Immunolocalization of natriuretic peptides and their receptors in goat (Capra hircus) heart

Natriuretic peptides are structurally similar, but genetically distinct, hormones that participate in cardiovascular homeostasis by regulating blood and extracellular fluid volume and blood pressure. We investigated the distribution of natriuretic peptides and their receptors in goat (Capra hircus) heart tissue using the peroxidase-anti-peroxidase (PAP) immunohistochemical method. Strong staining of atrial natriuretic peptide (ANP) was observed in atrial cardiomyocytes, while strong staining for brain natriuretic peptide (BNP) was observed in ventricular cardiomyocytes. Slightly stronger cytoplasmic C-type natriuretic peptide (CNP) immunostaining was detected in the ventricles compared to the atria. Natriuretic peptide receptor-A (NPR-A) immunoreactivity was more prominent in the atria, while natriuretic peptide receptor-B (NPR-B) immunoreactivity was stronger in the ventricles. Cytoplasmic natriuretic peptide receptor-C (NPR-C) immunoreactivity was observed in both the atria and ventricles, although staining was more prominent in the ventricles. ANP immunoreactivity ranged from weak to strong in endothelial and vascular smooth muscle cells. Endothelial cells exhibited moderate to strong BNP immunoreactivity, while vascular smooth cells displayed weak to strong staining. Endothelial cells exhibited weak to strong cytoplasmic CNP immunoreactivity. Vascular smooth muscle cells were labeled moderately to strongly for CNP. Weak to strong cytoplasmic NPR-A immunoreactivity was found in the endothelial cells and vascular smooth muscle cells stained weakly to moderately for NPR-A. Endothelial and vascular smooth cells exhibited weak to strong cytoplasmic NPR-B immunoreactivity. Moderate to strong NPR-C immunoreactivity was observed in the endothelial and smooth muscle cells. Small gender differences in the immunohistochemical distribution of natriuretic peptides and receptors were observed. Our findings suggest that endothelial cells, vascular smooth cells and cardiomyocytes express both natriuretic peptides and their receptors.     

Natriuretic peptides cause relaxation of human esophageal mucosal muscle

Natriuretic peptides have been demonstrated to cause relaxation of the human gallbladder muscle through interaction with natriuretic peptide receptor-B (NPR-B/NPR2). Effects of natriuretic peptides in the human esophageal muscle were unknown. To investigate the effects of natriuretic peptides in the human esophagus, we measured relaxation of muscularis mucosae strips isolated from the human esophagus caused by C-type natriuretic peptide (CNP), brain natriuretic peptide (BNP), atrial natriuretic peptide (ANP) and des[Gln(18), Ser(19), Gly(20), Leu(21), Gly(22)]ANP(4-23) amide (cANP(4-23)), a selective natriuretic peptide receptor-C (NPR-C) agonist. In endothelin-1 or carbachol-contracted mucosal muscle strips, CNP caused moderate, sustained and concentration-dependent relaxation. BNP caused a very mild relaxation whereas ANP and cANP(4-23) did not cause any relaxation. CNP was much more potent than BNP and ANP in causing relaxation. These suggest the existence of NPR-B mediating relaxation. The CNP-induced relaxation was not affected by tetrodotoxin or atropine in endothelin-1-contracted esophageal strips and not by tetrodotoxin in carbachol-contracted strips, indicating a direct effect of CNP on the human esophageal muscularis mucosae. Taken together, these results demonstrate that natriuretic peptides cause relaxation of the muscularis mucosae of the human esophagus and suggest that the relaxation is through interaction with NPR-B. Natriuretic peptides may play an important role in the control of human esophageal motility.     

ANP and CNP activate CFTR expressed in Xenopus laevis oocytes by direct activation of PKA

Abstract

Context: Acting through different receptors, natriuretic peptides (atrial natriuretic peptide [ANP], brain type natriuretic peptide [BNP] and C-type natriuretic peptide [CNP]) increase intracellular cGMP, which then stimulates different pathways that activate fluid secretion. Objective: We used two-electrode voltage clamping to define the dominant pathway that is employed when natriuretic peptides activate cystic fibrosis transmembrane conductance regulator (CFTR) in the Xenopus oocyte expression system. Natriuretic peptides could activate CFTR by 1) cGMP cross-activation of protein kinase A (PKA), 2) cGMP activation of cGMP-dependent protein kinase II, 3) cGMP inhibition of phosphodiesterase type III (PDE3), or 4) direct activation of CFTR. Materials and Methods: cRNA-microinjected Xenopus laevis oocytes were perfused with diverse compounds that examined these pathways of natriuretic peptide signaling. Results and Discussion: ANP stimulated the shark CFTR (sCFTR)-mediated chloride conductance and this activation was inhibited by H-89, a specific inhibitor of PKA. After co-expression of the CNP receptor (NPR-B), sCFTR became stimulatable by CNP and was similarly inhibited by H-89, pointing to cross-activation of PKA. 8-pCPT-cGMP, a relatively cGKII-selective cGMP, failed to stimulate sCFTR. Another membrane-permeable and non-hydrolyzable analog of cGMP, 8-Br-cGMP, stimulated CFTR only at millimolar concentrations, consistent with cross-activation of PKA. The PDE inhibitors EHNA, rolipram, cilostamide, and amrinone did not significantly increase chloride conductance, arguing against a significant role for PDE2, PDE3 and PDE4 signaling in the oocyte. Sildenafil, a PDE5 inhibitor, caused a partial activation of sCFTR channels and this effect was again inhibited by H-89. Conclusion: From these experiments we conclude that in the Xenopus oocyte system, natriuretic peptides, 8-Br-cGMP, and PDE5 inhibitors activate CFTR by cross-activation of PKA.     

B-Type and C-Type Natriuretic Peptides Modify Norepinephrine Uptake in Discrete Encephalic Nuclei of the Rat

SUMMARY 1. We previously demonstrated that atrial natriuretic factor and B- and C-type natriuretic peptides (ANF, BNP, and CNP, respectively) modified catecholamine metabolism by increasing the neuronal uptake and decreasing the neuronal release of norepinephrine in the rat hypothalamus. The aim of the present work was to study the effects of natriuretic peptides BNP and CNP on norepinephrine uptake as an index of the amine metabolism in discrete areas and nuclei of the central nervous system (CNS) of the rat.2. Experiments were carried out in vitro using the punchout technique in diverse areas and nuclei of rat CNS. Results showed that 100 nM BNP and 1 nM CNP increased norepinephrine (NE) uptake in all brain areas and nuclei studied.3. Present results permit us to conclude that BNP and CNP regulate NE metabolism independently of the encephalic area or nucleus involved. In fact, NE uptake increased in nuclei related to the regullation of cardivascular activity as well as nuclei associated with endocrine metabolism and hydrosaline homeostasis. These observations suggest that BNP and CNP may be involved in the regulation of these physiological processes in an indirect manner through modifications of noradrenergic neurotransmission. Present findings provide futher support to the hypothesis that CNP would be the main natriuretic peptide in brain. Furthermore, previous as well as present results support the role of the natriureic peptides as neuromodulators of noradrenergic transmission at the presynaptic level.     

C-type natriuretic peptide receptor expression in pancreatic alpha cells

Atrial natriuretic peptide (ANP), brain type natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) comprise a family of natriuretic peptides that mediate their biological effects through three natriuretic peptide receptor subtypes, NPR-A (ANP, BNP), NPR-B (CNP) and NPR-C (ANP, BNP, CNP). Several reports have provided evidence for the expression of ANP and specific binding sites for ANP in the pancreas. The purpose of this study was to identify the ANP receptor subtype and to localize its expression to a specific cell type in the human pancreas. NPR-C immunoreactivity, but neither ANP nor NPR-A, was detected in human islets by immunofluorescent staining. No immunostaining was observed in the exocrine pancreas or ductal structures. Double-staining revealed that NPR-C was expressed mainly in the glucagon-containing alpha cells. NPR-C mRNA and protein were detected in isolated human islets by RT-PCR and Western blot analysis, respectively. NPR-C expression was also detected by immunofluorescent staining in glucagonoma but not in insulinoma. ANP, as well as BNP and CNP, stimulated glucagon secretion from perifused human islets (1,111 ± 55% vs. basal [7.3 fmol/min]; P < 0.001). This response was mimicked by cANP(4–23), a selective agonist of NPR-C. In conclusion, the NPR-C receptor is expressed in normal and neoplastic human alpha cells. These findings suggest a role for natriuretic peptides in the regulation of glucagon secretion from human alpha cells.     

Phosphorylation-dependent regulation of the guanylyl cyclase-linked natriuretic peptide receptors

Natriuretic peptides are a family of hormones/paracrine factors that regulate blood pressure, cardiovascular homeostasis and bone growth. The mammalian family consists of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). A family of three cell surface receptors mediates their physiologic effects. Two are receptor guanylyl cyclases known as NPR-A/GC-A and NPR-B/GC-B. Peptide binding to these enzymes stimulates the synthesis of the intracellular second messenger, cGMP, whereas a third receptor, NPR-C, lacks enzymatic activity and functions primarily as a clearance receptor. Here, we provide a brief review of how various desensitizing agents and/or conditions inhibit NPR-A and NPR-B by decreasing their phosphorylation state.     

Natriuretic peptides affect Pseudomonas aeruginosa and specifically modify lipopolysaccharide biosynthesis

Natriuretic peptides of various forms are present in animals and plants, and display structural similarities to cyclic antibacterial peptides. Pretreatment of Pseudomonas aeruginosa PAO1 with brain natriuretic peptide (BNP) or C-type natriuretic peptide (CNP) increases bacterium-induced glial cell necrosis. In eukaryotes, natriuretic peptides act through receptors coupled to cyclases. We observed that stable analogs of cAMP (dibutyryl cAMP) and cGMP (8-bromo-cGMP) mimicked the effect of brain natriuretic peptide and CNP on bacteria. Further evidence for the involvement of bacterial cyclases in the regulation of P. aeruginosa PAO1 cytotoxicity by natriuretic peptides is provided by the observed doubling of intrabacterial cAMP concentration after exposure to CNP. Lipopolysaccharide (LPS) extracted from P. aeruginosa PAO1 treated with both dibutyryl cAMP and 8-bromo-cGMP induces higher levels of necrosis than LPS extracted from untreated bacteria. Capillary electrophoresis and MALDI-TOF MS analysis have shown that differences in LPS toxicity are due to specific differences in the structure of the macromolecule. Using a strain deleted in the vfr gene, we showed that the Vfr protein is essential for the effect of natriuretic peptides on P. aeruginosa PAO1 virulence. These data support the hypothesis that P. aeruginosa has a cyclic nucleotide-dependent natriuretic peptide sensor system that may affect virulence by activating the expression of Vfr and LPS biosynthesis.     

Natriuretic peptides--their receptors and role in cardiovascular system

Natriuretic peptides belong to a family of small proteins that play a major role in modulation of natriuresis, diuresis and vasodilatation. They counteract the activity of renin-angiotensin-aldosterone system. They are also involved in the regulation of homeostasis, fat metabolism and long bone growth. Natriuretic peptides family in mammals consists of three main members: atrial natriuretic peptide (ANP) - secreted by the atrial myocardium; brain natriuretic peptide (BNP)--secreted mainly by the ventricular myocardium, and C-type natriuretic peptide (CNP)--produced and released by endothelial cells. Secretion of these peptides is stimulated by atrial and ventricular distension, increased blood pressure, hypoxia or renal dysfunction. Natriuretic peptides play their roles via interactions with NPR-A and NPR-B receptors which are transmembrane guanylyl cyclases. Their local concentrations, regulated by internalization and degradation, are mediated by the NPR-C receptor and by neutral endopeptidase. The paper presents the current knowledge of structure and biological function of natriuretic peptides.     

Cell Surface Protein Disulfide Isomerase Regulates Natriuretic Peptide Generation of Cyclic Guanosine Monophosphate

Rationale

The family of natriuretic peptides (NPs), including atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), exert important and diverse actions for cardiovascular and renal homeostasis. The autocrine and paracrine functions of the NPs are primarily mediated through the cellular membrane bound guanylyl cyclase-linked receptors GC-A (NPR-A) and GC-B (NPR-B). As the ligands and receptors each contain disulfide bonds, a regulatory role for the cell surface protein disulfide isomerase (PDI) was investigated.

Objective

We utilized complementary in vitro and in vivo models to determine the potential role of PDI in regulating the ability of the NPs to generate its second messenger, cyclic guanosine monophosphate.

Methods and Results

Inhibition of PDI attenuated the ability of ANP, BNP and CNP to generate cGMP in human mesangial cells (HMCs), human umbilical vein endothelial cells (HUVECs), and human aortic smooth muscle cells (HASMCs), each of which were shown to express PDI. In LLC-PK1 cells, where PDI expression was undetectable by immunoblotting, PDI inhibition had a minimal effect on cGMP generation. Addition of PDI to cultured LLC-PK1 cells increased intracellular cGMP generation mediated by ANP. Inhibition of PDI in vivo attenuated NP-mediated generation of cGMP by ANP. Surface Plasmon Resonance demonstrated modest and differential binding of the natriuretic peptides with immobilized PDI in a cell free system. However, PDI was shown to co-localize on the surface of cells with GC-A and GC-B by co-immunoprecpitation and immunohistochemistry.

Conclusion

These data demonstrate for the first time that cell surface PDI expression and function regulate the capacity of natriuretic peptides to generate cGMP through interaction with their receptors.     

Potential biological control of Erwinia tracheiphila by internal alimentary canal interactions in Acalymma vittatum with Pseudomonas fluorescens

Aims

We aim to determine if Pseudomonas fluorescens is a viable biological control for Erwinia tracheiphila within the insect vector, Acalymma vittatum.

Methods and Results

Pseudomonas fluorescens secreted fluorescein and inhibited growth of E. tracheiphila in disc diffusion assays. To determine if this antagonism was conserved within the insect vector, we performed in vivo assays by orally injecting beetles with bacterial treatments and fluorescent in situ hybridization to determine bacterial presence within the alimentary canal.

Conclusions

Pseudomonas fluorescens inhibited the growth of E. tracheiphila on a nutrient‐limiting medium. In situ experiments demonstrated that P. fluorescens is maintained within the alimentary canal of the beetle for at least 4 days, and co‐occurred with E. tracheiphila. When beetles were first presented with Pseudomonas and then challenged with E. tracheiphila, E. tracheiphila was not recovered via FISH after 4 days. These data suggest that P. fluorescens has potential as a biological control agent to limit E. tracheiphila within the insect vector.

Significance and Impact of the Study

This is a novel approach for controlling E. tracheiphila that has the potential to decrease reliance on insecticides, providing a safer environment for pollinators and growers.     

利钠肽家族基因与心血管疾病研究新进展

利钠肽家族是一组由心肌细胞分泌的激素, 主要包括A型、B型和C型利钠肽, 具有相似的基因结构和生理学效应, 可对心血管系统产生血压调节、抗心肌肥厚、抗心肌纤维化和抗心肌弛缓等保护作用。利钠肽受体A、B和C亦介导多种生理活性, 调节心血管稳态。利钠肽受体A选择性结合A型、B型利钠肽。利钠肽受体B结合C型利钠肽。利钠肽受体C结合各型利钠肽, 通过受体介导的内化和退化作用清除血液循环中利钠肽。对利钠肽家族及其受体基因单核甘酸多态性及功能研究显示, 其与多种心血管疾病(房颤、高血压、心力衰竭等)的易感性相关。利钠肽家族及其受体基因缺失的转基因小鼠表现为心肌肥厚、心肌纤维化, 与高血压、心肌病及心力衰竭的发生发展相关。各种导致心肌肥厚和缺血性损伤的刺激均参与利钠肽及其受体基因的表达调控。临床将脑钠肽作为左室功能障碍和心力衰竭失代偿的一个预测指标。静脉注射重组脑钠肽已经成为治疗急性心力衰竭的有效手段。深入了解利钠肽家族基因变异及其信号调控有助于探索心血管疾病的病理生理机制, 为临床诊疗开辟新思路。     

Natriuretic peptide receptors regulate cytoprotective effects in a human ex vivo 3D/bioreactor model

Introduction

The present study examined the effect of C-type natriuretic peptide (CNP) and biomechanical signals on anabolic and catabolic activities in chondrocyte/agarose constructs.

Methods

Natriuretic peptide (Npr) 2 and 3 expression were compared in non-diseased (grade 0/1) and diseased (grade IV) human cartilage by immunofluoresence microscopy and western blotting. In separate experiments, constructs were cultured under free-swelling conditions or subjected to dynamic compression with CNP, interleukin-1β (IL-1β), the Npr2 antagonist P19 or the Npr3 agonist cANF^4-23. Nitric oxide (NO) production, prostaglandin E₂ (PGE₂) release, glycosaminoglycan (GAG) synthesis and CNP concentration were quantified using biochemical assays. Gene expression of Npr2, Npr3, CNP, aggrecan and collagen type II were assessed by real-time qPCR. Two-way ANOVA and a post hoc Bonferroni-corrected t-test were used to analyse the data.

Results

The present study demonstrates increased expression of natriuretic peptide receptors in diseased or older cartilage (age 70) when compared to non-diseased tissue (age 60) which showed minimal expression. There was strong parallelism in the actions of CNP on cGMP induction resulting in enhanced GAG synthesis and reduction of NO and PGE₂ release induced by IL-1β. Inhibition of Npr2 with P19 maintained catabolic activities whilst specific agonism of Npr3 with cANF^4-23 had the opposite effect and reduced NO and PGE₂ release. Co-stimulation with CNP and dynamic compression enhanced anabolic activities and inhibited catabolic effects induced by IL-1β. The presence of CNP and the Npr2 antagonist abolished the anabolic response to mechanical loading and prevented loading-induced inhibition of NO and PGE₂ release. In contrast, the presence of the Npr3 agonist had the opposite effect and increased GAG synthesis and cGMP levels in response to mechanical loading and reduced NO and PGE₂ release comparable to control samples. In addition, CNP concentration and natriuretic peptide receptor expression were increased with dynamic compression.

Conclusions

Mechanical loading mediates endogenous CNP release leading to increased natriuretic peptide signalling. The loading-induced CNP/Npr2/cGMP signalling route mediates anabolic events and prevents catabolic activities induced by IL-1β. The CNP pathway therefore represents a potentially chondroprotective intervention for patients with OA, particularly when combined with physiotherapeutic approaches to stimulate biomechanical signals.     

利钠肽家族基因与心血管疾病研究新进展

利钠肽家族是一组由心肌细胞分泌的激素,主要包括A型、B型和C型利钠肽,具有相似的基因结构和生理学效应,可对心血管系统产生血压调节、抗心肌肥厚、抗心肌纤维化和抗心肌弛缓等保护作用。利钠肽受体A、B和C亦介导多种生理活性,调节心血管稳态。利钠肽受体A选择性结合A型、B型利钠肽。利钠肽受体B结合C型利钠肽。利钠肽受体C结合各型利钠肽,通过受体介导的内化和退化作用清除血液循环中利钠肽。对利钠肽家族及其受体基因单核甘酸多态性及功能研究显示,其与多种心血管疾病(房颤、高血压、心力衰竭等)的易感性相关。利钠肽家族及其受体基因缺失的转基因小鼠表现为心肌肥厚、心肌纤维化,与高血压、心肌病及心力衰竭的发生发展相关。各种导致心肌肥厚和缺血性损伤的刺激均参与利钠肽及其受体基因的表达调控。临床将脑钠肽作为左室功能障碍和心力衰竭失代偿的一个预测指标。静脉注射重组脑钠肽已经成为治疗急性心力衰竭的有效手段。深入了解利钠肽家族基因变异及其信号调控有助于探索心血管疾病的病理生理机制,为临床诊疗开辟新思路。     

Effect of natriuretic peptides on cerebral artery blood flow in healthy volunteers

The natriuretic peptides (NPs), atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP), have vasoactive functions that concern humans and most animals, but their specific effects on cerebral circulation are poorly understood. We therefore examined the responsiveness of cerebral arteries to different doses of the natriuretic peptides in animals and humans. We conducted a dose-response experiment in guinea pigs (in vitro) and a double-blind, three-way cross-over study in healthy volunteers (in vivo). In the animal experiment, we administered cumulative doses of NPs to pre-contracted segments of cerebral arteries. In the main study, six healthy volunteers were randomly allocated to receive two intravenous doses of ANP, BNP or CNP, respectively, over 20 min on three separate study days. We recorded blood flow velocity in the middle cerebral artery (V_MCA) by transcranial Doppler. In addition, we measured temporal and radial artery diameters, headache response and plasma concentrations of the NPs. In guinea pigs, ANP and BNP but not CNP showed significant dose-dependent relaxation of cerebral arteries. In healthy humans, NP infusion had no effect on mean V_MCA, and we found no difference in hemodynamic responses between the NPs. Furthermore, natriuretic peptides did not affect temporal and radial artery diameters or induce headache. In conclusion, natriuretic peptides in physiological and pharmacological doses do not affect blood flow velocity in the middle cerebral artery or dilate extracerebral arteries in healthy volunteers.     

<Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> biofilms subjected to phage phiIBB-PF7A

Background  

Pseudomonas fluorescens is an important food spoilage organism, usually found in the form of biofilms. Bacterial biofilms are inherently resistant to a variety of antimicrobial agents, therefore alternative methods to biofilm control, such as bacteriophages (phages) have been suggested. Phage behavior on biofilms is still poorly investigated and needs further understanding. Here we describe the application of phage ϕIBB-PF7, a newly isolated phage, to control P. fluorescens biofilms. The biofilms were formed under static or dynamic conditions and with or without renewal of medium.     

Involvement of a phospholipase C in the hemolytic activity of a clinical strain of <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis>

Background  

Pseudomonas fluorescens is a ubiquitous Gram-negative bacterium frequently encountered in hospitals as a contaminant of injectable material and surfaces. This psychrotrophic bacterium, commonly described as unable to grow at temperatures above 32°C, is now considered non pathogenic. We studied a recently identified clinical strain of P. fluorescens biovar I, MFN1032, which is considered to cause human lung infection and can grow at 37°C in laboratory conditions.     

A Comparison of the Lipolytic Activity of Different Natriuretic Peptides on Human Adipocytes

Atrial natriuretic peptide (ANP) was recently shown to promote triacylglycerol hydrolysis in human white adipocytes both in vitro and in vivo through a cGMP-dependent pathway. The ANP-stimulated lipolytic effect is known to be specific to primates. In this study, we compared the lipolytic effect of different natriuretic peptides obtained from several species, including ANP from human, rat, chicken, frog, and eel, brain natriuretic peptide (BNP) from porcine and rat, C-type natriuretic peptide (CNP) from human, chicken, and frog, Dendroaspis natriuretic peptide (DNP), urodilatin, and des-[Gln¹⁸, Ser¹⁹, Gly²⁰, Leu²¹, Gly²²]-ANP (C-ANP), on human and rat adipocytes. We also compared the amount of intracellular cGMP produced in both human and rat adipocytes that were treated with natriuretic peptides. Among these NPs, rat ANP, as well as porcine and rat BNP, DNP and urodilatin showed the ability to elevate intracellular cGMP and to stimulate lipolysis as human ANP. No natriuretic peptide showed the ability to stimulate lipolysis in rat adipocytes, though some of them induced significant elevation of intracelluar cGMP concentrations. These results suggest that ANP and BNP from species close to human have the ability to induce lipolysis in human adipocytes. Jiahua Yu and Yeon Jun Jeong contributed equally.     

C-type natriuretic peptide plasma levels are reduced in obese adolescents

The high prevalence of obesity in children may increase the magnitude of lifetime risk of cardiovascular disease (CD). At present, explicit data for recommending biomarkers as routine pre-clinical markers of CD in children are lacking. C-type natriuretic peptide (CNP) is assuming increasing importance in CD; in adults with heart failure, its plasma levels are related to clinical and functional disease severity. We have previously reported five different reference intervals for blood CNP as a function of age in healthy children; however, data on plasma CNP levels in obese children are still lacking. Aim of this study was to assess CNP levels in obese adolescents and verify whether they differ from healthy subjects. Plasma CNP was measured in 29 obese adolescents (age: 11.8 ± 0.4 years; BMI: 29.8 ± 0.82) by radioimmunoassay and compared with the reference values of healthy subjects. BNP was also measured. Both plasma CNP and BNP levels were significantly lower in the obese adolescents compared to the appropriate reference values (CNP: 3.4 ± 0.2 vs 13.6 ± 2.3 pg/ml, p < 0.0001; BNP: 18.8 ± 2.6 vs 36.9 ± 5.5 pg/ml, p = 0.003). There was no significant difference between CNP values in males and females. As reported in adults, we observed lower plasma CNP and BNP levels in obese children, suggesting a defective natriuretic peptide system in these patients. An altered regulation of production, clearance and function of natriuretic peptides, already operating in obese adolescents, may possibly contribute to the future development of CD. Thus, the availability of drugs promoting the action of natriuretic peptides may represent an attractive therapeutic option to prevent CD.     

C-type natriuretic peptide inhibits ANP secretion and atrial dynamics in perfused atria: NPR-B-cGMP signaling

The purpose of the present experiments was to define the role of C-type natriuretic peptide (CNP) in the regulation of atrial secretion of atrial natriuretic peptide (ANP) and atrial stroke volume. Experiments were performed in perfused beating and nonbeating quiescent atria, single atrial myocytes, and atrial membranes. CNP suppressed in a dose-related fashion the increase in atrial stroke volume and ANP secretion induced by atrial pacing. CNP caused a right shift in the positive relationships between changes in the secretion of ANP and atrial stroke volume or translocation of the extracellular fluid (ECF), which indicates the suppression of atrial myocytic release of ANP into the paracellular space. The effects of CNP on the secretion and contraction were mimicked by 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP). CNP increased cGMP production in the perfused atria, and the effects of CNP on the secretion of ANP and atrial dynamics were accentuated by pretreatment with an inhibitor of cGMP phosphodiesterase, zaprinast. An inhibitor of the biological natriuretic peptide receptor (NPR), HS-142-1, attenuated the effects of CNP. The suppression of ANP secretion by CNP and 8-BrcGMP was abolished by a depletion of extracellular Ca(2+) in nonbeating atria. Natriuretic peptides increased cGMP production in atrial membranes with a rank order of potency of CNP > BNP > ANP, and the effect was inhibited by HS-142-1. CNP and 8-BrcGMP increased intracellular Ca(2+) concentration transients in single atrial myocytes, and mRNAs for CNP and NPR-B were expressed in the rabbit atrium. From these results we conclude that atrial ANP release and stroke volume are controlled by CNP via NPR-B-cGMP mediated signaling, which may in turn act via regulation of intracellular Ca(2+).     

Natriuretic peptides reduce plasminogen activator inhibitor-1 expression in human endothelial cells

Plasma concentrations of natriuretic peptides increase in some pathological conditions, but very little is known about the effect of these vasodilator peptides on the regulation of the blood coagulation system. The fundamental role in the regulation of fibrinolysis is played by plasminogen activator inhibitor type 1 (PAI-1). Recent studies demonstrate that natriuretic peptides can modulate PAI-1 expression in bovine aortic smooth muscle cells and rat aortic endothelial cells. In this report, we tested the effect of natriuretic peptides on PAI-1 expression in the human endothelial cell line (EA.hy 926). For this purpose, we treated the cell cultures with ANP, BNP and CNP, and modulation of PAI-1 synthesis was evaluated. We compared the effect of natriuretic peptides on synthesis and release of PAI-1 in unstimulated cells, and after activation with tumour necrosis factor alpha (TNFalpha). Natriuretic peptides abolished TNFalpha - induced upregulation of PAI-1 expression at both the PAI-1 mRNA and the antigen levels. The inhibitory efficiency was higher in the case of CNP when compared to that produced by ANP and BNP, particularly when TNFalpha-stimulated cells were used. We observed an inhibition of stimulatory effect of TNFalpha on PAI-1 expression also at the level of the PAI-1 promoter in cells transfected with a PAI-1 promoter fragment (+71 to -800). The PAI-1 promoter activity was markedly inhibited by C-type natriuretic peptide, already at a very low (0.001 micro M) concentration of the peptide.