A selective endothelin ETA antagonist,BQ-123, inhibits125I-endothelin-1 (125I-ET-1) binding to human meningiomas and antagonizes ET-1-induced proliferation of meningioma cells

Summary 1. We studied the effects of BQ-123, a selective ET_A receptor antagonist, on¹²⁵I-endothelin-1 (¹²⁵I-ET-1) binding to cell surface receptors in surgically excised human meningiomas and on ET-1-induced DNA synthesis in cultured human meningioma cellsin vitro, using a quantitative receptor autoradiographic technique with radioluminography and³H-thymidine incorporation, respectively.2. All of the human meningiomas expressed high-affinity binding sites for¹²⁵I-ET-1, regardless of differences in histological subtypes (K _d=2.6±0.2 nM,B _max=374±93 fmol/mg; mean ± SE;n=9).3. BQ-123 competed for¹²⁵I-ET-1 binding to sections of meningiomas with IC₅₀s of 3.2±0.9×10^–7 M, and 10^–4 M BQ-123 displaced 80% of the binding.4. ET-1 significantly stimulated DNA synthesis in cultured human meningioma cells, up to 170% of the basal level in the presence of 10^–9 M ET-1. BQ-123 inhibited ET-1 (10^–9 M)-induced DNA synthesis in meningioma cells, in a dose-dependent manner, and 10^–5 M BQ-123 reduced it to 120% of the basal level.5. The number of meningioma cells determined after 4 days in culture was dose dependently increased in the presence of ET-1 (10^–9 and 10^–7 M). The growth rate of meningioma cells, incubated with 10^–9 M ET-1, was reduced by 50% in the presence of 10^–7 M BQ-123.6. Our data suggest that (a) human meningioma cells express a large number of ET_A endothelin receptors, with a small proportion of non-ET_A receptors linked to proliferation of the cells, and (b) ET receptor antagonists, including BQ-123, might prove to be effective treatment for patients with meningioma.     

Effects of Cl- channel blockers on endothelin-1-induced proliferation of rat vascular smooth muscle cells

The effects of Cl- channel blockers on endothelin-1 (ET-1)-induced proliferation of rat aortic vascular smooth muscle cells (VSMC) were examined. We found ET-1 concentration-dependently increased cell count and [3H]-thymidine incorporation into VSMC, with EC50 values of 24.8 and 11.4 nM, respectively. Both nifedipine and SK&F96365 inhibited 10 nM ET-1-induced [3H]-thymidine incorporation into VSMC with the maximal inhibitory concentrations of 1 and 10 microM, respectively. DIDS inhibited 10 nM ET-1-induced increase in cell count and [3H]-thymidine incorporation into VSMC in a concentration-dependent manner, whereas other Cl- channel blockers including IAA-94, NPPB, DPC, SITS and furosemide did not produce these effects. 3 microM DIDS reduced 10 nM ET-1-induced sustained increase in cytoplasmic Ca2+ concentration ([Ca2+]) by 52%. Pretreatment of VSMC with 1 microM nifedipine completely inhibited the DIDS effect on 10 nM ET-1-induced [3H]-thymidine incorporation into VSMC and sustained increase in [Ca2+]i, whereas pretreatment with 10 microM SK&F96365 did not completely block these effects of DIDS. DIDS did not affect ET-1-induced Ca2+ release and 30 mM KCl-induced increase in [Ca2+]i. Our data suggest that DIDS-sensitive Cl- channels mediate VSMC proliferation induced by ET-1 by mechanisms related to membrane depolarization and Ca2+ influx through voltage-dependent Ca2+ channels.     

Phorbol ester modulates serotonin-stimulated phosphoinositide breakdown in cultured vascular smooth muscle cells

Stimulation of cultured rabbit aortic vascular smooth muscle cells (VSMC) with serotonin (5HT) induced a rapid generation of inositol phosphates from receptor-mediated hydrolysis of inositol phospholipids. Pretreatment of these cells with 500ng/ml of pertussis toxin for 24h prior to addition of 5HT reduced 5HT-induced formation of inositol phosphates. Phorbol esters, such as 12-O-tetradecanoylphorbol-13-acetate (TPA) or phorbol-12,13-dibutyrate (PDBu), are known to activate protein kinase C (PKC), but their role on cultured VSMC stimulated by 5HT has not been defined. TPA exhibited a rapid inhibition of 5HT-stimulated phosphoinositide breakdown, although 4 alpha-phorbol-12,13-didecanoate (4 alpha PDD), an inactive phorbol ester, did not inhibit it. These data suggest that a guanine nucleotide inhibitory (Gi) protein couples 5HT receptor to phospholipase C and TPA modulates 5HT-stimulated hydrolysis of inositol phospholipids in cultured VSMC through activation of PKC.     

Nonselective ETA/ETB receptor antagonist blocks proliferation of rat vascular smooth muscle cells after balloon angioplasty

To elucidate the role of endothelin receptor subtypes in the abnormal proliferation of vascular smooth muscle cells (VSMC) associated with vascular injury, we have investigated the effects of a novel and potent nonselective ET_A/ET_B receptor antagonist (TAK-044) on the proliferation of rat VSMC in vitro and in vivo. TAK-044 dose-dependently inhibited DNA synthesis stimulated by 10⁻⁷ M ET-1 in cultured rat VSMC from the late passage with the approximate IC₅₀ of 6 × 10⁻⁸ M. After balloon angioplasty, the neointimal lesion in the injured carotid arteries in the TAK-044-treated group (0.052 ± 0.014 mm²) was significantly (p < 0.05) decreased compared to that in control group (0.26 ± 0.045 mm²), while the medial surface area was not affected. The intima/media ratio in the TAK-044 group (31 ± 6%) also significantly (p < 0.05) decreased from that of the control group (148 ± 25%). Our data suggest that nonselective ET_A/ET_B receptor antagonists may be therapeutic potential for prevention against the intimai thickening associated with vascular injury.     

Effect of endothelin-1 receptor antagonist BQ-123 on basilar artery diameter after subarachnoid hemorrhage (SAH) in rats.

Aim of the study was to quantify cerebral vasospasm in rats after subarachnoid hemorrhage (SAH) by morphometric examination of basilar artery and to evaluate the influence of endothelin receptor blocker BQ-123 on basilar artery constriction. The rat cisterna magna (CM) was cannulated and after 7 days SAH was developed by administration of 100 microl autologic, non-heparinized blood to the CM. The sham subarachnoid hemorrhage was developed by intracisternal administration of 100 microl of artificial cerebrospinal fluid. Endothelin receptor blocker BQ-123 was injected into the CM in a dose of 40 nmol diluted in 50 microl of cerebrospinal fluid 20 min. before SAH, and 24h and 48 h after SAH. After perfusion fixation the brains were removed from the skull and histological preparations of basilar artery were done. The internal diameter and wall thickness of basilar arteries was measured by interactive morphometric method. The most severe vasospasm was found in rats after SAH. The presence of numerous infiltrations composed of neutrophils and macrophages correlated with advanced vasospasm (index of constriction 5 times lower than in normal), suggesting the role of other factors participating in the late phase of vasospasms after SAH. Administration of BQ-123 in the late phase after SAH caused the dilatation of basilar artery. Following the administration of BQ-123 in the late phase (48 h after SAH) the basilar artery dilated, its wall became thinner, and the number of leukocyte infiltrations in the subarachnoid space decreased compared to the values after SAH alone.     

Phosphoramidon inhibits the generation of endothelin-1 from exogenously applied big endothelin-1 in cultured vascular endothelial cells and smooth muscle cells

When cultured porcine aortic endothelial cells (ECs) were incubated with porcine big endothelin-1 (bit ET-1(1-39)), there was a time-dependent increase in immunoreactive (IR)-ET in the culture supernatant, in addition to an endogenous IR-ET release fron the cells. Reverse-phase HPLC of the culture supernatant revealed one major IR-ET component corresponding to the elution position of synthetic ET-1, thereby indicating that the additional increase in IR-ET was due to the conversion of big ET-1 to mature ET-1(1-21). Phosphoramidon, a metalloproteinase inhibitor, strongly suppressed this increase in IR-ET as well as the endogenous IR-ET release. Cultured vascular smooth muscle cells (VSMCs) also released IR-ET. The apparent conversion of exogenously applied big ET-1 to ET-1 and its inhibition by phosphoramidon were observed using cultured VSMCs, although the enzyme inhibitor did not influence the basal secretion of IR-ET from VSMCs. These results suggest that both cultured ECs and VSMCs can generate ET-1 from exogenously applied big ET-1 via action of the same type of phosphoramidon-sensitive metalloproteinase, which is also involved in the endogenous ET-1 generation in ECs.     

Benzo(a)pyrene activates L1Md retrotransposon and inhibits DNA repair in vascular smooth muscle cells

Benzo(a)pyrene (BaP) modulates vascular smooth muscle cells (vSMCs) from a quiescent to proliferative phenotype, a shift associated with activation of L1Md retrotransposon [K.P. Lu, K.S. Ramos, Biochem. Biophys. Res. Commun. 253 (1998) 828-833]. The present studies were conducted to evaluate L1Md activation profiles in murine vSMCs treated with BaP or its oxidative metabolites, and to screen for possible insertional mutations into p53 and retinoblastoma (RB) genes. We also sought to examine the profile of DNA damage and repair in BaP-treated vSMCs. Northern analysis revealed that BaP (0. 03-3microM), and its major reactive 7,8-diol metabolite (0. 03-3microM), activate L1Md gene in a concentration-dependent manner. Two other metabolites, 3-OH BaP and 3,6-BaP quinone (0.03-3microM), as well as hydrogen peroxide (25-75microM) also activated L1Md. No insertional mutations into either p53 or RB genes were observed in vSMCs treated with BaP in vitro, although a slight elevation of p53 mRNA was observed as early as 4h after chemical challenge. Treatment of vSMCs with 3 or 30microM BaP for 4h increased unscheduled DNA synthesis (UDS) 1.4- and 2.5-fold, respectively. Challenge with 0. 3microM BaP for 24h inhibited DNA repair capacity in vSMCs for up to 48h. These results demonstrate that BaP and its oxidative metabolites activate L1Md retrotransposon in vSMCs, which coupled to DNA damage and inhibition of DNA repair are part of the atherogenic response elicited by BaP and related hydrocarbons.     

Effect of a new V1 antagonist (OPC-21268) on vascular action of vasopressin in cultured rat vascular smooth muscle cells.

The effect of 1-(1-[4-(3-acetylaminopropoxy)benzoyl]-4-piperidyl-3,4-dihydro-2(1 H)- quinolinone) (OPC-21268) on vascular action of arginine vasopressin (AVP) was examined in cultured rat vascular smooth muscle cells (VSMC) by the measurement of cytosolic free calcium concentration [( Ca2+]i) and the AVP V1 receptor study. The preincubation of cells with OPC-21268 for 10 min inhibited the AVP-induced mobilization of [Ca2+]i in a dose-dependent manner but did not affect the angiotensin II-induced mobilization of [Ca2+]i. The receptor study revealed that OPC-21268 blocks the binding of AVP to the receptor in VSMC in a similar way to the V1 structural antagonist [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid)-2-O-methyltyrosine]AVP: d(CH2)5Tyr(Me)AVP. Lineweaver-Burk plot showed that OPC-21268 is the competitive AVP V1 receptor antagonist. These results therefore indicate that OPC-21268 specifically blocks the vascular action of AVP mediated through the competitive inhibition of AVP binding to the receptors in VSMC.     

Endothelin-mediated stimulation of DNA synthesis in vascular smooth muscle cells

Effects of endothelin on DNA synthesis were investigated in two clones of vascular smooth muscle cells, 1YB4 and A7r5. The peptide stimulated DNA synthesis in both clones with apparent EC50 of less than 1 ng/ml. More than 17 h was required before initiating endothelin-stimulated DNA synthesis. The platelet-derived growth factor at a concentration which had no effects by itself on DNA synthesis enhanced the effect of low concentrations of endothelin. A calcium antagonist, nifedipine, inhibited endothelin-induced DNA synthesis. These data suggest that endothelin stimulates DNA synthesis in vascular smooth muscle cells through nifedipine-sensitive mechanisms that can be modulated by platelet-derived growth factor.     

一氧化氮抑制内皮素促血管平滑肌细胞增殖作用的信号转导途径

培养的家兔胸主动脉血管平滑肌细胞（ＶＳＭＣ）分别以内皮素（ＥＴ－１）、一氧化氮（ＮＯ）前体Ｌ－Ａｒｇ和ＮＯ供体ＳＩＮ－１刺激,或用ＥＴ－１＋Ｌ－Ａｒｇ、ＥＴ－１＋ＳＩＮ－１联合刺激,测ＶＳＭＣ＾３Ｈ－ＴｄＲ掺入、丝裂素活化蛋白激酶（ＭＡＰＫ）活性及蛋白激酶Ｃ（ＰＫＣ）活性的改变,以研究ＮＯ抑制ＥＴ－１促ＶＳＭＣ增殖作用的信号转导途径。结果表明：（１）ＥＴ－１ １０＾－８ｍｏｌ／Ｌ单独刺激,＾３Ｈ－     

Dexamethasone inhibits receptor-activated phosphoinositide breakdown in rat basophilic leukemia (RBL-2H3) cells

The activation of rat basophilic leukemia cells for histamine release is accompanied by Ca2+ influx and arachidonic acid release. IgE receptor but not A23187 ionophore stimulation of these cells also resulted in phosphoinositide breakdown. In these experiments, the culture of these cells with dexamethasone inhibited IgE- and ionophore-mediated histamine release. The concentration for 50% of maximal inhibition was 12 nM, and prolonged exposure to the drug was required, with maximal effect observed in 8 to 15 hr. The inhibitory effect of dexamethasone was reversible (t1/2 for recovery was 16 hr). Dexamethasone blocked the IgE-mediated 45Ca2+ influx and the release of [14C]-arachidonic acid (IC50 of 1 nM and 10 nM respectively). Dexamethasone inhibited the IgE receptor-mediated phosphoinositide breakdown (IC50 of 5 nM). It also decreased arachidonic acid release after A23187 stimulation demonstrating an effect on phospholipase A2. Therefore, exposure of the cells to dexamethasone results in the inhibition of both phospholipase A2 and phospholipase C pathways of arachidonic acid generation.     

内皮素-1对大鼠血管平滑肌细胞产生MCP-1的影响及其机制

目的：观察内皮素-1（ET-1）对大鼠血管平滑肌细胞（VSMCs）产生单核细胞趋化蛋白-1（MCP-1）的影响及其机制。方法：培养大鼠血管平滑肌细胞（VSMCs）。细胞分为2组：ET-1刺激组：以不同浓度ET-1刺激VSMCs不同时间;阻断剂干预组：VSMCs分别与不同阻断剂[ET_AR、ET_BR阻断剂BQ123、BQ788,抗氧化剂N-乙酰半胱氨酸（NAC）,ERK、p38MAPK、JNK及NF-κB抑制剂PD98059、SB203580、SP600125及PDTC]预先孵育30 min,再加入ET-1刺激24 h。在预定时间,以酶联免疫吸附（ELISA）法、逆转录聚合酶链反应（RT-PCR）法分别测定不同因素下VSMCs MCP-1蛋白质及mRNA表达量。VSMCs分别与不同阻断剂（BQ123、BQ788、NAC、PD98059、SB203580及SP600125预先孵育20 min,再加入ET-1刺激5 min,免疫印迹（WB）法测定VSMCs胞浆中细胞外调节蛋白激酶（ERK）、p38丝裂原活化蛋白激酶（p38MAPK）、c-Jun氨基末端激酶（JNK）及其各自磷酸化蛋白质的水平。各项检测均重复3次。结果：ET-1能刺激VSMCs MCP-1蛋白质及mRNA表达,其表达量随ET-1浓度及刺激时间的增加呈升高趋势（P<0.05,P<0.01）;BQ123、NAC、PD98059、SB203580及PDTC能显著抑制ET-1诱导的大鼠VSMCs MCP-1蛋白质及mRNA表达（P<0.01）,而BQ788及SP600125对此作用无明显影响。BQ123、NAC与PD98059或SB203580能分别抑制ET-1刺激后VSMCs胞浆内ERK及p38MAPK的磷酸化（P<0.05,P<0.01）,而ET-1对JNK的磷酸化无明显激活作用。结论：ET-1通过ET_AR、ROS、ERK、p38MAPK及NF-κB诱导大鼠VSMCs产生MCP-1。     

Butyrate inhibits proliferation-induced Proliferating Cell Nuclear Antigen expression (PCNA) in rat vascular smooth muscle cells

Arterial injury-induced vascular smooth muscle cell (VSMC) proliferation in intima is the important etiologic factor in vascular proliferative disorders such as atherosclerosis, hypertension and restenosis after balloon angioplasty. Butyrate, a naturally occurring short chain fatty acid, is produced by bacterial fermentation of dietary fiber and by mammary glands of certain mammals. Studies have shown that butyrate at millimolar concentrations, which are physiological, induces growth arrest, differentiation and apoptosis. We examined the effect of physiological concentrations of butyrate on rat VSMC proliferation and proliferation-induced PCNA expression to determine anti-atherogenic potential of butyrate. Butyrate concentrations, closer to physiological range, exhibited antiproliferative effects on both serum-induced proliferation of serum-starved quiescent VSMCs and actively proliferating non-confluent VSMCs. Treatment of serum-starved quiescent VSMCs with 1-8 mmol/l concentration of butyrate caused a concentration-dependent decrease in serum-induced VSMC proliferation and cell proliferation-associated increase in total cellular proteins and RNA levels. Similarly, exposure of actively growing VSMCs to 5 mmol/l butyrate resulted in the inhibition of cell proliferation and proliferation-induced increase in cellular proteins and RNA levels. Furthermore, cellular morphology was significantly altered. Analysis of cell cycle regulatory proteins indicated that levels of PCNA, an excellent marker for cell proliferation, was significantly altered by butyrate both in actively proliferating and serum-induced quiescent VSMCs. These observations suggest that butyrate exhibits potential antiatherogenic capability by inhibiting VSMC proliferation and proliferation-associated increase in PCNA expression and thus merits further investigations regarding therapeutic significance of butyrate in vascular proliferative disorders.     

S-adenosylmethionine inhibits ubiquitin-proteasome system in vitro and on rat vascular smooth muscle cells

S-adenosylmethionine is a metabolite regulating many biological processes; S-adenosylmethionine effect on ubiquitin-proteasome system (UPS) has not been studied yet. We investigated S-adenosylmethionine effects on UPS activity both in vitro, by inhibitor screening assay, and in rat vascular smooth muscle cells, by Western Blot of proteasomal targets. We found that S-adenosylmethionine inhibited UPS activity.     

Effects of calcium-antagonists and calmodulin inhibitors on DNA synthesis in cultured rat vascular smooth muscle cells

To investigate the role of intracellular Ca2+ in the mechanism of cellular proliferation of vascular smooth muscle cells (VSMC), the effects of Ca2+-antagonists and calmodulin (CaM) inhibitors on DNA synthesis stimulated by serum-derived growth factors were studied in cultured VSMCs derived from rat aorta. DNA synthesis assessed by incorporation of [3H]thymidine into the cells was significantly stimulated by epidermal growth factor (EGF), platelet-derived growth factor (PDGF) or fetal bovine serum (FBS), of which the effects were dose-dependently inhibited by a variety of Ca2+-antagonists, such as verapamil, diltiazem and nicardipine. Trifluoperazine and W-7, both specific CaM inhibitors, similarly inhibited DNA synthesis stimulated by EGF, PDGF or FBS in a dose-dependent manner, whereas W-5, a less specific CaM inhibitor, was minimally effective. These data suggest that the Ca2+-CaM system plays an important role in the mechanism of growth factor-induced DNA synthesis in VSMCs.     

Interferon-gamma inhibits proliferation of rat vascular smooth muscle cells by nitric oxide generation.

Interferon (IFN)-gamma inhibited the proliferation of rat vascular smooth muscle cells (VSMC) and increased the cyclic GMP (cGMP) concentration in the cells. The dose dependencies of the two effects were similar (IC50 = 4 U/ml for the anti-proliferation and EC50 = 3 U/ml for cGMP formation) and the effect of IFN-gamma was enhanced by tumor necrosis factor-alpha treatment. Furthermore, NG-nitro-L-arginine, a nitric oxide (NO) synthase inhibitor, inhibited both activities induced by IFN-gamma. These findings show that the anti-proliferation and cGMP formation are closely related and that IFN-gamma inhibits the proliferation of rat VSMC by generation of NO through the induction of an NO synthase.