Bradykinin (B2 independent effect of captopril on the development of pressure overload cardiac hypertrophy

Besides the reduction of angiotensin II formation, locally increased kinins may play a role in the cardiovascular action of angiotensin converting enzyme (ACE) inhibitors.To characterize the contribution of bradykinin to the effects of ACE inhibition by captopril on the development of pressure overload hypertrophy, sham-operated rats and rats with ascending aortic constriction were treated with captopril (80 mg/kg/day) or captopril and B₂-kinin receptor antagonist HOE 140 (0.5 mg/kg/day) for 7 weeks. Left ventricular mass and geometry, hydroxyproline concentration and myosin isozymes (marker of a fetal phenotype) were assessed. Rats with aortic constriction exhibited a marked increase in left ventricular weight and diastolic pressure-volume relationship was shifted to smaller volumes. Signs of congestive heart failure were not apparent. The hydroxyproline concentration remained unaltered. However, the proportion of isomyosin V3 was increased (p < 0.05). Administration of captopril reduced (p < 0.05) systolic blood pressure, body and cardiac weight in all treated rats. The reduction of left ventricular weight was disproportionally higher in pressure overloaded rats, thus the relative left ventricular weight decreased by 15% (p < 0.05). Captopril augmented the isomyosin V1 expression (p < 0.05) in sham operated as well as pressure overloaded rats. The isomyosin V1 percentage was inversely related to the relative left ventricular weight. Two different (p < 0.05) correlation lines were detected for untreated and captopril treated rats. None of captopril associated effects were removed by simultaneously administered B₂ kinin receptor antagonist HOE 140.Thus, stimulation of bradykinin B2 receptor appears not to mediate the effects of captopril on cardiac growth and contractile proteins during the development of pressure overload hypertrophy.     

The effects of enalapril maleate and cold stress exposure on tyrosine hydroxylase activity in some rat tissues

Enalapril is a highly specific and competitive inhibitor of angiotensin-I converting enzyme (ACE) and thus belongs to the category of ACE inhibitors. The beneficial effects of ACE inhibitors appear to result primarily from the suppression of the plasma renin-angiotensin-aldesterone system. This study was designed to detect the effects of enalapril maleate and cold stress on tyrosine hydroxylase (TH) activity in adrenal medulla, heart and hypothalamus in rat. In cold stress treatment (exposed to 8 degrees C cold for 48 h) TH activity was found to be raised significantly (p < 0.05) in adrenal medulla, hypothalamus and heart tissues. In the adrenal medulla, hypothalamus and heart tissues, TH activity of enalapril maleate treated rats (10 mg kg(-1) body weight) group was not raised significantly (p > 0.05). Following intraperitoneal injection of enalapril maleate (10 mg kg(-1) body weight) the rats were exposed to 8 degrees C cold for 48 h. After cold stress and enalapril maleate treatment no statistically significant change in tyrosine hydroxylase activity was detected in adrenal medulla, hypothalamus or heart (p > 0.05). The results of our studies show that enalapril maleate blocks the effect of cold stress on the regulation of TH activity.     

The role of Jak/STAT signaling in heart tissue renin-angiotensin system

The involvement of the Renin Angiotensin System (RAS) and the role of its primary effector, angiotensin II (Ang II), in etiology of myocardial hypertrophy and ischemia is well documented. In several animal models, the RAS is activated in cardiac cell types that express the receptor AT₁, and/or AT₂, through which the Ang II mediated effects are promoted. In this article, we briefly review recent experimental evidence on the critical role of a prominent signaling pathway, the Jak/Stat pathway in activation and maintenance of the local RAS in cardiac hypertrophy and ischemia. Recent studies in our laboratory document that the promoter of the prohormone angiotensinogen (Ang) gene serves as the target site for STAT proteins, thereby linking the Jak/Stat pathway to activation of heart tissue autocrine Ang II loop. Stat5A and Stat6, are selectively activated when the heart is subjected to ischemic injury, whereas activation of Stat3 and Stat5A is involved in myocardial hypertrophy. Blockage of RAS activation by treatment with specific inhibitor promotes a remarkable recovery in functional hemodynamics of the myocardium. Thus, activation of selective sets of Stat proteins constitutes the primary signaling event in the pathogenesis of myocardial hypertrophy and ischemia.     

Presence of angiotensin-converting enzyme in follicular fluids of porcine ovaries and its possible involvement in the intrafollicular breakdown of bradykinin

The follicular fluid of porcine ovaries contains a metalloenzyme capable of hydrolyzing the synthetic substrate, benzyloxycarbonyl-Val-Lys-Met-MCA. This enzyme was purified by ammonium sulfate fractionation followed by column chromatography on DEAE-cellulose, CM-cellulose, Zn(2+)-chelating Cellulofine, and Diol-300 gel-filtration columns. The molecular weight of the purified enzyme was estimated to be 170,000 by SDS-PAGE and 400,000 by gel-filtration analysis, suggesting that the native enzyme is a dimer of the 170-kDa subunit polypeptide. The enzyme activity was drastically enhanced by the presence of chloride ion, and strongly inhibited by captopril and bradykinin potentiator B. A 9-residue peptide containing a processing site of human amyloid precursor protein was degraded by its dipeptidyl carboxypeptidase activity. Furthermore, the purified protein was recognized by specific antibody raised against human angiotensin-converting enzyme. The enzyme rapidly degraded bradykinin in vitro. These results indicate that benzyloxycarbonyl-Val-Lys-Met-MCA-hydrolyzing enzyme is a porcine angiotensin-converting enzyme, and that the enzyme may play a role in bradykinin turnover within the follicles of porcine ovaries.     

Conformational basis for the biological activity of TOAC-labeled angiotensin II and bradykinin: electron paramagnetic resonance, circular dichroism, and fluorescence studies

N-Terminally and internally labeled analogues of the hormones angiotensin (AII, DRVYIHPF) and bradykinin (BK, RPPGFSPFR) were synthesized containing the paramagnetic amino acid 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC). TOAC replaced Asp1 (TOAC1-AII) and Val3 (TOAC3-AII) in AII and was inserted prior to Arg1 (TOAC0-BK) and replacing Pro3 (TOAC3-BK) in BK. The peptide conformational properties were examined as a function of trifluoroethanol (TFE) content and pH. Electron paramagnetic resonance spectra were sensitive to both variables and showed that internally labeled analogues yielded rotational correlation times (tauC) considerably larger than N-terminally labeled ones, evincing the greater freedom of motion of the N-terminus. In TFE, tauC increased due to viscosity effects. Calculation of tau(Cpeptide)/tau(CTOAC) ratios indicated that the peptides acquired more folded conformations. Circular dichroism spectra showed that, except for TOAC1-AII in TFE, the N-terminally labeled analogues displayed a conformational behavior similar to that of the parent peptides. In contrast, under all conditions, the TOAC3 derivatives acquired more restricted conformations. Fluorescence spectra of AII and its derivatives were especially sensitive to the ionization of Tyr4. Fluorescence quenching by the nitroxide moiety was much more pronounced for TOAC3-AII. The conformational behavior of the TOAC derivatives bears excellent correlation with their biological activity, since, while the N-terminally labeled peptides were partially active, their internally labeled counterparts were inactive [Nakaie, C. R., et al., Peptides 2002, 23, 65-70]. The data demonstrate that insertion of TOAC in the middle of the peptide chain induces conformational restrictions that lead to loss of backbone flexibility, not allowing the peptides to acquire their receptor-bound conformation.     

Role of hydroxyl containing residues in the intracellular region of rat bradykinin B(2) receptor in signal transduction, receptor internalization, and resensitization

In past reports we illustrated the importance of Y131, Y322, and T137 within the intracellular (IC) face of the rat bradykinin B2 receptor (rBKB2R) for signal transduction and receptor maintenance (Prado et al. [1997] J. Biol. Chem. 272:14638-14642; Prado et al. [1998] J. Biol. Chem. 273:33548-33555). In this report, we mutate the remaining hydroxyl possessing residues located within the rBKB2R IC region. Exchange of S139A (IC2) or T239V (IC3) did not affect BK activated phosphatidylinositol (PI) turnover or receptor internalization. Chimeric exchange of the last 34 amino acids of BKB2R C-terminus with the corresponding 34 amino acids of the rat angiotensin II AT1a receptor (rAT1aR), both containing an S/T cluster, resulted in a mutant with normal endocytosis and BK activated PI turnover. A more selective chimera of these S/T clusters, with an exchange of BKB2R (333-351) with a rAT1aR fragment (326-342), resulted in a receptor with a retarded internalization but a normal BK activated PI turnover. Subsequent mutation of rBKB2R T344V showed little change in receptor uptake but a pronounced loss of BK activated PI turnover. The mutation of S335A, S341A, S348A, and S350A resulted in very poor receptor internalization and loss of activated PI turnover. Closer examination of this serine cluster illustrated that the replacement of S348A led to poor internalization; whereas the retention of S348 and mutation of S341A resulted in a receptor with a much greater internalization than WT. These and other results suggest that the presence of S348 promotes internalization while the presence of S341 dampens it. Conversely, S341 and S350 proved important for receptor signaling. In sum, our results illustrate that the distal C-terminus including its S/T cluster is important for both rBKB2R internalization and signal transduction. Individual S/T residues within this cluster appear involved in either signal transmission or receptor uptake capacity. However, replacement of the entire distal tail region with the corresponding rAT1aR sequence, also containing an S/T cluster, enables the BKB2R/AT1aR chimera to act in a very similar manner to wild type rBKB2R.     

Functional role of the conserved proline in helix 6 of the human bradykinin B2 receptor

Pro258 in transmembrane domain (TMD) 6 of the bradykinin (BK) B₂ receptor (B₂R) is highly conserved among G-protein coupled receptors (GPCRs). Using mutagenesis, we show that Pro258 is required for normal trafficking of the receptor to the plasma membrane and that mutation of Pro258 to Ala or Leu but not Gly, enhances BK efficacy to induce receptor activation. Furthermore, P258A mutation suppresses the constitutive activity of a constitutively activated N113A-B₂R mutant but preserves the antagonist to agonist efficacy shift previously observed on the N113A single mutant. Our data suggest that Pro258 in TMD6 is required for agonist-independent activation of the B₂R and that straightening of TMD6 at the Pro-kink might favor G-protein coupling. It is also shown that Asn113 is a contact point of BK interaction and it is proposed that the release of a TMD3-TMD6 interaction involving Asn113 is crucial for the efficacy shift from antagonism toward agonism.     

Contribution of bradykinin and nitric oxide to AT2 receptor-mediated differentiation in PC12 W cells

We investigated the effect of angiotensin II on intracellular cyclic GMP content and neurite outgrowth as an indicator of cell differentiation in PC12 W cells. Neurite outgrowth was examined by phase-contrast microscopy. Outgrown neurites were classified as small, medium and large, and were expressed as neurites per 100 cells. Angiotensin II (10-7 m) increased the outgrowth of medium and large neurites by mean +/- SEM 20.2 +/- 2.3 and 6.6 +/- 1.4 compared with 1.66 +/- 0.5 and 0.1 +/- 0.06 neurites per 100 cells in control. Cellular cyclic GMP content increased by 50-250% with angiotensin II at concentrations of 10-6-10-4 m. Both blockade of AT2 receptors and of nitric oxide synthase markedly reduced angiotensin II-induced neurite outgrowth and cyclic GMP production. In contrast, B2 receptor blockade had no effect or even increased these angiotensin II effects. Sodium nitroprusside and 8-bromo-cyclic GMP both mimicked the effects of angiotensin II on cell differentiation. The protein kinase G inhibitor KT-5823 inhibited the neurite outgrowth induced by both angiotensin II and 8-bromo-cyclic GMP. Our results demonstrate that angiotensin II can stimulate cell differentiation in PC12 W cells by nitric oxide-related and cyclic GMP-dependent mechanisms. The effects of angiotensin II on cell differentiation and cyclic GMP production were mediated via the AT2 receptor and further enhanced by bradykinin B2 receptor blockade.     

The effects of Zn2+ on guinea pig isolated heart preparations

Isolated guinea pig hearts were perfused, by the Langendorff technique, with 30, 15, 7.5, and 1.5 μM Zn²⁺ in Chenoweth solution. Contractile force, coronary flow, and heart rate were recorded by means of Narco IV Physiograph. Calcium inhibitor (Verapamil 1 μM) and anion inhibitor (DIDS: 0.1, 1, and 5 μM) were used subsequently in the perfusing solutions in order to distinguish some of the possible mechanisms that Zn²⁺ uses to exert its action on cardiac myocytes. Isomolar to zinc concentration of Pb (II) and Co (II) were used to elucidate whether zinc effects on heart are specific for this metal. All hearts were used to estimate their zinc and calcium content by means of AAS (Atomic Absorption Spectrometry). Our findings suggest that the higher the Zn²⁺ concentration, the more toxic effects on heart are expressed by rapid reversible contractile force reduction and reversible specific changes of heart rate and flow. Zinc 1.5 μM in the perfusing solution benefits heart performance, but not significantly. Furthermore, the metal exerts specific effects on guinea pig heart, and it is rather possible that these effects on cardiac myocytes are held through cell membrane receptors.     

Reduction of cardiac hypertrophy in TGR(mREN2)27 by angiotensin II receptor blockade

TGR(mREN2)27 is a transgenic rat harboring the murine Ren-2 gene and exhibit fulminant hypertension and marked heart hypertrophy. In order to study the role of angiotensin II in the increase of cardiac mass, these animals were treated with anti-hypertensive and non-antihypertensive doses of the angiotensin II receptor AT₁ antagonist Telmisartan for 9 weeks. All doses led to significant reductions of heart hypertrophy detected by the evaluation of the diameter of cardiac muscle bundles. We conclude from this study that cardiac hypertrophy in TGR(mREN2)27 is characterized by an increased volume of cardiomyocytes and an unchanged amount of fibrous tissue and that angiotensin II plays an important role in the mechanisms leading to this phenotype.     

Determination of enalapril maleate and atenolol in their pharmaceutical products and in biological fluids by flow‐injection chemiluminescence

A chemiluminescent method using flow injection (FI) was investigated for rapid and sensitive determination of enalapril maleate and atenolol, which are used in the treatment of hypertension. The method is based on the sensitizing effect of these drugs on the Ce(IV)–sulfite reaction. The different experimental parameters affecting the chemiluminescence (CL) intensity were carefully studied and incorporated into the procedure. The method permitted the determination of 0.01–3.0 µg mL^?1 of enalapril maleate in bulk form with correlation coefficient r = 0.99993, lower limit of detection (LOD) 0.0025 µg mL^?1 (S/N = 2) and lower limit of quantitation (LOQ) 0.01 µg mL^?1. The linearity range of atenolol in bulk form was 0.01–2.0 µg mL^?1 (r = 0.99989) with LOD of 0.0003 µg mL^?1 (S/N = 2) and LOQ of 0.01 µg mL^?1. In biological fluids the linearity range of enalapril maleate was 0.1–2.0 µg mL^?1 in both urine and serum, and for atenolol the linearity range was 0.1–1.0 µg mL^?1 in both urine and serum. The method was also applied to the determination of the drugs in their pharmaceutical preparations. Copyright © 2009 John Wiley & Sons, Ltd.     

Partial prevention of changes in SR gene expression in congestive heart failure due to myocardial infarction by enalapril or losartan

Although activation of the renin-angiotensin system (RAS) is known to produce ventricular remodeling and congestive heart failure (CHF), its role in inducing changes in the sarcoplasmic reticulum (SR) protein and gene expression in CHF is not fully understood. In this study, CHF was induced in rats by ligation of the left coronary artery for 3 weeks and then the animals were treated orally with or without an angiotensin converting enzyme inhibitor, enalapril (10 mg/kg/day) or an angiotensin II receptor antagonist, losartan (20 mg/kg/day) for 4 weeks. Sham-operated animals were used as control. The animals were hemodynamically assessed and protein content as well as gene expression of SR Ca²⁺-release channel (ryanodine receptor, RYR), Ca²⁺-pump ATPase (SERCA2), phospholamban (PLB) and calsequestrin (CQS) were determined in the left ventricle (LV). The infarcted animals showed cardiac hypertrophy, lung congestion, depression in LV +dP/dt and –dP/dt, as well as increase in LV end diastolic pressure. Both protein content and mRNA levels for RYR, SERCA2 and PLB were decreased without any changes in CQS in the failing heart. These alterations in LV function as well as SR protein and gene expression in CHF were partially prevented by treatment with enalapril or losartan. The results suggest that partial improvement in LV function by enalapril and losartan treatments may be due to partial prevention of changes in SR protein and gene expression in CHF and that these effects may be due to blockade of the RAS.     

缓激肽在骨骼肌缺血预适应对猪心肌坏死和凋亡中的作用

目的：研究缓激肽在骨骼肌缺血预适应对心肌坏死和心肌凋亡保护中可能的作用：方法：采用非开胸法建立猪心脏缺血／再灌注（I／R）模型,通过球囊堵塞左股动脉造成骨骼肌短暂缺血,使用缓激肽（BK）的B2受体拮抗剂烟酸已可碱（HOE-140）以及外源BK进行干预。分别观察各组对心肌坏死和凋亡的影响。结果：远端预处理后心肌坏死范围明显缩小,凋亡率明显降低：预处理前使用HOE-140可使对坏死范围的保护作用明显减弱;心肌I／R前使用外源BK注射,可缩小心肌梗死范围。但HOE-140及外源BK对以上凋亡指标无影响．结论：骨骼肌远端预适应可减少心肌坏死和心肌凋亡、BK可能参与对坏死面积的保护．但不参与对凋亡的保护作用。     

Investigation of bradykinin metabolism in human and rat plasma in the presence of the dual ACE/NEP inhibitors GW660511X and omapatrilat.

Several studies have suggested that the accumulation of bradykinin, or that of one its metabolites BK1-8, is involved in the occurrence of side effects such as AE associated with the use of various ACEi. In this work a novel approach combining HPLC-UV on-line with oaTOF-MS and ICPMS was applied to investigate in human and rat plasma the metabolism of labelled BK (79/81 Br-Phe5) BrBK in the presence of two new dual ACE/NEP inhibitors (GW660511X and omapatrilat) currently under clinical trial. In human plasma the BrBK half-life values in the absence or in the presence of GW660511X (3.8 microM) or omapatrilat (32 nM) were 38.7 +/- 2.4, 51.2 +/- 4.7 and 114.7 +/- 9.3 min, respectively and BrBK was degraded into BrBK1-8, BrBK1-7, BrBK1-5 and Br-Phe. In the presence of inhibitors, however, the levels of these resultant metabolites were different. Unlike GW660511X, omapatrilat abolished the production of BrBK1-5 and BrBK1-7, suggesting a better ACE inhibition effect over GW660511X as no NEP activity was found. In addition the production of BrBK1-8 was enhanced in the presence of these inhibitors with a greater accumulation being observed with omapatrilat. The production of Br-Phe5 was reduced with GW660511X while no significant change was observed with omapatrilat after 4 h of incubation. In rat plasma the BrBK half-life values in the absence or in the presence of GW660511X (530 nM) or omapatrilat (50 nM) were 9.31 +/- 1.7, 22.06 +/- 3.1 and 25.3 +/- 1.7 min, respectively and BrBK was degraded into BrBK1-8, BrBK1-7, BrBK1-5 and Br-Phe5 plus BrBK2-9, BrBK4-8 and BrBK2-8 metabolites not found in human plasma. GW660511X and omapatrilat reduced the production of BrBK1-5 and BrBK1-7 with more effect being observed with omapatrilat. GW660511X and omapatrilat increased the production of both BrBK1-8 and Br-Phe5 but not that of BrBK4-8 and BrBK2-8. This study shows that the potency of GW660511X in comparison with omapatrilat is more than 100-fold lower in human, but less than 10-fold lower in rat plasma, suggesting that rat may not be a suitable in vivo model for the evaluation of ACE/NEP inhibition in relation to effects in humans.     

Bradykinin analogs containing the 4-amino-2-benzazepin-3-one scaffold at the C-terminus.

High affinity peptide ligands for the bradykinin (BK) B(2) subtype receptor have been shown to adopt a beta-turn conformation of the C-terminal tetrapeptide (H-Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)-Ser(6)-Pro(7)-Phe(8)-Arg(9)-OH). We investigated the replacement of the Pro(7)-Phe(8) dipeptide moiety in BK or the D-Tic(7)-Oic(8) subunit in HOE140 (H-D-Arg(0)-Arg(1)-Pro(2)-Hyp(3)-Gly(4)-Thi(5)-Ser(6)-D-Tic(7)-Oic(8)-Arg(9)-OH) by 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one templates (Aba). Binding studies to the human B(2) receptor showed a correlation between the affinities of the BK analogs and the propensity of the templates to adopt a beta-turn conformation. The L-spiro-Aba-Gly containing HOE140 analog BK10 has the best affinity, which correlates with the known turn-inducing property of this template. All the compounds did not modify basal inositolphosphate (IP) output in B(2)-expressing CHO cells up to 10 microM concentration. The antagonist properties were confirmed by the guinea pig ileum smooth muscle contractility assay. The new amino-benzazepinone (Aba) substituted BK analogs were found to be surmountable antagonists.     

Comparative effects of a vasopeptidase inhibitor vs. an angiotensin convertin enzyme inhibitor on cardiomyocyte apoptosis in rats with heart failure

Apoptosis is involved in ventricular remodeling after myocardial infarction (MI). We investigated the effects of the vasopeptidase inhibitor (VPI) omapatrilat on cardiomyocyte apoptosis and compared it to the angiotensin converting enzyme inhibitor (ACEI) captopril in the rat post-MI model and in cultured neonatal rat cardiomyocytes. Wistar males rats surviving 4 h post-MI were assigned to omapatrilat (40 or 80 mg/kg/day), captopril (160 mg/kg/day) or no treatment. After 56 days, hemodynamic measurements were performed (n = 96) and rats were sacrificed. One group had assessment of cardiac remodeling and detection of DNA fragments by in situ end labelling method (ISEL), while the other had morphologic measurements and DNA laddering assessed. In addition, cultured neonatal rat cardiomyocytes (n = 6) were treated for 72 h with vehicle, captopril or omapatrilat in the presence or absence of the apoptosis inducing agent H₂O₂. Omapatrilat and captopril resulted in similar improvements of hemodynamic measurements, ventricular weight and dilatation, cardiac fibrosis and myocardial cell cross-section in large MI rats. Omapatrilat increased scar thickness more than did captopril. All sham-operated groups had little evidence of apoptosis. In the large MI group, there was a significant increase in ISEL-positive cells in the control (0.095 ± 0.016%) and captopril (0.124 ± 0.024%) groups in comparison with control sham-operated (0.006 ± 0.006%), but this increase was limited to the peri-MI area. Omapatrilat (0.012 ± 0.012% for both doses) prevented the increase in apoptosis in the peri-MI area. Also, omapatrilat but not captopril reduced DNA laddering in large MI. Moreover, in cultured neonatal rat cardiomyocytes, omapatrilat but not captopril reduced apoptosis as assessed by DNA laddering. The VPI omapatrilat, with its combination of NEP and ACE inhibition, suppresses cardiomyocyte apoptosis post-MI and in neonatal cultured rat cardiomyocytes more than the ACEI captopril, but this does not result in significant hemodynamic or morphologic differences between omapatrilat and captopril.     

The inhibitory effects of rosiglitazone on cardiac hypertrophy through modulating the renin‐angiotensin system in diet‐induced hypercholesterolemic rats

Cardiac hypertrophy is not only an adaptational state before heart failure but also is an independent risk factor for ischemia, arrhythmia, and sudden death. However, the direct effects of hypercholesterolemia on the myocardium and mechanisms are not completely understood. It has been demonstrated that peroxisome proliferator‐activated receptor‐γ (PPARγ) ligand agonists attenuate cardiac hypertrophy through anti‐inflammatory effects. The present study investigated the effects of PPARγ agonists on hypercholesterolemia‐dependent, renin‐angiotensin‐system‐related cardiac hypertrophy. The findings showed that left ventricular hypertrophy, eminent cardiomyocyte hypertrophy, and lipid deposits in myocardium were observed in the rats fed a cholesterol‐rich diet for 6 months, while these characteristic pathological alterations and the increase in angiotensin II (ANG II) level and over‐expression of angiotensin II type 1 receptor (AT₁R) in the left ventricular tissues induced by the cholesterol‐rich diet were significantly suppressed to equal extents by rosiglitazone and irbesartan. In contrast, expression of angiotensin II type 2 receptor (AT₂R) was upregulated by these two drugs. In addition, lipid metabolism was markedly improved. The above findings suggest that the cardioprotection of the PPARγ agonist against cardiac hypertrophy evoked by hypercholesterolemia in rats is mediated partially by the improvement of lipid profile, the reduction of ANG II level in the local tissue along with the downregulation of AT₁R expression, and upregulation of AT₂R expression. Copyright © 2009 John Wiley & Sons, Ltd.     

The effect of angiotensin II on myosin heavy chain expression in cultured myocardial cells

Summary Angiotensin II (AII), the principal mediator of the renin-angiotensin system, is an important regulator of vascular and cardiac homeostasis. AII has also been shown to be a regulator of cardiac hypertrophy and of the corresponding changes in amount and composition of certain tissue proteins. We examined the trophic effects of AII on cultured myocytes derived from neonatal rat ventricles and followed, by Northern blot analysis and polyacrylamide gel electrophoresis, the expression of α- and β-myosin heavy chain iso-mRNAs and isoproteins. Our findings show that a single administration of AII is sufficient to induce a trophic response in cultured beating myocytes and to enhance the expression of β-myosin heavy chain iso-mRNA and isoprotein, having no effect on α-myosin heavy chain. Induction of α-myosin heavy chain expression by thyroid hormone before AII was administered showed that AII could not potentiate a shift from α- to β-myosin heavy chain predominance. We suggest that the potency of AII to regulate the expression of myosin heavy chain isogenes is restricted to the β isoform and is overridden by thyroid hormone.     

Structural insight into the role of the second intracellular loop of the bradykinin 2 receptor in signaling and internalization.

The second cytoplasmic loop (IC2) of the bradykinin B2 receptor plays a vital role in its dynamic life cycle including the activation, internalization, desensitization, and resensitization of this receptor. Here, we probe the structure and function of the IC2, with particular emphasis on threonine-137, which is crucial for signal transduction and internalization. Mutation of this threonine to proline (T137P) produces wild type (WT) signaling and complete inhibition of internalization. Incorporation of aspartate (T137D) leads to a marked reduction in receptor signaling but with WT receptor uptake. The T137D mutation coupled with serine to alanine substitution of S335 and S341 within the distal C-terminus recovers signaling, leading to an actually enhanced arachidonic acid release and phosphoinositide turnover compared to WT bradykinin B2 receptor (BKB2R). To provide a structural basis for the actions of this mutant, the conformational features of IC2 (both WT and mutant) were investigated by high-resolution NMR. The NMR analysis illustrated two prominent alpha-helices at the N- (L123-M138) and C-termini (A149-I156) of the IC2 receptor domain. Incorporating these structural characteristics into a model of BKB2R, we determined that the entire N-terminal helix of IC2 is incorporated as TM3, placing Y131 1.5 helical turns into TM3 and T137 at the membrane surface. The NMR data indicated no structural changes upon substitution of T137D. These results suggest that the altered signaling of the T137D mutant can be attributed to the introduction of a negative charge, indicating that phosphorylation of this residue takes place and participates in the life cycle of this receptor. Additionally, the return to WT signal capacity of the mutation T137D/S335A/S341A, to overcome the deleterious T137D substitution points to a functional interaction between the IC2 and the C-terminus.