Multisite phosphorylation of Bcl‐2 via protein kinase Cδ facilitates apoptosis of hypertrophic cardiomyocytes

Activated protein kinase Cδ (PKCδ) associated with cardiac hypertrophy moves from the cytoplasm to the mitochondria and subsequently triggers the apoptotic signalling pathway. The underlying mechanisms remain unknown. The aim of the present study was to investigate whether mitochondrial translocation of PKCδ phosphorylates multiple sites of Bcl‐2, resulting in an imbalance between Bcl‐2 and Bak or Bax, thus enhancing the susceptibility of hypertrophic cardiomyocytes to angiotensin II (AngII)‐induced apoptosis. Chronic pressure overload was induced by transverse aortic constriction (TAC) in rats. The apoptotic rate increased in hypertrophied cardiomyocytes. In AngII‐treated hearts (10 nmol/L, 60 min), there was an increase in the number of TERMINAL deoxyribonucleotidyl transferase‐mediated dUTP–digoxigenin nick end‐labelling (TUNEL)‐positive cells; PKCδ inhibition with 500 nmol/L δV1‐1 for 60 min prevented the AngII‐induced increase in apoptosis. In the hypertrophied myocardium, PKCδ expression increased, whereas that of Bcl‐2 decreased compared with the synchronous control. Treatment of hearts with 10 nmol/L AngII for 60 min activated PKCδ and induced translocation of PKCδ to the mitochondria, where activated PKCδ facilitated the phosphorylation of Bcl‐2 at serine‐87 and serine‐70 sites. The multisite phosphorylated Bcl‐2 was released from the mitochondria, and exhibited reduced affinity for Bak and Bax. The imbalance between Bcl‐2 and Bak/Bax induced the release of mitochondrial cytochrome c and then activated the caspase 3 apoptotic pathway during AngII stimulation (10 nmol/L, 60 min) of hypertrophied cardiomyocytes. Inhibition of PKCδ reduced these effects of AngII. The results suggest that PKCδ can counteract the anti‐apoptotic effect of Bcl‐2 and may promote cardiomyocyte apoptosis through multisite phosphorylation of Bcl‐2 in hypertrophied cardiomyocytes.     

Angiotensin‐(1–7) treatment ameliorates angiotensin II‐induced apoptosis of human umbilical vein endothelial cells

相似文献   

Role of α1D‐adrenoceptors in vascular wall hypertrophy during angiotensin II‐induced hypertension

相似文献   

Ghrelin protects H9c2 cardiomyocytes from angiotensin II-induced apoptosis through the endoplasmic reticulum stress pathway

Ghrelin, a gastric hormone, exerts cardioprotective function by increasing myocardial contractility and vasodilation. Previous studies have reported that angiotensin II (Ang II) production increased in heart failure, which can induce cardiomyocyte apoptosis. In this study, we investigated the effect of ghrelin on Ang II-induced H9c2 cardiomyocyte apoptosis. The results showed that Ang II inhibited H9c2 cell viability, which was blocked by ghrelin. By annexin V-propidium iodide dual staining and 2'-deoxyuridine 5'-triphosphate nick end-labeling analysis, we found that Ang II induced H9c2 cell apoptosis, whereas coincubation of ghrelin with Ang II significantly reduced H9c2 cell apoptosis induced by Ang II. Simultaneously, the results revealed that ghrelin regulated the Ang II-induced imbalance of Bax and Bcl-2 expression and reduced Ang II-induced caspase-3 expression. Moreover, mRNA expressions of endoplasmic reticulum stress-related molecules GRP78, caspase-12, and C/EBP homologous protein were significantly upregulated by Ang II. However, their expressions were significantly inhibited by ghrelin. In addition, we found that ghrelin markedly inhibited Ang II-induced Ang II type 1 receptor expression. These data suggest that ghrelin may play an antagonistic role in Ang II-induced cardiomyocyte apoptosis via decreasing Ang II type 1 receptor expression and inhibiting the activation of endoplasmic reticulum stress pathway.     

Polygalasaponin F inhibits neuronal apoptosis induced by oxygen‐glucose deprivation and reoxygenation through the PI3K/Akt pathway

Cerebral ischaemia is a common cerebrovascular disease and often induces neuronal apoptosis, leading to brain damage. Polygalasaponin F (PGSF) is one of the components in Polygala japonica Houtt, and it is a triterpenoid saponin monomer. This research focused on anti‐apoptotic effect of PGSF during oxygen‐glucose deprivation and reoxygenation (OGD/R) injury in rat adrenal pheochromocytoma cells (PC12) and primary rat cortical neurons. OGD/R treatment reduced viability of PC12 cells and primary neurons. This reduced viability was prevented by PGSF, as shown by MTT assay. OGD/R insult decreased expression of Bcl‐2/Bax both in PC12 cells and primary neurons but elevated levels of caspase‐3 in primary neurons. However, PGSF may up‐regulate expression of Bcl‐2/Bax and down‐regulate caspase‐3 in these particular cells. Furthermore, Bcl‐2/Bax and the ratio between phosphorylated Akt and total Akt were decreased in PC12 cells treated with OGD/R, and both were increased by PGSF. Moreover, increase in the ratios of Bcl‐2/Bax and phosphorylated Akt/total Akt in PC12 cells was suppressed by phosphatidylinositol 3‐kinase (PI3K) inhibitor. Data suggest PGSF might prevent OGD/R‐induced injury via activation of PI3K/Akt signalling. The ability of PGSF to block the effects of OGD/R appears to involve regulation of Bcl‐2, Bax and caspase‐3, which are related to apoptosis.     

AlCl3 induces lymphocyte apoptosis in rats through the mitochondria–caspase dependent pathway

To investigate apoptosis mechanisms in lymphocytes induced by aluminum trichloride (AlCl₃) through the mitochondria–caspase dependent pathway, the spleen lymphocytes of rats were cultured with RPMI‐1640 medium and exposed to AlCl₃·6H₂O in the final concentrations of 0 (control group, CG), 0.3 (low‐dose group, LG), 0.6 (mid‐dose group, MG), and 1.2 (high‐dose group, HG) mmol·L^?1 for 24 h, respectively. Mitochondrial transmembrane potential (ΔΨm), cytochrome C (Cyt C) protein expression in cytoplasm, Caspase‐3 and Caspase‐9 activity, Bcl‐2, Bax, Caspase‐3 and Caspase‐9 mRNA expressions, DNA ladder and lymphocytes apoptosis index were detected. The results showed that Cyt C protein expression in cytoplasm, Caspase‐3 and Caspase‐9 activity, Bcl‐2, Bax, Caspase‐3 and Caspase‐9 mRNA expressions, the ratio of Bcl‐2 and Bax mRNA expression, lymphocytes apoptosis index increased, while ΔΨm decreased in the AlCl₃‐treated groups compared with those in CG. The results indicate that AlCl₃ induces lymphocyte apoptosis in rats through the mitochondria–caspase dependent pathway. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 385–394, 2016.     

Role of 5‐HT2B receptors in cardiomyocyte apoptosis in noradrenaline‐induced cardiomyopathy in rats

1. Serotonin (5‐hydroxytryptamine; 5‐HT) plays important roles in the development of cardiac hypertrophy via activation of 5‐HT receptors. The aim of the present study was to investigate the role of 5‐HT_2B receptors in the development of cardiomyocyte apoptosis and hypertrophy associated with noradrenaline (NA) overload. 2. Cardiac hypertrophy was induced in rats by intraperitoneal injection of 1.5 mg/kg NA for 4 weeks. Starting from Day 15, 5‐HT2B receptor antagonist SB 204741 (i.p., 0.5 or 2 mg/kg) or SDZ SER 082 (i.p., 1 mg/kg) was injected twice daily for another 14 days. Whole‐cell patch‐clamp techniques were used to record ionic currents in freshly isolated ventricular cardiomyocytes. Western blot and terminal deoxyribonucleotidyl transferase‐mediated dUTP–digoxigenin nick end‐labelling (TUNEL) assays were used to assess myocardial apoptosis. 3. Expression of 5‐HT_2B receptors was enhanced in the hypertrophic left ventricle induced by NE overload in vivo. The 5‐HT_2B receptor antagonist SB 204741 partially reversed cardiac hypertrophy induced by NE overload (P < 0.05) and decreased L‐type calcium currents in ventricular cardiomyocytes (P < 0.05). In addition, SB 204741 notably attenuated myocardial apoptosis, as evidenced by downregulation of Bax and caspase 3 (P < 0.05) and upregulation of the anti‐apoptotic Bcl‐2 protein (P < 0.05). 4. In conclusion, the data suggest an involvement of 5‐HT_2B receptors in the generation of apoptotic events associated with cardiac remodelling during increased adrenergic stimulation.     

PROTECTIVE EFFECTS OF LEONURINE IN NEONATAL RAT HYPOXIC CARDIOMYOCYTES AND RAT INFARCTED HEART

• 1 Previous studies have shown that extracts of Herba leonuri, predominantly containing the phytochemical components leonurine and stachydrine, provide protective effects in the ischaemic myocardium by acting as free radical scavengers and inhibiting the formation of reactive oxygen species.
• 2 The present study was designed to investigate the cardioprotective effects of 10^?6 mol/L leonurine on neonatal rat cardiomyocytes treated with hypoxia plus serum deprivation, a component of ischaemia, and to determine the mechanisms underlying the protective effects with regard to cardiac anti‐oxidant enzymes and apoptosis genes. Cardiomyocytes were treated with leonurine 8 h prior to exposure to hypoxia. In addition, we investigated the effects of 7.5 and 15 mg/kg leonurine, administered to rats i.p. for 7 days prior to left coronary artery ligation, on subsequent infarct size of the ischaemic heart.
• 3 Leonurine significantly increased the viability of cardiomyocytes injured by hypoxia. In the leonurine‐treated group, gene expression levels of pro‐apoptotic genes, namely Bax and Fas, were significantly downregulated (by 0.95‐ and 0.72‐fold, respectively; P < 0.001) compared with the hypoxic control group, whereas the expression of Bcl‐2 and Bcl‐xl was upregulated following leonurine treatment (by 1.03‐ and 1.07‐fold, respectively; P < 0.05). Correspondingly, leonurine treatment increased Bcl‐2 protein levels and decreased Bax protein levels. Assays investigating cardiac anti‐oxidant enzymes provided further evidence for a protective effect of leonurine, as indicated by the induction of the anti‐oxidant enzymes superoxide dismutase and catalase. Furthermore, leonurine decreased infarct size in ischaemic rat heart.
• 4 The results of the present study suggest that the mechanisms of action of leonurine in hypoxic neonatal rat cardiomyocytes and infarcted rat heart may be related to its anti‐oxidant and anti‐apoptotic properties.

     

Simvastatin inhibits angiotensin II-induced cardiac cell hypertrophy: Role of Homer 1a

• 1 The scaffolding protein Homer 1a is constitutively expressed in the myocardium, although its function in cardiomyocytes remains poorly understood. The aim of the present study was to investigate Homer 1a expression in hypertrophic cardiac cells and its role in angiotensin (Ang) II‐induced cardiac hypertrophy.
• 2 After serum starvation for 24 h, cells were treated with 1 μmol/L simvastatin, 100 nmol/L angiotensin (Ang) II or their combination added to Dulbecco's modified Eagle's medium containing 0.5% serum. For combination treatment with AngII plus simvastatin, cells were exposed to simvastatin 12 h before the addition of AngII to the medium and cells were then incubated in the presence of both drugs for a further 24 h. Western blotting was used to determine Homer 1a protein expression. Hypertrophy was evaluated by determining the protein content per cell.
• 3 Homer 1a protein levels were upregulated following AngII‐induced hypertrophy in H9C2 cells and neonatal rat cardiomyocytes, and these increases were augmented by simvastatin pretreatment. Concomitantly, simvastatin pretreatment inhibited extracellular signal‐regulated kinase (ERK) 1/2 phosphorylation and AngII‐induced hypertrophy.
• 4 The inhibitory effects of simvastatin against AngII‐induced hypertrophy were attenuated by Homer 1a silencing, suggesting that simvastatin suppresses cardiac hypertrophy in a Homer 1a‐dependent manner. Furthermore, AngII‐induced hypertrophy and ERK1/2 phosphorylation in neonatal rat cardiomyocytes were significantly inhibited following the overexpression of Homer 1a using an adenovirus.
• 5 These results suggest a possible role for Homer 1a in inhibiting cardiac hypertrophy perhaps in part through inhibition of ERK1/2 activation.

     

Baicalin protects H9c2 cardiomyocytes against hypoxia/reoxygenation‐induced apoptosis and oxidative stress through activation of mitochondrial aldehyde dehydrogenase 2

Baicalin, a flavonoid glycoside separated from Scutellaria baicalensis, has cardioprotection against ischaemia/reperfusion (I/R) injury. Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is considered as an endogenous protective mechanism against I/R injury depending on its anti‐oxidant and anti‐apoptotic characteristics. The present study demonstrates whether ALDH2 contributes to the cardioprotection of baicalin against hypoxia/reoxygenation (H/R)‐inudced H9c2 cardiomyocytes injury. Our results observed that H/R treatment resulted in a significant decrease in cells viability and obvious increases in caspase‐3 activity and apoptosis rate in H9c2 cells, while these alterations were evidently reversed by baicalin pretreatment. Simultaneously, baicalin mitigated H/R‐induced the decreases in the levels of ALDH2 mRNA and protein as well as the activity of ALDH2 in H9c2 cells. However, we found that daidzin, an ALDH2 antagonist, remarkably attenuated baicalin‐elicited inhibitory action on H/R‐induced the downregulation of cells viability and Bcl‐2 protein expression, and the upregulations of caspase‐3 activity, apoptosis rate, cytochrome c and Bax proteins expressions in H9c2 cells. In addition, baicalin reversed H/R‐induced oxidative stress as evidenced by the downregulation of malondialdehyde (MAD) and 4‐hydroxy aldehydes (4‐HNE) levels, the inhibition of endogenous reactive oxygen species (ROS) generation, and the downregulation of superoxide dismutase (SOD) activity induced by H/R treatment, while these effects were also blocked by daidzin. Furthermore, we found that Alda‐1, an ALDH2 agonist, also abolished H/R‐induced cytotoxicity, apoptosis, and oxidative stress, indicating that ALDH2 mediated H/R‐induced H9c2 cell injury. Overall, these results suggested that baicalin prevents H/R‐induced apoptosis and oxidative stress through enhancing ALDH activity and expression in H9c2 cardiomyocytes.     

Overproduction of reactive oxygen species and activation of MAPKs are involved in apoptosis induced by PM2.5 in rat cardiac H9c2 cells

Epidemiological studies show a positive correlation between the air levels of fine particulate matter (PM_2.5) and cardiovascular disorders, but how PM_2.5 affects cardiomyocytes has not been studied in great deal. The aim of the present study was to obtain an insight into the links among intracellular levels of reactive oxygen species (ROS), apoptosis and mitogen‐activated protein kinases (MAPKs) in rat cardiac H9c2 cells exposed to PM_2.5. H9c2 cells were incubated with PM_2.5 at 100–800 µg ml^–1 to evaluate the effects of PM_2.5 on cell viability, cell apoptosis, intracellular levels of ROS and expression of apoptosis‐related proteins as well as activation of MAPKs. PM_2.5 decreased cell viability, increased the cell apoptosis rate and intracellular ROS production in a concentration‐dependent manner. PM_2.5 decreased the Bcl‐2/Bax ratio and increased cleaved caspase‐3 levels. A Western blots study showed up‐regulation of phosphorylated MAPKs including extracellular signal‐regulated protein kinases (ERKs), c‐Jun NH₂‐terminal kinases (JNKs) and p38 MAPK in the PM_2.5‐treated cells. The p38 MAPK inhibitor SB239063 attenuated whereas the ERKs inhibitor PD98059 augmented the effects of PM_2.5 on apoptosis and the expression of related proteins. In conclusion, PM_2.5 decreases cell viability and increases apoptosis by enhancing intracellular ROS production and activating the MAPKs signaling pathway in H9c2 cells. The MAPKs signaling pathway could be a new promising target for clinical therapeutic strategies against PM_2.5‐induced cardiac injury. Copyright © 2015 John Wiley & Sons, Ltd.     

Apoptotic‐related protein changes induced by hexavalent chromium in mice liver

Although it has been reported that hexavalent chromium (Cr(VI)) could induce apoptosis in a variety of cell types, the molecular mechanisms underlying this process is still largely unknown. This study was undertaken to determine effects of single oral 0, 25, 50, and 100 mg/kg body weight doses of potassium dichromate on the expression level of p53, Bcl‐2, Bax, cytochrome c, and caspase‐3, which are vital regulators of apoptosis, in mice liver. The results showed that Cr(VI) could upregulate the protein expression of p53, Bax, cytochrome c, and caspase‐3 and downregulate the expression of Bcl‐2 in mice liver. All these results suggested that p53, Bcl‐2, Bax, cytochrome c, and caspase‐3 may be involved in the regulation of Cr(VI) induced apoptosis in vivo. © 2009 Wiley Periodicals, Inc. Environ Toxicol, 2010.     

Ouabain induces apoptotic cell death in human prostate DU 145 cancer cells through DNA damage and TRAIL pathways

Ouabain, a cardiotonic steroid and specific Na⁺/K⁺‐ATPase inhibitor, has a potential to induce cancer cell apoptosis but the mechanisms of apoptosis induced by ouabain are not fully understand. The aim of this study was to investigate the cytotoxic effects of ouabain on human prostate cancer DU 145 cells in vitro. Cell morphological changes were examined by phase contrast microscopy. Cell viability, cell cycle distribution, cell apoptosis, DNA damage, the production of ROS and Ca²⁺, and mitochondrial membrane potential (ΔΨ_m) were measured by flow cytometry assay. Results indicated that ouabain induced cell morphological changes, decreased total cell viability, induced G0/G1 phase arrest, DNA damage, and cell apoptosis, increased ROS and Ca²⁺ production, but decreased the levels of ΔΨ_m in DU 145 cells. Ouabain also increased the activities of caspase‐3, ‐8, and ‐9. Western blotting was used for measuring the alterations of apoptosis‐associated protein expressions in DU 145 cells and results indicated that ouabain increased the expression of DNA damage associated proteins (pATM^Ser1981, p‐H2A.X^Ser139, and p‐p53^Ser15) and ER‐stress‐associated proteins (Grp78, ATF6β, p‐PERK^Thr981, PERK, eIF2A, GADD153, CaMKIIβ, and caspase‐4) in time‐dependently. Furthermore, ouabain increased apoptosis‐associated proteins (DR4, DR5, Fas, Fas Ligand, and FADD), TRAIL pathway, which related to extrinsic pathway, promoted the pro‐apoptotic protein Bax, increased apoptotic‐associated proteins, such as cytochrome c, AIF, Endo G, caspase‐3, ‐8, and ‐9, but reduced anti‐apoptotic protein Bcl‐2 and Bcl‐x in DU 145 cells. In conclusion, we may suggest that ouabain decreased cell viability and induced apoptotic cell death may via caspase‐dependent and mitochondria‐dependent pathways in human prostate cancer DU 145 cells.     

槲皮素抗阿霉素诱导的培养心肌细胞的凋亡

目的观察槲皮素对阿霉素(adriamycin,ADR)致大鼠心肌细胞凋亡的影响。方法原代培养乳鼠心肌细胞,随机分为正常对照组、阿霉素损伤组、槲皮素对照组、阿霉素+槲皮素(低、中、高浓度)组。比色法检测培养液中乳酸脱氢酶(LDH)的活性,MTT法测定心肌细胞存活率,电镜观察细胞超微结构,免疫细胞化学法检测Bcl-2和Bax蛋白的表达,用RT-PCR和Western blot检测caspase-3 mRNA和蛋白的表达。结果与正常对照组相比,槲皮素对照组各指标无明显改变,阿霉素组LDH活性增强,细胞存活率降低,Bcl-2表达减少,Bax表达增加,心肌细胞超微结构损伤明显,caspase-3 mRNA和蛋白的表达均升高;高中低剂量槲皮素均可减轻阿霉素所致的损伤。结论槲皮素对ADR致培养心肌细胞凋亡具有保护作用,其机制与调节细胞内凋亡相关蛋白caspase-3、Bcl-2和Bax的表达有关。     

氯沙坦对血管紧张素II致培养的牛脑微血管内皮细胞损伤的保护作用

目的观察氯沙坦对血管紧张素II(Ang II)致牛脑微血管内皮细胞(BCMECs)损伤的保护作用。方法用分光光度计测定培养的BCMECs乳酸脱氢酶(LDH)的漏出量,流式细胞仪测定BCMECs细胞间粘附分子-1(ICAM-1)的表达量,硝酸还原酶法和放射免疫分析法分别测定BCMECs上清液中一氧化氮(NO)和内皮素-1(ET₁)的含量。结果Ang II呈剂量依赖性增加BCMECs LDH漏出、NO和ET₁释放及ICAM-1表达,氯沙坦对此均有明显抑制作用。结论氯沙坦抑制Ang II致体外培养BCMECs的损伤。     

The Protective Effect of Bee Venom against Ethanol‐Induced Hepatic Injury via Regulation of the Mitochondria‐Related Apoptotic Pathway

Abstract: Alcohol consumption increases apoptosis of hepatocytes. Death of hepatocytes is a characteristic feature of chronic liver disease for various causes. Bee venom (Apis mellifera) has been traditionally used for the treatment of various chronic diseases, such as chronic inflammatory arthritis and chronic liver disease. However, the precise mechanism for bee venom in chronic liver disease is not still cleared. To assess the effects of bee venom in chronic liver disease, we investigated the potential role of the bee venom in the ethanol‐induced hepatocyte apoptosis. Bee venom treatment inhibited the apoptotic cell morphology and increased the cell viability in ethanol‐induced hepatocyte apoptosis. With ethanol treatment, bee venom‐treated hepatocytes increased activity of Bcl‐2 and Bcl‐xL, reduced activity of Bax, Caspase and PARP. In conclusion, bee venom treatment in ethanol‐induced hepatocyte apoptosis occurred through the regulation of Bcl family with subsequent inactivation of the Caspase and PARP. These results suggest that bee venom could be an effective agent to reduce ethanol‐induced hepatocyte apoptosis.     

Losartan Inhibits Vascular Smooth Muscle Cell Proliferation through Activation of AMP-Activated Protein Kinase

Losartan is a selective angiotensin II (Ang II) type 1 (AT₁) receptor antagonist which inhibits vascular smooth muscle cells (VSMCs) contraction and proliferation. We hypothesized that losartan may prevent cell proliferation by activating AMP-activated protein kinase (AMPK) in VSMCs. VSMCs were treated with various concentrations of losartan. AMPK activation was measured by Western blot analysis and cell proliferation was measured by MTT assay and flowcytometry. Losartan dose- and time-dependently increased the phosphorylation of AMPK and its downstream target, acetyl-CoA carboxylase (ACC) in VSMCs. Losartan also significantly decreased the Ang II- or 15% FBS-induced VSMC proliferation by inhibiting the expression of cell cycle associated proteins, such as p-Rb, cyclin D, and cyclin E. Compound C, a specific inhibitor of AMPK, or AMPK siRNA blocked the losartan-induced inhibition of cell proliferation and the G₀/G₁ cell cycle arrest. These data suggest that losartan-induced AMPK activation might attenuate Ang II-induced VSMC proliferation through the inhibition of cell cycle progression.     

The role of angiotensin antagonism in stroke prevention in patients with hypertension: focus on losartan

Summary

Optimal management of hypertension has been shown to reduce the risk of stroke. In recent years, newer classes of antihypertensive such as the angiotensin II (Ang II) antagonists have become available. Results from the Losartan Intervention for Endpoint reduction in hypertension (LIFE) study suggest the utility of this particular Ang II antagonist in stroke prevention. Treatment with a losartan-based regimen or an atenolol-based regimen produced similar reductions in blood pressure during almost 5 years of follow up. Losartan, however, reduced the risk of stroke by 25% compared with atenolol (p = 0.001). For a subgroup of patients with isolated systolic hypertension, losartan reduced the risk of stroke by 40% (p = 0.02). As well as blocking the Ang II type 1 receptor, losartan also acts as an antagonist at the thromboxane A₂ receptor and has uricosuric effects, which may provide additional mechanisms by which losartan provides protective benefits beyond its antihypertensive action. The relevance of these molecular properties of losartan over other Ang II antagonists is further supported by comparison of the outcomes obtained in clinical trials employing two other Ang II antagonists, valsartan and candesartan.     

Ventricular PKC‐ϵ and humoral signaling in DOCA‐Salt rats treated with labedipinedilol‐A

Effects of oral antihypertensive monotherapy with labedipinedilol‐A, labetalol, atenolol, amlodipine, prazosin (20 mg kg^?1 day^?1), and short‐acting nifedipine (3 mg kg^?1 day^?1) on DOCA‐salt‐induced translocation of ventricular protein kinase C‐?(PKC‐?), humoral signaling, and the cardiovascular system were investigated in rats for 4 weeks. The triple blocking activities of labedipinedilol‐A (α/β‐adrenoceptor blockade and calcium entry blockade) were compared with single blocking activities of selective drugs. Cytosolic PKC‐? immunoreactivity was decreased by labedipinedilol‐A, short‐acting nifedipine, amlodipine, prazosin, labetalol, atenolol, and losartan. Membranous PKC‐? immunoreactivity was significantly decreased by labedipinedilol‐A, amlodipine, prazosin, labetalol, and atenolol. Labedipinedilol‐A and prazosin more potently decreased membranous than cytosolic PKC‐? expression. Labedipinedilol‐A, labetalol, and atenolol effectively inhibited DOCA‐salt‐induced increases in angiotensin II (Ang II). All antihypertensive agents reduced endothelin‐1 (ET‐1) levels in urine and cardiac weight growth. Treatments with labedipinedilol‐A, labetalol, atenolol, and amlodipine normalized DOCA‐salt‐induced ANP increases. Prazosin did not decrease ANP. Short‐acting nifedipine elevated ANP. During long‐term antihypertensive therapy in DOCA‐salt hypertensive rats, single blockade drugs did not fully inhibit ventricular PKC‐? translocation or Ang II, ET‐1, and ANP humoral signaling. However, triple blockade labedipinedilol‐A therapy had a wide range of α/β‐adrenergic receptor and calcium channel inhibitory activities, including diminished reflux tachycardia, inhibition of PKC‐? translocation, and reduction of Ang II, ET‐1, and ANP formation. Drug Dev. Res. 59:307–315, 2003. 2003 Wiley‐Liss, Inc.