國立中山大學學位論文典藏.PDF

Transcription

1 立 論 量 粒 離 參 令 年

2 錄 錄

3 錄 論 料 論 38 六 論 46 來 49 參

4 acsf BH4 cgmp CVLM DETC EDRF GABA GFP HR IML L-NAME MnTBAP MSAP NADH NO NOS NTS O 2 - ODQ ONOO - pfu ROS RVLM SD sgc SHR SOD SP-SHR Tempol WKY artificial cerebrospinal fluid tetrahydrobiopterin quanosine 3,5 -cyclic monophosphate caudal ventrolateral medulla diethyldithiocarbamic acid endothelium-derived relaxing factor gamma-aminobutyric acid green fluorescent protein heart rate intermediolateral cell column N w -nitro-l-arginine methyl ester Mn(III)tetrakis(4-benzoic acid) porphyrin chloride mean systemic arterial pressure nicotinamide adenine dinucleotide hydrogen nitric oxide nitric oxide synthase nucleus of tractus solitarii superoxide anion 1-H-[1,2,4]oxadiaolol[4,3-α]quinoxalin-1-one peroxynitrite plague forming unit reactive oxygen species rostral ventrolateral medulla Sprague-Dawley soluble guanylyl cyclase spontaneously hypertensive rat superoxide dismutase stroke-prone spontaneously hypertensive rat 4-hydroxytetramethyl piperidine-1-oxyl Wistar-Kyoto 3

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6 類 見 神 神 兩 來 力 離 力 類 離 精 NADPH 神 神 參 力 參 兩 參 了 量 AdeNOS (Adenoviral vectors encoding endothelial nitric oxide synthase) AdSOD2 (Adenoviral vectors encoding superoxide dismutase 2) AdeNOS AdSOD2 Wistar-Kyoto (WKY) spontaneously hypertensive rats (SHR) 1. 量 AdeNOS SHR WKY 兩 降 兩 enos GABAergic 神 降 神 SHR 5

7 降 度 WKY SHR 量 WKY SHR 28 SHR 離 度 2. 量 AdSOD2 SHR WKY 兩 SHR 降 WKY SHR WKY SHR 離 度 力 類 不 來 SOD2 離 力 降 3. 量 AdeNOS AdSOD2 SHR AdeNOS AdSOD2 更 度 降 降 28 論 SOD2 離 離 不 論 SHR 力 論 離 參 降 不 6

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9 The dissection of etiology of hypertension is a medical imperative. In the central nervous system, rostral ventral lateral medulla (RVLM) plays an essential role in the maintenance of arterial pressure and heart rate through tonic activation of the sympathetic vasomotor activity and regulation of baroreflex response. Oxidative stress of an enhanced cellular content of the reactive oxygen species, in particular the superoxide anion (O - 2 ), has been implicated in hypertension. Superoxide dismutase (SOD) is one of the most important defense enzymes against the oxidative stress through catalysis of O - 2 into O 2 and H 2 O 2. SOD treatment has been demonstrated to decrease arterial pressure. Moreover, in addition to its peripheral vasodilatory effect, nitric oxide (NO) plays an active role in central regulation of arterial pressure and heart rate via modulation of the autonomic system. In the RVLM, both O 2 - and NO have been demonstrated to be involved in hypertension. Interactions between these two molecules, however, are not understood. The aims of this study are therefore to establish the significance of O 2 - and NO in the RVLM on blood pressure regulation in hypertension and to examine whether O 2 - interacts with NO to participate in the pathogenesis of hypertension. To examine their long term effects on mean systemic arterial pressure (MSAP) and heart rate (HR), SOD and/or NO was over-expressed by microinjection of the adenoviral vectors encoding the endothelial NO synthase (AdeNOS) and/or mitochondrial SOD (AdSOD2) into RVLM of the normotensive Wistar-Kyoto (WKY) rats or the spontaneously hypertensive rats (SHR). I found that microinjection of AdeNOS in the RVLM of SHR or WKY rats significantly decreased MSAP or HR that lasted for around 10 days postinjection. The hypotensive effect of AdeNOS was significantly greater in SHR than WKY rats. The AdeNOS-promoted hypotension in SHR, but not WKY rats, was followed by a rebound hypertension, detected in 28 days after the gene transfer. In the AdeSOD2-treated animals, I found a significant decrease in the MSAP in SHR, but not WKY rats, that lasted for about 7 days postinjection. On the other hand, no change in HR was detected in either SHR or 8

10 WKY rats after the AdSOD2 gene transfer into the RVLM. In animals that received co-microinjection into the bilateral RVLM of AdeNOS and AdSOD2, there was a further prolonged decrease in MSAP or HR in SHR. The rebound hypertension observed in the AdeNOS-treated SHR was reversed to hypotension in the AdeNOS+AdSOD2-treated SHR. There was no difference in the hypotensive or bradycardiac effects in WKY rats that received the AdeNOS+AdSOD2 or AdeNOS gene transfer. Together these results suggest that (1) NO in RVLM plays an important role in central regulation of arterial pressure and heart rate under both normotensive and hypertensive conditions. A greater reduction in MSAP in the AdeNOS-treated SHR further indicates a reduced action of NO at the RVLM in the pathogenesis of hypertension. (2) An excessive oxidative stress of a reduced function of SOD2 in RVLM may be an important factor in neural mechanism of hypertension in SHR. The same mechanism, at the same time, may underlie the rebound hypertensive observed in the AdeNOS-treated SHR. (3) The excessive oxidative stress in the RVLM contributes to hypertension by at least two mechanisms. One is to cause oxidative injury in the RVLM and the other is to interact with NO to decrease already insufficient activity of NO in central cardiovascular regulation. 9

11 論 10

12 1-1 類 見 塞 類 神 兩 : (baroreceptor reflex) (tonic) 神 來 力 神 (medulla oblongata) (rostral ventrolateral medulla, RVLM) 兩 都 神 (Colombari et al., 2001) 神 (facial nucleus) (inferior olivary nucleus) (nucleus ambiguus) (Calaresu et al., 1988) RVLM glycine 降 (Dean et al., 1986) 刺 (Dampney et al., 1980) RVLM glutamate RVLM glutamatergic synapse 降 (Bergamaschi et al., 1995) RVLM gamma-aminobutyric acid (GABA) 降 RVLM GABA bicuculline (Benarroch et al., 1986) RVLM vasomotor neurons (Dampney et al., 1982; Granata et al., 1985) 理 RVLM 神 11

13 (nucleus of tractus solitarius, NTS) (forebrain) (caudal ventrolateral medulla, CVLM) (Colombari et al., 2001) 論 RVLM 神 (intermediolateral cell column, IML) 神 神 神 神 來 1-2 (reactive oxygen species, ROS) 離 (superoxide anion, O - 2 ) hydroxyl radical (HO - ) (nitric oxide, NO) (lipid radicals) (H 2 O 2 ) (peroxynitrite, ONOO - ) (hypochlorous acid, HOCl) 不 力 力 (oxidative stress) 類 ROS : mitochondrial respiratory complexes NADH/NADPH oxidase 參 arachidonic acid pathways lipoxygenase cyclooxygenase cytochrome P450s xanthine oxidase NO synthase peroxidase 類 12

14 . Mitochondrial respiratory complexes 理狀 粒 90% 1.2% 離 (Turrens, 1997) 粒 complex I (NADH dehydrogenase) complex III (ubiquinone-cytochrome c reductase) 離 complex III 來 complex I complex II 量 ubisemiquinone ubiquinone 離 (Finkel et al., 2000). Xanthine oxidase Xanthine oxidoreductase purine hypoxanthine xanthine 兩 xanthine dehydrogenase xanthine oxidase xanthine dehydrogenase NAD + 離 xanthine oxidase (Tarpey et al., 2001) xanthine oxidase inhibitor oxypurinol 降 降 (Suzuki et al., 1995) 論 xanthine oxidase 度 (Nakazona et al., 1991). NADH/NADPH oxidase 13

15 NADH/NADPH oxidase growth factor cytokine 刺 離 angiotensin II NADH/NADPH oxidase 離 (Griendling et al., 1994) angiotensin II NADH/NADPH oxidase (Griendling et al., 2000). Endothelial nitric oxide synthase Endothelial NO synthase (enos) 類 P450 (cytochrome P450 reductase-like enzyme) 粒 NADPH (heme) enos tetrahydrobiopterin (BH4) L-arginine L-citrulline NO tetrahydrobiopterin L-arginine enos enos uncoupling (Cai et al., 2000) enos O - 2 NO (peroxynitrite, ONOO - ), 力 (Cai et al., 2000) stroke-prone spontaneous hypertensive rat (SP-SHR) 離 若 enos inhibitor L-NAME 離 量 降 (Kerr et al., 1999) 1-3 (superoxide dismutase, SOD) 力 14

16 類 離 O 2 SOD 不 類 Cupper/Zinc SOD (Cu/Zn-SOD SOD1) Manganese SOD (Mn-SOD SOD2) Extracellular SOD (EC-SOD SOD3) 類 SOD :. Cytosolic Cu/Zn-SOD SOD 年 (McCord and Fridovich, 1969) SOD1 兩 16 KDa 離 離 SOD (Borders et al., 1985) 來 Mn-SOD Cu/Zn-SOD H 2 O 2 度 (Yarom et al., 1988) 類 Cu/Zn SOD 21 21q22.1 (De LA Torre et al., 1996) (Down syndrome) 3 21 Cu/Zn SOD 50%(De La Torre et al., 1996) 率 (Morrison et al., 1996). Mitochondrial Mn-SOD SOD 年 粒 (Weisiger and Fridovich, 1973) 96 Kda 都 離 粒 Mn-SOD (dilated cardiomyopathy) Mn-SOD 15

17 量 (Li et al., 1995) 類 Mn-SOD 六 6q25 數 3 列 錄 NF- B NF- B (up-regulation)mn-sod (McIntyre et al., 1999). Extracellullar Cu/Zn SOD SOD 年 Marklund 類 EC-SOD 4 量 130 KDa 30 KDa 離 離 (fibroblast) 神 (glial cell) (Marklund, 1990) 糖 易 glycosaminoglycan 例 heparin heparin sulfate 4 露 量 力 EC-SOD (Carlsson et al., 1995) 1-4 離 離 年來 in vitro 滑 NADPH oxidase 來刺 離 (Sindhu et al., 2005) 若利 liposome Cu/ZnSOD angiotensin II 16

18 都 復 (Laursen et al., 1997) 若 angiotensin II NADPH oxidase p22 phox mrna (Fukui et al., 1997) salt-sensitive hypertension (up-regulation)angiotensin II 離 (Zhou et al., 2003) 列 angiotensin II 力 離 力 NO ONOO - NO (Rubanyi et al., 1986) SHR SOD 類 Tempol MnTBAP 錄 MnSOD 降 (Kishi et al., 2004; Tai et al., 2005) 降 離 年 (endothelium-derived relaxing factor, EDRF) (Ignarro et al., 1987) 精 (L-arginine) (NOS) NADPH 兩 類 : (constitutive) (inducible) NOS 神 (neuronal nitric oxide synthase, nnos NOSI) 17

19 (inducible nitric oxide synthase, inos NOSII) (endothelial nitric oxide synthase, enos NOS III)(Bredt and Snyder, 1990) 神 神 神 狀 (Murphy et al., 1993) (Forstermann et al., 1995) nnos enos 離 - (calcium-calmodulin) (Moncada et al., 1991) inos 不 離 - (calcium-calmodulin independent NOS) 量 SD RVLM inos 神 (Chang et al., 2003) 參 (Chan et al., 2001a, 2003) 都 行 力 (Nathan, 1992) 神 soluble guanylyl cyclase (sgc) 路 滑 (Forstermann et al., 1986) 1-6 神 (for Review, see Patel et al., 2001; Zanzinger, 2002; Hirooka et al., 2003; Hirooka, 2004) nnos inos NO 18

20 (dynamic) (Chan SHH et al., 2001) nnos NO glutamatergic neurotransmission 神 inos NO GABA neurotransmission 神 降 (Chan et al., 2003) enos 神 不參 (Chang et al., 2003) RVLM inos mnra 都 inos NO (Chan JYH et al., 2001; Kishi et al., 2002) RVLM enos RVLM 降 WKY (Kishi et al., 2001; Tai et al., 2004) enos RVLM 降 norepinephrine RVLM GABA RVLM GABA 神 (Kishi et al., 2001) 不良 離 離 度 離 度 (Thomson et al., 1995) 離 易 離 (ONOO - ) (Cai et al., 2000; Kishi et al., 2004) 19

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22 2-1 狀 神 神 神 神 參 行 理 SOD catalase 不 量 若 力 力 (Zalba et al., 2001) 狀 神 參 (Chan et al., 2001a, 2001b; Chan et al., 2003; Tai et al., 2004, 2005) 年來 離 NO peroxynitrite (ONOO - ) (Grunfeld et al., 1995) RVLM WKY 離 SOD SOD mimetic 降 (Tai et al., 2004) SOD2 降 (Kishi et al., 2004) NO RVLM 都 參 兩 數 21

23 2-2 論 論 < > enos NO 量 降 神 降 < > 力 力 SOD2 降 < > enos SOD2 降 神 力 降 兩 降 神 力 更 22

24 2-3 論 : 1. 狀 NO NO 量 2. 了 狀 SOD2 參 3. 狀 NO SOD2 離 狀 參 23

25 料 24

26 3-1 論 Wistar-Kyoto (WKY) spontaneously hypertensive rat (SHR) 兩 數 4~6 飼 (24-25 O C) 飼料 5 ( 飼 領 度 飼 不 ) 12 不 殺 若 不 13 殺 飼 行 列 3-2 量 3-4 狀 量 (MK-2000, Muromachikikai, Japan) 量 量 (sensor) 25

27 狀 量 量 25 mmhg 4-5 mmhg 數 數 (mean arterial pressure, MAP) 1/3 +2/3 (heart rate, HR) 20 數 若 量 量不 錄 量 異 10 mmhg 錄 數 列 便 數 3-3 類 enos SOD2 : SOD2 enos pjm pjm17 E1 E3 5 利 calcium phosphate 行 (Graham and Prevec, 1995) cytopathic effect (CPE) polymerase chain reaction Western blotting 行 293 量 CsCl gradient 離 兩 10 mm Tris ph mm MgCl 2 10% glycerol buffer 4 C 行 -80 C 26

28 260nm 量 293 來 量 行 列 in vivo 3-4 狀 量 SHR WKY (pentobarbital sodium, 50 mg/kg) 狀 立 (stereotaxic head holder) 切 lambda( ) 4.0 mm 4.5 mm 1.8 mm 8.0 mm 8.5 mm RVLM (Tai et al., 2005) RVLM 4 RVLM 兩 2 4 RVLM 兩 8 5O nl 留 RVLM 切 累 切 量 RVLM 3-5 SHR WKY 飼 行 狀 量 量兩 量兩 行 enos SOD2 27

29 狀 量 行 狀 量 10 SHR WKY 狀 量 acsf(artificial cerebrospinal fluid) 量 綠 AdGFP(Adenoviral vectors encoding green fluorescence protein) 量 enos SOD2 enos SOD2 量 6 6 不 3-6 數 (standard error of mean, SEM) one-way analysis of variance (ANOVA) 異 Scheff or Tukey multiple range test 異 P<

30 29

31 . 1. SHR AdeNOS a) : SHR AdeNOS(5x10 8 pfu/50 nl, 量 400 nl) 降 降 (-34.7±3.6 mm Hg, n = 9, P<0.05) (Figure 1) 14 復 28 (+24.4±4.1 mm Hg, n = 9, P<0.05) b) : AdeNOS (5x10 8 pfu/50 nl, 量 400 nl) 降 降 (-58±9 bpm, n = 9, P<0.05) (Figure 1) 14 復 2. SHR AdSOD2 a) : AdSOD2 (5x10 8 pfu/50 nl, 量 400 nl) 降 降 (-27.8±4.8 mm Hg, n = 8, P<0.05) (Figure 2) 復 11 復. 30

32 b) : AdSOD2 (5x10 8 pfu/50 nl, 量 400 nl) 28 (-10±6 bpm, n = 8, P>0.05) (Figure 2) 異 3. SHR AdeNOS AdSOD2 a) : AdeNOS AdSOD2 (2.5x x10 8 pfu/100 nl, 量 400 nl) 降 (-46.3±5.7 mm Hg, n = 6, P<0.05) (Figure 3) 降 28 數 (-19.8±4.9 mm Hg, n = 6, P<0.05) b) : AdeNOS AdSOD2 (2.5x x10 8 pfu/100 nl, 量 400 nl) 降 降 (-59±10 bpm, n = 6, P<0.05) (Figure 3) 28 數 (-23±8 bpm, n = 6, P<0.05) 4. WKY AdeNOS a) : AdeNOS (5x

33 pfu/50 nl, 量 400 nl) 降 降 (-21.9±3.7 mm Hg, n = 8, P<0.05) (Figure 4) 11 復 b) : AdeNOS (5x10 8 pfu/50 nl, 量 400 nl) 降 降 度 (-48±9 bpm, n = 8, P<0.05) (Figure 4) 21 復 5. WKY AdSOD2 a) : AdSOD2 (5x10 8 pfu/50 nl, 量 400 nl) (-6.2±2.9 mm Hg, n = 6, P>0.05) (Figure 5) b) : AdSOD2 (5x10 8 pfu/50 nl, 量 400 nl) (-11±4 bpm, n = 6, P>0.05) (Figure 5) 6. WKY AdeNOS AdSOD2 a) : AdeNOS 32

34 AdSOD2 (2.5x x10 8 pfu/100 nl, 量 400 nl) 降 降 (-27.8±5.4 mm Hg, n = 6, P<0.05) (Figure 6) 21 復 b) : AdeNOS AdSOD2(2.5x x10 8 pfu/100 nl, 量 400 nl) 降 5-7 降 (-56±10 bpm, n = 6, P<0.05) (Figure 6) 21 復. 異 1. SHR WKY AdeNOS 異 a) : AdeNOS 兩 都 降 降 度 SHR WKY (-23.1±3.1% vs ±2.4%, P<0.05) SHR AdeNOS 28 WKY (Figure 7) b) : AdeNOS 兩 都 降 降 度 33

35 異 (-15.3±5.1% vs ±4.8%, P>0.05) (Figure 8) 2. SHR WKY AdSOD2 異 a) : AdSOD2 SHR 降 (-18.7±4.6%, n =8) WKY 降 (-2.6±1.7%, n = 6) (Figure 9) b) : AdSOD2 SHR (-6.4±2.6%, n = 8) WKY (+2.8±2.1%, n = 6) 兩 降 都 (Figure 10) 3. SHR WKY AdeNOS AdSOD2 異 a) : AdeNOS AdSOD2 兩 都 降, 降 度 SHR WKY 異 (29.1±2.4% vs. 25.3±2.1%, P<0.05) SHR 28 (-12.3±3.2%, n = 6, P<0.05) 異 WKY 21 復 (Figure 11) 34

36 b) : AdeNOS AdSOD2 兩 都 降, 降 度 不 異 (-14.8±2.9% vs ±3.4%, P>0.05) (Figure 12) 4. SHR AdeNOS AdSOD2 AdeNOS AdSOD2 異 a) : AdeNOS AdSOD2 AdeNOS AdSOD2 SHR 都 降 降 度 AdeNOS AdSOD2 異 AdeNOS AdSOD2 AdeNOS ADSOD2 (P<0.05) 28 AdeNOS (Rebound hypertension) AdSOD2 AdeNOS AdSOD2 28 理 SHR 28 異 (P<0.05) (Figure 13) b) : AdeNOS AdeNOS AdSOD2 降 AdeNOS AdeNOS AdSOD2 兩 降 度 異 (P>0.05) AdSOD2 28 AdeNOS 復 AdSOD2 35

37 AdeNOS AdSOD2 28 (Figure 14) 5. WKY AdeNOS AdSOD2 AdeNOS AdSOD2 異 a) : AdeNOS AdeNOS AdSOD2 降 AdeNOS AdSOD2 降 度 AdeNOS 降 異 (P>0.05) AdSOD2 WKY (Figure 15) b) : AdeNOS AdeNOS AdSOD2 WKY 降 兩 降 度 異 (P>0.05) AdSOD2 WKY (Figure 16) 36

38 論 37

39 5-1 量 AdeNOS 精 (L-arginine) (nitric oxide synthase) NADPH soluble guanylate cyclase (sgc) cyclic 3-5 guanosine monophosphate (cgmp) 離 離 路 (Hawkins et al., 1998) (Forstermann et al., 1995) 度 (Gathwaite and Boulton, 1995) angiotensin II 降 (Tagawa et al., 1999) 神 神 (Prast and Philippu, 2001) 例 nnos glutamate 神 inos GABA 神 降 (Chan et al., 2003) nnos inos NO (dynamic) (Chan et al., 2001a) enos 神 不參 (Chang et al., 2003) 神 nnos inos AdeNOS 量 38

40 易 量 神 量 AdeNOS SHR (Fig. 1) WKY (Fig. 4) 兩 降兩 AdeNOS 神 力 降 (Tai et al., 2005) 論 AdeNOS NO 神 降 GABAergic 神 降 神 (Chan et al., 2003) AdeNOS 量 sgc ODQ NO-sGC-cGMP 參 AdeNOS 降 (Tai et al., 2005) 度 WKY enos 降 (Malinski et al., 1993) AdeNOS SHR 降 度 WKY (Fig. 7) SHR (endogenous) 量 WKY AdeNOS SHR 更 降 NAD(P)H oxidase ROS 量 降 (Sindhu et al., 2005) 論 神 ROS 量 SHR SHR 39

41 AdeNOS 降 AdeNOS 28 (Figs. 1 7) (Tai et al., 2005) 1 (Tai et al., 2005) 2 量 enos BH4 NADH L-arginine NO 量不 enos uncouple 離 3 SHR 力 1 WKY RVLM AdeNOS 不 NO 量不 來 AdeNOS SOD 類 MnTBAP 論 SHR RVLM 離 量 AdeNOS (Tai et al., 2005) 離 度 離 度 (Thomson et al., 1995) 離 易 離 (ONOO - ) (Cai et al., 2000; Kishi et al., 2004) WKY 力 AdeNOS (Figs. 4 7) SHR 40

42 RVLM AdeNOS 28 量 NO superoxide anion SOD peroxynitrite 度 更 5-2 量 AdSOD2, (ROS) 度 參 理 (Yin et al., 1995) 力 例 糖尿 神 (Taniyama et al., 2003) (superoxide dismutase, SOD) 力 類 離 O 2 SHR SOD 類 M40403 降 (Cuzzocrea et al., 2004) Sprague-Dawley SOD DETC (Makino et al., 2003) angiotensin II liposome-encapsulated SOD 降 (Laursen et al., 1997) SHR 量 AdSOD2 降 (Fig. 2) WKY AdSOD2 都 (Fig. 5) WKY 力 不 WKY 41

43 離 度不 力 類 離 AdSOD2 WKY 都 SHR 力 SHR 離 度 力 類 不 (Tai et al., 2005) 離 來 AdSOD2 離 力 降 (Kishi et al., 2004) 論 SHR 力 SHR 粒 離 AdSOD2 參 力 類 李 論 AdSOD1 SHR 了粒 力 類 SHR 力 AdeNOS SHR 都 (Figs. 1 and 2) SHR AdSOD2 降 (Figs. 3 and 4) 異 論 NO 參 神 兩 離 參 神 42

44 5-3 量 AdeNOS AdSOD2, 度 滑 enos 度 度 不 (McIntyre et al., 1997) WKY (Malinski et al., 1993) SHR SOD (Tschudi et al., 1996) SHR 度 落 SOD (Grunfeld et al., 1995) 離 (Grunfeld et al.,1995) 來 離 易 離 (ONOO - ) (Cai et al., 2000; Kishi et al., 2004) Arachidonic acid F 2 -isoprostanes (Romero and Reckelhoff, 1999) 暴露 刺 離 (Grover et al., 2003) SHR nitrotyrosine ( ) WKY (Cuzzocrea et al., 2004) SOD 類 M40403 nitrotyrosine (Cuzzocrea et al., 2004) 都 離 43

45 降 SHR AdeNOS AdSOD2 更 度 降 降 28 (Figs. 3 13) WKY AdeNOS AdSOD2 降 AdeNOS 降 (Figs. 6 15) SHR 更 度 更 降 論 AdSOD2 離 離 不 AdeNOS SHR 更 度 降 降 SHR 28 AdeNOS AdSOD2 降 AdeNOS (Fig. 13) AdeNOS SHR 力 WKY AdeNOS AdSOD2 降 AdeNOS 力 AdSOD2 不 AdeNOS 降 不 AdSOD2 了 數 都 度 便 AdeNOS AdSOD2 AdeNOS AdSOD2 度 (5x10 8 pfu/50 nl) 度 (2.5x x10 8 pfu/100 nl) 44

46 度 (5x x10 8 pfu/100 nl) 降 度 降 更 5-4 利 enos SOD2 參 量 兩 量 力 量不 量 離 了 路 了 例 量 Cu/Zn-SOD H 2 O 2 度 (Yarom et al., 1988) 來 來 參 量 更 料 45

47 六 論 46

48 SHR WKY 量 AdeNOS AdSOD2 AdeNOS AdSOD2 1. SHR WKY 量 AdeNOS 降 enos GABAergic 神 降 神 降 SHR 降 度 WKY SHR 度 SHR 28 SHR 離 度 2. SHR WKY AdeSOD2 SHR 降 WKY SHR 力 力 SHR 降兩 更 度 降 降 47

49 離 離 不 降 4. 論 SHR 力 論 離 參 降 不 48

50 來 49

51 離 來 1. 降 2. 離 度 降 離 3. 離 ( SOD1 SOD3 ) 力 50

52 參 51

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60 59

61 MEAN SYSTEMIC ARTERIAL PRESSURE (mmhg) * acsf AdGFP AdeNOS * * * * * P O S T IN J E C T IO N T IM E (d a y ) * HEART RATE (bpm) * * * * * * POSTINJECTION TIME (day) Figure 1. Temporal changes in mean systemic arterial pressure (MSAP) or heart rate (HR) after microinjection of AdeNOS into the bilateral RVLM of SHR. Values are mean ± SE, n = 9 animals per each group. *P<0.05 vs corresponding acsf group in the Scheffé multiple-range test. 60

62 200 MEAN SYSTEMIC ARTERIAL PRESSURE (mmhg) acsf AdGFP AdSOD * * * P O S T IN JE C T IO N T IM E (d a y) HEART RATE (bpm) P O S T IN JE C T IO N T IM E (d a y) Figure 2. Temporal changes in mean systemic arterial pressure (MSAP) or heart rate (HR) after microinjection of AdSOD2 into the bilateral RVLM of SHR. Values are mean ± SE, n = 8 animals per each group. *P<0.05 vs corresponding acsf group in the Scheffé multiple-range test. 61

63 2 0 0 MEAN SYSTEMIC ARTERIAL PRESSURE (mmhg) ac SF A d G F P A d e N O S + A d S O D * * * * * * * P O S T IN J E C T IO N T IM E (d a y ) HEART RATE (bpm) * * * * * * * P O S T IN J E C T IO N T IM E (d a y ) Figure 3. Temporal changes in mean systemic arterial pressure (MSAP) or heart rate (HR) after co-microinjection of AdeNOS+AdSOD2 into the bilateral RVLM of SHR. Values are mean ± SE, n = 6 animals per each group. *P<0.05 vs corresponding acsf group in the Scheffé multiple-range test. 62

64 MEAN SYSTEMIC ARTERIAL PRESSURE (mmhg) acsf AdGFP A deno S * * * POSTINJECTION TIME (day) 450 * HEART RATE (bpm) * * * POSTINJECTION TIME (day) Figure 4. Temporal changes in mean systemic arterial pressure (MSAP) or heart rate (HR) after microinjection of AdeNOS into the bilateral RVLM of WKY rats. Values are mean ± SE, n = 8 animals per each group. *P<0.05 vs corresponding acsf group in the Scheffé multiple-range test. 63

65 MEAN SYSTEMIC ARTERIAL PRESSURE (mmhg) ac SF A d G F P A den O S A d S O D 2 + P O S T IN J E C T IO N T IM E (d a y ) HEART RATE (bpm) P O S T IN J E C T IO N T IM E (d a y ) Figure 5. Temporal changes in mean systemic arterial pressure (MSAP) or heart rate (HR) after microinjection of AdSOD2 into the bilateral RVLM of WKY rats. Values are mean ± SE, n=6 animals per each group. No significant difference existed among groups in two-way ANOVA. 64

66 MEAN SYSTEMIC ARTERIAL PRESSURE (mmhg) acsf AdGFP AdeNOS + AdSOD * * * * P O S T IN J E C T IO N T IM E (d a y ) HEART RATE (bpm) * * * P O S T IN J E C T IO N T IM E (d a y ) Figure 6. Temporal changes in mean systemic arterial pressure (MSAP) or heart rate (HR) after co-microinjection of AdeNOS+AdSOD2 into the bilateral RVLM of WKY rats. Values are mean ± SE, n = 6 animals per each group. *P<0.05 vs corresponding acsf group in the Scheffé multiple-range test. 65

67 MEAN SYSTEMIC ARTERIAL PRESSURE (mmhg) SHR-AdeNOS WKY-AdeNOS POSTINJECTION TIME (day) Figure 7. Comparison in changes in mean systemic arterial pressure (MSAP) after microinjection of AdeNOS into the bilateral RVLM of SHR or WKY rats. Values are mean ± SE, n = 8-9 animals per each group. Significant difference existed in the maximal decrease in MSAP between AdeNOS-treated SHR and WKY group in the Student s t test. 66

68 HEART RATE (bpm) SHR-AdeNOS WKY-AdeNOS POSTINJECTION TIME (day) Figure 8. Comparison in changes in heart rate (HR) after microinjection of AdeNOS into the bilateral RVLM of SHR or WKY rats. Values are mean ± SE, n = 8-9 animals per each group. No significant difference existed between groups in two-way ANOVA. 67

69 MEAN SYSTEMIC ARTERIAL PRESSURE (mmhg) SHR-SOD2 WKY-SOD2 SHR-AdSOD2 WKY-AdSOD2 * POSTINJECTION TIME (day) Figure 9. Comparison in changes in mean systemic arterial pressure (MSAP) after microinjection of AdSOD2 into the bilateral RVLM of SHR or WKY rats. Values are mean ± SE, n = 6-8 animals per each group. Significant difference existed in the maximal decrease in MSAP between AdSOD2-treated SHR and WKY group in the Student s t test. 68

70 HEART RATE (bpm) SHR-AdSOD2 WKY-AdSOD POSTINJECTION TIME (day) Figure 10. Comparison in changes in heart rate (HR) after microinjection of AdSOD2 into the bilateral RVLM of SHR or WKY rats. Values are mean ± SE, n = 6-8 animals per each group. No significant difference existed between groups in two-way ANOVA. 69

71 MEAN SYSTEMIC ARTERIAL PRESSURE (mmhg) SHR-AdeNOS+AdSOD2 WKY-AdeNOS+AdSOD POSTINJECTION TIME (day) Figure 11. Comparison in changes in mean systemic arterial pressure (MSAP) after co-microinjection of AdeNOS+AdSOD2 into the bilateral RVLM of SHR or WKY rat. Values are mean ± SE, n = 6 animals per each group. Significant difference existed in the maximal decrease in MSAP between AdeNOS+AdDOS2-treated SHR and WKY group in the Student s t test. 70

72 HEART RATE (bpm) SHR-AdeNOS+AdSOD2 WKY-AdeNOS+AdSOD POSTINJECTION TIME (day) Figure 12. Temporal changes in heart rate (HR) after co-microinjection of AdeNOS +AdSOD2 into the bilateral RVLM of SHR or WKY rats. Values are mean ± SE, n = 6 animals per each group. No significant difference existed between groups in two-way ANOVA. 71

73 200 MEAN SYSTEMIC ARTERIAL PRESSURE (mmhg) acsf AdeNOS AdGFP AdSOD2 AdeNOS+AdSOD POSTINJECTION TIME (day) Figure 13. Comparison in changes in mean systemic arterial pressure (MSAP) after microinjection of AdeNOS, AdSOD2 or AdeNOS +AdSOD2 into the bilateral RVLM of SHR. Values are mean ± SE, n = 6-9 animals per each group. Significant difference existed between AdeNOS+AdSOD2 vs AdeSOD2 or AdeNOS group in the Tukey multiple-range test. 72

74 HEART RATE (bpm) AdeNOS AdSOD2 AdeNOS+AdSOD POSTINJECTION TIME (day) Figure 14. Comparison in changes in heart rate (HR) after microinjection of AdeNOS, AdSOD2 or AdeNOS+AdSOD2into the bilateral RVLM of SHR. Values are mean ± SE, n = 6-9 animals in each group. Significant difference existed between AdeNOS+AdSOD2 vs AdeSOD2 group in the Tukey multiple-range test. No significant difference existed between AdeNOS+AdSOD2 and AdeNOS groups in two-way ANOVA. 73

75 MEAN SYSTEMIC ARTERIAL PRESSURE (mmhg) AdeNOS AdSOD2 AdeNOS+AdSOD POSTINJECTION TIME (day) Figure 15. Comparison in changes in mean systemic arterial pressure (MSAP) after microinjection of AdeNOS, AdSOD2 or AdeNOS +AdSOD2 into the bilateral RVLM of WKY rats. Values are mean ± SE, n = 6-9 animals per each group. Significant difference existed between AdeNOS+AdSOD2 vs AdeSOD2 group in the Tukey multiple-range test. No significant difference existed between AdeNOS+AdSOD2 and AdeNOS groups in two-way ANOVA. 74

76 AdeNOS AdSOD2 AdeNOS+AdSOD2 HEART RATE (bpm) POSTINJECTION TIME (day) igure 16. Comparison in changes in heart rate (HR) after microinjection of AdeNOS, AdSOD2 or AdeNOS+AdSOD2into the bilateral RVLM of WKY rats. Values are mean ± SE, n = 6-9 animals in each group. Significant difference existed between AdeNOS+AdSOD2 vs AdeSOD2 group in the Tukey multiple-range test. No significant difference existed between AdeNOS+AdSOD2 and AdeNOS groups in two-way ANOVA. 75