大鼠肺动脉平滑肌细胞增殖和有丝分裂的5-HT_（1B/1D）受体机制
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摘要
目的
肺血管构型重建是肺动脉高压的基本病理改变。肺血管平滑肌细胞增殖和肥大是肺血管构型重建主要特征。5-HT在肺动脉高压的发病过程中有非常重要的作用。研究发现野百合碱所致肺动脉高压大鼠的肺血流中有5-HT水平升高并且肺血管平滑肌有5-HT_(1B)受体mRNA的表达增加。5-HT_(1B)受体基因敲除小鼠能抵抗缺氧诱导的肺动脉高压和肺血管构型重建。5-HT是一种重要的血管活性物质，5-HT不仅刺激肺动脉平滑肌细胞收缩还可造成其增生和肥大，并且能与多种细胞生长因子发生协同作用。很多研究表明5-HT的促有丝分裂作用是通过细胞表面的受体，但是5-HT对肺动脉平滑肌细胞的促有丝分裂作用机制涉及的受体亚型至今未能够完全阐明。为此我们假设5-HT_(1B)受体在肺动脉平滑肌细胞增殖和肺血管构型重建中有重要作用，并做以下研究：
1．建立大鼠血管平滑肌细胞体外培养方法，分析流式细胞术与~3HTdR掺入法在观察细胞增殖指标上的相关性，为研究药物对细胞增殖和有丝分裂的影响和作用机制建立可靠的实验方法。
2．观察5-HT和5-HT_(1B／1D)受体激动剂舒马曲坦对肺动脉平滑肌细胞的促有丝分裂作用，研究选择性5-HT_(1B)受体拮抗剂SB_(224289)和选择性5-HT_(1D)受体拮抗剂BRL_(15572)对5-HT诱导的肺动脉平滑肌细胞增殖的影响，探讨5-HT对肺动脉平滑肌细胞促有丝分裂作用的5-HT_(1B)受体机制。探讨5-HT_(1B)受体和5-HT_(1D)受体在肺血管构型重建中的作用机制。
3．观察5-HT和5-HT_(1B／1D)受体激动剂舒马曲坦对肺动脉平滑肌细胞和主动脉平滑肌细胞的促有丝分裂作用的差异。比较选择性5-HT_(1B)受体拮抗剂和选择性5-HT_(1D)受体拮抗剂对5-HT诱导的肺动脉平滑肌细胞增殖和主动脉平滑肌细胞增殖的影响的差异，探讨5-HT对主动脉平滑肌细胞的促有丝分裂作用机制。根据主动脉和肺动脉平滑肌细胞对5-HT的促有丝分裂反应的差异性和5-HT_(1B)受体拮抗剂对主动脉和肺动脉平滑肌细胞增殖的影响的差异，探讨5-HT_(1B)受体拮抗剂成为高选择性的抗肺
动脉高压新药先导物的可能性。
方法
1.采用贴块法分离培养大鼠肺动脉平滑肌细胞和主动脉平滑肌细胞。
用含20%FBS的DMEM培养液原代培养平滑肌细胞，用含5%FBS的
DMEM培养液作Vehicle传代培养平滑肌细胞。用不同浓度的FBS刺激细
胞生长，分析流式细胞术与’H一TdR掺人法在细胞增殖指标上的相关性。
2.施加5一HT、舒马曲坦和选择性5一HTIB受体拮抗剂SB224289、选择性
5一HTID受体拮抗剂BRL15572等处理因素。用四哇盐(M，I’r)比色法计数平
滑肌细胞增殖，观察5一HT、舒马曲坦、SB224289和BRL15572对平滑肌细胞增殖
的影响。
3.用，H一胸腺嗜咙掺人法观察细胞DNA合成情况，比较实验组与对
照组的cPm值，分析特异性的5一HT受体激动剂和5一HT受体拮抗剂对肺
动脉和主动脉平滑肌细胞的DNA合成的促进和抑制作用，证明5一HTI。受
体是5一HT诱导肺动脉平滑肌细胞有丝分裂的作用点之一。探讨5一HT
对主动脉平滑肌细胞的促有丝分裂作用机制。研究选择性5一HT;B受体拮
抗剂SB2242s9、选择性5一HT，。受体拮抗剂BRL15572对5一HT诱导的平滑肌细
胞有丝分裂的影响。
4.用流式细胞术法分析细胞周期，配合M，rr检测观察细胞增殖，并探
讨5一HT对细胞分裂周期的影响以及选择性5一HT受体拮抗剂影响平滑
肌细胞有丝分裂所作用的细胞周期。
结果
1.大鼠血管平滑肌细胞培养及细胞增殖的考察方法
流式细胞仪指标(PI和SPF)与，H一TdR掺人实验指标(cPm)有极为
显著的相关关系。流式细胞仪技术与MTr活细胞染色技术联合应用考察
细胞增殖和有丝分裂比单独应用一种指标更能准确反映细胞增殖和有丝分
裂情况。
2.5一HT对肺动脉平滑肌细胞的促有丝分裂作用机制
(1)5一HT和舒马曲坦对PASMC的促有丝分裂作用
M竹法检测5一HT10一6 moFL一10一‘moFL可促进肺动脉平滑肌细胞
的增殖，增殖率分别为256%士18%;228%土29%;201%土17%。舒马曲
坦10一6 moFL一10一8 moFL也可促进肺动脉平滑肌细胞增殖，增殖率分别
为从5%士2一%;220%士25%和199%士25%。5一HT从20一6一10一‘moF
L和舒马曲坦从10一6一10一”moFL均可以使肺动脉平滑肌细胞的增殖率增
加，并呈剂量依赖关系。
流式细胞仪分析5一HT10一5 moFL一10一7 moFL的细胞增殖指数(Pro-
liferation Index)为23 .7%土1 .1%，22.6%土0.7%，20.5%土0.8%;S期
细胞分数(S一Phase Cell Fraetion)为8 .5%土1 .3%，6.9%土0.4%，5.9%
土0.5%。舒马曲坦10一，，10一6 moUL的增殖指数为23.9%土0.8%和21.
2%士0.8%;S期细胞分数为8.8%土0.3%和6.6%士0.5%。空白对照组
的增殖指数和S期细胞分数分别为17.59%士0.5%和4.68%土0.7%。5
一HT和舒马曲坦能够促进肺动脉平滑肌细胞从Go/G，期进入S期，然后进
人GZ/M期。5一HT对肺动脉平滑肌细胞的促有丝分裂作用可能有5-
HT:二1。受体的参与。
(2)SB2242s9和BRL15572对5一HT诱导的PASMC增殖的影响
MTr法检测SB22428，从10一’moFL到10一‘“moFL的浓度能剂量依赖地
抑制5一?
Objective
Serotonin (5 - hydroxytryptamine, 5 - HT) plays an important role in pulmonary hypertension. It was reported that there was an increased expression of 5 - HT1B receptor mRNA in monocrotaline (MCT) induced pulmonary hypertension rats. Also, hypoxia increased pulmonary vascular remodeling in wild - type mice and this was reduced in the 5 - HT1B receptor knockout mice. Furthermore , hypoxia increased 5 - HT1 receptor - mediated constriction of pulmonary arteries from the wild - type mice and this was attenuated in the 5 - HT1B receptor knockout mice. These evidences suggested 5 - HT1B receptor plays an important role in the development of PHT. Serotonin is one of vasoactive substances and has been recognized to cause proliferation of a variety of cells. Several studies have shown that the mitogenic action of serotonin is initiated through its binding to cells surface receptors. Yet, the 5 - HT receptor mechanism of pulmonary artery smooth muscle cell proliferation and mitogenesis has not yet been fully e-licited. So, the objetive of the present study covers the following aspects.
1. To investigate the mitogenetic effects of 5 - HT and, a 5 - HT1B/1D receptor agonist sumatriptan (Sum) on pulmonary artery smooth muscle cells (PASMC) , to identify the role of 5 - HT1B/1D receptors in PASMC proliferation
and pulmonary remodeling.
2. To observe the effects of selective 5 - HT1B receptor antagonist SB224289 and selective 5 - HT1D receptor antagonist BRL15572 on PASMC proliferation induced by 5 - HT and sumatriptan, investigate the role of 5 - HT1B receptor in PASMC proliferation and pulmonary remodeling.
3. To observe the effect of 5 - HT and 5 - HT1B/1D receptor agonist sumatriptan on ASMC proliferation and DNA synthesis, observe the difference reaction of PASMC and ASMC to 5 - HT and 5 - HT1B/1D receptor agonist. Study the effect of selective 5 - HT1B receptor antagonist SB224289 and selective 5 - HT1D receptor antagonist BRL15572 on ASMC proliferation induced by 5 - HT, investigate the mitogenic mechanism of ASMC.
4. According to the different mitogenic mechanism between ASMC and PASMC, research high selectivity and efficiency new medicine for the treatment of pulmonary remodeling and pulmonary hypertension, especially from the point of 5 - HT1B receptor and 5 - HT1B receptor antagonists.
Methods
1. PASMC and ASMC were isolated from rat pulmonary artery and aorta. DMEM culture media with 20% FBS were used in primary culture; DMEM culture media with 5% FBS were used as vehicle; 5-HT, sumatriptan, 5 - HT1B
receptor antagonist SB22489 and selective 5 - HT1D receptor antagonist BRL15572 were used as treatments.
2. Proliferation of cultured PASMC and ASMC were evaluated by MTT assay , cells DNA synthesis were evaluated by 3 H - TdR incorporation, and cell cycle analysis, proliferation index (PI) , s - phase cell fraction (SPF) were performed by flow cytometry (FCM).
3. All data were expressed as mean value standard deviation. Statistic a-nalysis was done by Statistical Program for Social Sciences (SPSS) Software. One Way ANOVA analyze multi - groups comparisons. Comparisons between groups were tested by LSD (least significant difference) test. P < 0. 05 were
considered to be statistical significant.
Results
1. SMC culture and evaluation of cell proliferation method
PI and SPF performed by FCM were closely correlated with cpm that was acquired from 3H -TdR incorporation tests. It is a better way to use MTT assay and FCM method in observing cells proliferation and mitogenesis than to use them alone.
2. Mechanism of PASMC mitogenesis induced by 5 - HT
(1) Effect of 5 - HT and sumatriptan on PASMC
It was found that 5 - HT 10-6 - 10-8 mole/L stimulated proliferation of PASMC by MTT assay. The proliferation rates ( PR) of 5 - HT 10 -6-10-8mol/ L treatments were 256%±18% ; 228%±29% and 201% ±17% respectively. Sumatriptan 10 -6-10-8mol/L mimicked the mitogenesis effect of 5 - HT. The proliferation rates of Sum were 245%±21% ; 220%±25% ; and 199%±25
肺血管构型重建是肺动脉高压的基本病理改变。肺血管平滑肌细胞增殖和肥大是肺血管构型重建主要特征。5-HT在肺动脉高压的发病过程中有非常重要的作用。研究发现野百合碱所致肺动脉高压大鼠的肺血流中有5-HT水平升高并且肺血管平滑肌有5-HT_(1B)受体mRNA的表达增加。5-HT_(1B)受体基因敲除小鼠能抵抗缺氧诱导的肺动脉高压和肺血管构型重建。5-HT是一种重要的血管活性物质，5-HT不仅刺激肺动脉平滑肌细胞收缩还可造成其增生和肥大，并且能与多种细胞生长因子发生协同作用。很多研究表明5-HT的促有丝分裂作用是通过细胞表面的受体，但是5-HT对肺动脉平滑肌细胞的促有丝分裂作用机制涉及的受体亚型至今未能够完全阐明。为此我们假设5-HT_(1B)受体在肺动脉平滑肌细胞增殖和肺血管构型重建中有重要作用，并做以下研究：
1．建立大鼠血管平滑肌细胞体外培养方法，分析流式细胞术与~3HTdR掺入法在观察细胞增殖指标上的相关性，为研究药物对细胞增殖和有丝分裂的影响和作用机制建立可靠的实验方法。
2．观察5-HT和5-HT_(1B／1D)受体激动剂舒马曲坦对肺动脉平滑肌细胞的促有丝分裂作用，研究选择性5-HT_(1B)受体拮抗剂SB_(224289)和选择性5-HT_(1D)受体拮抗剂BRL_(15572)对5-HT诱导的肺动脉平滑肌细胞增殖的影响，探讨5-HT对肺动脉平滑肌细胞促有丝分裂作用的5-HT_(1B)受体机制。探讨5-HT_(1B)受体和5-HT_(1D)受体在肺血管构型重建中的作用机制。
3．观察5-HT和5-HT_(1B／1D)受体激动剂舒马曲坦对肺动脉平滑肌细胞和主动脉平滑肌细胞的促有丝分裂作用的差异。比较选择性5-HT_(1B)受体拮抗剂和选择性5-HT_(1D)受体拮抗剂对5-HT诱导的肺动脉平滑肌细胞增殖和主动脉平滑肌细胞增殖的影响的差异，探讨5-HT对主动脉平滑肌细胞的促有丝分裂作用机制。根据主动脉和肺动脉平滑肌细胞对5-HT的促有丝分裂反应的差异性和5-HT_(1B)受体拮抗剂对主动脉和肺动脉平滑肌细胞增殖的影响的差异，探讨5-HT_(1B)受体拮抗剂成为高选择性的抗肺
动脉高压新药先导物的可能性。
方法
1.采用贴块法分离培养大鼠肺动脉平滑肌细胞和主动脉平滑肌细胞。
用含20%FBS的DMEM培养液原代培养平滑肌细胞，用含5%FBS的
DMEM培养液作Vehicle传代培养平滑肌细胞。用不同浓度的FBS刺激细
胞生长，分析流式细胞术与’H一TdR掺人法在细胞增殖指标上的相关性。
2.施加5一HT、舒马曲坦和选择性5一HTIB受体拮抗剂SB224289、选择性
5一HTID受体拮抗剂BRL15572等处理因素。用四哇盐(M，I’r)比色法计数平
滑肌细胞增殖，观察5一HT、舒马曲坦、SB224289和BRL15572对平滑肌细胞增殖
的影响。
3.用，H一胸腺嗜咙掺人法观察细胞DNA合成情况，比较实验组与对
照组的cPm值，分析特异性的5一HT受体激动剂和5一HT受体拮抗剂对肺
动脉和主动脉平滑肌细胞的DNA合成的促进和抑制作用，证明5一HTI。受
体是5一HT诱导肺动脉平滑肌细胞有丝分裂的作用点之一。探讨5一HT
对主动脉平滑肌细胞的促有丝分裂作用机制。研究选择性5一HT;B受体拮
抗剂SB2242s9、选择性5一HT，。受体拮抗剂BRL15572对5一HT诱导的平滑肌细
胞有丝分裂的影响。
4.用流式细胞术法分析细胞周期，配合M，rr检测观察细胞增殖，并探
讨5一HT对细胞分裂周期的影响以及选择性5一HT受体拮抗剂影响平滑
肌细胞有丝分裂所作用的细胞周期。
结果
1.大鼠血管平滑肌细胞培养及细胞增殖的考察方法
流式细胞仪指标(PI和SPF)与，H一TdR掺人实验指标(cPm)有极为
显著的相关关系。流式细胞仪技术与MTr活细胞染色技术联合应用考察
细胞增殖和有丝分裂比单独应用一种指标更能准确反映细胞增殖和有丝分
裂情况。
2.5一HT对肺动脉平滑肌细胞的促有丝分裂作用机制
(1)5一HT和舒马曲坦对PASMC的促有丝分裂作用
M竹法检测5一HT10一6 moFL一10一‘moFL可促进肺动脉平滑肌细胞
的增殖，增殖率分别为256%士18%;228%土29%;201%土17%。舒马曲
坦10一6 moFL一10一8 moFL也可促进肺动脉平滑肌细胞增殖，增殖率分别
为从5%士2一%;220%士25%和199%士25%。5一HT从20一6一10一‘moF
L和舒马曲坦从10一6一10一”moFL均可以使肺动脉平滑肌细胞的增殖率增
加，并呈剂量依赖关系。
流式细胞仪分析5一HT10一5 moFL一10一7 moFL的细胞增殖指数(Pro-
liferation Index)为23 .7%土1 .1%，22.6%土0.7%，20.5%土0.8%;S期
细胞分数(S一Phase Cell Fraetion)为8 .5%土1 .3%，6.9%土0.4%，5.9%
土0.5%。舒马曲坦10一，，10一6 moUL的增殖指数为23.9%土0.8%和21.
2%士0.8%;S期细胞分数为8.8%土0.3%和6.6%士0.5%。空白对照组
的增殖指数和S期细胞分数分别为17.59%士0.5%和4.68%土0.7%。5
一HT和舒马曲坦能够促进肺动脉平滑肌细胞从Go/G，期进入S期，然后进
人GZ/M期。5一HT对肺动脉平滑肌细胞的促有丝分裂作用可能有5-
HT:二1。受体的参与。
(2)SB2242s9和BRL15572对5一HT诱导的PASMC增殖的影响
MTr法检测SB22428，从10一’moFL到10一‘“moFL的浓度能剂量依赖地
抑制5一?
Objective
Serotonin (5 - hydroxytryptamine, 5 - HT) plays an important role in pulmonary hypertension. It was reported that there was an increased expression of 5 - HT1B receptor mRNA in monocrotaline (MCT) induced pulmonary hypertension rats. Also, hypoxia increased pulmonary vascular remodeling in wild - type mice and this was reduced in the 5 - HT1B receptor knockout mice. Furthermore , hypoxia increased 5 - HT1 receptor - mediated constriction of pulmonary arteries from the wild - type mice and this was attenuated in the 5 - HT1B receptor knockout mice. These evidences suggested 5 - HT1B receptor plays an important role in the development of PHT. Serotonin is one of vasoactive substances and has been recognized to cause proliferation of a variety of cells. Several studies have shown that the mitogenic action of serotonin is initiated through its binding to cells surface receptors. Yet, the 5 - HT receptor mechanism of pulmonary artery smooth muscle cell proliferation and mitogenesis has not yet been fully e-licited. So, the objetive of the present study covers the following aspects.
1. To investigate the mitogenetic effects of 5 - HT and, a 5 - HT1B/1D receptor agonist sumatriptan (Sum) on pulmonary artery smooth muscle cells (PASMC) , to identify the role of 5 - HT1B/1D receptors in PASMC proliferation
and pulmonary remodeling.
2. To observe the effects of selective 5 - HT1B receptor antagonist SB224289 and selective 5 - HT1D receptor antagonist BRL15572 on PASMC proliferation induced by 5 - HT and sumatriptan, investigate the role of 5 - HT1B receptor in PASMC proliferation and pulmonary remodeling.
3. To observe the effect of 5 - HT and 5 - HT1B/1D receptor agonist sumatriptan on ASMC proliferation and DNA synthesis, observe the difference reaction of PASMC and ASMC to 5 - HT and 5 - HT1B/1D receptor agonist. Study the effect of selective 5 - HT1B receptor antagonist SB224289 and selective 5 - HT1D receptor antagonist BRL15572 on ASMC proliferation induced by 5 - HT, investigate the mitogenic mechanism of ASMC.
4. According to the different mitogenic mechanism between ASMC and PASMC, research high selectivity and efficiency new medicine for the treatment of pulmonary remodeling and pulmonary hypertension, especially from the point of 5 - HT1B receptor and 5 - HT1B receptor antagonists.
Methods
1. PASMC and ASMC were isolated from rat pulmonary artery and aorta. DMEM culture media with 20% FBS were used in primary culture; DMEM culture media with 5% FBS were used as vehicle; 5-HT, sumatriptan, 5 - HT1B
receptor antagonist SB22489 and selective 5 - HT1D receptor antagonist BRL15572 were used as treatments.
2. Proliferation of cultured PASMC and ASMC were evaluated by MTT assay , cells DNA synthesis were evaluated by 3 H - TdR incorporation, and cell cycle analysis, proliferation index (PI) , s - phase cell fraction (SPF) were performed by flow cytometry (FCM).
3. All data were expressed as mean value standard deviation. Statistic a-nalysis was done by Statistical Program for Social Sciences (SPSS) Software. One Way ANOVA analyze multi - groups comparisons. Comparisons between groups were tested by LSD (least significant difference) test. P < 0. 05 were
considered to be statistical significant.
Results
1. SMC culture and evaluation of cell proliferation method
PI and SPF performed by FCM were closely correlated with cpm that was acquired from 3H -TdR incorporation tests. It is a better way to use MTT assay and FCM method in observing cells proliferation and mitogenesis than to use them alone.
2. Mechanism of PASMC mitogenesis induced by 5 - HT
(1) Effect of 5 - HT and sumatriptan on PASMC
It was found that 5 - HT 10-6 - 10-8 mole/L stimulated proliferation of PASMC by MTT assay. The proliferation rates ( PR) of 5 - HT 10 -6-10-8mol/ L treatments were 256%±18% ; 228%±29% and 201% ±17% respectively. Sumatriptan 10 -6-10-8mol/L mimicked the mitogenesis effect of 5 - HT. The proliferation rates of Sum were 245%±21% ; 220%±25% ; and 199%±25
引文
1. Wen JK, Shi Y. New approaches on regulatory mechanisms of vascular smooth muscle cell proliferation. Proceedings of the physiology, 27(2): 149-56, 1996
2．韩启德，文允镒．血管生物学．北京医科大学、中国协和医科大学联合出版社1997 3-23
3. Wang Huai-Liang, Stephen A Kilfeather, Graeme R Martin et al Effects of heparin on growth factor- induced mitogenic and proliferative responses of rat pulmonary artery smooth muscle cells. Acta Pharmacologica Sinica Sep; 18(5): 397-400, 1997
4. WANG Huai-Liang, ZHANG Xin-Hua, JIN Xin, et al. Tetrandrine inhibited chronic "inflammatory" pulmonary hypertension in rats Acta Pharmacologica Sinica Sep; 18(5): 401-404, 1997
5. Lee SL, Wang WW, Finlay GA, Fanburg BL Serotonin stimulates mitogen-activated protein kinase activity through the formation of superoxide anion. Am J Physiol Aug; 277(2 Pt 1): L282-91, 1999
6. Lee SL, Wang WW, Lanzillo JJ and Fanburg BL Serotonin produce both hyperplasia and hypertrophy of bovine pulmonnry artery smooth muscle cells in culture. Am. J. Physiol. 266(Lung cell. Mol. Physiol. 10): L46-L52, 1994.
7. Lee SL, Wang WW and Fanburg BL Nitro Prusside inhibit serotonin-induced mitogenesis and tyrosine phosphory-lation of smooth muscle cells. Am J Phystol 270(Lung Cell. mol. Physiol. 14): L362-L367, 1996.
8. Lee SL, Wang WW and Fanburg BL Association of tyrosine phosphorylation of GTPase activating protein with mitogenic action of serotonin Am. J. Physiol. 272(Cell Physiol. 41): C223-CA30, 1997
9. Seuwen, K., I. Magnaldol, and J. Pouyssegur. Serotonin stimulates DNA Synthesis in fibroblasts acting through 5-HT1B receptors coupled to a Giprotein. Nature 335: 254-256, 1988
10. Ishizuka, J., R. D. Beauchamp, C. M. Townsend, Jr., G. H. Greeley, Jr., and J. C. Thompson. Receptor-mediated autocrine growth-stimulatory effect of 5-hydroxytryptamine on human pancreatic carcinoid cells. J. Cell. Physiol 150: 1-7, 1992.
11. Pitt B. R., W Wang, A. R. Steve, K. D. Blakely, I. Reynolds, and R Davies. Serotonin increases DNA synthesis in rat proximal and distal pulmonary vascular smooth muscle cells in culture. Am. J Physiol. 266: (Lung Cell. Mol. Physiol. 10): L178-L186. 1994.
12. Parrott D. P., R. M. lockey and C. P. Bright. Comparison of the mitogenic activity of angiotension Ⅱ and serotonin on porcine arterial smooth muscle cell Atherosclerosis 88: 213-218, 1991.
13. Corson, M. A., R. W. Alexander, and B. C. Berk. 5-HT2 receptor mRNA is over expressed in cultured rat aortic smooth muscle cells relative to normal aorta. Am. J. Physiol. 262(Cell Physiol. 31): C309-C315, 1992
14. Crowley. S. T., E. C. Dempsey., k. B. Horwitz, and L. D. Horwitz. Platelet-induced vascular smooth muscle cell proliferation is modulated by the growth amplification factors serotonin and adenosine diphosphate. Circulation 90: 1908-1918, 1994
15. Raymond, J. R., F. J. Alberts, J. P. Middleton, R. J. Lefkowitz, M. G. Caron, L. M. Obeid, and V. W. Dennis. 5- HT1A and histamine H1 receptors in HeLa cells stimulate phosphoinositide hydrolysis and phosphate uptake via distinct G protein pools. J. biol. Chem. 266: 372-379, 1991
16. S. Eddahibi, V. Fabre, C. Boni, M. P. Martres, B. Raffestin, M. Hamon, S. Adnot Induction of serotonin transporter by hypoxia in pulmonary vascular smooth muscle cells Relationship with the mitogenic action of serotonin Circ Res Feb 19; 84(3): 329-336, 1999
17. Sushil K. Sharma, Peter Zahradka, Donald Chapman, Hideo Kumamoto, Nobuakira Takeda2 and Naranjan S. Dhalla Inhibition of Serotonin-Induced Vascular Smooth Muscle Cell Proliferation by Sarpogrelatel J Pharmacol Exp Ther Sep; 290(3): 1475-81, 1999
18. Dashou Wang, Martin Post, and Ernest Cutz Expression of Serotonin Receptor 2c in Rat Type Ⅱ Pneumocytes Am. J. Respir Cell Mol. Biol Volume 20, Number 6, June 1175-1180, 1999
19. Freitag, A., I. Wessler, and K. Racke. Characterization of 5-hydroxytryptamine releases from isolated rabbit and rat trachea: the role of neuroendocrine epithelial cells and mast cells. Naunyn Schmiedeberg's Arch. Pharmacol. 353: 55-63, 1995
20. Koenig J. A., and J. M. Edwardson. Intracellular trafficking of the muscarinic acetylcholine receptor: importance of subtype and cell type. Mol. Pharmacol. 49: 351-359, 1996
21. Watanabe T, Pakala R, Katagiri T, Benedict CR. Synergistic effect of urotensin Ⅱ with serotonin on vascular smooth muscle cell proliferation J Hypertens Dec; 19(12): 2191-6, 2001
22. Seuwen K. and J. Pouyssegur. Serotonin as a growth factor Biochem Pharmacol. 39: 985-990, 1990
23. I. Massberg S, Sausbier M, Klatt P, Bauer M, Pfeifer A, Siess W, Fassler R, Ruth P, Krombach F, Hofmann F. Increased adhesion and aggregation of platelets lacking cyclic guanosine 3', 5'-monophosphate kinase J Exp Med Apr 19; 189(8): 1255-64, 1999
24. Watanabe T, Pakala R, Katagiri T, Benedict CR. Serotonin potentiates angiotensin Ⅱ-induced vascular smooth muscle cell proliferation. Atherosclerosis Dec; 159(2): 269-79, 2001
25. Pakala R, Katagiri T, Benedict CR. Monocyte chemotactic protein 1 amplifies serotonin-induced vascular smooth muscle cell proliferation. Watanabe T J Vasc Res Jul-Aug; 38(4): 341-9, 2001
26. Nilsson T, Longmore J, Shaw D, Pantev E, Bard JA, Branchek T, Edvinsson L Characterisation of 5-HT receptors in human coronary arteries by molecular and pharmacological techniques. Eur J Pharmacol May 7; 372(1): 49-56, 1999
27. Ullmer C, Schmuck K, Kalkman HO, Lubbert H. Expression of serotonin receptor mRNAs in blood vessels FEBS Lett Aug 21; 370(3): 215-21,
1995
28. Wang HL, Dong X, Zhang XH, Xing J 5-HT(1B) receptor augmented 5-HT vasoconstrictor response of pulmonary artery in monocrotaline-induced pulmonary hypertensive rats. Acta Pharmacol Sin Mar; 22(3): 269-73, 2001
2．韩启德，文允镒．血管生物学．北京医科大学、中国协和医科大学联合出版社1997 3-23
3. Wang Huai-Liang, Stephen A Kilfeather, Graeme R Martin et al Effects of heparin on growth factor- induced mitogenic and proliferative responses of rat pulmonary artery smooth muscle cells. Acta Pharmacologica Sinica Sep; 18(5): 397-400, 1997
4. WANG Huai-Liang, ZHANG Xin-Hua, JIN Xin, et al. Tetrandrine inhibited chronic "inflammatory" pulmonary hypertension in rats Acta Pharmacologica Sinica Sep; 18(5): 401-404, 1997
5. Lee SL, Wang WW, Finlay GA, Fanburg BL Serotonin stimulates mitogen-activated protein kinase activity through the formation of superoxide anion. Am J Physiol Aug; 277(2 Pt 1): L282-91, 1999
6. Lee SL, Wang WW, Lanzillo JJ and Fanburg BL Serotonin produce both hyperplasia and hypertrophy of bovine pulmonnry artery smooth muscle cells in culture. Am. J. Physiol. 266(Lung cell. Mol. Physiol. 10): L46-L52, 1994.
7. Lee SL, Wang WW and Fanburg BL Nitro Prusside inhibit serotonin-induced mitogenesis and tyrosine phosphory-lation of smooth muscle cells. Am J Phystol 270(Lung Cell. mol. Physiol. 14): L362-L367, 1996.
8. Lee SL, Wang WW and Fanburg BL Association of tyrosine phosphorylation of GTPase activating protein with mitogenic action of serotonin Am. J. Physiol. 272(Cell Physiol. 41): C223-CA30, 1997
9. Seuwen, K., I. Magnaldol, and J. Pouyssegur. Serotonin stimulates DNA Synthesis in fibroblasts acting through 5-HT1B receptors coupled to a Giprotein. Nature 335: 254-256, 1988
10. Ishizuka, J., R. D. Beauchamp, C. M. Townsend, Jr., G. H. Greeley, Jr., and J. C. Thompson. Receptor-mediated autocrine growth-stimulatory effect of 5-hydroxytryptamine on human pancreatic carcinoid cells. J. Cell. Physiol 150: 1-7, 1992.
11. Pitt B. R., W Wang, A. R. Steve, K. D. Blakely, I. Reynolds, and R Davies. Serotonin increases DNA synthesis in rat proximal and distal pulmonary vascular smooth muscle cells in culture. Am. J Physiol. 266: (Lung Cell. Mol. Physiol. 10): L178-L186. 1994.
12. Parrott D. P., R. M. lockey and C. P. Bright. Comparison of the mitogenic activity of angiotension Ⅱ and serotonin on porcine arterial smooth muscle cell Atherosclerosis 88: 213-218, 1991.
13. Corson, M. A., R. W. Alexander, and B. C. Berk. 5-HT2 receptor mRNA is over expressed in cultured rat aortic smooth muscle cells relative to normal aorta. Am. J. Physiol. 262(Cell Physiol. 31): C309-C315, 1992
14. Crowley. S. T., E. C. Dempsey., k. B. Horwitz, and L. D. Horwitz. Platelet-induced vascular smooth muscle cell proliferation is modulated by the growth amplification factors serotonin and adenosine diphosphate. Circulation 90: 1908-1918, 1994
15. Raymond, J. R., F. J. Alberts, J. P. Middleton, R. J. Lefkowitz, M. G. Caron, L. M. Obeid, and V. W. Dennis. 5- HT1A and histamine H1 receptors in HeLa cells stimulate phosphoinositide hydrolysis and phosphate uptake via distinct G protein pools. J. biol. Chem. 266: 372-379, 1991
16. S. Eddahibi, V. Fabre, C. Boni, M. P. Martres, B. Raffestin, M. Hamon, S. Adnot Induction of serotonin transporter by hypoxia in pulmonary vascular smooth muscle cells Relationship with the mitogenic action of serotonin Circ Res Feb 19; 84(3): 329-336, 1999
17. Sushil K. Sharma, Peter Zahradka, Donald Chapman, Hideo Kumamoto, Nobuakira Takeda2 and Naranjan S. Dhalla Inhibition of Serotonin-Induced Vascular Smooth Muscle Cell Proliferation by Sarpogrelatel J Pharmacol Exp Ther Sep; 290(3): 1475-81, 1999
18. Dashou Wang, Martin Post, and Ernest Cutz Expression of Serotonin Receptor 2c in Rat Type Ⅱ Pneumocytes Am. J. Respir Cell Mol. Biol Volume 20, Number 6, June 1175-1180, 1999
19. Freitag, A., I. Wessler, and K. Racke. Characterization of 5-hydroxytryptamine releases from isolated rabbit and rat trachea: the role of neuroendocrine epithelial cells and mast cells. Naunyn Schmiedeberg's Arch. Pharmacol. 353: 55-63, 1995
20. Koenig J. A., and J. M. Edwardson. Intracellular trafficking of the muscarinic acetylcholine receptor: importance of subtype and cell type. Mol. Pharmacol. 49: 351-359, 1996
21. Watanabe T, Pakala R, Katagiri T, Benedict CR. Synergistic effect of urotensin Ⅱ with serotonin on vascular smooth muscle cell proliferation J Hypertens Dec; 19(12): 2191-6, 2001
22. Seuwen K. and J. Pouyssegur. Serotonin as a growth factor Biochem Pharmacol. 39: 985-990, 1990
23. I. Massberg S, Sausbier M, Klatt P, Bauer M, Pfeifer A, Siess W, Fassler R, Ruth P, Krombach F, Hofmann F. Increased adhesion and aggregation of platelets lacking cyclic guanosine 3', 5'-monophosphate kinase J Exp Med Apr 19; 189(8): 1255-64, 1999
24. Watanabe T, Pakala R, Katagiri T, Benedict CR. Serotonin potentiates angiotensin Ⅱ-induced vascular smooth muscle cell proliferation. Atherosclerosis Dec; 159(2): 269-79, 2001
25. Pakala R, Katagiri T, Benedict CR. Monocyte chemotactic protein 1 amplifies serotonin-induced vascular smooth muscle cell proliferation. Watanabe T J Vasc Res Jul-Aug; 38(4): 341-9, 2001
26. Nilsson T, Longmore J, Shaw D, Pantev E, Bard JA, Branchek T, Edvinsson L Characterisation of 5-HT receptors in human coronary arteries by molecular and pharmacological techniques. Eur J Pharmacol May 7; 372(1): 49-56, 1999
27. Ullmer C, Schmuck K, Kalkman HO, Lubbert H. Expression of serotonin receptor mRNAs in blood vessels FEBS Lett Aug 21; 370(3): 215-21,
1995
28. Wang HL, Dong X, Zhang XH, Xing J 5-HT(1B) receptor augmented 5-HT vasoconstrictor response of pulmonary artery in monocrotaline-induced pulmonary hypertensive rats. Acta Pharmacol Sin Mar; 22(3): 269-73, 2001