Inhibitory effect of antisense oligodeoxynucleotide to p44/p42 MAPK on angiotensin Ⅱ-induced hypertrophic response in cultured neonatal rat cardiac myocyte

AIM: To explore the inhibitory effect of antisense oligonucleotide (ODN) to mitogen activated protein kinase (MAPK) on cardiomyocyte hypertrophy induced by angiotensin Ⅱ (Ang Ⅱ). METHODS: A 17-mer phosphorothioate protected antisense ODN directed against the initiation of translation sites of the p42 and p44 MAPK isoforms by liposomal transfection was applied to inhibit the translation of p44/p42 MAPK mRNA. The sense and random ODNs to p44/p42MAPK were used as sequence controls. Neonatal cardiac myocytes were exposed to Ang Ⅱ (10 nmol/L) for 5 min and then harvested in lysis buffer for the measurement of the activity and the phosphorylated protein content of p44/p42MAPK that were tested by P-81 phosphocellulose filter paper method and Western blotting, respectively. The     

Inhibitory effects of nifedipine on DNA and protein synthesis in cultured cardiac nonmyocytes of neonatal rats

观察硝苯地平（Ｎｉｆｅｄｉｐｉｎｅ，Ｎｉｆ）对非心肌细胞生长和增殖的抑制作用．方法：以培养乳鼠非心肌细胞作为模型，用［３Ｈ］胸腺嘧啶核苷结合及［３Ｈ］亮氨酸结合的方法．结果：在血管紧张素Ⅱ存在的情况下，Ｎｉｆ１μｍｏｌ·Ｌ－１作用细胞７２ｈ，细胞的总蛋白及细胞数目明显减少．测得血管紧张素Ⅱ１，１０，１００，１０００ｎｍｏｌ·Ｌ－１在４８ｈ内，增加ＤＮＡ合成及蛋白合成．Ｎｉｆ１－１０μｍｏｌ·Ｌ－１均能够减少１００ｎｍｏｌ·Ｌ－１血管紧张素Ⅱ诱导的细胞ＤＮＡ合成及蛋白合成．结论：Ｎｉｆ可以直接抑制非心肌细胞的生长，该种作用与其抑制心肌肥厚有关．     

Inhibitory effect of antisense basic fibroblast growth factor oligonucleotides on proliferation of cultured aortic smooth muscle cells induced by angiotensin Ⅱ in SHR rats

目的：研究转染反义碱性成纤维细胞生长因子（ｂＦＧＦ）寡核苷酸（ＯＤＮ）对培养的自发性高血压大鼠（ＳＨＲ）主动脉平滑肌细胞（ＳＭＣ）生长的影响．方法：用脂质体介导法将反义ｂＦＧＦＯＤＮ转入ＳＭＣ内，用Ｎｏｒｔｈｅｒｎ杂交检测ｂＦＧＦ基因表达，并测定［３Ｈ］ｔｈｙｍｉｄｉｎｅ掺入和细胞计数．结果：转染反义ｂＦＧＦＯＤＮ（５μｍｏｌ·Ｌ－１）几乎完全抑制血管紧张素Ⅱ（ＡｎｇⅡ，１μｍｏｌ·Ｌ－１）诱导增高的ｂＦＧＦｍＲＮＡ表达和明显抑制ＳＭＣ增殖，在基础状态和ＡｎｇⅡ刺激条件下，［３Ｈ］ｔｈｙｍｉｄｉｎｅ掺入分别被抑制２６５％（Ｐ＜００１）和４２０％（Ｐ＜００１），细胞数分别被抑制１７３％（Ｐ＜００１）和２２２％（Ｐ＜００１）．结论：反义ｂＦＧＦＯＤＮ能有效抑制ＡｎｇⅡ诱导的ｂＦＧＦ基因表达和ＳＭＣ增殖．     

Bradykinin B_2receptor antagonist icatibant reduces inhibitory effect of captopril on growth of cultured neonatal rat cardiomyocytes

目的：观察内源性激肽对培养新生大鼠心肌细胞生长的影响及其机制．方法：［３Ｈ］尿嘧啶和［３Ｈ］亮氨酸参入法检测ＲＮＡ和蛋白质合成速率，Ｎｏｒｔｈｅｒｎ杂交检测ｃｍｙｃ和ｃｆｏｓｍＲＮＡ表达．结果：卡托普利１００μｍｏｌ·Ｌ－１孵育４８ｈ显著抑制［３Ｈ］尿嘧啶和［３Ｈ］亮氨酸参入，孵育２ｈ明显抑制ｃｍｙｃ和ｃｆｏｓ基因表达．ＡｎｇⅡ１μｍｏｌ·Ｌ－１处理４８ｈ刺激ＲＮＡ和蛋白质合成，１ｈ可上调ｃｍｙｃ和ｃｆｏｓ表达．Ｃａｐ１００μｍｏｌ·Ｌ－１部分抑制ＡｎｇⅡ上述作用．缓激肽Ｂ２受体拮抗剂Ｉｃａ（０１－１０μｍｏｌ·Ｌ－１）剂量依赖性阻断Ｃａｐ作用．结论：内源性激肽经ＢＫＢ２受体介导对心肌细胞的生长起负调节作用     

Protective effect of Salvia miltiorrhiza on angiotensin II-induced hypertrophic responses in neonatal rat cardiac cells

The effect of the root of Salvia miltiorrhiza (SM) on angiotensin II (Ang II)-induced hypertrophic responses was examined in cultured neonatal rat cardiac cells (cardiomyocytes and non-cardiomyocytes). The methanol eluate fraction (SM2) of the water extract and the ethyl acetate-insoluble fraction (SM3) and its soluble fraction (SM4) partitioned from the methanol extract were prepared. Treatment with SM4 (5-80 microg/ml), not SM2 and SM3, for 24 h produced dose-dependent cytotoxicity against cardiac cells relative to the reduction in viability and the morphological injury of cardiomyocytes. SM2 or SM3 in the absence of Ang II affected neither hyperplastic nor hypertrophic growth of both cell types. However, SM3 (40 microg/ml) attenuated the positive chronotropic responsiveness of cardiomyocytes to Ang II (1 nM) stimulation, whereas Ang II-induced increase in non-cardiomyocyte number was decreased only by SM2 (40 microg/ml) treatment. Furthermore, SM3 suppressed Ang II-induced enlargement of cell size by preceding Ang II-induced induction of immediate early response gene (c-jun) expression in cardiomyocytes, while SM2 decreased Ang II-induced DNA synthesis in non-cardiomyocytes. Moreover, three phenolic compounds and tanshinone IIA that differed quantitatively among three SM fractions were identified by reverse-phase high performance liquid chromatography. Thus, the present findings indicate that the root of SM is an effective inhibitor of Ang II action and has a plural effective constituent, which possess different pharmacological activities on Ang II-induced hypertrophy and hyperplasia in cultured neonatal rat cardiac cells.     

Lovastatin prevents angiotensin II-induced cardiac hypertrophy in cultured neonatal rat heart cells.

Angiotensin II activates p21ras, and mediates cardiac hypertrophic growth through the type 1 angiotensin II receptor in cardiac myocytes. An inhibitor of 3-hydroxy-3-methyglutaryl-coenzyme A (HMG-CoA) reductase has been shown to block the post-translational farnesylation of p21ras and inhibit protein synthesis in several cell types. Primary cultures of neonatal cardiac myocytes were used to determine whether HMG-CoA reductase inhibitors, lovastatin, simvastatin and pravastatin inhibit the angiotensin II-induced hypertrophic growth. Angiotensin II (10(-6) M) significantly increased protein-DNA ratio, RNA-DNA ratio, ratios of protein synthesis and mitogen-activated protein (MAP) kinase activity. Lipid-soluble HMG-CoA reductase inhibitors, lovastatin (10(-6) M) and simvastatin (10(-6) M) partially and significantly inhibited the angiotensin II-induced increases in these parameters, but a water-soluble HMG-CoA reductase inhibitor, pravastatin (10(-6) M) did not. Mevalonate (10(-4) M) overcame the inhibitory effects of lovastatin and simvastatin on angiotensin II-induced increases in these parameters. A selective protein kinase C inhibitor, calphostin C (10(-6) M) partially and significantly prevented angiotensin II-induced increases in these parameters, and treatment with both lovastatin and calphostin C inhibited completely. Angiotensin II increased p21ras activity and membrane association, and lovastatin inhibited them. These studies demonstrate that a lipid-soluble HMG-CoA reductase inhibitor, lovastatin, may prevent angiotensin II-induced cardiac hypertrophy, at least in part, through p21ras/MAP kinase pathway, which is linked to mevalonate metabolism.     

丝裂素活化蛋白激酶反义寡核苷酸对血管紧张素Ⅱ诱导的大鼠心肌？…

AIM: To investigate the inhibitory effect of down-regulating mitogen activated protein kinase (MAPK) on c-myc gene expression and further on cardiac fibroblast proliferation. METHODS: Cultured neonatal rat cardiac fibroblasts was pretreated with a phosphorothioate-protected 17-mer antisense MAPK oligodeoxynucleotide (ODN) directed against the initiation of translation sites of the p42 and p44 MAPK isoforms by liposomal transfection. A 17-mer sense and mismatch sequence MAPK ODN were used as controls. After liposomal transfecting, cells were exposed to angiotensin II (Ang II) 10 nmol.L-1 for 5 min and then harvested in lysis buffer. MAPK activity was measured by Western blot and P-81 phosphocellulose filter paper method by using [gamma-32P]ATP and myelin basic protein as substrates. c-myc mRNA expression stimulated by Ang II for 30 min was measured by Northern blot. DNA synthesis and collagen protein synthesis induced by Ang II for 24 h were measured by [3H]thymidine incorporation and [3H]Proline incorporation, respectively. RESULTS: Antisense ODN 0.2 mumol.L-1 reduced Ang II-induced MAPK activities by 72%, MAPK protein expression by 80%, and suppressed c-myc mRNA expression by 97%, respectively. [3H]thymidine incorporation and [3H]proline incorporation in Ang II-induced cardiac fibroblast were inhibited by 59% and 58%, respectively. CONCLUSION: A 17-mer MAPK antisense oligonucleotide directed againsts the initiation of translation sites of MAPK could specifically inhibit Ang II-stimulated cultured neonatal rat cardiac fibroblast proliferation through down-regulating MAPK activity and further depleting c-myc mRNA expression.     

槲皮素对血管紧张素Ⅱ致培养乳鼠心肌细胞肥大的抑制作用

目的:研究槲皮素(Quercetin,Que)对血管紧张素Ⅱ(Aug Ⅱ)诱发培养乳鼠心肌细胞肥大的抑制作用及机制。方法:分别用[~3H]胸苷、[~(14)C]尿苷和[~3H]酪氨酸标记测定DNA、RNA和蛋白质合成;用Lowry法测定蛋白质含量;用组蛋白ⅢS、[γ-~(32)P]ATP与蛋白激酶C(PKC)酶液一起保温测定PKC活性;用聚谷氨酸·酪氨酸(4:1)多肽、[γ-~(32)P]ATP与酪氨酸蛋白激酶(TPK)酶液一起保温测TPK活性。结果:Aug Ⅱ作用于心肌细胞24h后,心肌细胞总蛋白含量明显增加(P<0.01),[~(14)C]尿苷和[~3H]酪氨酸掺入量明显增加(P<0.01),而[~3H]胸苷掺入量未见增加(P>0.05);Aug Ⅱ作用30min后,心肌细胞PKC和TPK活性明显增加。Que(1-100μmol/L)能剂量依赖性地抑制Ang Ⅱ所致心肌细胞总蛋白含量、[~(14)C]尿苷和[~3H]酪氨酸掺入量、PKC及TPK活性的增加。结论:Que可抑制Aug Ⅱ致培养乳鼠心肌细胞肥大,该作用与抑制PKC及TPK活性有关。     

丝裂素活化的蛋白激酶反义寡核苷酸对p42/p44,p38和JNK激酶的靶向选择性及其对血管平滑肌细胞DNA合成的抑制作用

目的:探讨丝裂素活化的蛋白激酶(MAPK)反义寡核苷酸对血清诱导的培养大鼠血管平滑肌细胞增殖的选择性及序列依赖性抑制作用。方法:用脂质体将p42-和p44-MAPK反义寡核苷酸转染入大鼠血管平滑肌细胞,设正义及随机寡核苷酸对照,20％血清刺激后,用Western Blot法测定总p44/p42-MAPK、p38 MAPK及JNKs蛋白水平及磷酸化MAPK表达。[~3H]胸腺嘧啶核苷酸掺入测定平滑肌细胞DNA合成。结果:MAPK反义寡核苷酸能明显抑制血清诱导的血管平滑肌细胞总MAPK蛋白水平及磷酸化MAPK蛋白表达,对p38 MAPK及JNKs表达无影响,并能明显抑制[~3H]胸腺嘧啶核苷酸掺入。结论:针对p42-和p44-MAPK起始部位设计的17-mer反义寡核苷酸能选择性及序列依赖性地抑制血清诱导的血管平滑肌细胞的增殖。     

Target selectivity of MAPK phosphorothioate antisense ODN on p42/p44, p38 MAPK, and JNK protein expression and its inhibitory effect on VSMC DNA synthesis.

AIM: To analyze the target selective and sequence-specific inhibitory effect of mitogen-activated protein kinase (MAPK) phosphorothioate antisense oligodeoxynucleotides (ODN) on p42/p44, p38 MAPK, c-jun NH2-terminal protein kinases (JNK) protein expression, and DNA synthesis in vascular smooth muscle cell (VSMC). METHODS: Using a phosphorothioate-protected 17-mer antisense MAPK ODN directed against the initiation of translation sites of the p42/p44 MAPK isoforms by liposomal transfection to deplete cultured rat, rabbit, and fetal calf VSMC MAP kinases. The 17-mer sense and random sequence MAPK ODN were used as controls. After liposomal transfection, cells were exposed to 20% serum for 24 h, and then harvested in lysis buffer. P42/p44, p38 MAPK, and p46/p58 JNK protein expression were measured by Western blot. DNA synthesis was measured by [3H]thymidine incorporation. RESULTS: Treatment with MAPK antisense ODN (0.1-0.8 mumol.L-1) for 48 h reduced phosphored p42/p44 MAPK protein expression but without effect on p38 MAPK and JNK expression, and inhibited cultured rat, rabbit, and fetal calf VSMC [3H]thymidine incorporation stimulated by 20% serum in a concentration-dependent manner. CONCLUSION: The MAPK antisense ODN target-selectively and sequence-specifically reduces the p42/p44 MAPK protein expression and concentration-dependently inhibits proliferation of rat, rabbit and fetal calf VSMC.     

Inhibitory effect of trilinolein on angiotensin II-induced cardiomyocyte hypertrophy

The myocardial protective effects of trilinolein, isolated from the Chinese herb Sanchi (Panax notoginseng), may be related to its antioxidant effects. In the present study, we investigated the effects of trilinolein on angiotensin II-induced cardiomyocyte hypertrophy. Cultured neonatal rat cardiomyocytes were stimulated with angiotensin II, [3H]leucine incorporation and the beta-myosin heavy chain promoter activity were examined. We also examined the effects of trilinolein on angiotensin II-induced intracellular reactive oxygen species generation. Trilinolein significantly inhibited angiotensin II-increased protein synthesis, beta-myosin heavy chain promoter activity, and intracellular reactive oxygen species generation. Antioxidant N-acetylcysteine also decreased angiotensin II-increased protein synthesis and beta-myosin heavy chain promoter activity. Furthermore, trilinolein and N-acetylcysteine decreased angiotensin II- or hydrogen peroxide (H2O2)-activated mitogen-activated protein kinases (MAPKs) phosphorylation, and activator protein-1 (AP-1)- [or nuclear factor-kappaB (NF-kappaB)]-reporter activities. These data indicate that trilinolein inhibits angiotensin II-induced cardiomyocyte hypertrophy and beta-myosin heavy chain promoter activity via attenuation of reactive oxygen species generation.     

Inhibitory effect of resveratrol on angiotensin II-induced cardiomyocyte hypertrophy

Resveratrol is proposed to account in part for the protective effect of red wine on the cardiovascular system. Angiotensin II (Ang II) is a potent hypertrophic stimulus in cardiomyocytes. In this study, we determined the effect of resveratrol on Ang II-induced cardiomyocyte hypertrophy. Cultured neonatal rat cardiomyocytes were stimulated with Ang II, and [³H]leucine incorporation and -myosin heavy chain (-MyHC) promoter activity were examined. Intracellular reactive oxygen species (ROS) were measured by a redox-sensitive fluorescent dye, 2 7-dichlorofluorescin diacetate, and the extracellular signal-regulated kinase (ERK) phosphorylation was examined by Western blotting. Resveratrol inhibited Ang II-increased intracellular ROS levels. Furthermore, resveratrol, as well as the antioxidant N-acetyl-cysteine, decreased Ang II- or H₂O₂-increased protein synthesis, -MyHC promoter activity, and ERK phosphorylation. In summary, we demonstrate for the first time that resveratrol inhibits Ang II-induced cardiomyocyte hypertrophy via attenuation of ROS generation.Abbreviations Ang II Angiotensin II - MAPKs Mitogen-activated protein kinases - ERK Extracellular signal-regulated kinase - JNK c-Jun N-terminal kinase - p38 MAPK p38 mitogen-activated protein kinases - MEK MAPK or ERK kinase - NAC N-acetylcysteine - DCF-DA Dichlorodihydrofluorescein diacetate - DCF Dichlorofluorescein     

Dopamine D2 receptor stimulation inhibits angiotensin II-induced hypertrophy in cultured neonatal rat ventricular myocytes

1. Myocardial hypertrophy is a common pathological change that accompanies cardiovascular disease. Dopamine D2 receptors have been demonstrated in cardiovascular tissues. However, the pathophysiological involvement of D2 receptors in myocardial hypertrophy is unclear. Therefore, the effects of the D2 receptor agonist bromocriptine and the D2 receptor antagonist haloperidol on angiotensin (Ang) II- or endothelin (ET)-1-induced hypertrophy of cultured neonatal rat ventricular myocytes were investigated in the present study. 2. Protein content and protein synthesis, determined by examining [(3)H]-leucine uptake, were used as estimates of cardiomyocyte hypertrophy. The expression of D2 receptor protein in neonatal rat ventricular myocytes was determined using western blotting. Changes in [Ca(2+)](i) in cardiomyocytes were observed by laser scanning confocal microscopy. 3. Angiotensin II and ET-1, both at 10 nmol/L, induced myocyte hypertrophy, as demonstrated by increased protein content and synthesis, [Ca(2+)](i) levels, protein kinase C (PKC) activity and phosphorylation of extracellular signal-regulated kinase, c-Jun N-terminal kinase and mitogen-activated protein kinase (MAPK) p38 (p38). Concomitant treatment of cells with 10 nmol/L AngII plus 10 micromol/L bromocriptine significantly inhibited cardiomyocyte hypertrophy, MAPK phosphorylation and PKC activity in the membrane, as well as [Ca(2+)](i) signalling pathways, compared with the effects of AngII alone. In addition, 10 micromol/L bromocriptine significantly inhibited cardiomyocyte hypertrophy induced by 10 nmol/L ET-1. However, pretreatment with haloperidol (10 micromol/L) had no significant effects on cardiomyocyte hypertrophy induced by either AngII or ET-1. 4. In conclusion, D2 receptor stimulation inhibits AngII-induced hypertrophy of cultured neonatal rat ventricular myocytes via inhibition of MAPK, PKC and [Ca(2+)](i) signalling pathways.     

17beta-estradiol inhibits angiotensin II-induced collagen synthesis of cultured rat cardiac fibroblasts via modulating angiotensin II receptors

Circulating endogenous estrogen is considered to be cardiovascular protective, but the underlying mechanisms remain obscure. The cardiac fibroblasts, the most abundant cell type present in the heart, are responsible for the deposition of extracellular matrix. Angiotensin II has been known to stimulate cardiac collagen gene expression. The present study was designed to investigate the effect of 17beta-estradiol on the angiotensin II-induced proliferation and collagen synthesis in cultured cardiac fibroblasts by using real-time polymerase chain reaction (PCR), Western blot and 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide proliferation assay. Angiotensin II increased the cell proliferation and synthesis of collagen types I and III. Angiotensin II up-regulated the gene expression of the angiotensin AT(1) receptor and down-regulated the gene expression of the angiotensin AT(2) receptor in cardiac fibroblasts. The effects of angiotensin II was abolished by the angiotensin AT(1) receptor antagonist, losartan, but not by the angiotensin AT(2) receptor antagonist, PD 123319. 17beta-estradiol prevented increases in proliferation and attenuated the collagen synthesis in response to angiotensin II. The increased AT(1) receptor mRNA levels and decreased AT(2) receptor mRNA levels were partially reversed by 17beta-estradiol treatment. In conclusion, the down-regulation of angiotensin AT(1) receptor expression and function is likely to be an important mechanism accounting for the inhibitory effect of 17beta-estradiol on angiotensin II-stimulated proliferation and collagen synthesis in cardiac fibroblasts. This effect may confer at least in part the cardiac protective action of 17beta-estradiol under pathological conditions with increased activity of the rennin-angiotensin system.