Gene expression analysis in LLC-PK1 renal tubular cells by atrial natriuretic peptide (ANP): correlation of homologous human genes with renal response

We used human DNA microarray to explore the differential gene expression profiling of atrial natriuretic peptide (ANP)-stimulated renal tubular epithelial kidney cells (LLC-PK1) in order to understand the biological effect of ANP on renal kidney cell's response. Gene expression profiling revealed 807 differentially expressed genes, consisting of 483 up-regulated and 324 down-regulated genes. The bioinformatics tool was used to gain a better understanding of differentially expressed genes in porcine genome homologous with human genome and to search the gene ontology and category classification, such as cellular component, molecular function and biological process. Four up-regulated genes of ATP1B1, H3F3A, ITGB1 and RHO that were typically validated by real-time quantitative PCR (RT-qPCR) analysis serve important roles in the alleviation of renal hypertrophy as well as other related effects. Therefore, the human array can be used for gene expression analysis in pig kidney cells and we believe that our findings of differentially expressed genes served as genetic markers and biological functions can lead to a better understanding of ANP action on the renal protective system and may be used for further therapeutic application.     

Continuos intravenous infusion of atrial natriuretic peptide (ANP) prevented liver fibrosis in rat

The atrial natriuretic peptide (ANP) are used as the acute heart failure treatment in clinical and reported the suppression of fibrosis in the heart, lung recently. The aim of this study was to analyze the suppressive effect of liver fibrosis about ANP. In vitro, rat hepatic stellate cell line (HSC-T6) were treated with ANP. In vivo, Wister rats were injected with dimethylnitrosamine (DMN) twice a week via intra-peritoneal for 4 weeks. ANP group was given by continuance intravenous dosage system used 24 h infusion pump for 3 weeks after 1 week of DMN administration. In vitro, ANP suppressed α-SMA expression and was inhibited the growth of HSC, and reduced the expression of type 1 procollagen, TIMP-1, -2 expression. In vivo, The ANP group showed lower serum AST, ALT, HA level. Liver fibrosis was suppressed by ANP. ANP also decreased gene expression of type 1 procollagen, TIMP-1, -2 and α-SMA, TGF-β1 expression. Our results showed that continuous ANP infusion has the specific capacity of inhibiting HSC activation and protecting hepatocytes and the useful capacity to suppress the liver fibrosis.     

Intracellular trafficking and metabolic turnover ofligand-bound guanylyl cyclase/atrial natriuretic peptide receptor-A into subcellular compartments

Atrial natriuretic peptide (ANP) is the first described member of the natriuretic peptide hormone family. ANP elicits natriuretic, diuretic, vasorelaxant and antiproliferative effects, important factors in the control of blood pressure homeostasis. One of the principal loci involved in the regulatory action of ANP is the guanylyl cyclase-linked ANP-receptor which has been designated as NPRA, also referred to as GC-A, whose ANP-binding efficiency and guanylyl cyclase activity vary remarkably in different target tissues. However, the cellular and molecular basis of these activities and the functional expression and regulation of NPRA are not well understood. The mature form of receptor resides in the plasma membrane and consists of an extracellular ligand-binding domain, a single transmembrane-spanning region, and intracellular protein kinase-like homology and guanylyl cyclase catalytic domains. In this review, emphasis has been placed on the interaction of ANP with NPRA, the ligand-mediated endocytosis, trafficking, and subcellular distribution of ligand-receptor complexes from cell surface to the intracellular compartments. Furthermore, it is implicated that after internalization, the ANP/NPRA complexes dissociate into the subcellular compartments and a population of receptor recycles back to the plasma membrane. This is an interesting area of research in the natriuretic peptide receptor field because there is currently debate over whether ANP/NPRA complexes internalize at all or whether cell utilizes some other mechanisms to release ANP from the bound receptor molecules. Indeed, controversy exist since it has been previously reported by default that among the three natriuretic peptide receptors only NPRC internalizes with bound ligand. Hence, from a thematic standpoint it is clearly evident that there is a current need to review this subject and provide a consensus forum that establishes the cellular trafficking, sequestration and processing of ANP/NPRA complexes in intact cells. Towards this aim the cellular life-cycle of NPRA will be described in the context of ANP-binding, internalization, metabolic processing, and/or inactivation, down-regulation, and degradation of ligand-receptor complexes in model cell systems.     

血管钠肽、 C型钠尿肽和心房钠尿肽舒血管作用的对比

本实验采用离体血管灌流方法,观察和比较血管钠肽（ＶＮＰ）,Ｃ型钠尿肽（ＣＮＰ）和心房钠尿肽（ＡＮＰ）对大鼠肺动脉,腹主动脉和腹腔静脉的舒张作用。．结果表明,ＶＮＰ,ＣＮＰ和ＡＮＰ对离体大鼠的保留内皮与去内皮的肺动脉,腹主动脉和腹腔静脉均有浓度依赖性舒张作用。     

Chicken atrial natriuretic peptide (chANP) and its secretion

Summary An immunohistochemical study using antiserum raised against synthetic chicken natriuretic polypeptide was used to investigate the distribution of this peptide in the chicken heart. Immunoreactive cells, both in the atrial and ventricular walls, were identified by electron microscopy, and electron-dense granules in the atrial and ventricular cardiocytes were revealed to be storage sites of the peptide. The electron-dense material, thought to be the peptide, was found in the sarcoplasmic reticulum, and it is suggested that a secretory pathway of the peptide through the latter to extracellular space, may be present, in addition to an exocytotic one.     

Expression of atrial natriuretic peptide in thymic macrophages after dexamethasone treatment of rats

Summary The rat thymus represents a site of synthesis of atrial natriuretic peptide (ANP); the immunosuppressor dexamethasone strikingly increases ANP-expression in this immune organ. The presented data suggest that this increase can be attributed to macrophages. By means of immunohistochemistry and Northern blot analysis these immune cells were found to express ANP-immunoreactivity as well as mRNA coding for ANP. In contrast, macrophages of control thymi displayed only weak ANP-immunoreactivity. Thus, ANP appears to be a constituent of rat thymic macrophages, and its synthesis in the thymus is strongly elevated by acute exposure of the animals to glucocorticoids.     

重组人心房利钠肽 (ANP) 的原核表达、纯化及鉴定

为提高重组人心房利钠肽(Atrial natriuretic peptide,ANP)的表达量,将3个ANP通过赖氨酸(Lysine,K)串联,并构建相对应的重组表达载体p ET28a(+)/ANP3。转染大肠杆菌进行诱导表达,目的蛋白约占菌体总蛋白的60%。经过包涵体变复性,赖氨酸酶(Lys-C)和羧肽酶(CPB)水解,以及一系列层析纯化,每升培养液可获得约16 mg的ANP蛋白。最终,纯化后的ANP经UPLC及Tricine SDS-PAGE鉴定,纯度大于90%,LC-MS鉴定显示其分子量为3 080 Da,且为二硫键正确形成的ANP单体,通过ELISA试剂盒检测,其具有和参比品一致活性。本研究为ANP的大规模制备打下了基础。同时,所采用的串联表达技术也为其他多肽类药物的重组表达提供了新的思路。     

Human plasmacytoid dendritic cells express an atrial natriuretic peptide receptor, guanylyl cyclase-A

Atrial natriuretic peptide is a cardiovascular hormone secreted mainly by the cardiac atria and regulates the volume–pressure homeostasis. The action of ANP is mediated by GC-A. We previously reported that human monocyte-derived dendritic cells express GC-A and respond to ANP with polarization toward a Th2-inducing phenotype. In the present study, we explored the possibility that pDC are subjected to immunoregulation via the ANP/GC-A system. We examined GC-A expression on blood pDC and found that GC-A was not expressed on fresh pDC but was induced after stimulation with CpG-oligodeoxynucleotide AAC-30, IL-3, or interleukin-3 plus CD40 ligand. Activated pDC responded to ANP with an increase in cGMP production, indicating that GC-A expressed on pDC was functional. We investigated whether tonsillar pDC express GC-A by immunohistochemistry and immunofluorescence staining. We found that GC-A⁺ HLA-DR⁺ cells were present in the T-cell areas and the perivascular areas. Flow cytometric analysis with tonsillar cells confirmed that lineage⁻ CD123^high pDC express GC-A. These results indicate that the ANP/GC-A system is involved in immune regulation through pDC in secondary lymphoid organs.     

Alterations in atrial natriuretic peptide and its receptors in streptozotocin-induced diabetic rat kidneys

In this study the effect of diabetes mellitus on atrial natriuretic peptide (ANP) receptors in streptozotocin- (STZ-) induced diabetic rat kidneys was studied. Moreover, plasma ANP concentration was evaluated in diabetic and control rats by using radioimmunoassay. In addition, the expression of ANP in the kidneys of control and diabetic rats was evaluated by immunohistochemistry. Body-weight loss and increased glucose levels were used as indices of diabetes mellitus in the STZ-induced rats. There was a significant loss in the body weight of the diabetic rats compared to controls. The efficacy of STZ administration was confirmed by rising blood glucose levels, which were significantly higher in diabetic rats compared to controls. Plasma ANP concentration was significantly greater in the diabetic rats in comparison with controls. Moreover, our immunohistochemical results show that the expression of ANP in diabetic rats was higher than that in age-matched controls. ANP was observed in the cells lining the proximal convoluted tubules in the cortex. The distribution and levels of ANP receptors in the kidneys of diabetic rats and age-matched controls were investigated using quantitative receptor autoradiography. Our results demonstrate significant decrease in ANP receptors in the kidneys of the diabetic rats compared to controls. The significant decrease was found in the juxtaglomerular medulla, inner medulla, and the papillae. The decrease in ANP receptors observed in the diabetic kidneys could have pathological consequences resulting in renal resistance to ANP in diabetes. (Mol Cell Biochem 261: 3–8, 2004)     

血管钠肽对低氧大鼠心脏钠尿肽C受体表达的影响

目的 :探讨低氧对大鼠心脏钠尿肽C受体 (NPR C)表达的调节作用 ,以及血管钠肽 (VNP)对这一过程的影响。方法 :将大鼠随机分为 3组 :对照组、低氧组 (3～ 2 8d)和VNP(2 5～ 75 μg/kgbw) 低氧组 ,采用放射免疫的方法测定大鼠血浆心房钠尿肽 (ANP)的浓度 ,并采用定量PCR的方法分析NPR C的mRNA水平。结果 :低氧 2 8d大鼠血浆ANP浓度显著高于正常大鼠 (P <0 .0 5 ) ,而且每天注射 75 μg/kgbw的VNP使ANP浓度进一步升高 (P <0 .0 1)。低氧 3d对大鼠心脏NPR C的mRNA的量没有显著影响 ;低氧 7d使大鼠心脏NPR C的mRNA的拷贝数显著升高 (P <0 .0 5 ) ;低氧 14d、2 8d使大鼠心脏NPR C的mRNA的拷贝数进一步升高 (P <0 .0 1)。每日注射 2 5μg/kgbw的VNP对低氧诱导的大鼠心脏NPR C表达没有显著影响 ;5 0 μg/kgbw的VNP显著降低低氧大鼠心脏NPR C的表达 (P <0 .0 5 ) ;75 μg/kgbw的VNP进一步降低低氧大鼠心脏NPR C的表达 (P <0 .0 1)。 结论 :VNP可以升高低氧大鼠的血浆ANP水平 ;低氧可以使大鼠心脏NPR C表达增加 ,而且具有时间依赖性 ,而VNP对这一过程有抑制作用 ,并且呈剂量依赖性     

三种钠尿肽抑制大鼠肺动脉平滑肌细胞增殖效应的比较

本文比较了心房钠尿肽（ＡＮＰ）、Ｃ－型钠尿肽（ＣＮＰ）、血管钠肽（ＶＮＰ）抑制肺动脉平滑肌细胞（ＰＡＳＭＣｓ）增殖的效应。用蛋白激酶Ｃ激动剂佛波酯（ＰＭＡ）刺激体外培养大鼠ＰＡＳＭＣｓ的增殖,以总蛋白含量和ＭＴＴ比色ＯＤ值为指标,观察三种钠尿肽对ＰＭＡ刺激大鼠ＰＡＳＭＣｓ增殖的影响。结果表明,ＰＭＡ（１０＾－９－１０＾－７ｍｏｌ／Ｌ）显著升高（Ｐ＜０．０５）ＰＡＳＭＣｓ的总蛋白含量和ＭＴＴＯＤ值,     

心房颤动患者血清脑钠肽水平变化的临床研究

目的：探讨患者心房颤动（房颤,AF）发作时对血清脑钠肽水平的影响。方法：选择阵发性房颤组、持续性房颤组、对照组（窦性心律）患者各30例,观察各组血清脑钠肽水平;并对阵发性房颤组中心室率≤100 beats/min与心室率〉100 beats/min的患者进行亚组分析;观察阵发性房颤组复律后24 h和30 d血清脑钠肽水平。结果：阵发性房颤组和持续性房颤组血清脑钠肽水平明显高于对照组（P〈0.01）,房颤复律后血清脑钠肽水平很快下降。结论：血清脑钠肽水平在房颤发作时明显升高,血清脑钠肽水平的升高与房颤的发作有关。     

Spatial and temporal distribution of atrial natriuretic factor in the rat testis.

The atrial natriuretic factor (ANF) is a cardiac hormone whose gene and receptor are widely expressed in extracardiac tissues and organs. ANF induces its biological effects by binding to its specific guanylyl-cyclase-A receptor, which synthesizes the intracellular second messenger cGMP. Increasing evidences indicate that the testis shows the highest reactivity of stimulation of guanylate cyclase by ANF. The well-established functionally active ANF receptors in seminiferous tubules raise the question of the origin and function of ANF in the testis. Therefore, the current study was carried out to investigate the spatial and temporal distribution of ANF in the rat testis by use of immunocytochemistry. Our immunocytochemical results showed that at different pre- and postnatal ages of testicular development ANF was constantly expressed in Leydig cell cytoplasm. However, the intensity of immunoreaction varied between the different Leydig cell populations (fetal, progenitor and immature) and apparently depends on the acquisition of testosterone producing ability. In seminiferous tubules ANF staining was established in the cytoplasm of the developing spermatocytes, in degenerating germ cells (23-day of age) in the cytoplasm of Sertoli cells, cap phase of acrosomal development and in the spermatids (55-day of age). The observed staining patterns suggest a broader spectrum of ANF activities and a possible participation in the whole process of regulation of germ cell development. Our data provide additional support for the hypothesis that ANF plays a major role in autocrine/paracrine regulation of the rat male gonad.     

Atrial natriuretic peptide regulates lipid mobilization and oxygen consumption in human adipocytes by activating AMPK

Atrial natriuretic peptide (ANP) has been shown to regulate lipid and carbohydrate metabolism providing a possible link between cardiovascular function and metabolism by mediating the switch from carbohydrate to lipid mobilization and oxidation. ANP exerts a potent lipolytic effect via cGMP-dependent protein kinase (cGK)-I mediated-stimulation of AMP-activated protein kinase (AMPK). Activation of the ANP/cGK signaling cascade also promotes muscle mitochondrial biogenesis and fat oxidation.Here we demonstrate that ANP regulates lipid metabolism and oxygen utilization in differentiated human adipocytes by activating the alpha2 subunit of AMPK. ANP treatment increased lipolysis by seven fold and oxygen consumption by two fold, both of which were attenuated by inhibition of AMPK activity. ANP-induced lipolysis was shown to be mediated by the alpha2 subunit of AMPK as introduction of dominant-negative alpha2 subunit of AMPK attenuated ANP effects on lipolysis. ANP-induced activation of AMPK enhanced mitochondrial oxidative capacity as evidenced by a two fold increase in oxygen consumption and induction of mitochondrial genes, including carnitine palmitoyltransferase 1A (CPT1a) by 1.4-fold, cytochrome C (CytC) by 1.3-fold, and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) by 1.4-fold. Treatment of human adipocytes with fatty acids and tumor necrosis factor α (TNFα) induced insulin resistance and down-regulation of mitochondrial genes, which was restored by ANP treatment. These results show that ANP regulates lipid catabolism and enhances energy dissipation through AMPK activation in human adipocytes.     

Comparative study of cardiac electrophysiological effects of atrial natriuretic peptide

The effects of atrial natriuretic peptide (ANP) on action potential characteristics were studied in various (human, rabbit, guinea-pig) atrial and guinea-pig right ventricular papillary muscles. ANP (1–100 nM) did not modify the resting membrane potential nor the maximum rate of depolarization phase (V_max). Up to 10 nM, ANP dose-dependently decreased the action potential amplitude both in guinea-pig atrial and ventricular muscles, but it did not affect this parameter in the other atrial preparations. ANP caused a dose-dependent, marked decrease of action potential duration (APD) in practically every cardiac preparation studied (exception of guinea-pig left atrium). The strongest effect on APD can be observed in human atrial and guinea-pig ventricular fibers. The K⁺ channel blocker 4-aminopyridine (1 mM) and the ATP-dependent K⁺ channel inhibitor glibenclamide (10Nl) prevented the effect of ANP on APD in both ventricular atrial preparations. ANP prevented the appearance of isoprenaline (0.5 M) induced slow AP in K⁺ depolarized myocardium. The present data suggest that ANP may inhibit the slow inward Ca²⁺ channel activity and facilitate the K⁺ channel activity.     

The atrial natriuretic peptide (ANP) system in the pronephros and mesonephros of Bufo bufo larvae

In this study on the excretory apparatus of the Bufo bufo larvae, the ultrastructural features and the atrial natriuretic peptide (ANP)-system were examined using cytochemical and immunocytochemical methods. The early embryonic kidney, the pronephros, is replaced by a later stage, the mesonephros. The pronephros degenerates at the time of metamorphosis and the mesonephros becomes the functional kidney in the adult. Both these organs are targets for ANP, demonstrated by the presence of the specific receptors, indirectly highlighted by the cytochemical localization of the guanylate cyclase in the presence of exogenous atrial natriuretic peptide. This study concluded that the mesonephros produces ANP and thus clusters of cells containing ANP-like granules, positive to the anti-α ANP immunolocalization, were present along the mesonephric proximal tubule. The atrial natriuretic peptide system carries out an important osmoregulatory role in the excretory apparatus.     

Immunolocalization of ANP in mast cells of rat and human pericardium

It is known that many heart diseases are accompanied by a significant increase in the level of atrial natriuretic peptide (ANP), a regulator of cardiovascular homeostasis, in the pericardial fluid. Cellular sources of ANP in pericardial cavity remain uncertain. By EM immunocytochemistry, we have examined the presence and localization of ANP in rat and human pericardium. ANP-immunoreactive material was revealed in granules of mast cells (MCs) situated in connective tissue of the pericardium. MCs have an oval form and measure about 6.5 x 12.5 and 9.1 x 13.6 microm in the rat and human pericardium, respectively. Density of MC population makes up about 50 and 10 cells/mm2 in the rat and human pericardium, respectively. Our data suggest possible participation of the pericardial MCs in endocrine function of pericardium and in control of the ANP level in pericardial cavity.