Angiotensin II stimulates PGF(2 alpha)release in cultured neonatal rat ventricular myocytes via L-type calcium channels.

Angiotensin II (Ang II) has been shown to cause Prostaglandin F(2 alpha)(PGF(2 alpha)) release in neonatal rat ventricular myocytes and smooth muscle cells. In these cells, Ang II has also been shown to regulate growth. We used neonatal rat ventricular myocytes to investigate the role of calcium in maintenance of Ang II-induced PGF(2 alpha)release. The amount of PGF(2 alpha)produced was determined by radioimmunoassay. Ang II-induced PGF(2 alpha)release. Pretreatment of neonatal rat ventricular myocytes with different doses (10(-8)M, 10(-7)M, 10(-6)M and 10(-5)M) of diltiazm (voltage-sensitive L-type calcium channel blocker) produced significant inhibition in Ang II-induced PGF(2 alpha)release. Inhibition was first noted at 10(-8)M and was complete at 10(-6)M. Conversely, pretreatment of neonatal rat ventricular myocytes with different doses (10(-8)M, 10(-7)M, 10(-6)M and 10(-5)M) of calcium channel blockers (conotoxin; voltage-sensitive N-type calcium channel blocker or thapsigargin; intracellular calcium channel blocker) produced no changes in Ang II-induced PGF(2 alpha)release. These results strongly suggest that Ang II-induced PGF(2 alpha)release in neonatal rat ventricular myocytes is maintained, at least in part, via increase in extracellular calcium influx.     

Angiotensin II-induced ET and PGI2 release in rat aortic endothelial cells is mediated by PKC.

Angiotensin II (Ang II) has been shown to stimulate the release of immunoreactive endothelin (ET) from cultured bovine ECs. Also, Ang II activates phospholipase A2 (PLA2) in various tissues, resulting in the release of arachidonic acid and formation of prostaglandins. We used rat aortic endothelial cells to investigate the role of protein kinase C (PKC) in Ang II-induced release of both ET and prostacyclin (PGI2). The amount of ET and PGI2 produced were determined by radioimmunoassay. Ang II-induced the release of both ET and PGI2. Pretreatment with 10(-6) M of any one of the PKC inhibitors: 1-(5-isoquinolinesulfonyl) piperazine(CL), staurosporine, 1-(5-isoquinolinesulfonylmethyl)piperazine(H7), and calphostin C blocked AII-induced release of both ET and PGI2. In rat aortic endothelial cells that were treated with either AII or PDBu, PKC enzyme assay showed PKC was translocated from the cytosol to the membrane which indicates activation. This suggests that PKC mediates AII-induced ET and PGI2 release. In summary, AII activates PKC which inhibits rat aortic endothelial cells ET and PGI2 formation, and this inhibition can be overcome by pretreatment with PKC inhibitors.     

Inhibition of Angiotensin II-Induced Cardiac Hypertrophy and Associated Ventricular Arrhythmias by a p21 Activated Kinase 1 Bioactive Peptide

Cardiac hypertrophy increases the risk of morbidity and mortality of cardiovascular disease and thus inhibiting such hypertrophy is beneficial. In the present study, we explored the effect of a bioactive peptide (PAP) on angiotensin II (Ang II)-induced hypertrophy and associated ventricular arrhythmias in in vitro and in vivo models. PAP enhances p21 activated kinase 1 (Pak1) activity by increasing the level of phosphorylated Pak1 in cultured neonatal rat ventricular myocytes (NRVMs). Such PAP-induced Pak1 activation is associated with a significant reduction of Ang II-induced hypertrophy in NRVMs and C57BL/6 mice, in vitro and in vivo, respectively. Furthermore, PAP antagonizes ventricular arrhythmias associated with Ang II-induced hypertrophy in mice. Its antiarrhythmic effect is likely to be involved in multiple mechanisms to affect both substrate and trigger of ventricular arrhythmogenesis. Thus our results suggest that Pak1 activation achieved by specific bioactive peptide represents a potential novel therapeutic strategy for cardiac hypertrophy and associated ventricular arrhythmias.     

Rho plays an important role in angiotensin II-induced hypertrophic responses in cardiac myocytes

Angiotensin II (Ang II) evokes a variety of hypertrophic responses such as activation of protein kinases, reprogramming of gene expressions and an increase in protein synthesis in cardiac myocytes. In this study, we examined the role of Rho family small GTP binding proteins (G proteins) in Ang II-induced cardiac hypertrophy. Ang II strongly activated extracellular signal-regulated protein kinases (ERKs) in cardiac myocytes of neonatal rats. Although Ang II-induced activation of ERKs was completely suppressed by an Ang II type 1 receptor antagonist, CV-11974, this activation was not inhibited by the pretreatment with C3 exoenzyme, which abrogates Rho functions. Overexpression of Rho GDP dissociation inhibitor (Rho-GDI), dominant negative mutants of Rac1 (D.N.Rac1), or D.N.Cdc42 had no effects on Ang II-induced activation of transfected ERK2. The promoter activity of skeletal a-actin and c-fos genes was increased by Ang II, and the increase was partly inhibited by overexpression of Rho-GDI and the pretreatment with C3 exoenzyme. Ang II increased phenylalanine incorporation into cardiac myocytes by approximately 1.4 fold as compared with control, and this increase was also significantly suppressed by the pretreatment with C3 exoenzyme. These results suggest that the Rho family small G proteins play important roles in Ang II-induced hypertrophic responses in cardiac myocytes.     

钙调神经磷酸酶依赖的信号通路参与血管紧张素Ⅱ刺激的心肌细胞肥大

本研究观察了钙调神经磷酸酶依赖的信号通路在血管紧张素Ⅱ诱导的大鼠心肌细胞肥大中的作用。在ＡｎｇⅡ刺激的大鼠心肌细胞肥大模型上,应用环孢素Ａ（ＣｓＡ）阻断ＣａＮ通路,观察心肌细胞＾３Ｈ－亮氨酸掺入,ＣａＮ,ＭＡＰＫ及ＰＫＣ活性的变化。结果表明,ＡｎｇⅡ（１０＾－７ｍｏｌ／Ｌ）刺激大鼠心肌细胞＾３Ｈ－亮氨酸掺入较对照组增高４６％（Ｐ〈０．０１）,ＣｓＡ（０．５－５μｇ／ｍｌ）可以浓度依赖性方式抑制Ａｎ     

Effects of triiodo-thyronine on angiotensin-induced cardiomyocyte hypertrophy: reversal of increased beta-myosin heavy chain gene expression

Thyroid hormone-induced cardiac hypertrophy is similar to that observed in physiological hypertrophy, which is associated with high cardiac contractility and increased alpha-myosin heavy chain (alpha-MHC, the high ATPase activity isoform) expression. In contrast, angiotensin II (Ang II) induces an increase in myocardial mass with a compromised contractility accompanied by a shift from alpha-MHC to the fetal isoform beta-MHC (the low ATPase activity isoform), which is considered as a pathological hypertrophy and inevitably leads to the development of heart failure. The present study is designed to assess the effect of thyroid hormone on angiotensin II-induced hypertrophic growth of cardiomyocytes in vitro. Cardiomyocytes were prepared from hearts of neonatal Wistar rats. The effects of Ang II and 3,3',5-triiodo-thyronine (T3) on incorporations of [3H]-thymine and [3H]-leucine, MHC isoform mRNA expression, PKC activity, and PKC isoform protein expression were studied. Ang II enhanced [3H]-leucine incorporation, beta-MHC mRNA expression, PKC activity, and PKCepsilon expression and inhibited alpha-MHC mRNA expression in cardiomyocytes. T3 treatment prevented Ang II-induced increases in PKC activity, PKCepsilon, and beta-MHC mRNA overexpression and favored alpha-MHC mRNA expression. Thyroid hormone appears to be able to reprogram gene expression in Ang II-induced cardiac hypertrophy, and a PKC signal pathway may be involved in such remodeling process.     

Phorbol ester stimulates cyclooxygenase-2 expression and prostanoid production in cardiac myocytes

Phorbol-12-myristate- 13-acetate (PMA) has been shown to induce hypertrophy of cardiac myocytes. The prostaglandin endoperoxide H synthase isoform 2 (cyclooxygenase-2, COX-2) has been associated with enhanced growth and/or proliferation of several types of cells. Thus we studied whether PMA induces COX-2 and prostanoid products PGE(2) and PGF(2alpha) in neonatal ventricular myocytes and whether endogenous COX-2 products participate in their growth. In addition, we examined whether PMA affects interleukin-1beta (IL-1beta) stimulation of COX-2 and PGE(2) production. PMA (0.1 micromol/l) stimulated growth, as indicated by a 1.6-fold increase in [(3)H]leucine incorporation. PMA increased COX-2 protein levels 2. 8-fold, PGE(2) 3.7-fold, and PGF(2alpha) 2.9-fold. Inhibition of either p38 kinase or protein kinase C (PKC) prevented PMA-stimulated COX-2. Inhibition of COX-2 with either indomethacin or NS-398 had no effect on PMA-stimulated [(3)H]leucine incorporation. Exogenous administration of PGF(2alpha), but not PGE(2), stimulated protein synthesis. Treatment with IL-1beta (5 ng/ml) increased COX-2 protein levels 42-fold, whereas cotreatment with IL-1beta and PMA stimulated COX-2 protein only 32-fold. IL-1beta did not affect control or PMA-stimulated protein synthesis. These findings indicate that: 1) PMA, acting through PKC and p38 kinase, enhances COX-2 expression, but chronic treatment with PMA partially inhibits IL-1beta stimulation of COX-2; and 2) exogenous PGF(2alpha) is involved in neonatal ventricular myocyte growth but endogenous COX-2 products are not.     

Angiotensin II modulates gene expression of adrenomedullin receptor components in rat cardiomyocytes

Both adrenomedullin (AM) and angiotensin II (Ang II) are locally-acting hormones in the cardiac ventricles. Previously we reported that AM inhibits Ang II-induced hypertrophy of cultured rat neonatal cardiomyocytes. In this study, we examined whether Ang II affects the gene expression of the AM receptor components of calcitonin-receptor-like receptor (CRLR) and receptor-activity-modifying protein (RAMP) in rat cardiomyocytes. The mRNA levels of RAMP1 and RAMP3 were significantly elevated following 24-h treatment with Ang II without a change of those of RAMP2 and CRLR. AM increased the intracellular cAMP level and the cAMP accumulation by AM was significantly amplified by the 24-h preincubation with Ang II. The effects of Ang II on RAMP1 and RAMP3 expression were abolished by an Ang II type 1 (AT1) receptor antagonist, but not by an AT2 receptor antagonist. Thus, Ang II modulates gene expression of the AM receptor components via AT1 receptor, suggesting alteration of AM actions by Ang II in cultured rat cardiomyocytes.     

Effects of all-trans retinoic acid on angiotensin II-induced myocyte hypertrophy.

We used cultured neonatal rat cardiac myocytes to test the hypothesis that all-trans retinoic acid (atRA) may act to modulate ANG II actions in inducing myocyte hypertrophy. Our observations were as follows. 1) atRA (10(-7) to approximately 10(-5) M ) inhibited ANG II-induced hyperplasia of fibroblasts in a dose-dependent manner. 2) Treatment of atRA attenuated the ANG II-induced increase in total cell protein content. 3) Treated with ANG II (10(-7) M) for 5 days, the cultured neonatal rat cardiac myocytes demonstrated an apparent accumulation of sarcomeric fiber proteins and Golgi's complex, as well as reorganization of the sarcomeric unit within individual myocytes. atRA (10(-6) M) treatment reduced the accumulation of contractile proteins and Golgi's complex without affecting the ANG II-induced reorganization of the sarcomeric unit. 4) atRA attenuated the ANG II-induced increase in intracellular Ca2+. Our results show that atRA inhibits some effects of ANG II on neonatal rat cardiac myocytes and suggest that atRA may be a therapeutic candidate for the prevention and therapy of cardiac hypertrophy and remodeling.     

Adiponectin Upregulates MiR-133a in Cardiac Hypertrophy through AMPK Activation and Reduced ERK1/2 Phosphorylation

Adiponectin and miR-133a are key regulators in cardiac hypertrophy. However, whether APN has a potential effect on miR-133a remains unclear. In this study, we aimed to investigate whether APN could regulate miR-133a expression in Angiotensin II (Ang II) induced cardiac hypertrophy in vivo and in vitro. Lentiviral-mediated adiponectin treatment attenuated cardiac hypertrophy induced by Ang II infusion in male wistar rats as determined by reduced cell surface area and mRNA levels of atrial natriuretic peptide (ANF) and brain natriuretic peptide (BNP), also the reduced left ventricular end-diastolic posterior wall thickness (LVPWd) and end-diastolic interventricular septal thickness (IVSd). Meanwhile, APN elevated miR-133a level which was downregulated by Ang II. To further investigate the underlying molecular mechanisms, we treated neonatal rat ventricular myocytes (NRVMs) with recombinant rat APN before Ang II stimulation. Pretreating cells with recombinant APN promoted AMP-activated protein kinase (AMPK) phosphorylation and inhibited ERK activation. By using the inhibitor of AMPK or a lentiviral vector expressing AMPK short hairpin RNA (shRNA) cancelled the positive effect of APN on miR-133a. The ERK inhibitor PD98059 reversed the downregulation of miR-133a induced by Ang II. These results indicated that the AMPK activation and ERK inhibition were responsible for the positive effect of APN on miR-133a. Furthermore, adiponectin receptor 1 (AdipoR1) mRNA expression was inhibited by Ang II stimulation. The positive effects of APN on AMPK activation and miR-133a, and the inhibitory effect on ERK phosphorylation were inhibited in NRVMs transfected with lentiviral AdipoR1shRNA. In addition, APN depressed the elevated expression of connective tissue growth factor (CTGF), a direct target of miR-133a, through the AMPK pathway. Taken together, our data indicated that APN reversed miR-133a levels through AMPK activation, reduced ERK1/2 phosphorylation in cardiomyocytes stimulated with Ang II, revealing a previously undemonstrated and important link between APN and miR-133a.     

新生大鼠心室成纤维细胞条件培养液的促心肌细胞肥大作用

利用新生大鼠心室成纤维细胞条件培养液孵育心肌细胞,证实成纤维细胞条件培养液能明显增大心肌细胞的表面积,增加心肌细胞的蛋白含量和「＾３Ｈ」－亮氨酸「＾３Ｈ」－Ｌｅｕ）的掺入,上述作用以第３天的条件培养液作用最强,具有剂量依赖性。ＥＴＡ受体拮抗剂ＢＱ１２３能部分阻断成纤维细胞条件培养液促进心肌细胞肥大的作用,而ＡＴ１受体拮抗剂ＣＶ１１９７４和α－肾上腺素受体拮抗剂ｒｅｇｉｔｉｎ却无此效果。结果提示：成     

血管紧张素II诱导培养的成年大鼠心肌细胞凋亡

研究血管紧张素Ⅱ（ＡｎｇⅡ）诱导培养的成年大鼠心肌细胞（ＡＲＶＭｓ）凋亡。酶灌流消化法分离培养ＡＲＶＭｓ,不同处理后,光镜观察形态改变,琼脂糖凝胶电泳定性分析ＤＮＡ降解程度。结果发现培养的ＡＲＶＭｓ经ＡｎｇⅡ１０μｍｏｌ／Ｌ处理４８ｈ后,大部分细胞变圆,胞浆浓缩;电泳显示核酸断裂片段“梯形”结构,上述改变在７２ｈ更为明显。上述作用可被氯沙坦、维拉帕米和ｓｔａｕｒｏｓｐｏｒｉｎｅ所取消。这表明Ａｎｇ     

The effects of thiazolidinediones on vascular smooth muscle cell activation by angiotensin II

Angiotensin II (Ang II) stimulates the activation of extracellular signal-regulated kinase (ERK), a subgroup of the mitogen-activated protein kinase (MAPK) family, in cultured vascular smooth muscle cells (VSMC). This ERK activation was recently shown to be a critical regulatory factor for Ang II-mediated migration and growth. It has been demonstrated that the thiazolidinedione troglitazone (TRO) blocked Ang II-induced DNA synthesis and migration in VSMC. Here we provide evidence for TRO to inhibit Ang II-induced ERK activation which was suggested to constitute the mechanism by which this agent blocks Ang II-induced VSMC growth and migration. We have found that pretreatment with PD98059, which selectively blocks the activity of ERK pathway at the level of MAPK kinase, decreased Ang II-induced AP-1 activation and that TRO is capable of inhibiting Ang II-induced AP-1 activation. On the other hand, the other thiazolidinediones pioglitazone (PIO) and rosiglitazone (ROSI) had little effect on Ang II-induced activation of ERK or AP-1, suggesting the inhibitory effects of TRO on VSMC activation by Ang II be independent of the peroxisome proliferator-activated receptor-gamma (PPARgamma) for which thiazolidinediones are ligands. Ang II-induced ERK activation was inhibited by protein kinase C (PKC)-specific inhibitor GF109203X, while TRO was also able to block PKC activator phorbol 12 myristate 13-acetate (PMA)-induced ERK activation. Accordingly, TRO may inhibit Ang II-induced MAPK activation at least partly by an inhibition of PKC. These results support the assumption that by targeting MAPK activation, TRO may inhibits the critical signaling steps leading to restenosis and atherosclerosis that may result in part from dysregulated VSMC growth and migration induced by Ang II.     

Angiotensin IV protects against angiotensin II-induced cardiac injury via AT4 receptor

Angiotensin II (Ang II) is an important regulator of cardiac function and injury in hypertension. The novel Ang IV peptide/AT4 receptor system has been implicated in several physiological functions and has some effects opposite to those of Ang II. However, little is known about the role of this system in Ang II-induced cardiac injury. Here we studied the effect of Ang IV on Ang II-induced cardiac dysfunction and injury using isolated rat hearts, neonatal cardiomyocytes and cardiac fibroblasts. We found that Ang IV significantly improved Ang II-induced cardiac dysfunction and injury in the isolated heart in response to ischemia/reperfusion (I/R). Moreover, Ang IV inhibited Ang II-induced cardiac cell apoptosis, cardiomyocyte hypertrophy, and proliferation and collagen synthesis of cardiac fibroblasts; these effects were mediated through the AT4 receptor as confirmed by siRNA knockdown. These findings suggest that Ang IV may have a protective effect on Ang II-induced cardiac injury and dysfunction and may be a novel therapeutic target for hypertensive heart disease.     

Inhibitory effects of interferon-gamma on myocardial hypertrophy

Prostaglandin F(2alpha) (PGF(2alpha)) plays an important role in pathologic cardiac growth. After testing several immune cytokines, we found that interferon-gamma (IFN-gamma) inhibited responsiveness of adult myocytes to PGF(2alpha). The present study was designed to test the hypothesis that IFN-gamma inhibits cardiac hypertrophy induced by PGF(2alpha). Incubation of cultured adult rat cardiac myocytes with PGF(2alpha) caused cell spreading, which was inhibited by IFN-gamma. The inhibitory effect was not affected by nitric oxide (NO) synthase inhibitors. In addition, administration of fluprostenol, a more selective agonist at the PGF(2alpha) receptor, induced cardiac hypertrophy in rats. Chronic treatment with IFN-gamma inhibited this myocardial growth, and the inhibitory effect of IFN-gamma was not accompanied by an increase in myocardial NO synthase gene expression. Further, abdominal aortic constriction resulted in a substantial increase in heart, ventricular and left ventricular weights to BW ratio that was significantly attenuated by treatment with IFN-gamma. The results demonstrate that IFN-gamma inhibits the in vitro and in vivo effects of PGF(2alpha) on cardiac hypertrophy, and that the mechanism of action is likely independent of NO production. IFN-gamma also attenuated cardiac hypertrophy induced by pressure overload, suggesting that PGF(2alpha) plays a role in the pathogeneses of this severe type of cardiac hypertrophy.     

Angiotensin II inhibits inward rectifier K+ channels in rabbit coronary arterial smooth muscle cells through protein kinase Calpha

We investigated the effects of the vasoconstrictor angiotensin (Ang) II on the whole cell inward rectifier K(+) (Kir) current enzymatically isolated from small-diameter (<100 microm) coronary arterial smooth muscle cells (CASMCs). Ang II inhibited the Kir current in a dose-dependent manner (half inhibition value: 154 nM). Pretreatment with phospholipase C inhibitor and protein kinase C (PKC) inhibitors prevented the Ang II-induced inhibition of the Kir current. The PKC activator reduced the Kir currents. The inhibitory effect of Ang II was reduced by intracellular and extracellular Ca(2+) free condition and by G?6976, which inhibits Ca(2+)-dependent PKC isoforms alpha and beta. However, the inhibitory effect of Ang II was unaffected by a peptide that selectively inhibits the translocation of the epsilon isoform of PKC. Western blot analysis confirmed that PKCalpha, and not PKCbeta, was expressed in small-diameter CASMCs. The Ang II type 1 (AT(1))-receptor antagonist CV-11974 prevented the Ang II-induced inhibition of the Kir current. From these results, we conclude that Ang II inhibits Kir channels through AT(1) receptors by the activation of PKCalpha.     

Angiotensin II interacts with prostaglandin F2alpha and endothelin-1 as a local luteolytic factor in the bovine corpus luteum in vitro

Recent findings suggest that the ovarian renin-angiotensin system may regulate ovarian function through the paracrine/autocrine actions of angiotensin II (Ang II). In this study, we have examined and characterized the local effects of Ang II as a luteolytic factor and its interaction with prostaglandin F2alpha (PGF2alpha) and endothelin-1 (ET-1) in the bovine corpus luteum (CL) of the mid-luteal phase, by using an in vitro microdialysis system (MDS). Ang II was detected in the MDS perfusate (4 pg/ml), and infusion of PGF2alpha (10(-6) M) for 2 h increased the Ang II release by 50-100% during the following experimental period, in addition to its stimulation of ET-1 release. Two 2-h infusions of Ang II (10(-7)-10(-5) M) separated by a 2-h interval induced a dose- and time-dependent decrease of progesterone (P4) release by 41-66%. When the luteal explants were pre-perfused with PGF2alpha (10(-6) M) for 2 h, two consecutive perfusions of Ang II (10(-6) M) at a 2-h interval rapidly reduced the P4 release (by 50%). This reduction occurred 6 h earlier than those of infusions of PGF2alpha or Ang II alone. The simultaneous infusion of either 1) Ang II (10(-6) M) with PGF2alpha (10(-6) M), 2) ET-1 (10(-7) M) with PGF2alpha, or 3) Ang II + ET-1 with PGF2alpha (10(-6) M) for 2 h also induced a rapid and pronounced (60%) decrease in P4 release. Perfusion with the Ang II antagonist blocked the P4-suppressing activity of Ang II alone or PGF2alpha + Ang II infusion. Ang II stimulated the release of ET-1 and oxytocin during infusion but inhibited them after infusion. These results show that Ang II is released in the bovine midcycle CL in vitro, and this peptide, either alone or together with PGF2alpha, can suppress the release of P4. As PGF2alpha directly stimulated Ang II release, Ang II may influence the critical period for starting the cascade of functional luteolysis in vivo and might lead to structural luteolysis with ET-1 as a major vasoconstrictor. The overall results suggest that Ang II may have an important role at luteolysis in the bovine CL.     

Hypertrophic response to angiotensin II is mediated by protein kinase D-extracellular signal-regulated kinase 5 pathway in human aortic smooth muscle cells

Angiotensin II plays a critical role in hypertrophy of vascular smooth muscle cells, however, the molecular underpinnings remain unclear. The present study indicated that AT₁/PKC/PKD pathway was able to regulate downstream ERK5, affecting pro-hypertrophic responses to Ang II. Ang II-stimulated phosphorylation of ERK5 in a time- and dose-dependent manner in human aortic smooth muscle cells (HASMCs). The pharmacological inhibitors for AT₁ and PKCs significantly inhibited Ang II-induced ERK5 activation, suggesting the involvement of the AT₁/PKC pathway. In particular, PKD was critical for Ang II-induced ERK5 activation since silencing PKD by siRNA markedly inhibited Ang II-induced ERK5 activation. Consequently, we found that Losartan, Gö 6983 and PKD siRNA significantly attenuated ERK5 activated translocation and hypertrophy of HASMCs by Ang II. Taken together, we demonstrated for the first time that Ang II activates ERK5 via the AT₁/PKC/PKD pathway and revealed a critical role of ERK5 in Ang II-induced HASMCs hypertrophy.