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)     

Effect of endothelin on angiotensin converting enzyme activity in cultured vascular smooth muscle cells

We evaluated the effect of endothelin-1(ET) on the angiotensin converting enzyme (ACE) activity in rat aortic smooth muscle cells (VSMCs). ACE activity was determined by radioimmunoassay of the amount of angiotensin II generated after the addition of angiotensin I (500 pg/ml) to cultured VSMCs. The antibody used had less than 0.1% cross-reactivity with angiotensin 1. ACE activity increased 1.9-fold 5 hr after the addition of 10⁻⁶ M ET under serum-free conditions. This stimulatory effect of ET on ACE activity in VSMCs was completely inhibited by 10^-7 M captopril. Results suggested that the ACE present in SMCs is stimulated by ET.     

Nitric oxide regulates angiotensin II receptors in vascular smooth muscle cells

The objective of this study was to determine whether an enhanced generation of nitric oxide (NO) causes regulation of angiotensin II receptors in vitro using rat vascular smooth muscle cells in culture. Chronic treatment of cells with a series of NO-generating drugs, sodium nitroprusside, S-nitroso-N-acetylpenicillamine and isosorbide dinitrate for 18h dose and time-dependently decreased [¹²⁵I]-angiotensin II binding to cells without any significant change in affinity. Induction of nitric oxide synthase following lipopolysaccharide (10 and 100 ng/ml) treatment of cells for 18 h increased basal nitric oxide synthase activity with a concomitant increase of nitrite and cyclic cGMP levels in the conditioned media. LPS treatment significantly (P < 0.05) decreased [¹²⁵I]-angiotensin II binding to these cells, an effect that was significantly (P < 0.05) attenuated in the presence of N^G-nitro-L-arginine methyl ester. In contrast, treatment of cells with atrial natriuretic factor, dibutyryl cGMP, 8-bromo-cGMP, NaNO₂ or NaNO₃ failed to significantly alter the affinity or number of [¹²⁵I]-angiotensin II binding sites. These results suggest that NO regulates angiotensin II receptors in vitro through a cGMP-independent mechanism.     

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.     

罗格列酮对血管平滑肌细胞血管紧张肽Ⅱ受体2表达的影响

目的:观察罗格列酮对血管紧张肽Ⅱ(AngⅡ)诱导大鼠血管平滑肌细胞(VSMCs)血管紧张肽Ⅱ受体2(AT_2R)mRNA和蛋白表达的影响及其机制。方法:1μmol·L~(-1) AngⅡ孵育体外培养大鼠VSMCs 6h,然后随机分成7组,另设空白对照组。其中3组分别加入终浓度为20、30、50μmol·L~(-1)罗格列酮干预12h;另3组加入30μmol·L~(-1)的罗格列酮分别再干预6、12、24h;余1组为AngⅡ模型组。采用RT- PCR和免疫组织化学方法检测VSMCs AT_2R mRNA及蛋白的表达水平。结果:空白对照组AT_2R mRNA和蛋白表达量为[(57±s5)％,1.094±0.012],AngⅡ可下调AT_2R mRNA及蛋白的表达[(41±4)％,0.91±0.05](P<0.01,P<0.05);与AngⅡ组相比,罗格列酮20、30、50μmol·L~(-1)干预12h或30μmol·L~(-1)罗格列酮再干预6、12、24h均能使AT_2R mRNA和蛋白的表达明显上调[(63±6)％,1.18±0.04;(94±10)％, 1.35±0.03;(110±12)％,1.58±0.03;(40±4)％,1.23±0.03;(57±6)％,1.372±0.023;(90±10)％,1.64±0.03](P<0.05,P<0.01)。结论:基础状态下,体外培养的大鼠VSMCs AT_2R少量表达, AngⅡ下调AT_2R mRNA及蛋白的表达;一定浓度范围内,罗格列酮可剂量、时间依赖性地上调AngⅡ诱导的大鼠VSMCs AT_2R mRNA和蛋白的表达。     

Effect of calcitonin gene-related peptide on angiotensin II-induced proliferation of rat vascular smooth muscle cells

The present study was designed to examine the effect of calcitonin gene-related peptide (CGRP) on angiotensin II-induced proliferation of cultured rat vascular smooth muscle cells. Vascular smooth muscle cells were grown from explants of Sprague-Dawley rat aorta. Vascular smooth muscle cells (between passages 5 and 10) were incubated with 0.1% neonatal calf serum for 48 h, and then treated with angiotensin II (100 nM) in the absence or presence of CGRP for 24 h. The viability, DNA synthesis and cell cycle of vascular smooth muscle cells were measured. Western blotting was used to determine the activity of intracellular extracellular regulated kinase (ERK1/2). Angiotensin II significantly decreased the viability and proliferation of vascular smooth muscle cells, decreased the proliferation index, and increased the activity of ERK1/2; the effects of angiotensin II were inhibited by CGRP (1-100 nM) in a concentration-dependent manner. In conclusion, CGRP significantly inhibits angiotensin II-induced proliferation of vascular smooth muscle cells, an effect related to a decrease in the activation of mitogen-activated protein kinase pathway.     

Effect of capsaicin on membrane currents in cultured vascular smooth muscle cells of rat aorta

The application of capsaicin (1 μM) produced a minor relaxant effect in endothelium-denuded rat aortae. However, capsaicin caused a greater relaxation of blood vessels precontracted with high K⁺ or phenylephrine. The effects of capsaicin on the ionic currents were also examined in A7r5 vascular smooth muscle cells. The tight-seal whole-cell voltage clamp technique was used. Capsaicin inhibited the Ba²⁺ inward current (I_Ba) through the voltage-dependent L-type Ca²⁺ channel in a concentration-dependent fashion, whereas calcitonin gene-related peptide and phenylephrine produced a minor increase in I_Ba. Capsaicin did not alter the overall shape of current-voltage relationship of I_Ba. However, capsaicin (3 μM) shifted the quasi-steady-state inactivation curve of I_Ba to more negative membrane potential by about 5 mV. These effects of capsaicin on I_Ba were reversible. In addition, capsaicin had inhibitory effects on voltage dependent K⁺ currents. These results suggest that inhibition of the voltage-dependent L-type Ca²⁺ channel is involved in the capsaicin-induced relaxation of the vascular smooth muscle, whereas capsaicin-induced inhibition of voltage-dependent K⁺ channels might produced an increase in cell excitability.     

Importance of intracellular angiotensin II in vascular smooth muscle cell apoptosis: inhibition by the angiotensin AT1 receptor antagonist irbesartan

The intracellular uptake of Angiotensin II has been described, although its physiological role is not yet understood. We aimed to study the role of Angiotensin II internalization in Angiotensin II-induced apoptosis. Vascular smooth muscle cells were cultured from male Wistar-Kyoto rats and treated with Angiotensin II (1 microM, 48 h). Apoptosis was assessed by DNA fragmentation, cell cytometry and caspase-3 activity. The Angiotensin AT(1) receptor antagonist irbesartan (0.1-10 microM) and the inhibitors of Angiotensin II internalization phenylarsine oxide (PAO, 20 microM), but not the AT(2) receptor antagonist PD123319 (S-(+)-1-[(4-(Dimethylamino)-3-methylphenyl)methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid di(trifluoroacetate) salt), decreased Angiotensin II-mediated apoptosis. Pre-treatment with irbesartan, but not with PD123319, blocked Angiotensin II internalization. We found a strong correlation between intracellular Angiotensin II staining and Angiotensin II-induced apoptosis for all compared groups. We therefore conclude that internalization of Angiotensin II is involved in apoptosis of vascular smooth muscle cells induced by this peptide.     

血管紧张素转化酶和血管紧张素Ⅱ对低氧促培养的肺内小动脉平滑肌细胞增殖作用的影响

目的：研究局部血管紧张素转化酶及血管紧张素Ⅱ和低氧促进肺动脉平滑肌细胞增殖作用之间的关系。方法：分离培养肺内小动脉平滑肌细胞,测定[^3H]thymidine掺入和细胞计数作为细胞增殖的指标。结果：低氧显著促进培养的肺内小动脉平滑肌细胞增殖,其[^3H]thymidine掺入和细胞计数分别增加166．6％（P＜0．01）和52．0％（P＜0．01）。captopril和losartan预处理可显著抑制低氧对肺内小动脉平滑肌细胞增殖的促进作用,[^3H]thymidine掺入分别被抑制51．3％（P＜0．01）和49．8％（P＜0．01）,细胞计数分别被抑制22．2％（P＜0．01）和17．％（P＜0．01）。而PD－123319基本无明显作用。结论：肺内小动脉平滑肌细胞局部血管紧张素转化酶的过度表达及血管紧张素Ⅱ在低氧促平滑肌细胞增殖中发挥重要作用。     

Signal transduction mechanisms mediating the physiological and pathophysiological actions of angiotensin II in vascular smooth muscle cells

Until recently, the signaling events elicited in vascular smooth muscle cells by angiotensin II (Ang II) were considered to be rapid, short-lived, and divided into separate linear pathways, where intracellular targets of the phospholipase C-diacylglycerol-Ca(2+) axis were distinct from those of the tyrosine kinase- and mitogen-activated protein kinase- dependent pathways. However, these major intracellular signaling cascades do not function independently and are actively engaged in cross-talk. Downstream signals from the Ang II-bound receptors converge to elicit complex and multiple responses. The exact adapter proteins or "go-between" molecules that link the multiple intracellular pathways await clarification. Ang II induces a multitude of actions in various tissues, and the signaling events following occupancy and activation of angiotensin receptors are tightly controlled and extremely complex. Alterations of these highly regulated signaling pathways in vascular smooth cells may be pivotal in structural and functional abnormalities that underlie vascular pathological processes in cardiovascular diseases such as hypertension, atherosclerosis, and post-interventional restenosis.     

ATP release by angiotensin II from segments and cultured smooth muscle cells of guinea-pig taenia coil

Effects of angiotensin II on ATP release were evaluated in segments and cultured smooth muscle cells from the guinea-pig taenia coli. In the segments, angiotensin II (0.3–3 M) elicited release of ATP which was blocked by losartan and SC-52458, non-peptide angiotensin II type-1 receptor (AT₁)-antagonists, but not by PD-123319, an AT₂-antagonist. In superfused cultured cells, 10M angiotensin II likewise elicited release of ATP Again the response was blocked by losartan and SC-52458 but not by PD-123319. These findings suggest that angiotensin II releases ATP from the smooth muscles by activation of angiotensin II-, presumably ATE₁-, receptors.     

Effect of different calcium channel blockers on angiotensin II- and vasopressin-induced prostacyclin biosynthesis in vascular smooth muscle cells.

To gain insight with regard to the mode of action of calcium antagonists on the vasculature, we examined the effects of nifedipine, isradipine, felodipine, verapamil, gallopamil, and amlodipine on vasoconstrictor-induced prostacyclin synthesis in vitro. Cultured rat aortic smooth muscle cells were seeded after two to four passages in multiwell plates. After washing of the culture medium and a preincubation period, the cells were exposed for 1 h to either angiotensin II (Ang II) or arginine-vasopressin (AVP) at increasing concentrations between 10(-10)-10(-6) M with or without each calcium antagonist tested at 10(-6) M. At the end of the incubation period, the medium was aspirated, centrifuged, and assayed for its content of protein and of 6-keto-PGF1 alpha by radioimmunoassay. Ang II induced a 15-fold increase and AVP induced a fivefold increase of 6-keto-PGF1 alpha at 10(-6) M. None of the various calcium channel blockers tested showed a significant effect on this agonist-stimulated production of 6-keto-PGF1 alpha. Consequently, calcium-channel blockers with different chemical structure, although known to inhibit agonist-induced vasoconstriction, appear to preserve vasoconstrictor-induced production of prostacyclin, a potent vasodilator and an inhibitor of platelet aggregation.     

Evidence for a regulatory site in the angiotensin II receptor of smooth muscle

The homologous desensitization induced by angiotensin II analogues in the guinea-pig isolated ileum was studied. Desensitization assessed by the loss of response on repeated treatment showed [Sar1]angiotensin II to be a strong desensitizer whereas no desensitization to [Lys2]angiotensin II was detected. However, prolonged treatment with either analogue desensitized the tissue, indicating that [Lys2]angiotensin II-induced desensitization was reversed faster. A correlation was found between the degree of desensitization caused by repeated treatment and the time for half-relaxation after washout of the first treatment, but the relaxation after washout became faster in the desensitized state. In experiments designed to study competition between the agonistic and desensitizing properties of angiotensin II analogues, high concentrations of [Lys2]angiotensin II blocked the agonistic but not the desensitizing effect of lower concentrations of [Sar1]angiotensin II. It is concluded that desensitization is due to the interaction of angiotensin II with a regulatory site on the receptor.     

Interaction of dimercaptosuccinic acid (DMSA) with angiotensin II on calcium mobilization in vascular smooth muscle cells

Dimercaptosuccinic acid (DMSA) was shown to lower blood pressure in rat models of arterial hypertension. Thus, there is evidence that-besides its chelating properties-DMSA has a direct vascular effect, e.g. through scavenging of reactive oxygen species (ROS). We speculated that, in addition, intracellular calcium mobilization may be involved in this action. Therefore, the present study examined the effects of DMSA on Ca(2+) mobilization in cultured vascular smooth muscle cells (VSMCs) from rat aorta. Intracellular free Ca(2+) concentration ([Ca(2+)](i)) was measured with fura-2 AM. In a first series of experiments DMSA, 10(-11) to 10(-6)M, induced an immediate dose-dependent up to 4-fold rise of [Ca(2+)](i) (P<0.001) which was almost completely blunted by the calcium channel blocker verapamil or the intracellular calcium release blocker TMB-8. In a second series of experiments, when VSMCs were exposed acutely to DMSA (10(-11) to 10(-6)M), the angiotensin (ANG) II (10(-8)M)-induced rise in [Ca(2+)](i) to 295+/-40nM was attenuated at the average by 49% independent of the dose of DMSA. Preincubation of VSMCs with DMSA (10(-6)M) for 60min reduced basal [Ca(2+)](i) by 77% (P<0.001) and dose-dependently attenuated the ANG II (10(-8)M)-induced rise in [Ca(2+)](i) between 28 and 69% at concentrations between 10(-9) and 10(-5)M DMSA, respectively (P<0.05 and <0.01). In the presence of TMB-8, which attenuated the ANG II (10(-8)M)-induced rise in [Ca(2+)](i) by 66%, DMSA (10(-6)M) had no additional suppressive effect on [Ca(2+)](i). The results suggest that DMSA acutely raises [Ca(2+)](i) by stimulating transmembrane calcium influx via L-type calcium channels and by calcium release from intracellular stores followed by a decrease in [Ca(2+)](i) probably due to cellular calcium depletion. Thus, in addition to its action as scavenger of ROS, which in part mediate the vasoconstrictor response, e.g. to ANG II, DMSA may exert its hypotensive effect through decreasing total cell calcium, thereby attenuating the vasoconstrictor-induced rise in [Ca(2+)](i) in VSMCs.     

Phosphatidylinositol 3-kinase in angiotensin II-induced hypertrophy of vascular smooth muscle cells

Activation of 4E-binding protein 1 (4E-BP1) by growth factors regulates protein synthesis in vascular smooth muscle cells. The interaction between G protein-coupled receptors and activated 4E-BP1 is unclear. We examined phosphadityl inositol (PI) 3-kinase in angiotensin II-induced 4E-BP1 phosphorylation in cultured rat vascular smooth muscle cells. Angiotensin II time and dose dependently stimulated phosphorylation of 4E-BP1 through the angiotensin AT(1) receptor. Pretreatment with wortmannin or 2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), a PI 3-kinase inhibitor, suppressed angiotensin II-induced phosphorylation, but a mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases (ERK) kinase-1 (MEK-1) inhibitor, 2'-Amino-3'-methoxyflavone (PD98059), and a p38 MAPK inhibitor, 4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580), had no effect. With regard to the involvement of mammalian target of rapamycin (mTOR) and p70 S6 kinase, angiotensin II-induced phosphorylation was abolished by pretreatment with rapamycin, but not by tosylphenylalanine chloromethyl ketone or tosyllysine chloromethyl ketone. Ca(2+) was involved, since intracellular Ca(2+) chelation inhibited angiotensin II-induced phosphorylation while a Ca(2+) ionophore, A23187, stimulated phosphorylation. Thus, angiotensin II induces the phosphorylation of 4E-BP1 via the PI 3-kinase/mTOR pathway, but not via ERK or p70 S6 kinase.     

镁离子对大鼠血管平滑肌细胞钙化的影响 , 镁离子对大鼠血管平滑肌细胞钙化的影响

目的 探讨不同浓度镁离子对大鼠血管平滑肌细胞(VSMCs)钙化的影响。方法 原代培养获取 VSMCs,进行形态学及免疫细胞鉴定,后将VSMCs随机分为阴性对照组、高磷组、镁干预组。阴性对照组采用含10%胎牛血清培养,高磷组采用高磷培养基培养,镁干预组在高磷培养基的基础上分别加入不同浓度氯化镁,使镁离子终浓度分别为1、2、3 mmol/L（镁干预组1~3）,刺激7 d后行钙化检测,测定钙含量及碱性磷酸酶（ALP）活性,并行反转录聚合酶链反应（RT-PCR）检测细胞内核心结合因子α1（Cbfα1）mRNA的表达。结果 高磷组和镁干预组VSMCs均有钙盐沉积,其钙含量均高于阴性对照组;镁干预组随镁离子浓度增大钙化结节逐渐缩小,除镁干预组1钙含量与高磷组无差异外,镁干预组2和镁干预组3均低于高磷组（均P＜0.05）。VSMCs ALP活性和Cbfα1 mRNA的表达除镁干预组3与阴性对照组无差异外,其余组均高于阴性对照组（P＜0.05）。镁干预组随镁离子浓度增大,ALP活性和Cbfα1 mRNA的表达水平均逐渐降低,且均低于高磷组（P＜0.05）。结论 镁离子可在一定程度上抑制高磷诱导的VSMCs钙化和成骨样转分化,其可能是通过降低VSMCs中Cbfα1的表达来实现的。