Inhibition of angiotensin Ⅱ and blockade of endothelin receptors reduce arterial calcification in rats

Objective To examine whether the two vascular paracrine/autocrine factors, angiotensin Ⅱ (Ang Ⅱ) and endothelin, participate in the pathogenesis of arterial calcification. Methods Nicotine and vitamin D3 treated rats were studied. Vascular calcification was confirmed by using Von Kossa staining, measurement of calcium content,45Ca2+ uptake assay and alkaline phosphatase (ALP) activity. The plasma and vascular Ang Ⅱ and endothelin levels were measured by using radioimmunoassay. Angiotensinogen and endothelin mRNA levels were determined by RTPCR. Results The arterial calcium content, 45Ca2+ uptake and ALP activity were increased in calcification groups compared with control ( P ＜ 0.01 ). Administration of the angiotensin receptor antagonist losartan, the endothelin receptor antagonist bosentan, and the angiotensin-converting enzyme inhibitor captopril reduced significantly the arterial calcium content, 45Ca2+ uptake and ALP activity. In addition, the plasma and aortic Ang Ⅱ and endothelin contents, and vascular angiotensinogen and endothelin mRNA expression were significantly up-regulated ( P ＜0.05).Conclusions These findings suggest that functional renin-angiotensin system and endothelin pathway are involved in vascular calcification, and that activation of these systems could potentiate pathogenesis of arterial calcification. ( J Geriatr Cardiol 2004;1(2) :108-113. )     

Angiotensin II signaling pathways mediate expression of cardiac T-type calcium channels

Recent studies indicate that cardiac T-type Ca2+ current (ICaT) reappears in hypertrophied ventricular cells. The aim of this study was to investigate the role of angiotensin II (Ang II), a major inducer of cardiac hypertrophy, in the reexpression of T-type channel in left ventricular hypertrophied myocytes. We induced cardiac hypertrophy in rats by abdominal aorta stenosis for 12 weeks and thereafter animals were treated for 2 weeks with losartan (12 mg/kg per day), an antagonist of type 1 Ang II receptors (AT1). In hypertrophied myocytes, we showed that the reexpressed ICaT is generated by the CaV3.1 and CaV3.2 subunits. After losartan treatment, ICaT density decreased from 0.40+/-0.05 pA/pF (n=26) to 0.20+/-0.03 pA/pF (n=27, P<0.01), affecting CaV3.1- and CaV3.2-related currents. The amount of CaV3.1 mRNA increased during hypertrophy and retrieved its nonhypertrophic level after losartan treatment, whereas the amount of CaV3.2 mRNA was unaffected by stenosis. In cultured newborn ventricular cells, chronic Ang II application (0.1 micromol/L) also increased ICaT density and CaV3.1 mRNA amount. UO126, a mitogen-activated protein kinase kinase-1/2 (MEK1/2) inhibitor, reduced Ang II-increased ICaT density and CaV3.1 mRNA amount. Bosentan, an endothelin (ET) receptor antagonist, reduced Ang II-increased ICaT density without affecting the amount of CaV3.1 mRNA. Finally, cotreatment with bosentan and UO126 abolished the Ang II-increased ICaT density. Our results show that AT1-activated MEK pathway and autocrine ET-activated independent MEK pathway upregulate T-type channel expression. Ang II-increased of ICaT density observed in hypertrophied myocytes may play a role in the pathogenesis of Ca2+ overload and arrhythmias seen in cardiac pathology.     

Sarcosine1, glycine8 angiotensin II is a functional AT1 angiotensin receptor antagonist

Sarcosine¹, glycine⁸ angiotensin II (SG Ang II) displayed unusual characteristics in early pharmacological studies. It was a potent antagonist of the dipsogenic actions of intracerebroventricularly administered Ang II in the rat, but showed low affinity for bovine cerebellar Ang II receptors. It has recently been shown that SG Ang II binds preferentially to the AT₁ receptor, To determine if SG Ang II is a functional antagonist of the AT₁ receptor-mediated calcium signaling, CHO cells stably transfected with AT₁ receptors were exposed to Ang II in the presence and absence of SG Ang II. At concentrations of 10–100 nM, SG Ang II completely inhibited Ang II-stimulated intracellular Ca²⁺ mobilization in AT_1A and AT_1B receptor-transfected cells. SG Ang II and ¹²⁵I-SG Ang II bound to AT_1A and AT_1B receptor-transfected cells with K _D and K ₁ values of 2–4 nM. In contrast, SG Ang II bound to AT₂ transfected cells with a K ₁ value of 7.86 μM. These results demonstrate that SG Ang II is a selective and functional peptide antagonist of the AT₁ angiotensin II receptor subtype.     

肾上腺髓质素对大鼠血管钙化的影响

目的　观察肾上腺髓质素 (ADM)对血管钙化的影响。方法　维生素D3 (VitD3 )和尼古丁制备大鼠血管钙化模型 ,分别测定血管、心肌钙含量和血管碱性磷酸酶 (ALP)活性 ,血浆和血管ADM含量 ,12 5I ADM与ADM受体的结合力及血管环磷酸腺苷 (cAMP)含量。结果　与对照组相比 ,钙化组 (VDN组 )的血管钙含量和ALP活性明显升高 ;血管及血浆ADM的含量亦升高 ,但12 5I ADM与血管质膜ADM受体结合的位点减少 ,Kd值升高 ,提示亲合力降低 ,同时伴钙化血管的cAMP合成减少。提示钙化血管对ADM的反应减弱。空载脂质体对血管钙化无影响。用脂质体包裹ADM组 (VDN ADM组 )与钙化组相比 ,其血管的钙含量、ALP的活性均明显降低 ;12 5　I ADM与血管质膜ADM受体结合的位点增加 ,Kd值减少 ,cAMP的合成也增加 ,提示该组血管对ADM的反应增强。结论　血管钙化时ADM ADM受体 cAMP途径发生变化 ,外源性ADM通过改善ADM ADM受体 cAMP途径 ,发挥抑制血管钙化的作用     

Angiotensin II relaxations of bovine adrenal cortical arteries: role of angiotensin II metabolites and endothelial nitric oxide

Angiotensin (Ang) II regulates adrenal steroidogenesis and adrenal cortical arterial tone. Vascular metabolism could decrease Ang II concentrations and produce metabolites with vascular activity. Our goals were to study adrenal artery Ang II metabolism and to characterize metabolite vascular activity. Bovine adrenal cortical arteries were incubated with Ang II (100 nmol/L) for 10 and 30 minutes. Metabolites were analyzed by mass spectrometry. Ang (1-7), Ang III, and Ang IV concentrations were 146+/-21, 173+/-42 and 58+/-11 pg/mg at 10 minutes and 845+/-163, 70+/-14, and 31+/-3 pg/mg at 30 minutes, respectively. Concentration-related relaxations of U46619-preconstricted cortical arteries to Ang II (maximum relaxation=29+/-3%; EC(50)=3.4 pmol/L) were eliminated by endothelium removal and inhibited by the NO synthase inhibitor, nitro-L-arginine (30 micromol/L; maximum relaxation=14+/-7%). Ang II relaxations were enhanced by the angiotensin type-1 receptor antagonist losartan (1 micromol/L; maximum relaxation=41+/-3%; EC(50)=11 pmol/L). Losartan-enhanced Ang II relaxations were inhibited by nitro-L-arginine (maximum relaxation=18+/-5%) and the angiotensin type-2 receptor antagonist PD123319 (10 micromol/L; maximum relaxation=27+/-5%). Ang (1-7) and Ang III caused concentration-related relaxations with less potency (EC(50)=43 and 24 nmol/L, respectively) but similar efficacy (maximum relaxations=39+/-3% and 48+/-5%, respectively) as losartan-enhanced Ang II relaxations. Ang (1-7) relaxations were inhibited by nitro-L-arginine (maximum relaxation=16+/-4%) and the Ang (1-7) receptor antagonist 7(D)-Ala-Ang (1-7) (1 micromol/L; maximum relaxation=10+/-3%) and eliminated by endothelium removal. Thus, Ang II metabolism by adrenal cortical arteries to metabolites with decreased vascular activity represents an inactivation pathway possibly decreasing Ang II presentation to adrenal steroidogenic cells and limits Ang II vascular effects.     

Losartan reduces the vasorelaxant effect of atrial natriuretic peptide on basal tone in aorta

The Ca2+ -and receptor-dependencies of the basal tone seen in angiotensin II (Ang II)-conditioned rabbit thoracic aortic rings were investigated. Ca2+ -free Krebs significantly and partially reversibly reduced basal tone in aortic rings that had recovered from an earlier challenge with Ang II; rings not previously exposed to Ang II were unaffected. The effect of Ca2+ -free Krebs was similar to the reduction in basal tone evoked by atrial natriuretic peptide (ANP), but was smaller than that seen with exposure to Ca2+ -free Krebs+EGTA+sodium nitroprusside (SNP). Pretreating rings with Ca2+ free Krebs blocked the vasorelaxant effects of ANP and Ca2+ -free Krebs+EGTA+SNP. Losartan, an AT1 receptor antagonist, significantly attenuated ANP-induced relaxation, but did not otherwise alter basal tension in either unstimulated or Ang II-conditioned rings. The AT2 receptor antagonist, PD 123319, had no effect. These data suggest that transient exposure to Ang II induces prolonged, AT1-dependent increases in intracellular free Ca2+ which are antagonized by ANP.     

Effects of adrenomedullin on vascular calcification in rats

OBJECTIVE: The aim of the present study was to investigate the effect of adrenomedullin (ADM) on vascular calcification. METHODS: The vascular calcification model was established in rats (VND group) by using vitamin D3 (300,000 IU/kg) and nicotine (25 mg/kg, two doses). The effect of liposome-encapsulated ADM was observed. Vascular calcium content, alkaline phosphatase (ALP) activity, ADM in aortic tissue and plasma, binding ability of 125I-ADM for ADM receptor on vascular plasma membrane and content of cAMP in vessels were measured. RESULTS: Compared with control rats, the aortic calcium content and vascular ALP activity in rats of the VDN group was obviously increased; in addition ADM concentrations in plasma and vessels of rats in VDN group were increased. But the maximum binding sites of 125I-ADM for ADM receptor (Bmax) on vascular plasma membrane in rats of VDN group were significantly decreased compared with control rats. The affinity of 125I-ADM for the ADM receptor was reduced, as shown by the Kd value and vascular cAMP content being reduced in rats of the VDN group compared to the control group. The in vitro response of isolated vessels to ADM incubation was weakened. Administration of empty liposome had no effect on vascular calcification. But administration of ADM significantly decreased vascular calcium content and ALP activity. The Bmax of 125I-ADM for ADM receptors on vascular plasma membrane increased by 17.7% (p < 0.01), and the value of Kd decreased by 36.2% (P < 0.01) in rats treated with ADM as compared with rats of the VDN group. In addition, the vascular cAMP content and the response to ADM in isolated aorta were markedly increased. CONCLUSION: Vascular calcification induced an alteration of the vascular ADM-ADM receptor-cAMP pathway. Treatment with exogenous ADM inhibited vascular calcification by improving the vascular ADM-ADM receptor-cAMP pathway.     

Angiotensin II stimulates contraction and growth of testicular peritubular myoid cells in vitro

Seminiferous tubule contraction, an important step in the regulation of spermatogenesis and testicular sperm output, is regulated by several agonists. In the present paper, we investigated whether angiotensin II (Ang II) may have a place among them. In binding experiments performed to assess the presence of specific receptors in rat peritubular myoid cells (TPMC), binding of (125)I-Ang II to TPMC was saturable in a time-dependent manner. Competition binding experiments performed with Losartan and PD 123319 showed that Losartan was able to inhibit the binding of (125)I-Ang II, whereas PD 123319 was ineffective. Ang II induced a dose-dependent rise in intracellular Ca(2+). Depletion of intracellular calcium stores by thapsygargin resulted in a lower rise of intracellular calcium, and the L-type voltage-operated calcium channel (VOCC-L) blocker verapamil abolished the Ca(2+) influx in rat TPMC. Altogether, these findings indicate that the Ang II-induced increase in [Ca(2+)](i) involves both extracellular influx and Ca(2+) release from intracellular stores. Ang II induced a dose-dependent TPMC contraction, and Losartan and not PD 123319 inhibited the response. Ang II-induced contraction was inhibited by adrenomedullin, previously shown to antagonize endothelin 1-provoked contraction in those cells. Ang II elicited (3)H-thymidine DNA incorporation and proliferation in a dose-dependent manner in TPMC. Losartan and both MAPK inhibitor PD 98059 and tyrosine kinase inhibitor AG18 were able to inhibit Ang II-induced (3)H-thymidine uptake and cell proliferation. In conclusion, the present study documents that angiotensin II, the active mediator of the tissue and circulating renin-angiotensin system present in the mammalian testis, induces contraction, growth and rise in intracellular calcium in rat peritubular myoid cells via angiotensin II type 1 receptors, and suggests that Ang II is involved in the paracrine regulation of the seminiferous tubule function.     

吡格列酮对糖尿病大鼠血管钙化的影响及机制

目的研究吡格列酮对糖尿病大鼠血管钙化的影响及其可能机制。方法将36只SD雄性大鼠随机平均分为6组:对照组、糖尿病组、钙化组、糖尿病+钙化组、钙化+吡格列酮组、糖尿病+钙化+吡格列酮组;建立大鼠血管钙化模型(维生素D3+华法林)和糖尿病模型(链尿佐菌素);并对血管组织进行Von Kossa染色、钙含量和碱性磷酸酶活性检测,qRT-PCR检测mRNA表达,免疫组织化学法检测骨保护素蛋白表达。结果钙化组血管平滑肌细胞及其间质内有大量黑色颗粒沉积;糖尿病+钙化组较糖尿病组和钙化组血管组织钙含量、碱性磷酸酶活性分别升高3.63倍、1.35倍和3.69倍、1.30倍(P<0.05),骨保护素mRNA含量及其蛋白表达降低(P<0.05);糖尿病+钙化+吡格列酮组较糖尿病+钙化组钙含量、碱性磷酸酶活性分别下调13.70%、18.04%(P<0.05),骨保护素mRNA含量及其蛋白表达升高(P<0.05)。结论吡格列酮可以减轻血管钙化程度并上调骨保护素mRNA含量及蛋白表达,骨保护素可能是抑制血管钙化主要因素之一。     

Critical role of Rho-kinase and MEK/ERK pathways for angiotensin II-induced plasminogen activator inhibitor type-1 gene expression

Plasminogen activator inhibitor type-1 (PAI-1) plays an integral role not only in the regulation of fibrinolytic activity but also in the pathogenesis of atherosclerosis and hypertension. We investigated the signaling pathways of angiotensin II (Ang II) leading to PAI-1 gene expression. Ang II increased the PAI-1 mRNA and protein levels in a time- and dose-dependent manner through the Ang II type 1 receptor in vascular smooth muscle cells. PAI-1 gene promoter activity measured by luciferase assay was significantly increased by Ang II. PAI-1 mRNA stability was also increased by Ang II. Ang II-induced PAI-1 mRNA upregulation was inhibited by BAPTA-AM, genistein, and AG1478, suggesting that intracellular calcium, tyrosine kinase, and epidermal growth factor receptor transactivation are involved. Furthermore, PD98059, an inhibitor of extracellular signal-regulated kinase (ERK) kinase (MEK), almost completely suppressed Ang II-induced PAI-1 upregulation. Adenovirus-mediated overexpression of the dominant-negative form of Rho-kinase or Y27632, a Rho-kinase inhibitor, also completely prevented PAI-1 induction by Ang II without affecting Ang II-induced ERK activation. These data suggest that activation of MEK/ERK and Rho-kinase pathways plays a pivotal role in PAI-1 gene upregulation by Ang II. The Rho-kinase pathway may be a novel target to inhibit Ang II signaling, and its inhibition may be useful in the treatment of hypertension as well as atherosclerosis.     

Na+/K+ATPase activity inhibition and isoform-specific translocation of protein kinase C following angiotensin II administration in isolated eel enterocytes

In the eel, angiotensin II (Ang II) has a role at the level of both gill chloride and kidney tubular cells, regulating sodium balance and therefore osmoregulation. The present study extends these findings to another important osmoregulatory organ - the intestine. Enterocytes were obtained from sea-water (SW)-acclimated eels to investigate the role of Ang II on the intestinal Na+/K+ATPase activity, because in SW-acclimated animals the intestine represents an important site of water and NaCl transport from the mucosal to the serosal side. This paper demonstrates that isolated enterocytes stimulated with increasing Ang II concentrations (0.01-100 nM) showed a dose-dependent inhibition of the Na+/K+ATPase activity. The threshold decrease was at 0.01 nM Ang II; it reached a maximum at 10 nM (81.5% inhibition) and did not decrease further with the use of higher hormone doses. These hormonal effects were blocked by a specific competitive antagonist of the AT1 receptor subtype, DuP-753 (100% inhibition at 10 microM), indicating that these effects are mediated by an AT1-like receptor. Isolated enterocytes stimulated with 10 nM Ang II showed a transient increase in intracellular calcium ([Ca2+]i), followed by a lower sustained phase. Removal of extracellular Ca2+ did not reduce the initial transient response and completely abolished the plateau phase. The inhibition of the Na+/K+ATPase activity was dependent on protein kinase C (PKC) activation since PKC antagonists (calphostin C and staurosporine) abolished the inhibitory effect of Ang II, and the PKC activator phorbol 12-myristate 13-acetate reduced transporter activity. Western blot analysis with antibodies to PKC alpha, beta I, beta II, gamma, delta, epsilon, iota, eta and zeta isoforms showed that eel enterocytes expressed the conventional isoforms (alpha and beta I), the novel isoforms (delta and eta) and the atypical isoforms (zeta and iota). Ang II stimulated the translocation from the cytosol to the plasma membrane of PKC alpha, PKC delta and PKC eta isoforms. In conclusion, our results suggest that Ang II modulates intestinal Na+/K+ATPase in SW-acclimated eels via calcium mobilization and PKC activation.     

The positive inotropic effect of angiotensin II: role of endothelin-1 and reactive oxygen species

Many effects believed to be because of angiotensin II (Ang II) are attributable to the action of endothelin (ET)-1, which is released/produced by Ang II. We investigated whether Ang II elicits its positive inotropic effect (PIE) by the action of endogenous ET-1, in addition to the role played by reactive oxygen species (ROS) in this mechanism. Cat cardiomyocytes were used for: (1) sarcomere shortening measurements; (2) ROS measurements by epifluorescence; (3) immunohistochemical staining for preproET-1, BigET-1, and ET-1; and (4) measurement of preproET-1 mRNA by RT-PCR. Cells were exposed to 1 nmol/L Ang II for 15 minutes. This low concentration of Ang II increases sarcomere shortening by 29.2+/-3.7% (P<0.05). This PIE was abrogated by Na+/H+ exchanger or Na+/Ca2+ exchanger reverse mode inhibition. The production of ROS increased in response to Ang II treatment (DeltaROS respect to control: 68+/-15 fluorescence units; P<0.05). The Ang II-induced PIE and ROS production were blocked by the Ang II type 1 receptor blocker losartan, the nonselective ET-1 receptor blocker TAK044, the selective ETA receptor blocker BQ-123, or the ROS scavenger N-(2-mercapto-propionyl)glycine. Exogenous ET-1 (0.4 nmol/L) induced a similar PIE and increase in ROS production to those caused by Ang II. Immunostaining for preproET-1, BigET-1, and ET-1 was positive in cardiomyocytes. The preproET-1 mRNA abundance increased from 100+/-4.6% in control to 241.9+/-39.9% in Ang II-treated cells (P<0.05). We conclude that the PIE after exposure to 1 nmol/L Ang II is due to endogenous ET-1 acting through the ETA receptor and triggering ROS production, Na+/H+ exchanger stimulation, and Na+/Ca2+ exchanger reverse mode activation.     

Angiotensin II stimulation of Na+/K+ATPase activity and cell growth by calcium-independent pathway in MCF-7 breast cancer cells

Here we demonstrated, by RT-PCR analysis, the expression of both angiotensin II (Ang II) receptor subtypes, AT1 and AT2, in a breast cancer epithelial cell line, MCF-7. Ang II was not able to affect the intracellular Ca2+ concentration in Fura-2 loaded cells suggesting that AT1-mediated phospholipid hydrolysis is not involved in its intracellular transduction pathway. Ang II modulated the activity of the Na+/K+ATPase in a dose- and time-dependent manner and was mitogenic, with a dose-dependent (1-1000 nM) proliferative effect and a maximal response at 100 nM. Both Na+/K+ATPase activation and stimulation of proliferation were mediated by binding of Ang II to AT1, as the effects were completely blocked by DuP 753, a specific AT1 antagonist. CGP 42112, an AT2 antagonist, did not affect Ang II actions. The main conclusion of this study is that Ang II exerts its effects on cell proliferation and Na+/K+ATPase in breast cancer epithelial cells, MCF-7, via AT1 activation independently of the Ca(2+) signalling mechanism.     

依那普利和氯沙坦对心肌梗死大鼠心室重构影响的比较

目的 研究比较血管紧张素转换酶抑制剂(ACEI)及选择性血管紧张素Ⅱ受体拮抗剂(AT_1受体拮抗剂)对心肌梗死大鼠心室重构的影响.方法Wistar大鼠冠脉结扎制成心肌梗死模型,24小时后随机分为依那普利(enalapril)治疗组,氯沙坦(losartan)治疗组,安慰剂(placebo)组,治疗6周.假手术组为对照.6周后测定体重、梗死区重量、心脏重量/体重、血浆及心肌的血管紧张素Ⅱ(AngⅡ)浓度、心肌胶原含量及血浆内皮素浓度.结果enalapril及losartan治疗组中心脏重量/体重及心脏胶原含量高于假手术组,而低于安慰剂组.在三个梗死组中,梗死区的重量无显著差异.enalapril治疗组血浆AngⅡ浓度降低,而losartan治疗组中血浆AngⅡ浓度升高.梗死后安慰剂组心脏局部AnsⅡ浓度明显高于假手术组、enalanril和losartan治疗组.enalanril及losartan可降低血浆ET-1浓度.结论血管紧张素转换酶抑制剂及血管紧张素Ⅱ受体拮抗剂对阻止心室重构的发展具有相同作用.     

糖尿病大鼠钙化血管中Msx2和Wnt3a的表达

目的 研究糖尿病对血管钙化的影响及Msx2和wnt3a基因在钙化血管中的表达变化.方法 将48只雄性wistar大鼠随机分为四组:维生素D3和尼古丁诱导的单纯血管钙化组(n=12)、链脲佐菌素诱导的单纯糖尿病组(n=12)、链脲佐菌素联合维生素D3和尼古丁诱导的糖尿病合并血管钙化组(n=12)和正常雄性Wistar大鼠为正常对照组(n=12).测定大鼠血糖、血清胰岛素、总胆固醇和甘油三酯水平,以血管von Kossa染色、血管钙含量和碱性磷酸酶活性作为判断血管钙化程度的指标,测定大鼠血管中Msx2和wnt3a mRNA 的表达.结果 与正常对照组相比,单纯血管钙化组大鼠血管中Msx2和wnt3a mRNA 相对表达量有所升高(P<0.05),但血管钙含量和碱性磷酸酶活性无明显变化.与正常对照组及单纯血管钙化组相比,糖尿病合并血管钙化组大鼠的血管中,可见沿中膜弹力层内广泛分布的钙盐沉积,大鼠血管中钙含量和碱性磷酸酶活性以及血管内Msx2和wnt3amRNA 相对表达量明显增高(P<0.05).结论 糖尿病可以明显加速血管钙化的发生和发展.在糖尿病大鼠钙化血管中,骨形成过程中的转录因子Msx2和Wnt3a表达增高,提示血管钙化是一个类似于骨形成的过程,Msx2和Wnt3a 参与血管钙化病变的发生.     

Issue-specific Regulation of Angiotensin-Converting Enzyme by Angiotensin II and Losartan in the Rat

Angiotensin II (Ang II) may regulate the release of components of the renin-angiotensin system in a tissue-specific manner. In order to study: (1) the effect of Ang II on gene expression and tissue levels of angiotensin-converting enzyme (ACE), and (2) the mechanism of the possible Ang 11 effect, we treated normal rats with Ang II and Losartan, an angiotensin AT,-receptor antagonist. Forty normal rats received Ang II (n = 20) at a rate of 200ngkg¹ min¹ or 0.9% NaCl (n = 20) subcutaneously for 3 days using osmotic Alzet minipumps. Ten rats in both groups received Losartan (15 mg kg^-1 day^-1) in their drinking water, while the rest received tap water. ACE activity and mRNA levels were measured from pulmonary, cardiac, and renal tissue. Ang II treatment resulted in significant increases in blood pressure and heart weight, as well as an increase in plasma Ang II concentration and a decrease in plasma renin activity. Simultaneous treatment with Losartan reduced the Ang II-induced effects on blood pressure and heart weight, and attenuated the Ang II-induced decrease in plasma renin activity. Pulmonary ACE activity and mRNA levels decreased during Ang II treatment, and these effects were not modified by simultaneous treatment with Losartan. Cardiac and kidney ACE activities and mRNA levels did not change significantly during Ang II treatment, but Losartan increased cardiac ACE activity (and decreased pulmonary ACE activity). The data indicate that Ang II regulates gene expression and activity of ACE in a tissue-specific manner in the rat, an effect probably involving angiotensin receptor subtype(s) different from the AT₁,-receptor.     

Effects of adrenomedullin on vascular calcification in rats

Objective The aim of the present study was to investigate the effect of adrenomedullin (ADM) on vascular calcification. Methods The vascular calcification model was established in rats (VND group) by using vitamin D₃ (300000IU/kg) and nicotine (25mg/kg, two doses). The effect of liposome-encapsulated ADM was observed. Vascular calcium content, alkaline phosphatase (ALP) activity, ADM in aortic tissue and plasma, binding ability of ¹²⁵I-ADM for ADM receptor on vascular plasma membrane and content of cAMP in vessels were measured. Results Compared with control rats, the aortic calcium content and vascular ALP activity in rats of the VDN group was obviously increased; in addition ADM concentrations in plasma and vessels of rats in VDN group were increased. But the maximum binding sites of ¹²⁵I-ADM for ADM receptor (Bmax) on vascular plasma membrane in rats of VDN group were significantly decreased compared with control rats. The affinity of ¹²⁵I-ADM for the ADM receptor was reduced, as shown by the Kd value and vascular cAMP content being reduced in rats of the VDN group compared to the control group. The in vitro response of isolated vessels to ADM incubation was weakened. Administration of empty liposome had no effect on vascular calcification. But administration of ADM significantly decreased vascular calcium content and ALP activity. The Bmax of ¹²⁵I-ADM for ADM receptors on vascular plasma membrane increased by 17.7% (p<0.01), and the value of Kd decreased by 36.2% (P<0.01) in rats treated with ADM as compared with rats of the VDN group. In addition, the vascular cAMP content and the response to ADM in isolated aorta were markedly increased. Conclusion Vascular calcification induced an alteration of the vascular ADM-ADM receptor-cAMP pathway. Treatment with exogenous ADM inhibited vascular calcification by improving the vascular ADM-ADM receptor-cAMP pathway.     

Gender-dependent difference in cell calcium handling in VSMC isolated from SHR: the effect of angiotensin II

OBJECTIVE: To investigate gender-dependent difference in the free cytosolic calcium concentration ([Ca2+ ]i ) response to angiotensin II (Ang II) in vascular smooth muscle cells (VSMC) isolated from spontaneously hypertensive rats (SHR). To further evaluate this gender-dependent difference by studying the role of thapsigargin-sensitive intracellular calcium stores and calcium influx in VSMC isolated from male and female SHR. DESIGN AND METHODS: Confluent primary cultures of VSMC isolated from male (n = 14) and female (n = 14) SHR aged 10 weeks were used in this study. [Ca2+ ]i was measured by image analysis of single myocytes loaded with Fura-2. [Ca2+ ]i response of VSMC to Ang II was measured in the presence and absence of extracellular Ca2+, to evaluate the influence of Ca2+ influx. To characterize inositol triphosphate (IP3 )-sensitive sarcoplasmic reticulum calcium stores, thapsigargin-sensitive calcium stores were measured in VSMC isolated from SHR of both genders. RESULTS: VSMC isolated from male SHR were characterized by an augmented [Ca2+ ]i response to angiotensin II in comparison with VSMC isolated from female SHR. Surprisingly, the thapsigargin-stimulated [Ca2+ ]i rise was found to be significantly greater in VSMC isolated from female SHR compared with VSMC isolated from male SHR. On the other hand, the gender-dependent difference in [Ca2+ ]i response to angiotensin II was abolished in the absence of extracellular calcium. CONCLUSIONS: We demonstrated in VSMC isolated from SHR of both genders that a greater [Ca2+ ]i response to angiotensin II in male than female VSMC is dependent on Ca2+ influx.