Effects of ATP on cultured smooth muscle cells from rat aorta.

1. Membrane ionic currents provoked by externally applied ATP were studied by patch-clamp techniques in cultured aortic smooth muscle cells of the rat. 2. Using standard bath and pipette solutions and whole-cell voltage-clamp, ATP evoked an inward current when the cell membrane potential was held at -50 mV and an outward current when the potential was held at 30 mV, with a reversal potential near -10 mV. 3. Application of ATP gamma S gave results similar to those obtained with ATP, while adenosine, AMP and alpha,beta-methylene ATP were ineffective. The ATP-activated current was inhibited by suramin, 100 microM. 4. ATP also induced a biphasic rise in internal free Ca levels as shown directly by Fura-2 measurements and by the increase in Ca-dependent K single-channel activity in cell-attached patches. 5. With outward current through K channels blocked by internal Cs and TEA, modification of the ionic composition of bath and pipette solutions revealed that the reversal potential for the ATP-induced whole-cell current closely followed ECl, the chloride equilibrium potential, and was insensitive to manipulations of the monovalent cation gradient. 6. These results indicate that in rat cultured aortic smooth muscle cells, ATP binding to P2-purinoceptors produces increases of internal free Ca levels and subsequent activation of both Ca-dependent K and Cl currents.     

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.     

Effects of heparin on growth factor-induced mitogenic and proliferative responses of rat pulmonary a

目的：探讨肝素是否能抑制生长因子诱导的大鼠肺动脉平滑肌细胞（ＰＡＳＭＣ）分裂和增殖．方法：应用含１０％ＦＢＳ的Ｍ１９９培养液培养大鼠ＰＡＳＭＣ．细胞分裂及细胞增殖分别用［ｍｅｔｈｙｌ３Ｈ］ＴｄＲ和细胞计数监测．结果：ＦＢＳ（１０％），以及ＦＢＳ（１％）与ＰＤＧＦ（５０μｇ·Ｌ－１），ＦＧＦ（５０μｇ·Ｌ－１），或ＩＬ１α（１００ｎｇ·Ｌ－１）联合应用均能增加大鼠ＰＡＳＭＣ分裂．肝素（１００ｍｇ·Ｌ－１）抑制１０％ＦＢＳ诱导的大鼠ＰＡＳＭＣ增殖（２８％±６％）和胸腺嘧啶摄取反应（２７％±７％），抑制ＦＢＳ（１％）与ＰＤＧＦ（５０μｇ·Ｌ－１），ＦＧＦ（５０μｇ·Ｌ－１），或ＩＬ１α（１００ｎｇ·Ｌ－１）联用诱导的大鼠ＰＡＳＭＣ增殖（２５％±６％，２７％±７％，２０％±４％），以及胸腺嘧啶摄取反应（２３％±７％，２６％±６％，２０％±６％）．结论：肝素抑制生长因子诱导的大鼠ＰＡＳＭＣ的分裂与增殖．     

The effect of heparin on the relative myosin content of cultured smooth muscle cells of the pig aorta

Heparin inhibits the proliferation of aortic smooth muscle cells cultured in serum containing culture medium and leads to an increase of their relative myosin content. Using plasma as medium supplement the effect of heparin is not observed. Thrombin is able to inhibit the increase of myosin content caused by heparin. Platelet mitogens are not the point of attack of heparin. The possible mechanism of action of heparin on smooth muscle cells will be discussed.     

Beta-adrenergic responsiveness in cultured aorta smooth muscle cells. Effects of subculture and aging.

beta-Adrenoceptor-mediated vasorelaxation is diminished in vessels from a variety of aged species including humans. This phenomenon was studied for the first time in cultured aorta smooth muscle cells (ASMC) from young (4- to 6-month) and old (24- to 26-month) F-344 rats. Cyclic AMP (cAMP) accumulation was assessed following isoproterenol and forskolin stimulations in primary cultures and after 1-4 passages of aorta smooth muscle cells. Isoproterenol and forskolin increased cAMP accumulation 6- and 10-fold, respectively, in primary cultures from young rats. Isoproterenol stimulation was reduced markedly in passaged cells. Forskolin stimulation was unaffected, indicating passage-related phenotypic changes in receptor-mediated stimulation, but not in post-receptor adenylate cyclase activation. The response to isoproterenol was diminished in old animals, but that to forskolin was unaltered. Thus, cultured ASMC from F-344 rats are highly responsive to beta-adrenoceptor stimulation and demonstrate age-related changes, but undergo phenotypic modulation during passage.     

Homologous desensitization of the effects of endothelin on rabbit aorta rings and on cultured rat aorta smooth muscle cells

The effects of endothelin-1 on normal and everted rabbit aorta rings and on cultured rat aortic smooth muscle cells were studied. Endothelin-1 (40 nM) contracted both normal and everted rings, and was still able to induce a prolonged contraction in Ca2(+)-free medium. Treatment of cultured cells with 100 nM endothelin-1 caused a sharp transient increase in 45Ca2+ efflux. The contractile and 45Ca2+ responses showed severe and specific desensitization towards endothelin-1; the responses to angiotensin II and adrenaline were not affected. The contractile responses to endothelin-1 were not affected by previous treatment with angiotensin II or with the angiotensin receptor antagonist, [Sar1,Ala8]angiotensin II. It is concluded that endothelin-1 acts on its own specific receptors to elicit Ca2+ mobilization from both intracellular and extracellular sources.     

肝素对生长因子诱导的大鼠肺动脉平滑肌细胞分裂和增殖的影响

目的：探讨肝素是否能抑制生长因子诱导的大鼠肺动脉平滑肌细胞（ＰＡＳＭＣ）分裂和增殖。方法：应用含１０％ＦＢＳ的Ｍ－１９９培养液培养大鼠ＰＡＳＭＣ。细胞分裂及细胞增殖分别用［ｍｅｔｈｙｌ－＾３Ｈ］ＴｄＲ和细胞计数监测。结果：ＦＢＳ（１０％），以及ＦＢＳ（１％）与ＰＤＧＦ（５０μｇ·Ｌ＾－１），ＦＧＦ（５０μｇ·Ｌ＾－１），或ＩＬ－１α（１００ｎｇ·Ｌ＾－１）联合应用均能增加大鼠ＰＡＳＭＣ分裂。肝素（     

Connective tissue growth factor induces apoptosis via caspase 3 in cultured human aortic smooth muscle cells

Connective tissue growth factor (CTGF) stimulates proliferation of fibroblasts and endothelial cells, but nothing is known about its role in smooth muscle cells. In this study, the effects of recombinant human CTGF (r-hCTGF, 0.5-10 microgram/ml) on cultured human aortic vascular smooth muscle cells were investigated. r-hCTGF significantly reduced cell viability, increased apoptosis, and augmented caspase 3 activity. Moreover, r-hCTGF-induced apoptosis was significantly inhibited by an antibody to CTGF and a caspase-3 inhibitor, Z-Asp(Ome)-Glu-(Ome)Val-Asp(Ome)-FMK. These results suggest that r-hCTGF activates caspase 3 and induces apoptosis.     

IGF对大鼠平滑肌细胞骨桥蛋白表达的影响

目的　研究胰岛素样生长因子 (IGF)对培养的大鼠胸主动脉平滑肌细胞骨桥蛋白表达的影响。方法　大鼠平滑肌细胞被随机分为对照组 ,IGF I不同浓度 (5、10、2 0、3 0μg·L-1)及时间 (3、2 4、48h)干预组 ,用RT PCR及Western blotting技术结合光密度扫描分析 ,观察IGF I对培养的大鼠胸主动脉平滑肌细胞内骨桥蛋白基因表达的影响。结果　IGF I 2 0 μg·L-1即能刺激平滑肌细胞内骨桥蛋白的表达 ,RT PCR结果分析骨桥蛋白与 β actinmRNA比值 3h干预组 1 0 2 9± 0 0 60 ,2 4h干预组 1 0 3 2± 0 0 71和 48h干预组1 13 2± 0 190分别较对照组提高了 3 9 70 % ,40 44 %和5 4 0 5 % ;Western blotting分析 3h干预组骨桥蛋白量 19 5 1± 16 983 ,2 4h后 167 98± 15 780和 48h后 175 64±9 913分别较对照组提高 5 3 3 0 %、1 15倍和 1 2 5倍。结论　说明IGF I能在基因水平上刺激大鼠平滑肌细胞内骨桥蛋白的表达     

Mechanisms of tolerance to sodium nitroprusside in rat cultured aortic smooth muscle cells.

1. While exposure of smooth muscle cells to sodium nitroprusside (SNP) leads to the development of tolerance to soluble guanylate cyclase (sGC) activation, the mechanisms responsible for this phenomenon in intact cells remain unclear. In the present study, possible mechanisms of tolerance were investigated in a cell culture model where sGC activity was estimated from the accumulation of cyclic GMP in response to 10 microM SNP over a 15 min period in the presence of a phosphodiesterase (PDE) inhibitor. 2. Pretreatment of rat aortic smooth muscle cells with 10-500 microM SNP led to a dose-dependent downregulation of cyclic GMP accumulation upon subsequent SNP stimulation. This effect was evident as early as 2 h following incubation with 10 microM SNP, reached a plateau at 4 h and was blocked by co-incubation with 30 microM oxyhaemoglobin. 3. Pretreatment of smooth muscle cells with the PDE inhibitor, zaprinast, resulted in downregulation of the SNP-induced cyclic GMP accumulation in a time- and concentration-dependent manner, that was first evident after 12 h. Moreover, while the zaprinast-induced downregulation of cyclic GMP accumulation was completely inhibited by the protein kinase A (PKA) inhibitor, H89, tolerance to SNP was partially reversed by H89. 4. beta 1 sGC steady state mRNA levels of S-nitroso N-acetylpenicillamine (SNAP)- or 8Br-cyclic GMP-pretreated cells were unchanged, as indicated by Northern blot analysis. However, Western blot analysis revealed that alpha 1 protein levels were decreased in zaprinast, but not in SNP, SNAP or 8Br-cyclic GMP pretreated cells. 5. While thiol depletion did not prevent the development of tolerance, pretreatment of cells with SNP in the presence of reducing agents partially or completely restored the ability of cells to respond to SNP. 6. We conclude that tolerance to SNP results from two distinct mechanisms: an early onset, NO-mediated event that is reversed by reducing agents and a more delayed, PKA-sensitive process that is mediated through increases in cyclic GMP and a decrease in sGC protein levels.     

Characterization of the thromboxane (TP-) receptor subtype involved in proliferation in cultured vascular smooth muscle cells of rat.

1. The effects of the thromboxane A2 (TxA2)-mimetic, U-46619, on the proliferation of vascular smooth muscle cells (VSMCs) were examined in a clonal smooth muscle cell line, A10, which was derived from foetal rat aorta. 2. [3H]-U-46619 bound to A10 cells of passages 18-20 (p18-20) with two classes of sites. The high affinity site showed a Bmax of 3.0 +/- 1.8 fmol mg-1 protein with a KD value 1.0 +/- 0.1 nM, while the low affinity site showed a Bmax of 43.0 +/- 6.0 fmol mg-1 protein and KD value of 129.0 +/- 7.9 nM. However, [3H]-U-46619 bound to A10 cells from passages 28-30 (p28-30) at a single class of site with a Bmax 111.0 +/- 9.0 fmol mg-1 protein and a KD value of 175.4 +/- 22.0 nM. 3. Cinnamophilin and SQ29548 inhibited specific [3H]-U-46619 binding to p18-20 A10 cells in a concentration-dependent manner with Ki values of 390.0 +/- 3.2 and 4.6 +/- 1.0 nM, respectively at a high affinity site, and 2.6 +/- 0.2 microM and 310.0 +/- 6.4 nM, respectively at the low affinity site. 4. U-46619 produced isometric contractions of rat aorta in a concentration-dependent manner with an EC50 7.0 +/- 1.2 nM. Cinnamophilin and SQ29548 antagonized U-46619-induced aortic contractions with pA2 values 6.3 +/- 0.1 and 8.2 +/- 0.2, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Etidronate influences growth and phenotype of rat vascular smooth muscle cells.

Bisphosphonates have been reported to exhibit antiarteriosclerotic and anticalcification effects. We investigated the effect of a bisphosphonate, etidronate, on growth and phenotype of vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR). Etidronate (10 microM) significantly decreased DNA synthesis evaluated by [3H]thymidine incorporation in VSMC cultured without serum, and 1 microM etidronate significantly inhibited DNA synthesis in the presence of 10% calf serum. Etidronate (10 microM) significantly inhibited VSMC proliferation after 72h incubation. Etidronate (100 microM) significantly increased the expression of SM22alpha mRNA and protein in VSMC, while 10 microM etidronate significantly decreased the expression of matrix Gla mRNA. These findings indicate that etidronate inhibits the exaggerated growth of VSMC from SHR, while altering their phenotype from synthetic to contractile one. These effects of etidronate may account for its antiarteriosclerotic action.     

Delphinidin and cyanidin inhibit PDGF(AB)-induced VEGF release in vascular smooth muscle cells by preventing activation of p38 MAPK and JNK

BACKGROUND AND PURPOSE: Red wine polyphenols (RWPs) inhibit the expression of vascular endothelial growth factor (VEGF), a major pro-angiogenic and pro-atherosclerotic factor, in vascular smooth muscle cells (VSMCs). The aim of this study was to identify which red wine polyphenols were inhibitory and to determine the mechanism underlying the inhibitory effects. EXPERIMENTAL APPROACH: Release of VEGF stimulated by platelet derived growth factor(AB) (PDGF(AB)), from human aortic VSMCs was measured by immunoassay and phosphorylation of kinases by Western blot analysis. The direct antioxidant properties of polyphenols were determined by electron paramagnetic resonance and the cellular formation of reactive oxygen species (ROS) by dichlorofluorescein. KEY RESULTS: The inhibitory effect of RWPs on PDGF(AB)-induced release of VEGF was mimicked by delphinidin but not by quercetin, catechins, resveratrol, gallic acid or caffeic acid. In the anthocyanin class, not only delphinidin but also cyanidin prevented VEGF release whereas malvidin and peonidin were without effect. RWPs, delphinidin and cyanidin directly scavenged ROS and prevented the PDGF(AB)-induced formation of ROS in VSMCs. Malvidin and peonidin did not scavenge ROS but prevented the cellular formation of ROS. Although the p38 MAPK, ERK1/2 and JNK pathways have been involved in the PDGF(AB)-induced expression of VEGF, in our experiments, only phosphorylation of p38 MAPK and JNK was inhibited by RWPs, delphinidin and cyanidin. CONCLUSIONS AND IMPLICATIONS: Anthocyanins presenting a hydroxyl residue at position 3' are able to inhibit PDGF(AB)-induced VEGF expression by preventing activation of p38 MAPK and JNK in VSMCs.     

Transforming growth factor-beta(1) induces apoptosis via connective tissue growth factor in human aortic smooth muscle cells

We examined the possible involvement of connective tissue growth factor (CTGF) in the apoptosis induced by transforming growth factor-beta(1) (TGF-beta(1)) in human aortic vascular smooth muscle cells (HASC). In quiescent HASC, TGF-beta(1) induced the mRNA and protein of CTGF. A CTGF antisense oligonucleotide inhibited this induction. TGF-beta(1) significantly reduced cell viability and induced DNA fragmentation, and the CTGF antisense oligonucleotide reversed these effects. Moreover, TGF-beta(1) activated caspase 3 in HASC, and the CTGF antisense oligonucleotide reduced this activation. These findings show that CTGF plays a key role in the TGF-beta(1)-induced apoptosis in HASC.     

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。     

Evidence for the involvement of platelet-derived growth factor in the angiotensin II-induced growth of rat vascular smooth muscle cells

This study examines the possible involvement of endogenous platelet-derived growth factor (PDGF) in the angiotensin II-induced growth of rat aortic smooth muscle cells. In quiescent confluent cells, anti-PDGF-AB neutralizing antibody inhibited angiotensin II-induced DNA synthesis and protein synthesis. PDGF-AA, -AB, and -BB produced concentration-dependent increases in DNA synthesis and protein synthesis. Genistein did not inhibit PDGF-AB-induced [3H]thymidine incorporation and [3H]leucine incorporation. PDGF-AB stimulated mitogen-activated protein (MAP) kinases, and PDGF-induced MAP kinase activation was inhibited by genistein. Angiotensin II induced PDGF-A chain messenger RNA expression, and genistein inhibited angiotensin-induced PDGF gene expression. These findings suggest that endogenous PDGF is, at least in part, involved in angiotensin II-induced cell growth in rat vascular smooth muscle cells. It appears that genistein inhibits angiotensin II-induced DNA synthesis partly by inhibiting PDGF-A gene expression.