (2000) Spectral analysis of systemic arterial pressure and heart rate signals of patients with acute respiratory failure induced by severe organophosphate poisoning. Crit Care Med 28: 2805-2811.

Yien HW, Hseu SS, Lee LC, Kuo TBJ, Lee TY and Chan SHH (1997) Spectral analysis of systemic arterial pressure and heart rate signals as a prognostic tool for the prediction of patient outcome in the intensive care unit. Crit Care Med 25: 258-266.

Zhao WQ, Alkon DL and Ma W (2003) c-Src protein tyrosine kinase activity is required for muscarinic receptor-mediated DNA synthesis and neurogenesis via ERK1/2 and c-AMP-responsive element-binding protein signaling in neural precursor cells. J Neurosci Res 72: 334-342.

Zou MH, Hou XY, Shi CM, Kirkpatick S, Liu F, Goldman MH, Cohen RA (2003) Activation of 5'-AMP-activated kinase is mediated through c-Src and phosphoinositide 3-kinase activity during hypoxia-reoxygenation of bovine aortic endothelial cells. Role of peroxynitrite. J Biol Chem 278: 34003-34010.