Characterization of the angiotensin II AT1 receptor subtype involved in DNA synthesis in cultured vascular smooth muscle cells.

1. This study was undertaken in cultured vascular smooth muscle cells to characterize the angiotensin II (AII) AT1 receptor subtype involved in DNA synthesis because (i) the AII receptor involved in vascular proliferation has previously been characterized in vitro in rat aortic cells and identified as an AT1 subtype and (ii) molecular cloning and biochemical studies have provided evidence for the existence of different AT1 receptor subtypes. 2. In cultured rat aortic vascular smooth muscle (VSMC), exposure to AII (0.1 to 100 nM) resulted in a concentration-dependent increase in [3H]-thymidine incorporation with an EC50 of 1.41 +/- 0.51 nM. Maximal stimulation was observed in the presence of 100 nM AII and corresponded to 271 +/- 40% of basal [3H]-thymidine incorporation. 3. To characterize the AII AT1 receptor subtype involved in this effect, cells were exposed to AII (3 nM) in the absence or presence of increasing concentrations of various AII receptor antagonists. The stimulatory effect of AII (3 nM) on [3H]-thymidine incorporation in VSMC was antagonized by the non-selective AT1/AT2 receptor antagonist, [Sar1, Ile8]-AII (IC50 = 5.6 nM), by the AT1A/AT1B receptor antagonist, losartan (IC50 = 10.5 nM) and the AT1 receptor antagonist, L-158809 (IC50 = 0.20 nM). The selective AT2 receptor ligand, CGP 42112A, antagonized AII-induced [3H]-thymidine incorporation with an IC50 of 6.3 +/- 1.3 microM while the AT2/AT1B receptor antagonist, PD 123319, was found to be almost inactive (IC50 > 10 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the muscarinic receptor subtype involved in phosphoinositide metabolism in bovine tracheal smooth muscle

1. The muscarinic receptor subtype involved in the methacholine-induced enhancement of phosphoinositide metabolism in bovine tracheal smooth muscle was identified by using the M2-selective antagonist AF-DX 116 and the M3-selective antagonist 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) methobromide, in addition to the M1-selective antagonist pirenzepine, in a classical Schild analysis. 2. All the antagonists shifted the methacholine dose-response curve to the right in a parallel and concentration-dependent fashion, yielding Schild plots with slopes not significantly different from unity. The pA2 values (6.94, 6.32 and 8.54 for pirenzepine, AF-DX 116 and 4-DAMP methobromide respectively) indicate that it is the M3 (smooth muscle/glandular), but not the M2 (cardiac) muscarinic receptor subtype, present in this tissue, that mediates phosphoinositide turnover, in accordance with our previous contractile studies. 3. The results provide additional evidence for the involvement of phosphoinositide turnover in the pharmacomechanical coupling between muscarinic receptor stimulation and contraction in (bovine tracheal) smooth muscle.     

Effect of endotoxin on the angiotensin II receptor in cultured vascular smooth muscle cells.

1. In some tissues, a decrease in the number of cell surface receptors and alterations of the receptor coupling have been proposed as possible mechanisms mediating the deleterious effects of bacterial endotoxin in septic shock. 2. The effects of bacterial lipopolysaccharide (Escherichia coli 0111-B4; LPS) on vascular angiotensin II and vasopressin receptors have been examined in cultured aortic smooth muscle cells (SMC) of the rat by use of radioligand binding techniques. 3. In vascular SMC exposed to 1 micrograms ml-1 endotoxin for 24 h, a significant increase in angiotensin II binding was found. The change in [125I]-angiotensin II binding corresponded to an increase in the number of receptors whereas the affinity of the receptors was not affected by LPS. In contrast, no change in [3H]-vasopressin binding was observed. 4. The pharmacological characterization of angiotensin II binding sites in control and LPS-exposed cells demonstrated that LPS induced an increase in the AT1 subtype of the angiotensin II receptors. Receptor coupling as evaluated by measuring total inositol phosphates was not impaired by LPS. 5. The effect of LPS on the angiotensin II receptor was dose-, time- and protein-synthesis dependent and was associated with an increased expression of the receptor gene. 6. The ability of LPS to increase angiotensin II binding in cultured vascular SMC was independent of the endotoxin induction of NO-synthase. 7. These results suggest that, besides inducing factors such as cytokines and NO-synthase, endotoxin may enhance the expression of cell surface receptors. The surprising increase in angiotensin II binding in LPS exposed VSM cells may represent an attempt by the cells to compensate for the decreased vascular responsiveness. It may also result from a non-specific LPS-related induction of genes.     

半胱氨酸对人血管平滑肌细胞增殖和凋亡的影响

目的 观察半胱氨酸(Cys)对人血管平滑肌细胞(VSMCs)增殖和凋亡的影响.方法 采用组织贴块法体外培养人VSMCs,用不同浓度的Cys培养液孵育细胞24h,用MTT法检测VSMCs增殖,流式细胞术检测VSMCs凋亡.RT-PCR法检测bax mRNA的表达.结果 体外培养的人VSMCs在终浓度为100、200、500和10001μmol/L Cys的培养液中孵育24h后的吸光度(A)值均高于对照组(P<0.05),表明Cys可诱导人VSMCs增殖;但同时.VSMCs凋亡细胞数明显高于对照组(P<0.05),bax的表达增强,表明Cys亦诱导了人VSMCs凋亡.结论 浓度较高时半胱氨酸可同时诱导VSMCs增殖和凋亡.     

Regulation of endothelin-1 production in cultured rat vascular smooth muscle cells

Endothelin-1 (ET-1) is secreted from all rat vascular smooth muscle cells (VSMCs) examined, in addition to endothelial cells (ECs). An average secretion rate of ET-1 from rat VSMCs was determined to be 10% that excreted from ECs. We examined the effects of 22 substances on ET-1 secretion from VSMCs and compared them with those from ECs. Transforming growth factor-beta1 (TGF-beta), acidic and basic fibroblast growth factors, epidermal growth factor, angiotensin II, and adrenaline stimulated ET-1 secretion from VSMCs, whereas forskolin, thrombin, and platelet-derived growth factor-BB reduced it. Only TGF-beta and phorbol ester elicited consistent effects on ET-1 secretion from VSMCs and ECs. Regulation of ET-1 and adrenomedullin secretion from VSMCs was distinctly different. These data suggest that ET- 1 production in VSMCs is regulated by a mechanism separate from that in ECs and from adrenomedullin production in VSMCs. Chromatographic analysis showed immunoreactive ET-1 secreted from VSMCs was mainly composed of big ET- 1, whereas approximately 90% of that from ECs was ET-1. By TGF-beta stimulation of VSMCs, the ratio of big ET-1 to ET-1 was further increased. Because big ET-1 is converted into ET-1 only on the surface of the ECs in the culture system, big ET-1 secreted from the VSMCs may function as a mediator transmitting a signal from VSMCs to ECs in vivo.     

牛磺酸对大鼠血管平滑肌细胞增殖的影响

观察了牛磺酸对自发性高血压大鼠（ＳＨＲ）和Ｗｉｓｔａｒ－Ｋｙｏｔｏ大鼠（ＷＫＹ）动脉血管平滑肌细胞（ＶＳＭＣ）增殖的影响．结果显示牛磺酸（１．０ｍｍｏｌＬ－１）虽然抑制ＷＫＹ的ＶＳＭＣ的增殖，对ＳＨＲ的ＶＳＭＣ的生长却无影响，同时发现牛磺酸（１０ｍｍｏｌＬ－１）也不能减低小牛血清所致的ＳＨＲ的ＶＳＭＣ内肌醇磷脂量的异常增加．提示牛磺酸虽然可以通过抑制ＷＫＹ的ＶＳＭＣ增殖进而在防治某些血管性病变中发挥作用，但是对于ＳＨＲ这一遗传性高血压大鼠，牛磺酸既不能抑制ＶＳＭＣ的异常增殖也未能阻止肌醇磷脂量的增加．     

普洛托品对兔胸主动脉平滑肌细胞增殖的影响

目的　观察普洛托品 (Pro)对正常和内皮素 (ET)诱导的血管平滑肌细胞 (VSMC)增殖的影响。方法　采用组织块贴壁法培养兔胸主动脉VSMC ,应用细胞计数、[3 H] TdR参入量、微量MTT比色测定及电镜法 ,观察Pro对VSMC增殖、DNA合成和细胞超微结构的变化。结果　ET对VSMC的增殖和DNA合成有显著的促进作用 ;1、10、10 0μmol·L-1浓度Pro能够逆转ET所致的 [3 H] TdR参入量、MTT比色的吸光度、细胞计数的增多及电镜下细胞超微结构的改变 ,且随着浓度的增加及时间的延长 ,抑制作用愈显著 ;10、10 0 μmol·L-1浓度Pro对未经ET处理的VSMC有抑制其增殖作用 ,但其抑制作用弱于ET处理组。结论　Pro可显著地抑制正常及ET诱导的VSMC增殖 ,并且对ET诱导的VSMC增殖抑制作用更明显 ,提示Pro可作为临床防治动脉粥样硬化的有效药物。     

Down-regulation of atrial natriuretic peptide receptor and cyclic GMP response in cultured rat vascular smooth muscle cells

Treatment of cultured rat vascular smooth muscle cells with human atrial natriuretic peptide (hANP) or Met(O)12hANP caused a similar and marked reduction (approximately 80%) of ANP receptor number (down-regulation). A second challenge with hANP stimulated the accumulation of intracellular cGMP in the down-regulated cells to the same extent as in control cells. These data suggest that ANP receptor sites are functionally heterogenous, the more abundant site being uncoupled from guanylate cyclase but susceptible to down-regulation.     

Cytokines and growth factors involved in apoptosis and proliferation of vascular smooth muscle cells

This review focuses on the role of cytokines and growth factors involved in the regulation of smooth muscle cells in an atherosclerotic plaque. As a plaque begins to develop, upon endothelial injury inflammatory cells within the lesion interact with the accumulating LDL, other inflammatory cells and smooth muscle cells and release cytokines and growth factors. The mediators released from the activated cells regulate the proliferation and/or survival of smooth muscle cells. This determines the stability and integrity of a plaque. New data emerging from various studies have provided novel insights into many of the cellular interactions and signaling mechanisms involving apoptosis of smooth muscle cells in the atherosclerotic plaques. A number of these studies, focusing on activation of inflammatory cells and the roles of chemokines, cytokines and growth factors, are addressed in this review.     

Ligustilide inhibits vascular smooth muscle cells proliferation

Proliferation and migration of vascular smooth muscle cells (VSMCs) are believed to develop atherosclerosis and venous bypass graft disease. Ligustilide is widely used to treat some pathological settings such as atherosclerosis and hypertension. The aim of this study was to examine the effect of ligustilide on VSMCs proliferation. The results show that ligustilide significantly inhibited VSMCs proliferation and cell cycle progression. Further analysis shows that ligustilide suppressed reactive oxygen species production and extracellular signal-related kinases (ERK), c-Jun N-terminal protein kinase (JNK), and p38 MAP kinase. Cells were treated with antioxidant, superoxide dismutase, catalase, and DPI, respectively, leading to repress ERK, JNK, and p38 activation. The inhibitors of mitogen activated protein kinase (MAPK), PD98059, SB203580, and Sp600125, inhibited cell proliferation. These findings suggest the antiproliferative effect of ligustilide was associated with the decrement of reactive oxygen species resulting in the suppression of MAPK pathway. Thus, ligustilide contribute to be the effective agent in preventing cardiovascular diseases.     

参麦注射液对培养大鼠主动脉平滑肌细胞增殖的影响和机理研究

目的 研究参麦注射液对血管平滑肌细胞增殖的影响和机理。方法 以培养的大鼠主动脉血管平滑肌细胞为模型，通过细胞计数及测量3H-胸腺嘧啶核苷掺入量观察细胞增殖情况，同时测定培养液中超氧化物歧化酶(SOD)活性、脂质过氧化物、前列环素(PGI2)的含量，比较分析加入不同剂量参麦注射液后上述指标的变化和相互关系。结果 不同剂量参麦注射液组的平滑肌细胞增殖及3H—胸腺嘧啶核苷掺入量均显著低于对照组，SOD活性及PGI2含量显著高于对照组，脂质过氧化程度显著降低。结论 参麦注射液对血管平滑肌细胞增殖具有抑制作用，并呈剂量依赖关系，其作用可能与增加SOD活性、降低脂质过氧化物水平，升高PGI2／TXA2有关。     

Human thrombin receptor-activating peptide-induced proliferation of cultured vascular smooth muscle cells exhibits species specificity

Thrombin receptor stimulation in vitro signals many cellular events that are associated with the response to vascular injury in vivo. Indeed, we have previously shown that human α-thrombin and the 14-amino acid human thrombin receptor-activating peptide (huTRAP-14) stimulate proliferation of cultured rat aortic smooth muscle cells (SMC). In the present studies, the mitogenic response of rabbit vascular SMC to thrombin and huTRAP-14 was assessed using [³H]thymidine incorporation and cell number. Results demonstrated that thrombin stimulated mitogenesis of rabbit vascular SMC in culture and that the thrombin response was dependent on proteolytic activity. However, huTRAP-14 was not mitogenic for rabbit vascular SMC. Thus, there are species differences in huTRAP-14 responsiveness. As rat and rabbit models continue to be used extensively to evaluate mechanisms and potential therapies for human restenosis, it is important to identify any species differences in the mechanism whereby thrombin exerts its biological effects. © 1995 Wiley-Liss, Inc.     

Calcineurin-NFAT signaling is involved in phenylephrine-induced vascular smooth muscle cell proliferation

Aim： Catecholamine-induced vascular smooth muscle cell （VSMC） proliferation is one of the major events in the pathogenesis of atherosclerosis and vascular remodeling. The calcineurin-NFAT pathway plays a role in regulating growth and differentiation in various cell types. We investigated whether the calcineurin-NFAT pathway was involved in the regulation of phenylephrine-induced VSMC proliferation. Methods： Proliferation of VSMC was measured using an MTT assay and cell counts. Localization of NFATcl was detected by immunofluorescence staining. NFATcl-DNA binding was determined by EMSA and luciferase activity analyses. NFATcl and calcineurin levels were assayed by immunoprecipitation. Results： Phenylephrine （PE, an α1-adrenoceptor agonist） increased VSMC proliferation and cell number. Prazosin （an α1-adrenoceptor antagonist）, cyclosporin A （CsA, an inhibitor of calcineurin） and chelerythrine （an inhibitor of PKC） decreased PE-induced proliferation and cell number. Additional treatment of VSMC with CsA or chelerythrine further inhibited proliferation and cell number in the chelerythrine-pretreatment group and the CsA-pretreatment group. CsA and chelerythrine alone had no effect on either absorbance or cell number. CsA decreased PE-induced calcineurin levels and activity. NFATcl was translocated from the cytoplasm to the nucleus upon treatment with PE. This translocation was reversed by CsA. CsA decreased the PE-induced NFATcl level in the nucleus. PE increased NFAT＇s DNA binding activity and NFAT-dependent reporter gene expression. CsA blocked these effects. Conclusion： CsA partially suppresses PE-induced VSMC proliferation by inhibiting calcineurin activity and NFATcl nuclear translocation. The calcineurin-NFATcl pathway is involved in the hyperplastic growth of VSMC induced by phenylephrine.     

Cicletanine and eicosanoids in cultured vascular smooth muscle cells

Cicletanine, a new antihypertensive, slightly diuretic, drug was tested for its effects on arachidonic acid (AA) metabolism in cultured smooth muscle cells from rat aorta. Cicletanine significantly enhanced the production of prostacyclin from both exogenously added arachidonic acid and endogenous sources. This effect is not mediated through inhibition of the lipoxygenase pathways and occurs despite a slight but definite inhibition of the activity of acyl hydrolase. Taken together, these results indicate that the antihypertensive properties of cicletanine might be associated, at least in part, with activation of the AA cascade through the cyclooxygenase pathway.     

Characterization of Ca(2+) channels involved in endothelin-1-induced mitogenic responses in vascular smooth muscle cells.

Ca(2+) channels involved in the endothelin-1-induced mitogenic response of cultured rat thoracic aorta smooth muscle cells, A7r5 cells, were characterized using the Ca(2+) channel blockers, LOE 908 and SK&F 96365. Stimulation of A7r5 cells with endothelin-1 induced a mitogenic response as well as a biphasic increase in the intracellular-free Ca(2+) concentration. Based on the sensitivity to nifedipine, a specific blocker of L-type voltage-operated Ca(2+) channel (VOCC), Ca(2+) influx through VOCC has a minor role in endothelin-1-induced mitogenic responses. On the other hand, Ca(2+) influx through voltage-independent Ca(2+) channels (VICCs) plays an important part in endothelin-1-induced mitogenesis. Moreover, based on their sensitivity to SK&F 96365 and LOE 908, VICCs consist of two types of Ca(2+)-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and a store-operated Ca(2+) channel (SOCC). Ca(2+) influx through NSCC-1, NSCC-2 and SOCC contributes to 35%, 30% and 35%, respectively, to the nifedipine-resistant component of the endothelin-1 mitogenic response.     

Characterization of the thromboxane receptor mediating prostacyclin release from cultured endothelial cells.

The thromboxane A2 (TXA2) mimetic, 9,11-dideoxy-11,9-epoxymethano-prostaglandin F 2 alpha (U46619), mobilized calcium in the bovine aortic endothelial cell line AG4762 and stimulated release of prostacyclin from these cells. The U46619-stimulated release of prostacyclin could be inhibited by TXA2 antagonists with the order of potency [Is-[1 less than a, 2 less than b(5z), 3 less than b, 4 less than a]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo- [2.2.1]hept-2-yl]-5- heptenoic acid (SQ29548) greater than 4-[2-(4-chlorobenzene-sulphonamido) ethyl]phenylacetic acid (BM13505) greater than 4-[2-(phenylsulphonamido)-ethyl]phenoxyacetic acid (BM13177), which was consistent with release being mediated by a TXA2 (TP) receptor. The TP receptor ligands, [3H]SQ29548 and 9,11-dimethylmethano-16(3-[125I]iodo-4-hydroxyphenyl)-13,14-dih ydr o-13-aza- 15-omega-o-tetranor-thromboxane ([125I]-PTA-OH), both appeared to bind to a homogenous population of sites in AG4762 cell membranes. The affinities of [3H]SQ29548 and [125I]PTA-OH were approximately 10 nM and approximately 0.3 nM, respectively, and the density of sites labelled by either ligand was approximately 25 fmol/mg protein. Under conditions where equilibrium was approached, the specific binding of [3H] SQ29548 or [125I]PTA-OH was displaced by SQ29548, BM13505 and BM13177 with the same order of potency and similar apparent affinities as in the functional assay, suggesting that these binding sites represent bona fide TP receptors.     

叶酸抑制同型半胱氨酸诱导大鼠血管平滑肌细胞增殖的研究

目的探讨叶酸抑制同型半胱氨酸致大鼠血管平滑肌细胞增殖的研究。方法培养大鼠胸主动脉血管平滑肌细胞(VSMC),流式细胞仪检测VSMC周期,[3H]TdR参入测定VSMC的DNA合成。结果同型半胱氨酸以剂量依赖关系使VSMC周期中的G0/G1期细胞比例明显减少,S期细胞比例显著增多,增加VSMC的[3H]TdR参入。叶酸可明显抑制同型半胱氨酸诱导的作用。结论同型半胱氨酸能诱导VSMC增殖,叶酸能抑制同型半胱氨酸诱导的VSMC增殖。     

多沙唑嗪光学异构体对大鼠主动脉平滑肌细胞增殖的选择性抑制作用

目的观察多沙唑嗪(racemic-doxazosin,rac-DOX)及其对映体(S-DOX、R-DOX)对大鼠血管平滑肌细胞(vascularsmooth muscle cells,VSMCs)增殖的影响。方法采用培养的大鼠主动脉血管平滑肌细胞,应用MTT比色法,测定VSMCs的增殖活力,Giemsa’s染色观察VSMCs的形态学改变。结果在3~30μmol.L-1浓度范围,S-DOX、R-DOX和rac-DOX作用于VSMCs 96 h,均抑制大鼠胸主动脉VSMCs增殖活动;但是,S-DOX的抑制作用强于同浓度R-DOX(P<0.05),与同浓度rac-DOX的抑制作用无差别。以30μmol.L-1浓度的药物处理VSMCs 48 h、72 h和96 h;S-DOX对主动脉VSMCs增殖的抑制率依次为28.67%、34.51%和56.89%,R-DOX为22.59%、26.66%和45.79%,rac-DOX为21.88%、32.84%和52.04%。以不同浓度的药物处理大鼠胸主动脉VSMCs 96 h,rac-DOX、S-DOX和R-DOX抑制VSMCs增殖达40%的浓度(IC40)分别为(12.1±2.6)、(10.2±1.3)和(20.9±2.2)μmol.L-1;R-DOX的IC40值大于rac-DOX和S-DOX(P<0.01)。S-DOX处理后,VSMCs出现细胞体积变小,核固缩,核边集等形态学改变。结论S-DOX和R-DOX对大鼠胸主动脉VSMCs的抗增殖作用具有化学结构的立体选择性,S-DOX的抗VSMCs增殖作用明显强于R-DOX。     

金粉蕨素抑制大鼠主动脉平滑肌增殖作用及机制

目的 :观察金粉蕨素对牛血清刺激的大鼠主动脉平滑肌细胞增殖的抑制作用 ,并对其作用机制进行初步探讨。方法 :体外培养大鼠主动脉平滑肌细胞 ,以终浓度为 10 %的新生牛血清 (NCS)作为刺激因素 ,用噻唑蓝 (MTT)比色法和细胞计数法观察细胞增殖状况 ,用流式细胞仪分析细胞周期 ,用Westernblot实验测定蛋白表达。结果 :与 10 %牛血清组相比 ,不同浓度金粉蕨素组的MTT测定值与细胞数目均明显下降 (P <0 .0 5 ) ,其下降幅度呈浓度依赖性 ;10 μmol·L-1时达峰值 (P <0 .0 1) ;细胞周期分析显示 ,金粉蕨素组G1期百分比 (85 .1% )高于10 %牛血清组 (70 .0 % ) ,而S期比例 (4 .3% )低于10 %牛血清组 (16.4 % ) ;Westernblot结果显示给药组P ERK1 2蛋白表达明显低于同时间点牛血清组。结论 :金粉蕨素能阻止细胞周期由G0 G1期向S期推进 ,抑制血管平滑肌细胞增殖 ,此作用与其抑制ERK1 2磷酸化、影响MAPK ERK通路激活有关。