Cellular physiology of rat cardiac myocytes in cardiac fibrosis: in vitro simulation using the cardiac myocyte/cardiac non-myocyte co-culture system

An understanding of the cellular physiology of cardiac myocytes (MCs) and non-myocytes (NMCs) may help to explain the mechanisms underlying cardiac hypertrophy. Despite numerous studies using MC/NMC co-culture systems, it is difficult to precisely evaluate the influence of each cell type because of the inherent cellular heterogeneity of such a system. Here we developed a co-culture system using Wistar rat neonatal MCs and NMCs isolated by discontinuous Percoll gradient and adhesion separation methods and cultured on either side of insert well membranes. Co-culture of MCs and NMCs resulted in significant increases in [3H]-leucine incorporation by MCs, in the amount of protein synthesized by MCs, and in the secretion of natriuretic peptides, while the addition of MCs to NMC cultures significantly reduced [3H]-thymidine incorporation by NMCs. Interestingly, the percentage of the brain natriuretic peptide (BNP) component of total natriuretic peptide secreted (atrial natriuretic peptide+BNP) increased as the number of NMCs placed in the MC/NMC co-culture system increased. However, MCs did not affect production of angiotensin II (Ang II) by NMCs or secretion of endothelin-1 and transforming growth factor-beta1 into the MC/NMC co-culture system. This finding was supported by the anti-hypertrophic and anti-fibrotic actions of RNH6270, an active form of olmesartan, on MCs in the MC/NMC co-culture system and on NMCs that may synthesize Ang II in the heart. The present data indicate that cardiac fibrosis may not only facilitate MC hypertrophy (possibly through the local angiotensin system) but may also change particular pathophysiological properties of MCs, such as the secretory pattern of natriuretic peptides.     

Benidipine induces thermogenesis in brown adipose tissue by releasing endogenous noradrenaline: a possible mechanism for the anti-obesity effect of calcium antagonists

BACKGROUND: Anti-obesity effects of calcium antagonists such as benidipine and nifedipine have been described in rodent obesity models, but the mode of action of the calcium antagonists as anti-obesity agents has not been established. OBJECTIVE: To examine whether the anti-obesity effects of calcium antagonists (here benidipine) could be ascribed to a direct stimulation of brown adipose tissue (BAT) thermogenesis. METHODS: Examination of the ability of benidipine to induce thermogenesis (increased rate of oxygen consumption) in isolated brown-fat cells from rats, mice and hamsters--and in intact cold-acclimated rats. RESULTS: Benidipine itself, or in combination with any dose of noradrenaline (NA), was totally unable to induce or augment thermogenesis in isolated brown-fat cells of any species tested. However, it markedly induced thermogenesis in intact animals (approx 60% increase over resting metabolic rate). This effect could be fully inhibited by propranolol. CONCLUSION: Benidipine is itself without thermogenic effect. The thermogenic response in-vivo (and thus presumably the anti-obesity effect) is probably secondary to a previously described general side-effect of calcium antagonists: a release of NA from sympathetic nerves, here most likely directly from nerves in the BAT. The anti-obesity effect of benedipine is thus probably not due to its calcium channel blocking effect. PERSPECTIVES: It is probable that the anti-obesity effects of calcium antagonists reported in several models of genetically obese rodents (MSG-obese and agouti mice, SHHF/Mcc-fa(cp) and JCR:LA-corpulent rats) are mediated via an indirect stimulation of BAT. To what extent calcium antagonists may induce similar effects in a clinical situation, is currently unknown.     

Benidipine inhibits expression of ET-1 and TGF-beta1 in Dahl salt-sensitive hypertensive rats.

Endothelin and growth factors such as transforming growth factor (TGF)-beta1 are important regulators of the cardiovascular system. Although increased production of endothelin-1 (ET-1) and TGF-beta1 have been reported in left ventricular hypertrophy, the detailed roles of these substances in hypertrophy remain to be determined. To elucidate the cardioprotective effects of calcium antagonists in left ventricular hypertrophy, we evaluated the effects of long-term treatment with benidipine, a long-acting calcium antagonist, on preproET-1, ET(A) receptor (ETAR) and TGF-beta1 expression in the left ventricle and evaluated the relations between these effects and myocardial remodeling in Dahl salt-sensitive hypertensive (DS) rats fed a high-salt diet. After 5 weeks of feeding an 8% NaCl diet to 6-week-old DS rats (i.e., at 11 weeks of age), a distinct stage of concentric left ventricular hypertrophy (DSLVH) was noted. Benidipine (DSLVH-B group, n= 8; 1 mg/kg/day, subdepressor dose) or vehicle (DSLVH-V group, n=8) was administered to 6-week-old DS rats for 5 weeks, or until the onset of DSLVH stage, and age-matched (11-week-old) Dahl salt-resistant rats fed the same diet served as a control group (DR-C, n=8). Blood pressure was similar between the DSLVH-B and DSLVH-V groups, but was significantly lower in DR-C rats. The preproET-1, ETAR and TGF-beta1 expressions in the left ventricle were significantly higher in DSLVH-V than in DR-C rats, and significantly lower in DSLVH-B than in DSLVH-V. Benidipine administration resulted in significant improvements in the wall-to-lumen ratio and perivascular fibrosis in the coronary arterioles, and in myocardial fibrosis. We therefore concluded that myocardial remodeling and left ventricular hypertrophy in DS hypertensive rats fed a high-salt diet were significantly ameliorated by a subdepressor dose of benidipine, and that this amelioration was partly due to decreases in the expression of ET-1 and TGF-beta1 in the left ventricle.     

高糖培养的系膜细胞肥大与周期素激酶抑制剂p27的关系

目的：研究周期素激酶抑制剂p27在高糖培养系膜细胞（MC）肥大中的作用。方法：蛋白印迹（western杂交）方法测定MC裂解液P27蛋白水平,[^3H]TdR）及[^3H]leu)掺入方法测定MC细胞的肥大情况,观察P27反义寡核苷酸（ODN）转染对高糖培养MC肥大的影响。结果：高糖（450mg/dl)无血清培养的MC同正常糖（100mg/dl)无血清培养的MC相比,P27水平增高,[^3H]leu掺入增加,[^3H]TdR掺入减少;p27反义ODN转染后高糖无血清培养MC[^3H]leu掺入减少,[^3H]TdR掺入增加,用甘露醇提高正常培养液渗透压并不增加MC中p27水平,结论：高糖培养的MC中P27水平明显增高,增高的P27在高糖培养MC细胞肥大中起重要作用。     

Effects of specific endothelin-1 receptor antagonists on proliferation and fibronectin production of glomerular mesangial cells stimulated with Angiotensin II

Angiotensin II (Ang-II) is a potent vasoactive hormone, which plays an important role in the pathogenesis of glomerulosclerosis. Ang-II activates many cytokine systems in the kidney. Recent studies indicate that Ang-II is closely related to the activation of the endothelin-1 (ET-1) system. The present study was designed to measure the [H3]-thymidine uptake and fibronectin production of cultured rat mesangial cells stimulated with Ang-II, and to evaluate the effects of specific ET-1 receptor antagonists, BQ123 (type A receptor antagonist) and IRL1038 (type B receptor antagonist) on the cells. ET-1 was measured by radioimmunoassay and fibronectin by Western blot analysis. The results were as follows: (1) Ang-II enhanced ET-1 production, [H3]-thymidine uptake, number of cells, and fibronectin production of mesangial cells; (2) all the baseline [H3]-thymidine uptake, number of cells, and fibronectin production of mesangial cells can be partly suppressed by BQ123, but not by IRL1038; (3) the increment of Ang-II-enhanced number of cells can be partly suppressed by BQ123, but not by IRL1038; and (4) the increment of Ang-II-enhanced fibronectin production can be partly suppressed by both BQ123 and IRL1038. Our results indicate that Ang-II is an active stimulant for the proliferation and fibronectin production of mesangial cells, and the effect is partly suppressed mainly by ET-1 type A receptor antagonists.     

Combination treatment with a calcium channel blocker and an angiotensin blocker in a rat systolic heart failure model with hypertension.

The mechanism and treatment of hypertensive systolic heart failure are not well defined. We compared the effect of an angiotensin-converting enzyme inhibitor (cilazapril, 10 mg/kg), an angiotensin receptor blocker (candesartan, 3 mg/kg), a calcium channel blocker (benidipine, 1, 3 or 6 mg/kg), and the same calcium channel blocker combined with renin-angiotensin blockers on systolic heart failure in Dahl salt-sensitive (DS) rats. DS rats were fed an 8% Na diet from 6 weeks of age and then subjected to the above drug treatments. Benidipine (1 mg/kg), cilazapril, and candesartan had compatible hypotensive effects and similar beneficial effects on cardiac hypertrophy, gene expression, and survival rate. The combination of benidipine with cilazapril or candesartan was found to have no additional beneficial effects on the above parameters, with the exception of a reduction in atrial natriuretic polypeptide gene expression. On the other hand, candesartan normalized serum creatinine, but serum creatinine was unaffected by either benidipine at 1 or 3 mg/kg or cilazapril. Further, the combined use of benidipine and either candesartan or cilazapril resulted in an additional reduction of urinary albumin excretion in DS rats. Thus systolic heart failure in DS rats is mainly mediated by hypertension, while renal dysfunction of DS rats is due to both hypertension and the AT1 receptor itself. These findings suggest that the combination of a calcium channel blocker with an AT1 receptor blocker or ACE inhibitor may be more effective in treating the renal dysfunction associated with systolic heart failure than monotherapy with either agent alone. However, further studies will be needed before reaching any definitive conclusion on the efficacy of this combination therapy in patients with heart failure.     

贝尼地平对冠心病患者一氧化氮和内皮素的影响

目的探讨钙离子拮抗剂贝尼地平对冠心病患者一氧化氮(NO)和内皮素(ET-1)的影响。方法将2006年1～7月对哈尔滨医科大学第四医院心内科36例冠心病患者给予贝尼地平2～4mg/d,治疗8周,测定用药前及服药后第4、8周NO和ET-1水平。以40名相匹配健康体检者为对照组给予安慰剂口服。结果冠心病患者的NO较对照组明显降低[(7.46±3.36)μmol/L对(33.49±12.45)μmol/L,P<0.05],ET-1升高[(79.60±10.04)ng/L对(59.50±6.28)ng/L,P<0.05]。贝尼地平治疗前后比较NO升高[(28.24±14.92)μmol/L对(7.46±3.36)μmol/L,P<0.01],ET-1降低[(60.01±13.84)ng/L对(79.60±10.04)ng/L,P<0.01]。结论贝尼地平能升高冠心病患者的NO水平,降低ET-1水平,从而证明贝尼地平具有内皮保护作用。     

Effects of short- and long-acting calcium channel blockers on the relationship between blood pressure and physical activity

Calcium channel blockers are widely used as antihypertensive drugs. However, there is some controversy as to how they should be used. Our first aim was to clarify how the dihydropyridine calcium channel blocker, benidipine, affects the quantitative relationship between blood pressure (BP) and physical activity. The second aim was to determine whether there is a relationship between systolic blood pressure (SBP) and physical activity in patients with hypertension when treating with a short-acting (nifedipine) or long-acting (benidipine) calcium channel blocker. In Study 1, ambulatory BP and physical activity were measured simultaneously in 27 patients with hypertension before and after 6 months with benidipine. In Study 2, ambulatory BP and physical activity were measured simultaneously in 16 patients with hypertension before (placebo) and after 6 weeks of crossover treatment with nifedipine and benidipine. In Study 1, there was no difference in the SBP change caused by physical activity between the pre- and posttreatment periods. In Study 2, SBP was significantly related to physical activity in the placebo (16/16) and benidipine (16/16) groups but not in the nifedipine (12/16) group. The lowest BP during day-time and nighttime in the nifedipine group were significantly lower than those in the benidipine group. Plasma renin activity (ng/mL/h) was significantly higher in the nifedipine group (1.20+/-1.05) than in the placebo (0.57+/-0.59) and benidipine (0.75+/-0.78) groups. These findings indicate that nifedipine might interfere with the adaptation mechanism of BP changed by physical activity and that the activated renin-angiotensin system might cause cardiac events.     

Benidipine dilates both pre- and post-glomerular arteriole in the canine kidney.

The aim of the present study was to determine the effects of benidipine on renal function and whether benidipine may dilate the efferent arteriole as well as the afferent arteriole of the canine kidney. The effects of benidipine on the renal segmental vascular resistance were estimated using Gomez's formula with some modification. The renal hemodynamic action of benidipine was also compared with that of amlodipine. Intrarenal arterial injection of benidipine at a dose of 3 microg/kg resulted in a significant increase in renal blood flow (RBF), urine flow and urinary excretion of sodium, but not in glomerular filtration rate (GFR). Amlodipine at a dose of 300 microg/kg also increased RBF, urine flow and urinary excretion of sodium to a significant degree equivalent to that by benidipine. However, in contrast to benidipine, amlodipine significantly increased GFR. After the administration of benidipine, autoregulation of RBF and GFR was relatively maintained and the renal perfusion pressure (RPP)-RBF relation shifted upward; that is, RBFs at 75 and 50 mmHg were maintained at a higher level than those of the control. In contrast to benidipine, amlodipine diminished the autoregulation of RBF and GFR. RBFs at 75 and 50 mmHg were not different from those of the control. The afferent and efferent arteriolar resistance (Ra and Re) were calculated based on the RPP-RBF and RPP-GFR relations. Benidipine reduced both Ra and Re, but amlodipine selectively reduced Ra. Benidipine increased RBF but not GFR via the dilation of both afferent and efferent arterioles. Thus, benidipine has unique renal hemodynamic actions which differ from those by most calcium antagonists.     

Benidipine, a long-acting calcium channel blocker, inhibits cardiac remodeling in pressure-overloaded mice

OBJECTIVE: The effects of long-acting calcium channel blockers (CCBs) on pressure overload-induced cardiac remodeling are seldom studied in animals. We evaluated the effects of benidipine, a long-acting CCB, on cardiac remodeling. METHODS: Rat neonatal cardiac myocytes were used to examine the influence of benidipine on protein synthesis. Cardiac remodeling was induced in C57 B6/J mice by transverse aortic constriction (TAC). Then the effects of benidipine (10 mg/kg/d) were assessed on myocardial hypertrophy and heart failure, cardiac histology, and gene expression. RESULTS: Benidipine significantly inhibited protein synthesis by cardiac myocytes stimulated with phenylephrine (PE), and this effect was partially abolished by cotreatment with a nitric oxide synthase (NOS) inhibitor [N(G)-nitro-l-arginine methylester (l-NAME)]. Four weeks after the onset of pressure overload, benidipine therapy potently inhibited cardiac hypertrophy and prevented heart failure. The heart to body weight ratio was 6.89+/-0.48 mg/g in treated mice vs. 8.76+/-0.33 mg/g in untreated mice (P<0.01), and the lung to body weight ratio was 7.39+/-0.93 mg/g vs. 10.53+/-0.99 mg/g, respectively (P<0.05). Left ventricular fractional shortening (LVFS) was improved on echocardiography. Plasma NO levels were increased, while B type natriuretic peptide, protein inhibitor of neuronal NOS, and procollagen IV alpha were down-regulated in benidipine-treated mice. CONCLUSION: These results indicate that benidipine inhibits cardiac remodeling due to pressure overload at least partly by acting on the nitric oxide signaling pathway.     

Paracrine hypertrophic factors from cardiac non-myocyte cells downregulate the transient outward current density and Kv4.2 K+ channel expression in cultured rat cardiomyocytes.

OBJECTIVES: Cardiac hypertrophy is characterized by a prolongation of action potential duration (APD) and a reduction of outward K+ currents, primarily the transient outward current (Ito). Since the interaction between cardiac non-myocyte cells (NMCs) and cardiomyocytes (MCs) plays a critical role during the process of myocardial hypertrophy, in the present study, we investigated the effects of NMCs on cell growth and K+ channel expression in cultured newborn rat ventricular cells. METHODS: Single MCs were isolated from day-old Wistar rat ventricles and cultured for a period of five days. The effects of NMCs were examined by MC-NMC co-culture or incubating pure MCs in NMC-conditioned growth medium (NCGM). Whole-cell voltage-clamp recording and Western blot analysis using a polyclonal antibody against rat Kv4.2 channel protein were performed. RESULTS: A marked increase in surface area and total cell protein concentration of MCs was observed in the MC-NMC co-culture. In the pure MC culture, this hypertrophic effect could be mimicked by a 72-h addition of NCGM, with a significant prolongation of APD25 (APD at 25% repolarization) and a 42% decrease in Ito density (at +30 mV). The rates of inactivation and recovery from inactivation of Ito were unchanged. In the NCGM-treated MC culture, Western blots of MC proteins also showed a 36% reduction of the Kv4.2 K+ channel protein level. In addition, the NCGM-induced MC hypertrophy was partially inhibited by anti-insulin-like growth factor-1 (IGF-1) antibody, while it revealed no effects on Ito density and Kv4.2 channel expression. CONCLUSIONS: These findings first demonstrate that some paracrine hypertrophic factors released from cardiac NMCs, although unidentified, downregulate cardiac K+ channel expression.     

Cardiac fibroblasts are major production and target cells of adrenomedullin in the heart in vitro

OBJECTIVE: Adrenomedullin (AM) is a potent vasodilator peptide. Plasma AM concentration is increased in patients with various heart diseases, and both myocytes (MCs) and non-myocytes (NMCs) secrete AM and express its receptors. These facts suggest that cardiac cells possess an autocrine/paracrine capability mediated by AM. METHODS: MCs and NMCs were prepared from cardiac ventricles of neonatal rats. AM and endothelin-1 concentrations were measured by radioimmunoassays, and interleukin-6 level by a specific bioassay. Total nitrite/nitrate contents were measured with a fluorescence assay kit. RESULTS: A basal secretion rate of AM from NMCs was 2.8-fold higher than that from MCs. Interleukin-1beta, tumor necrosis factor-alpha and lipopolysaccharide stimulated AM secretion from NMCs but not from MCs. AM stimulated interleukin-6 production in the presence of these cytokines or lipopolysaccharide, which was more prominent in NMCs. In the presence of interleukin-1beta, AM augmented nitric oxide synthesis 2.7-fold in NMCs, but slightly in MCs. NMCs secreted endothelin-1 at a rate nine times higher than MCs, and AM inhibited endothelin-1 secretion from NMCs. CONCLUSION: This in vitro study suggests that AM in the heart is mainly produced in NMCs and exerts its effects through NMCs, especially under inflammatory conditions.     

Cardioprotective effect of benidipine on cardiac performance and remodeling in failing rat hearts

BACKGROUND: We have known that endothelial nitric-oxide synthase (eNOS) and oxidative stress may play a key role in cardiac performance in failing rat hearts. However, the interactions between eNOS or oxidative stress and bradykinin (BK) under treatment of calcium channel blockers (CCBs) remain unknown. To elucidate the mechanism underlying the cardioprotective effect of long-acting dihydropyridine CCBs, we evaluated the effect of benidipine on the BK-eNOS and NAD(P)H oxidase pathway in Dahl salt-sensitive (DS) rats with heart failure. METHODS: 11-week-old DS rats were treated with one of the following drug combinations for 7 weeks until the onset of the failing stage: vehicle, BK B2 receptor antagonist (FR172357 (FR)) alone, hydralazine, benidipine, and benidipine plus FR. The left ventricular end-systolic pressure-volume relationship (ESPVR) (contractility: Ees) was evaluated using a conductance catheter. RESULTS: Downregulated Ees and per cent of fractional shortening (%FS) assessed by echocardiography and eNOS expression in the failing stage were both significantly increased by using benidipine; this result was not found, however, when using FR alone or hydralazine or benidipine plus FR. Upregulated expression of NAD(P)H oxidase p22phox and p47phox and lectin-like oxidized low-density lipoprotein receptor-1, and downregulated superoxide dismutase-1 (SOD-1) were significantly ameliorated by benidipine, but not by FR alone or by hydralazine or benidipine plus FR. Benidipine effectively inhibited vascular lesion formation and suppressed atrial natriuretic peptide (ANP) and transforming growth factor-beta1 (TGF-beta1), but this was not the case when using FR alone or hydralazine or benidipine plus FR. CONCLUSIONS: These results suggest that benidipine may be useful for cardioprotective agents in preventing the cardiac dysfunction and remodeling associated with the BK-eNOS and oxidative stress pathway.     

Dihydropyridine agonist Bay K 8644 inhibits platelet activation by competitive antagonism of thromboxane A2-prostaglandin H2 receptor

The dihydropyridine calcium channel antagonists, such as nifedipine, inhibit platelet aggregation in vitro and ex vivo, but the mechanism by which this occurs is uncertain. Bay K 8644 (BAY) is a substituted dihydropyridine that has effects on voltage-dependent calcium channels in cardiac and smooth muscle that are opposite the effects of nifedipine. To evaluate the mechanism responsible for dihydropyridine-induced inhibition of platelet function, we studied the in vitro effects of BAY on human platelet aggregation and secretion plus several related biochemical parameters, including cytoplasmic ionized calcium ([Ca2+]i). BAY exerted concentration-dependent effects on platelet aggregation and secretion of [14C]serotonin. BAY (1-10 microns) inhibited the second wave of platelet aggregation and secretion stimulated by adenosine diphosphate or epinephrine and blocked shape change, aggregation, and secretion induced by the thromboxane A2 (TXA2) mimic, U46619. BAY also inhibited U46619-induced phosphorylation of the approximately 40,000-dalton cytoplasmic protein substrate of protein kinase C (40K protein), formation of TXA2, and rise in [Ca2+]i, all biochemical consequences of platelet activation. The (+)-(R) enantiomer of BAY [BAY(+)] was predominantly responsible for the inhibitory effects of racemic BAY. Nifedipine had the same inhibitory effects on platelet function and biochemistry, except it was approximately 10 times less potent than BAY. Since these results suggested inhibition of the TXA2-prostaglandin H2 (PGH2) receptor, we measured binding of [3H]U46619 to intact platelets. BAY, BAY(+), and nifedipine all functioned as competitive antagonists of [3H]U46619 binding (BAY Ki = 1.47 microM). They did not inhibit binding of [3H]yohimbine to platelet alpha 2-adrenergic receptors. At 1-10 nM BAY, BAY(+) and the (-)-(S) enantiomer of BAY [BAY(-)] all resulted in slight stimulation of platelet function and biochemical events. No significant increase in [3H]U46619 binding was demonstrable, however. Therefore, dihydropyridines that function as either calcium channel agonists or antagonists in cardiac or smooth muscle exert concentration-dependent effects on platelet function. In nanomolar concentrations, they augment, and in micromolar concentrations, they inhibit platelet activation induced by TXA2 or U46619. These data indicate that dihydropyridines do not inhibit TXA2-induced platelet activation by an effect on voltage-dependent calcium channels; they define the mechanism of inhibition as competitive antagonism of the TXA2-PGH2 receptor. The mechanism responsible for augmentation of platelet activation is uncertain.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of benidipine on glomerular hemodynamics and proteinuria in patients with nondiabetic nephropathy.

Experimental studies suggest that some long-acting calcium antagonists decrease glomerular hypertension and suppress the progression of nephropathy, but clinical evidence is lacking. To investigate clinically whether a long-acting calcium antagonist, benidipine, lowers glomerular capillary hydraulic pressure via a decrease in efferent arteriolar resistance and decreases proteinuria, we examined hypertensive patients with nondiabetic nephropathy. The subjects were 7 patients with chronic glomerulonephritis or glomerulosclerosis. Before and during the administration of benidipine (4 mg/day), systemic pressure, glomerular hemodynamics, the sodium sensitivity index (reciprocal of the pressure-natriuresis curve), and urinary excretion of proteins (total protein, albumin, and immunoglobulin G) were investigated. The glomerular hemodynamics in terms of glomerular capillary hydraulic pressure and resistance of afferent and efferent arterioles were calculated from the renal clearance, plasma total protein concentration, and pressure-natriuresis relationship. Benidipine lowered the mean arterial pressure from 105 +/-5 to 99 +/- 4 mm Hg (p = 0.002; mean +/- SD) and glomerular pressure from 48 +/- 8 to 39 +/- 5 mmHg (p = 0.006) by decreasing the resistance of efferent arterioles. Benidipine made the pressure-natriuresis curve steeper and decreased the median sodium sensitivity index from 0.099 (0.084 and 0.117; 25th and 75th percentiles) to 0.048 (0.017 and 0.058; p = 0.018). Urinary excretion of proteins did not change. Our clinical study showed that benidipine lowered the glomerular pressure by decreasing the resistance of efferent arterioles and decreased the sodium sensitivity of blood pressure, but did not affect proteinuria in patients with nondiabetic nephropathy.     

α-烯醇化酶在内皮素-1诱导的肥大心肌细胞中的表达和调控通路

目的 观察内皮素-1(ET-1)诱导的心肌细胞肥大模型中细胞外信号调节激酶(ERK1/2)、磷酸化细胞外信号调节激酶(p-ERK1/2)、低氧诱导因子(HIF-1α),α-烯醇化酶(α-enolase)蛋白表达情况,探讨肥大心肌细胞α-enolase高表达的调控机制. 方法 建立ET-1诱导的心肌细胞肥大模型,从细胞表面积、细胞蛋白合成速率和肌原纤维的重排3方面进行验证;将原代培养心肌细胞随机分为4组:(1)对照组;(2)PD98059干预组;(3)ET-1刺激组;(4)PD98059+ET-1刺激组.免疫印迹方法 检测ERK1/2、p-ERK1/2、HIF-1α、α-enolase的蛋白表达. 结果 ET-1刺激后心肌细胞表面积为(1350.7±107.5)μm2,较对照组(896.1±70.2)μm2增加(P<0.05);ET-1刺激后心肌细胞[3H]亮氨酸掺入量较对照组增加[分别为(1387.9±14.8)dpm和(787.7±10.2)dpm,P<0.013;肌原纤维染色可见ET-1刺激屙心肌细胞肌原纤维排列较对照组紧密、染色浓,表明ET-1能诱导心肌细胞肥大.ERK1/2抑制剂PD98059预处理的心肌细胞在ET-1刺激后细胞表面积和细胞的[3H]亮氨酸掺入量均较ET-1刺激组减少[细胞表面积分别为(907.0±92.5)μm2和(1350.7±107.5)μm2,P<0.05;[3H]亮氨酸掺入量:(841.5±10.5)dpm和(1387.9±14.8)dpm,P<0.05].ET-1刺激后心肌细胞有p-ERK1/2表达,其抑制剂PD98059在抑制ERK1/2活化的同时也部分抑制了HIF-1α、α-enolase蛋白的表达. 结论 ERK1/2的活化与ET-1诱导的细胞肥大关系密切,在ET-1促心肌细胞肥大过程中,从MAPK/ERK1/2至HlF-1α及α-enolase这条信号通路町能参与α-enolase高表达的调控.     

A Ca channel blocker,benidipine, increases coronary blood flow and attenuates the severity of myocardial ischemia via NO-dependent mechanisms in dogs

Objectives. This study was undertaken to examine whether a dihydropyridine Ca channel blocker, benidipine, increases cardiac NO levels, and thus coronary blood flow (CBF) in ischemic hearts.Background. Benidipine protects endothelial cells against ischemia and reperfusion injury in hearts.Methods and Results. In open chest dogs, coronary perfusion pressure (CPP) of the left anterior descending coronary artery was reduced so that CBF decreased to one-third of the control CBF, and thereafter CPP was maintained constant (103 ± 8 to 42 ± 1 mmHg). Both fractional shortening (FS: 6.1 ± 1.0%) and lactate extraction ratio (LER: −41 ± 4%) decreased. Ten minutes after the onset of an intracoronary infusion of benidipine (100 ng/kg/min), CBF increased from 32 ± 1 to 48 ± 4 ml/100g/min during 20 min without changing CPP (42 ± 2 mmHg). Both FS (10.7 ± 1.2%) and LER (−16 ± 4%) also increased. Benidipine increased cardiac NO levels (11 ± 2 to 17 ± 3 nmol/ml). The increases in CBF, FS, LER and cardiac NO levels due to benidipine were blunted by L-NAME. Benidipine increased cyclic GMP contents of the coronary artery of ischemic myocardium (139 ± 13 to 208 ± 15 fmol/mg protein), which was blunted by L-NAME.Conclusion. Thus, we conclude that benidipine mediates coronary vasodilation and improves myocardial ischemia through NO-cyclic GMP-dependent mechanisms.     

Effects of tanshinone VI on insulin-like growth factor-1-induced hypertrophy of isolated cardiomyocytes from neonatal rats

The effects of tanshinone VI (Tan), a diterpene extracted from Salvia miltiorrhiza, on insulin-like growth factor-1 (IGF-1)-induced hypertrophy of cardiomyocytes were examined. Cultured cardiomyocytes were isolated from neonatal rat hearts. The incorporation of [³H]-leucine into the trichloroacetic acid (TCA)-insoluble fraction was measured as a marker of protein synthesis, which revealed cardiomyocyte hypertrophy. Various concentrations of IGF-1, ranging from 0.1 nM to 10 nM, increased [³H]-leucine incorporation into the TCA-insoluble fraction of cardiomyocytes in a dose-dependent manner. IGF-1 induced an increase in phosphorylated extracellular signal-regulated kinase 1/2 (ERK), but did not change ERK protein content in cardiomyocytes. When the cells were incubated in the presence of Tan (0.1 μM to 10 μM), [³H]-leucine incorporation into IGF-1-untreated cells was unaltered. When the cells were incubated with 10 μM Tan, IGF-1-induced increases in [³H]-leucine incorporation into the TCA-insoluble fraction and phosphorylated ERK were attenuated. These results suggest that Tan is a possible agent for the suppression of IGF-1-induced hypertrophy of cardiomyocytes via an attenuation of ERK activation.     

Chronic treatment with long-acting nifedipine reduces vasoconstriction to endothelin-1 in essential hypertension

Essential hypertension is associated with enhanced biological activity of endothelin-1 (ET-1) and impaired endothelium-dependent vasodilatation. Dihydropyridine calcium antagonists have antioxidant activity in vitro, and they improve endothelial function in vivo. We tested whether calcium antagonists also influence the biological activity of ET-1 in essential hypertensive (EH) patients in the presence and absence of hypercholesterolemia. In 9 healthy subjects (normotensive [NT] subjects, age: 48.3+/-7.6 years; blood pressure: 118+/-8.6/69+/-5.4 mm Hg) and 21 EH subjects (age: 50.0+/-7.8 years; blood pressure: 164.4+/-5.4/103.8+/-4.4 mm Hg), we studied forearm blood flow and its modification induced by intrabrachial administration of ET-1, phenylephrine, acetylcholine, and sodium nitroprusside at baseline and after 24 weeks of treatment with a nifedipine gastrointestinal therapeutic system (30 to 60 mg per day). At baseline, the first dose of ET-1 (0.5 microg/100 mL of forearm tissue per minute) caused a slight vasodilatation in NT but not in EH subjects, whereas the following higher doses caused a comparable dose-dependent vasoconstriction in EH and NT subjects. The effect of acetylcholine was significantly reduced in EH as compared with NT subjects. In contrast, sodium nitroprusside and phenylephrine had similar effects in NT and EH subjects. After chronic treatment with the nifedipine gastrointestinal therapeutic system, the vasoconstrictor effect induced by both ET-1 and phenylephrine was significantly blunted, whereas the response to acetylcholine was significantly increased and the vasodilation to sodium nitroprusside unchanged. Hypercholesterolemic EH subjects showed a further reduced response to acetylcholine compared with normocholesterolemic EH subjects, and the nifedipine gastrointestinal therapeutic system restored the vasodilation to acetylcholine in this subgroup. In conclusion, in EH subjects, chronic treatment with a long-acting dihydropyridine calcium antagonist not only exhibits a blood pressure-lowering effect but also reduces ET-1-induced vasoconstriction and improves endothelium-dependent vasodilation. Those vasculoprotective effects may importantly contribute to a reduction in major clinical events seen during treatment with these compounds.     

Antiatherosclerotic effects of calcium antagonists. Study in human aortic cell culture

To investigate the effects of calcium antagonists on atherosclerotic cellular indices, [3H]thymidine incorporation and intracellular cholesterol content, primary culture of cells isolated from subendothelial intima of human atherosclerotic aorta was used. Among tested drugs were: verapamil, nifedipine, diltiazem, papaverin, nicardipine, D-600, cinnarizine, PN 200 110 and PY 108 068. Verapamil proved to be the most effective. It significantly reduced the total intracellular cholesterol and sharply decreased the incorporation of [3H]thymidine. With respective efficacy verapamil was followed by nifedipine and PY 108 068. Neither beta-blocker (metoprolol) nor nitrate (nitroglycerin) modified antiatherosclerotic effects of calcium antagonist (nifedipine). Calcium agonist Bay K 8644 which facilitates the penetration of calcium into cells caused the accumulation of intracellular cholesterol and stimulated cell proliferation. Simultaneous addition of nifedipine and Bay 8644 led to the inhibition of the agonist's atherogenic effect. Thus, facilitation of calcium influx into cells causes atherosclerotic alterations in the arterial cells; atherogenic calcium effects are inhibited by calcium channel blockers. Furthermore, based on the results of application of blood plasma from patients treated with calcium antagonists or beta-blocker to primary cultures of atherosclerotic cells, it can be assumed that calcium antagonists affect an anti-atherosclerotic and beta-blockers an atherogenic action.