Angiotensin II enhances the afferent arteriolar response to adenosine through increases in cytosolic calcium

Aims: Angiotensin II (Ang II) is a strong renal vasoconstrictor and modulates the tubuloglomerular feedback (TGF). We hypothesized that Ang II at low concentrations enhances the vasoconstrictor effect of adenosine (Ado), the mediator of TGF. Methods: Afferent arterioles of mice were isolated and perfused, and both isotonic contractions and cytosolic calcium transients were measured. Results: Bolus application of Ang II (10^?12 and 10^?10 m ) induced negligible vasoconstrictions, while Ang II at 10^?8 m reduced diameters by 35%. Ang II at 10^?12, 10^?10 and 10^?8 m clearly enhanced the arteriolar response to cumulative applications of Ado (10^?11 to 10^?4 m ). Ado application increased the cytosolic calcium concentrations in the vascular smooth muscle, which were higher at 10^?5 m than at 10^?8 m . Ang II (10^?11 to 10^?6 m ) also induced concentration‐dependent calcium transients, which were attenuated by AT₁ receptor inhibition. Simultaneously applied Ang II (10^?10 m ) additively enhanced the calcium transients induced by 10^?8 and 10^?5 m Ado. The transients were partly inhibited by AT₁ or A₁ receptor antagonists, but not significantly by A₂ receptor antagonists. Conclusion: A low dose of Ang II enhances Ado‐induced constrictions, partly via AT₁ receptor‐mediated calcium increase. Ado increases intracellular calcium by acting on A₁ but not A₂ receptors. The potentiating effect of Ang II on Ado‐induced arteriolar vasoconstrictions may involve calcium sensitization of the contractile machinery, as Ang II only additively increased cytosolic calcium concentrations, while its effect on the arteriolar constriction was more than additive. The potentiating effect of Ang II might contribute to the resetting of TGF.     

Angiotensin II induced contraction of rat and human small intestinal wall musculature in vitro

Background: Angiotensin II (Ang II) is a well‐known activator of smooth muscle in the vasculature but has been little explored with regard to intestinal wall muscular activity. This study investigates pharmacological properties of Ang II and expression of its receptors in small‐intestinal smooth muscle from rats and humans. Methods: Isometric recordings were performed in vitro on small intestinal longitudinal muscle strips. Protein expressions of Ang II typ 1 (AT1R) and typ 2 (AT2R) receptors were assessed by Western blot. Results: Ang II elicited concentration‐dependent contractions of rat jejunal and ileal muscle preparations. The concentration–response curve (rat ileum, EC₅₀: 1.5 ± 0.9 × 10^?8 m ) was shifted to the right by the AT1R receptor antagonist losartan (10^?7 m ) but was unaffected by the AT2R antagonist PD123319 (10^?7 m ) as well as by the adrenolytic guanethidine (3 × 10^?6 m ) and the anticholinergic atropine (10^?6 m ). Human duodenal, jejunal and ileal longitudinal muscle preparations all contracted concentration‐dependently in response to Ang II. The concentration–response curve (human jejunum, EC₅₀: 1.5 ± 0.8 × 10^?8 m ) was shifted to the right by losartan (10^?7 m ) but was unaffected by PD123319 (10^?7 m ). Both AT1R and AT2R were detected in all segments of the rat small intestinal wall musculature, whereas only AT1R was readily detectable in the human samples. Conclusion: Ang II elicits contractions of small‐intestinal longitudinal muscle preparations from the small intestine of rats and man. The pharmacological pattern and protein expression analyses indicate mediation via the AT1R.     

Angiotensin II angiogenic effect in vivo involves vascular endothelial growth factor- and inflammation-related pathways

Although accumulating lines of evidence indicate the proangiogenic role of angiotensin II (Ang II), little is known about the molecular mechanisms associated with such an effect. This study aimed to identify molecular events involved in Ang II-induced angiogenesis in the Matrigel model in mice. C57Bl/6 female mice received a subcutaneous injection of either Matrigel or Matrigel with Ang II (10(-7) M) alone, with Ang II and an AT1 receptor antagonist (candesartan, 10(-6) M), or with Ang II and AT2 receptor antagonist (PD123319, 10(-6) M). After 14 days, angiogenesis was assessed in the Matrigel-plug by histological evaluation and cellular counting. Ang II increased by 1.9-fold the number of cells within the Matrigel (p < 0.01 versus control). Immunohistological analysis revealed the presence of macrophages, endothelial and smooth muscle cells, and the development of vascular-like structure. Such an angiogenic effect was associated with an increase in vascular endothelial growth factor (VEGF) (1.5-fold, p < 0.01), endothelial nitric oxide (eNOS) (1.7-fold, p < 0.01), and cyclooxygenase-2 (1.4-fold, p < 0.05) protein levels measured by Western blotting. Conversely, Ang II treatment did not affect MMP-9 and MMP-2 activity, assessed by zymography. Blockade of AT1 receptor completely prevented the Ang II-induced angiogenesis and protein regulations, whereas that of AT2 was ineffective. Administration of VEGF neutralizing antibody (2.5 microg ip twice a week) and cyclooxygenase-2 selective inhibitor (nimesulide, 30 mg/L) also hampered Ang II proangiogenic effect. In addition, Ang II-induced cell ingrowth was impaired by treatment with nitric oxide synthase inhibitor (L-NAME, 10 mg/kg/day) and in eNOS-deficient mice. Therefore, in an in vivo model, Ang II induced angiogenesis through AT1 receptor, which involved activation of VEGF/eNOS-related pathway and of the inflammatory process.     

AT1 receptor-mediated angiotensin II activation and chemotaxis of T lymphocytes

Angiotensin II (Ang II), a central renin–angiotensin system (RAS) effector molecule, and its receptors, AT₁ and AT₂, have been shown to be involved in the inflammatory aspects of different diseases, however the cellular mechanisms underlying the regulation of immunity are not fully understood. In this work, using spleen-derived CD4⁺ and CD8⁺ T lymphocytes activated in vitro, we tested the influence of Ang II on different aspects of the T cell function, such as activation and adhesion/transmigration through endothelial basal membrane proteins. The addition of 10⁻⁸ M Ang II did not change any of the parameters evaluated. However, 10⁻⁶ M losartan, an AT₁ receptor antagonist: (i) reduced the percentage of CD25⁺ and CD69⁺ cells of both subsets; (ii) inhibited adhesion of these cells to fibronectin or laminin by 53% or 76%, respectively and (iii) significantly reduced transmigration through fibronectin or laminin by 57% or 43%, respectively. In addition, 10⁻⁶ M captopril, an angiotensin-converting enzyme inhibitor had similar effects to Ang II, however its effects were reverted by exogenous Ang II (10⁻⁸ M). None of these responses was modified by 10⁻⁷ M PD123319, an AT₂ antagonist. These data reinforce the notion of endogenous production of Ang II by T cells, which is important for T cell activation, and adhesion/transmigration induced on interaction with basal membrane proteins, possibly involving AT₁ receptor activation. Moreover, AT₁ receptor expression is 10-fold higher in activated T lymphocytes compared with naive cells, but AT₂ receptor expression did not change after T cell receptor triggering.     

Proliferation of human lens epithelial cells (HLE-B3) is inhibited by blocking of voltage-gated calcium channels

Calcium, as an integral part of a large number of cellular regulatory pathways, is selective in the control of specific cell functions like the start of G1 phase in cell cycle. Cell proliferation has been suggested to depend on increasing intracellular calcium levels. A major regulatory pathway for intracellular calcium is the calcium influx into the cell via voltage-gated calcium channels. T-type and L-type calcium channels are substantially present in human lens epithelial cell (hLEC), and total calcium currents are inhibited by mibefradil. Here, the hypothesis was tested if calcium influx via Ca_v channels regulates proliferation in epithelial cells. Cell proliferation was determined by cell culture assays using the L- and T-type Ca_v channel blockers mibefradil and verapamil as modulators for calcium influx. Calcium influx was investigated using the Manganese quench technique. Western blot experiments were accomplished under standard conditions using antibodies against MAPK 3. Mibefradil as well as verapamil impaired cell proliferation, but in different concentration ranges. Furthermore, the activation of MAPK 3 was reduced by both antagonists. Calcium influx was also reduced in the presence of both blockers. We conclude that the transmembrane influx of Ca²⁺ through Ca_v channels contributes to the regulation of hLEC proliferation, identifying Ca_v channel blockers as potential therapeutic substances in ocular diseases.     

Modulation of low-threshold T-type calcium channels by the five muscarinic receptor subtypes in NIH 3T3 cells

The modulation of a transient T-type calcium current by the five muscarinic receptor subtypes, stably expressed in NIH 3T3 cells, was studied with the whole-cell patch-clamp technique. Voltage-step depolarizations applied to the NIH 3T3 cells revealed a low-voltage-activated (LVA) T-type calcium current that was inhibited by Ni2+ and unaffected by omega-conotoxin GVIA. In cells transfected with the m3 and m5 muscarinic receptors, application of acetylcholine (ACh) resulted in a pertussis-toxin-insensitive increase in peak T-type calcium current amplitude. The m3-induced atropine-sensitive increase in current amplitude was accompanied by a shift in the voltage dependence of activation to more hyperpolarized potentials. The increase in peak T-type calcium current amplitude and the shift in voltage dependence was mimicked by incubation with 500 microM 8-bromo-cAMP. Conversely, T-type calcium current amplitudes were reduced by incubation with 10 microM RpcAMPS, an inhibitor of cAMP-dependent protein kinase (PKA). Preincubation with 500 microM 8-bromo-cAMP or with 10 microM RpcAMPS abolished the increase in T-type calcium current amplitude previously noted on stimulation of the m3 muscarinic receptor by ACh. Application of ACh to NIH 3T3 cells stably transformed with the m1 muscarinic receptor resulted in no discernable change in T-type calcium current amplitude. However, on pre-incubation of the cells with calphostin C, an inhibitor of protein kinase C (PKC), application of ACh to the cells now resulted in a robust increase in T-type calcium current amplitude. Application of 500 nM PDBu, an activator of PKC, reduced the T-type calcium current amplitude. No significant changes in T-type calcium currents were observed on application of ACh to cells stably transfected with the m2 or m4 muscarinic receptors. However, after pre-incubation with forskolin, the m2 muscarinic receptor induced a decrease in T-type calcium current amplitude. Stimulation of the ml, m3 and m5 muscarinic receptors in the NIH 3T3 cell resulted in dose-dependent increases in the concentration of intracellular cAMP in comparison to control as determined by cAMP immunoassay. Conversely, stimulation of the m2 and m4 muscarinic receptors by carbachol resulted in a dose-dependent reduction in intracellular concentrations of cAMP, as compared with control basal levels. It is concluded that the m3 and m5 muscarinic receptors enhance T-type calcium channel activity. At least in the case of the m3 muscarinic receptor, the increased T-type channel activity appeared to be mediated via increased cAMP levels and subsequent activation of PKA. The lack of effect of the ml muscarinic receptor on the T-type calcium channel was probably due to the opposing actions of concomitant activation of both PKC and PKA. The physiological significance of these findings is discussed.     

T-type calcium channels blockers as new tools in cancer therapies

T-type calcium channels are involved in a multitude of cellular processes, both physiological and pathological, including cancer. T-type channels are also often aberrantly expressed in different human cancers and participate in the regulation of cell cycle progression, proliferation, migration, and survival. Here, we review the recent literature and discuss the controversies, supporting the role of T-type Ca²⁺ channels in cancer cells and the proposed use of channels blockers as anticancer agents. A growing number of reports show that pharmacological inhibition or RNAi-mediated downregulation of T-type channels leads to inhibition of cancer cell proliferation and increased cancer cell death. In addition to a single agent activity, experimental results demonstrate that T-type channel blockers enhance the anticancer effects of conventional radio- and chemotherapy. At present, the detailed biological mechanism(s) underlying the anticancer activity of these channel blockers is not fully understood. Recent findings and ideas summarized here identify T-type Ca²⁺ channels as a molecular target for anticancer therapy and offer new directions for the design of novel therapeutic strategies employing channels blockers. Physiological relevance: T-type calcium channels are often aberrantly expressed or deregulated in cancer cells, supporting their proliferation, survival, and resistance to treatment; therefore, T-type Ca²⁺ channels could be attractive molecular targets for anticancer therapy.     

AT1 receptor-mediated angiotensin II activation and chemotaxis of T lymphocytes

Angiotensin II (Ang II), a central renin-angiotensin system (RAS) effector molecule, and its receptors, AT(1) and AT(2), have been shown to be involved in the inflammatory aspects of different diseases, however the cellular mechanisms underlying the regulation of immunity are not fully understood. In this work, using spleen-derived CD4(+) and CD8(+) T lymphocytes activated in vitro, we tested the influence of Ang II on different aspects of the T cell function, such as activation and adhesion/transmigration through endothelial basal membrane proteins. The addition of 10(-8)M Ang II did not change any of the parameters evaluated. However, 10(-6)M losartan, an AT(1) receptor antagonist: (i) reduced the percentage of CD25(+) and CD69(+) cells of both subsets; (ii) inhibited adhesion of these cells to fibronectin or laminin by 53% or 76%, respectively and (iii) significantly reduced transmigration through fibronectin or laminin by 57% or 43%, respectively. In addition, 10(-6)M captopril, an angiotensin-converting enzyme inhibitor had similar effects to Ang II, however its effects were reverted by exogenous Ang II (10(-8)M). None of these responses was modified by 10(-7)M PD123319, an AT(2) antagonist. These data reinforce the notion of endogenous production of Ang II by T cells, which is important for T cell activation, and adhesion/transmigration induced on interaction with basal membrane proteins, possibly involving AT(1) receptor activation. Moreover, AT(1) receptor expression is 10-fold higher in activated T lymphocytes compared with naive cells, but AT(2) receptor expression did not change after T cell receptor triggering.     

Calcium influx through the TRPV1 channel of endothelial cells (ECs) correlates with a stronger adhesion between monocytes and ECs

PurposeAtherosclerosis is thought to be initiated by the transendothelial migration of monocytes. In the early stage of this process, the adhesion of monocytes to endothelial cells is supported by an increase in the intracellular concentration of calcium ion ([Ca²⁺]i) in endothelial cells. However, the main source of Ca²⁺ has been unclear. In this study, the changes in ionic transmittance and [Ca²⁺]i due to the adhesion of monocytes were continuously measured by an electrophysiological technique and fluorescent imaging. Especially, we focused on transient receptor potential vanilloid channel 1 (TRPV1) as a Ca²+ channel that could influence the adhesion of monocytes.Material and methodsWhole-cell current was continuously recorded in human umbilical vein endothelial cells (HUVECs) by a patch electrode.ResultsThe adhesion of monocytes (THP-1) induced a transient inward current in HUVECs, as well as an elevation of [Ca²+]i. This inward element was abolished by the application of 100nM SB366,791, a selective antagonist of TRPV1 channel. Furthermore, SB366,791 significantly decreased the number of THP-1 cells that adhered to HUVECs (control: 231 ± 38, SB366,791: 96 ± 16 cells/mm2).ConclusionThese results suggest that an inward calcium current via the TRPV1 channels of endothelial cells correlates with a stronger adhesion between monocytes and endothelial cells.     

血管活性物质对人内皮细胞C-型利钠利尿肽合成和释放的影响

目的:在培养的人内皮细胞上观察内皮素(ET)、血管紧张素Ⅱ(AngⅡ)和同型半胱氨酸(Hcy)对C-型利钠利尿肽(CNP)生成和释放的影响,以探讨CNP生成和释放的机制。方法:人内皮细胞培养;CNP放免测定。结果:ET、AngⅡ均可刺激内皮细胞CNP的生成,10^-9、10^-8、10^-7mol/L的ET和AngⅡ分别使细胞CNP含量较对照组高1%(P＞0.05),49%(P＜0.05),117%(P＜0.01)和137%(P＜0.01),165%(P＜0.01),201%(P＜0.01),大剂量ET、AngⅡ可刺激CNP的释放。Hcy对内皮细胞CNP的生成没有影响,但10 ^-9、10^-8、10^-7mol/LHcy可使其释放增加17%(P＞0.05),84%(P＜0.01)和555%(P＜0.01)。结论:ET、Ang和Hcy可调节CNP的释放和/或生成。     

Effects of motilin on intracellular free calcium in cultured smooth muscle cells from the antrum of neonatal rats

Aim: The aim of this study was to determine the effects of motilin on [Ca²⁺]_i regulation and its underlying molecular mechanism in cultured antral smooth muscle cells (ASMCs). Methods: Antral cells were isolated and cultured from neonatal rats, and then the [Ca²⁺]_i in these cells was evaluated by calcium fluorescent probe Fluo-3/AM on a laser scanning confocal microscope. Results: We show that motilin dose-dependently increased [Ca²⁺]_i concentration in cultured ASMCs. Pre-incubation of cells with either the calcium antagonist verapamil (10⁻⁵ mol L⁻¹) or the calcium chelator Egtazic (EGTA, 0.1 mmol L⁻¹) significantly suppressed motilin (10⁻⁶ mol L⁻¹) induced [Ca²⁺]_i increase as indicated by fluorescent intensity. Interestingly, after mixing with the non-selective intracellular calcium release blocker TMB-8 (10⁻⁵ mol L⁻¹), guanosine triphosphate regulatory protein antagonist NEM (10⁻⁵ mol L⁻¹), phospholipase C (PLC) inhibitor compound 48/80 (1.2 μg mL⁻¹) and ryanodine at high concentration (10⁻⁵ mol L⁻¹), the motilin-induced [Ca²⁺]_i increase was only partially blocked. The protein kinase C inhibitor d -sphingosine (10⁻⁶ mol L⁻¹), however, did not show any inhibitory effect on motilin-induced [Ca²⁺]_i elevation. Conclusions: Our study suggests that motilin-stimulated [Ca²⁺]_i elevation in ASMCs is probably due to sustained extracellular Ca²⁺ influx and Ca²⁺ release from Ca²⁺ stores via inositol tris-phosphate receptors and ryanodine receptors. Specifically, motilin-induced [Ca²⁺]_i release is accompanied with guanosine triphosphate-binding protein-coupled receptor–PLC–inositol tris-phosphate signalling cascades.     

Local renin-angiotensin system regulates hypoxia-induced vascular endothelial growth factor synthesis in mesenchymal stem cells

The use of mesenchymal stem cell (MSC) transplantation for ischemic heart disease has been reported for several years. The main mechanisms responsible for the efficacy of this technique include the differentiation of MSCs into cardiomyocytes and endothelial cells, as well as paracrine effects. However, the differentiation rates of MSCs are very low, and the differentiated cells are not mature. In addition, MSCs undergo massive cell death within a few days after transplantation to the ischemic myocardium. Paracrine effects may thus play a major role in MSCs transplantation. Angiotensin II (Ang II) is known to be produced locally in the ischemic myocardium, but the effects of hypoxia on the local renin-angiotensin system (RAS) in MSCs, and the role of the RAS in hypoxia-induced vascular endothelial growth factor (VEGF) secretion remain unknown. In this study, we demonstrated that hypoxia stimulated the local RAS in MSCs, while pretreatment with the Ang II type 1 (AT1) receptor antagonist losartan reduced hypoxia-induced hypoxia-inducible factor 1α (HIF-1α) and VEGF production. The ERK1/2 inhibitor U0126 and the Akt inhibitor LY294002 also inhibited hypoxia-induced HIF-1α and VEGF production. Overall, these results indicate that the local RAS in MSCs regulates hypoxia-induced VEGF production through ERK1/2, Akt and HIF-1α pathways via the AT1 receptor.     

Phenotype,and migration properties of three major subsets of tissue homing T cells in sheep

T cells show a bias in their migration pathways: some T cells preferentially migrate to peripheral lymph nodes (LN), some to mucosal tissues, and some to peripheral tissues such as skin. These recirculation pathways were examined in sheep by collecting lymph draining into and out of peripheral and intestinal LN, and using fluorescent dyes to trace the recirculation of the lymph cells. Monoclonal antibodies (mAb) to α4, β1, and β7 integrins, and L-selectin, were used to define three major populations of recirculating T cells. Naive-type T cells (L-selectin⁺, α4β1^lo β7^lo) migrated preferentially through peripheral LN. Two memory populations could be defined: α4β1^hi β7⁻ and α4β7^hiβ1^lo. α4β1^hi β7⁻ T cells were present in lymph draining from the skin. T cells migrating preferentially through intestinal LN were α4β7^hi β1^lo. Consistent with this migration pattern, the endothelial receptor for α4β7, mucosal addressin cell adhesion molecule-1 (MAdCAM-1) was detected on high endothelial venules within intestinal LN and Peyer's patches, but only weakly on high endothelial venules within peripheral LN. Thus, there are at least three easily definable subsets of T cells, based on integrin expression, which show distinct migration preferences.     

血管紧张素II对巨噬细胞(THP-1细胞)凝集素样氧化低密度脂蛋白受体表达的影响

目的:研究AngII对人单核/巨噬细胞(THP-1细胞)凝集素样氧化低密度脂蛋白受体(LOX-1)蛋白表达和基因转录的影响,从细胞蛋白、分子水平探讨AngII和巨噬细胞LOX-1相互之间的关系,以进一步了解两者在动脉粥样硬化中的地位。方法:将不同浓度AngII(1×10^-9-1×10^-5mol/L)与经0.1μmol/L佛波酯(PMA)诱导分化后的THP-1细胞共孵育24h,以及将1×10^-6mol/L浓度的AngII与诱导分化后的THP-1细胞作用不同时间0、3、6、12、24、48h后,用细胞酶联免疫法和半定量RT-PCR分别检测LOX-1蛋白和mRNA表达的情况。结果:未经诱导的THP-1细胞不表达LOX-1mRNA;而经PMA诱导后,THP-1细胞停止增殖,由单核细胞分化成为巨噬细胞,并表达LOX-1mRNA。不同浓度的AngII作用诱导分化后的THP-1细胞24h,细胞LOX-1蛋白和mRNA的表达呈浓度依赖性显著增加。同一浓度的AngII作用THP-1细胞,可呈时间依赖性诱导LOX-1蛋白和mRNA表达,其趋势是3h左右开始增加,24h左右至最高峰,之后逐渐减低。结论:经PMA诱导分化后的THP-1细胞表达LOX-1;AngII能明显增强分化后的THP-1细胞表达LOX-1蛋白和mRNA,并呈浓度和时间依赖性。AngII这种作用可能是促进动脉粥样硬化发生、发展的机制之一。     

Mibefradil reduces blood glucose concentration in db/db mice

OBJECTIVE:

Numerous recent studies suggest that abnormal intracellular calcium concentration ([Ca²⁺]_i) is a common defect in diabetic animal models and patients. Abnormal calcium handling is an important mechanism in the defective pancreatic β-cell function in type 2 diabetes. T-type Ca²⁺ channel antagonists lower blood glucose in type 2 diabetes, but the mechanism remains unknown.

METHODS:

We examined the effect of the Ca²⁺ channel antagonist mibefradil on blood glucose in male db/db mice and phenotypically normal heterozygous mice by intraperitoneal injection.

RESULTS:

Mibefradil (15 mg/kg, i.p., b.i.d.) caused a profound reduction of fasting blood glucose from 430.92±20.46 mg/dl to 285.20±5.74 mg/dl in three days. The hypoglycemic effect of mibefradil was reproduced by NNC 55-0396, a compound structurally similar to mibefradil but more selective for T-type Ca²⁺ channels, but not by the specific L-type Ca²⁺ channel blocker nicardipine. Mibefradil did not show such hypoglycemic effects in heterozygous animals. In addition, triglycerides, basal insulin and food intake were significantly decreased by mibefradil treatment in the db/db mice but not in the controls. Western blot analysis, immunohistochemistry and immunofluorescence staining showed a significantly increased expression of T-type Ca²⁺ channel α-subunits Cav3.1 and Cav3.2 in liver and brain tissues from db/db mice compared to those from heterozygous animals.

CONCLUSIONS:

Collectively, these results suggest that T-type Ca²⁺ channels are potential therapeutic targets for antidiabetic drugs.     

AngⅡ诱导血管内皮细胞衰老及凋亡相关基因的表达

目的： 探讨血管紧张素Ⅱ（AngⅡ）诱导人脐静脉内皮细胞(HUVECs)衰老及凋亡相关基因Bcl-2、Bax的表达。方法: 体外培养人脐静脉内皮细胞, 采用四甲基偶氮唑蓝比色法测定内皮细胞存活率，用AngⅡ(10^-6mol/L)及valsartan（AngⅡ1型受体特异性拮抗剂）干预,分为实验对照组、AngⅡ诱导组及valsartan组，采用β-半乳糖苷酶染色和流式细胞术鉴定细胞衰老，通过Hoechst33258荧光染色观察细胞形态学变化，并利用免疫细胞化学染色法、RT-PCR法和Western blotting分析各组细胞凋亡相关基因Bcl-2、Bax mRNA及蛋白的表达水平。结果: 与对照组相比，10^-6mol/L AngⅡ诱导组存活的细胞数为对照组的（81.90±0.04)%; (80.10±6.81)%的细胞呈现β-半乳糖苷酶阳性染色。流式细胞仪检测细胞周期停滞于G₀-G₁［(91.36±6.45)%］，证实细胞衰老；荧光显微镜可见明显的细胞凋亡［(31.84±2.86)%］。与AngⅡ诱导组相比，valsartan组Bcl-2mRNA及蛋白表达水平明显增高(P<0.05), Bax mRNA及蛋白表达水平降低（P<0.05）。结论: AngⅡ可诱导体外培养的HUVECs老化。经AngⅡ诱导的衰老HUVECs发生凋亡，提示细胞凋亡参与了AngⅡ诱导HUVECs细胞的衰老过程。AngⅡ诱导血管内皮细胞衰老的分子机制之一可能与Bcl-2、Bax mRNA及蛋白表达的失衡有关。缬沙坦对血管内皮细胞衰老有一定保护作用。     

PI3K/AKT signaling pathway plays a role in enhancement of eNOS activity by recombinant human angiotensin converting enzyme 2 in human umbilical vein endothelial cells

The aim of this study was to investigate the effect of PI3K/AKT signaling pathway in the activity of recombinant human angiotensin converting enzyme 2 (rhACE2) promoted the activity of endothelial nitric oxide synthase (eNOS). The human umbilical vein endothelial cells (HUVEC) were cultured in vitro. Then treated with Ang II (1×10^-6 mol/L) for 24 h. The rhACE2 (100 μmol/L) was added and incubated for 5, 10, 15, 30, 60 min respectively which was based on Ang II intervention. The effect of rhACE2 on phosphorylation eNOS level was also observed in the presence of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (10 μmol/L) (PI3K/AKT inhibitors). Griess reagent method was applied to measure NO contents in cell culture supernatant, RT-PCR to detect the expression of eNOSmRNA in HUVEC, and Western blot to detect the expression of eNOS and phosphorylated eNOS. In Ang II intervention group, NO contents were significantly lower than control group (P < 0.05). Through rhACE2 treatment, the NO contents in cell culture medium and the expression level of phosphorylated eNOS were significantly higher than in Ang II intervention group (P < 0.05), but eNOSmRNA and non-phosphorylated eNOS protein expression level showed no significant difference (P > 0.05). After HUVEC was intervened by PI3K/AKT pathway inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, the expression level of phosphorylated eNOS was significantly lower than that in the rhACE2 30 min treatment group (P < 0.05). rhACE2 may reduce the activity of Ang II inhibited endothelial cell eNOS, which can be blocked by PI3K/AKT pathway inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, suggesting PI3K/AKT signaling pathway plays an important role in rhACE2’s promotion of the activity of endothelial cell eNOS.     

Decline of the calcium response on successive stimulation of a rat brain endothelial cell P2U purinoceptor

A microfluorimetric method using Fura-2 as calcium indicator was used to study the mechanism of desensitization of the calcium response evoked by activation of a brain endothelial cell P_2U receptor. The study was mainly carried out on an immortalized rat brain endothelial cell line (RBE4), with some additional experiments on primary cultured rat brain microvascular endothelial cells. As previously described ( 17 ), ATP (100 μM , 20 s) caused a transient increase in intracellular calcium levels ([Ca²⁺]_i). This effect was dependent on the rate of filling of intracellular calcium stores, since a large inhibition of the ATP-mediated response was seen in the presence of cyclopiazonic acid, an inhibitor of the store Ca²⁺-ATPase. Application of repeated pulses of extracellular ATP led to a desensitization of the response, as measured by a decline in the release of intracellular calcium ( 17 ). This desensitization was partially reversed after 300 s of incubation in agonist-free medium. Extracellular phosphorylation of the purinergic receptor appeared not to be involved in the desensitization process, since a similar rate of desensitization was obtained with the non-hydrolysable ATP analogue ATP gammaS. Oxidation of the purinergic receptor cannot account for the desensitization, since the decline of the ATP-mediated response was unchanged in the presence of 3 m M dithiothreitol. In the presence of ATP together with UTP, two equally potent activators of the P_2U receptor, the desensitization was less than in the presence of only one of the agonists. The desensitization was greater when ATP was applied for longer (150 s) periods. Although these results do not exclude the participation of post-receptor events in the desensitization process, they suggest that desensitization is governed at least in part by agonist–receptor interaction.     

Essential role for Smad3 in angiotensin II‐induced tubular epithelial–mesenchymal transition

Angiotensin II (Ang II) is a key mediator of chronic kidney disease, in which epithelial–mesenchymal transition (EMT) is a critical process mediated by the TGFβ/Smad signalling pathway. The present study examined the specific role of Smads in Ang II‐induced EMT in vitro and in vivo. We found that Ang II signalled through the receptor of AT1, not AT2, to activate Smad2/3 and induce EMT in a normal rat tubular epithelial cell line (NRK52E). Activation of Smads by Ang II was attributed to degradation of an inhibitory Smad7, which was mediated by the AT1‐Smurf2‐dependent ubiquitin degradation mechanism because blockade of AT1 receptor or knockdown of Smurf2 inhibited Smad7 loss, thereby reducing Smad2/3 activation and EMT in response to Ang II. In contrast, over‐expression of Smad7 inhibited Ang II‐induced Smad2/3 activation and EMT in NRK52E cells and in a rat model of remnant kidney disease. Moreover, knockdown of Smad3, not Smad2, attenuated Ang II‐induced EMT. In conclusion, Ang II activates Smad signalling to induce EMT, which is mediated by a loss of Smad7 through the AT1‐Smurf2‐dependent ubiquitin degradation pathway. Smad3, but not Smad2, may be a mediator of EMT, while Smad7 may play a protective role in EMT in response to Ang II. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.