NPR-C Receptor Research Articles

Dysfunctions of the C-type natriuretic peptide signaling pathway involved in the development of thoracic aortic aneurysm in Marfan patients

Marfan Syndrome (MFS) is an autosomal dominant inherited connective tissue disorder affecting the cardio-vascular system. Development of thoracic aortic aneurysms (TAA) leading to dissections and ruptures is the primary cause of morbidity and mortality in these patients. No treatment really cures Marfan patients. Interestingly, in humans, mutations in the gene coding for the C-type natriuretic peptide (CNP) or its receptors lead to a “Marfan-like syndrome”. CNP is a local regulator of skeletal growth and of vascular homeostasis, remodeling and angiogenesis. CNP binds to NPR-B or NPR-C receptors, both expressed on endothelial and vascular smooth muscle cells (vSMCs) and modulates the intracellular cGMP and cAMP levels. The aim of this project is to determine whether altered CNP signaling pathway contributes to TAA development and progression to rupture in Marfan patients. Plasma and aorta biopsies were taken from Marfan patients and from Fbn1C1039G/+ mice. The CNP, TGF-beta, cAMP and cGMP concentrations were measured in the plasma. mRNA and protein levels evaluated by qRT-PCR, and Western blot analysis in aortic tissue. Male and female mice studied and alterations of the aorta structure analyzed by ultrasound imaging. Plasma proCNP level in Marfan patients depends on the age and the TAA development. Increased levels found in young patients presenting no or reduced TAA and decreased levels in patients with TAA compared to age and sex-matched non-Marfan patients. CNP and NPR-C decreased at the mRNA and protein levels in the aorta of Marfan patients when compared to the same aorta in non-Marfan patients. In Fbn1+/- mice, aortic structure alterations are detected from 10 weeks in males and 34 weeks in females. CNP and NPR-C mRNA and protein levels drastically decreased in the thoracic aorta in 2–7 week-old male mice and in 34 week-old female mice. In 52-week old Fbn1+/- mice, the plasma cAMP level is increased in males and not in females, whereas the plasma cGMP protein level is increased in females but not in males. CNP injections in 3-week old Fbn1+/- male mice partially prevented the aorta structure alterations. A deficit in CNP protein level or in the expression of the NPR-C receptor contributes to the development and progression of TAA.

Molecular dynamics simulations and in vitro studies of hybrid decellularized leaf-peptide-polypyrrole composites for potential tissue engineering applications

Tissue engineering (TE) aims to repair and regenerate damaged tissue by an assimilation of optimal combination of cells specific to the tissue with an appropriate biomaterial. In this work, a new biomaterial for potential cardiac TE applications was developed by utilizing a combination of in silico studies and in vitro experiments. Molecular dynamics (MD) simulations for the formation of the novel composite prepared from the decellularized leaf components cellulose and pectin along with the VEGF derived peptide (NYLTHRQ) and polypyrrole (PPy) was carried out to assess self-assembly, mechanical properties, and interactions with integrin and NPR-C receptors which are commonly found in cells of cardiac tissue. Results of molecular dynamics simulations indicated the successful formation of stable assemblies. MD simulations also revealed that the scaffold successfully interacted with integrin and NPR-C receptors. As a proof of concept, beet leaves were decellularized (DC) and cross-linked with NYLTHRQ and PPy using layer-by-layer assembly. Decellularization (DC) was confirmed by DNA and protein quantification. Incorporation of the NYLTHRQ peptide and polypyrrole was confirmed by FTIR spectroscopy and SEM imaging. The DC-NYLTHRQ-PPy scaffold was seeded with co-cultured cardiomyocytes and vascular smooth muscle cells. The scaffold promoted cell proliferation and adhesion. Actin and Troponin T immunofluorescence staining showed the presence of these critical cardiomyocyte markers. Thus, for the first time we have developed a decellularized leaf-peptide-PPy composite scaffold by a combination of in silico studies and laboratory analyses that may have potential applications in cardiac TE. Communicated by Ramaswamy H. Sarma

The role of Natriuretic Peptide Receptor C in atrial electrophysiological remodelling in hypertensive heart disease

Atrial fibrillation (AF) is prevalent in hypertension in association with alterations in angiotensin II (Ang II) signaling. Natriuretic peptides (NPs) are a family of hormones with potent effects on atrial electrophysiology mediated in part through the NPR-C receptor. The goal of this study was to determine the role of NPR-C in atrial electrophysiological remodelling in Ang II treated mice (3 mg/kg/day for 3 weeks). Ang II treatment increased the susceptibility to AF in WT (P = 0.001) and was more severe in NPR-C-/- (P = 0.03) mice vs. saline controls. Action potential (AP) upstroke velocity (Vmax; P = 0.01) and peak Na+ current (INa; P < 0.001) were reduced in left atrial (LA) myocytes from Ang II treated WT mice, which was attributed to enhanced PKC⍺ signalling. Ang II differentially prolonged the AP in right atrial (RA; APD50; P < 0.001) and LA (P < 0.001) myocytes. In addition, Ang II reduced Ito in RA myocytes (P < 0.001) and to a greater extent in LA myocytes (P = 0.045). In the RA of NPR-C-/‑ mice, Ang II prolonged (P < 0.05) APD50 to a similar extent vs. WT. In contrast, Vmax was reduced and APD50 was prolonged by Ang II to a greater extent (P < 0.05) in the LA of NPR-C-/- vs. WT mice. Ito was similarly reduced (P<0.05) in the RA and LA of Ang II treated WT and NPR-C-/- mice. Co-treatment with cANF (NPR-C agonist) reduced the susceptibility to AF (P = 0.04) in WT mice vs. Ang II alone. Strikingly, APD50 was reduced (P = 0.01) and Ito was increased (P = 0.001) in the RA, but not LA, by cANF co-treatment. Collectively, these data demonstrate that NPR-C plays a critical role in modulating disease progression in the atria elicited by Ang II treatment.

Bi-allelic Loss-of-Function Mutations in the NPR-C Receptor Result in Enhanced Growth and Connective Tissue Abnormalities

The natriuretic peptide signaling pathway has been implicated in many cellular processes, including endochondral ossification and bone growth. More precisely, different mutations in the NPR-B receptor and the CNP ligand have been identified in individuals with either short or tall stature. In this study we show that the NPR-C receptor (encoded by NPR3) is also important for the regulation of linear bone growth. We report four individuals, originating from three different families, with a phenotype characterized by tall stature, long digits, and extra epiphyses in the hands and feet. In addition, aortic dilatation was observed in two of these families. In each affected individual,we identified a bi-allelic loss-of-function mutation in NPR3. The missense mutations (c.442T>C [p.Ser148Pro] and c.1088A>T [p.Asp363Val]) resulted in intracellular retention of the NPR-C receptor and absent localization on the plasma membrane, whereas the nonsense mutation (c.1524delC [p.Tyr508∗]) resulted in nonsense-mediated mRNA decay. Biochemical analysis of plasma from two affected and unrelated individuals revealed a reduced NTproNP/NP ratio for all ligands and also high cGMP levels. These data strongly suggest a reduced clearance of natriuretic peptides by the defective NPR-C receptor and consequently increased activity of the NPR-A/B receptors. In conclusion, this study demonstrates that loss-of-function mutations in NPR3 result in increased NPR-A/B signaling activity and cause a phenotype marked by enhanced bone growth and cardiovascular abnormalities.

Hormones natriurétiques et syndrome métabolique : mise au point

Natriuretic peptides are a group of hormones including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C type (CNP), urodilatin and guanilyn. ANP (half-life: 2-4 min), is secreted by the atrium, BNP (half-life: 20 min) by the ventricle, CNP by the vascular endothelium, urodilatin by the kidney and guanylin by the intestine. These natriuretic peptides prevent water and salt retention through renal action, vasodilatation and hormonal inhibition of aldosterone, vasopressin and cortisol. These peptides also have a recently demonstrated metabolic effect through an increase of lipolysis, thermogenesis, beta cell proliferation and muscular sensitivity to insulin. Blood levels of these natriuretic peptides depend on "active NPR-A receptors/clearance NPR-C receptors", the last ones being abundant on adipocytes. Therefore, natriuretic peptides act as adipose tissue regulator and constitute a link between blood pressure and metabolic syndrome. They are used as markers and treatment of cardiac failure. Other applications are on going. BNP and NT-proBNP (inactive portion de la pro-hormone) are used as markers of cardiac failure since they have a longer half-life than ANP. BNP decrease is quicker and more important than that one of NT-ProBNP in case of improvement of cardiac failure. Chronic renal insufficiency and beta-blockers increase BNP levels. BNP measurement is useless under treatment with neprilysine inhibitors such as sacubitril, one of the neutral endopeptidases involved in catabolism of natriuretic peptides. The association sacubitril/valsartan is a new treatment of chronic cardiac failure, acting through the decrease of ANP catabolism.

C53: A Novel CNP-Based Therapeutic Strategy Targeting Cardiac Fibrosis and Heart Failure

Background: Cardiac fibrosis is a characteristic feature of the failing heart for which there is an unmet need of fibro-inhibiting therapies targeting heart failure (HF). C-type natriuretic peptide (CNP), a human endothelial derived 22 amino acid (AA) peptide, is a guanylyl cyclase B (GC-B) activator that mediates potent anti-fibrotic actions via its second messenger, cGMP. However, the endogenous anti-fibrotic actions of CNP are limited, in part, due to its rapid metabolic clearance by the clearance receptor (NPRC), which has been reported to be up regulated in HF patients. Considering the fibro-suppressing properties of GC-B activation, we engineered a novel 53 AA peptide (C53) based upon the CNP and proCNP sequence with the goal of advancing C53‘s therapeutic potential including reduced NPRC binding. Hypothesis: We hypothesized C53 would have enhanced cGMP generating actions in vivo due to reduced NPRC binding compared to CNP and C53 would activate cGMP in human cardiac fibroblasts (hCFs). Methods: Therapeutic dose of CNP, equimolar dose of C53 and vehicle were IV infused into normal anesthetized rats (n = 12) and in vivo CNP and cGMP levels were assessed. NPRC binding with C53 and CNP were assessed using a modified AlphaScreen luminescent proximity assay (ALPA). In vitro cGMP activating property of C53 (10−8M and 10−6M) compared to no treatment in hCFs was assessed. Data are mean ± SE and significance was achieved with P < .05. Results: In vivo, C53 significantly increased plasma and urinary cGMP compared to equimolar CNP and vehicle (P < .05), which correlated with higher plasma CNP levels with C53 IV infusion (P < .05). Binding studies using the ALPA demonstrated that C53 had reduced binding to NPRC compared to CNP. In hCFs, C53 generated cGMP at 10−8M and markedly activated cGMP at 10−6M compared to no treatment (no treatment: 0.00 ± 0.00; 10−8M: 0.17 ± 0.03; 10−6M: 1.79 ± 0.02, pmol/well, P < .05). Conclusion: We report a newly engineered CNP-based peptide, C53, has cGMP activating actions in hCFs and has superior in vivo cGMP generation than CNP which may, in part, be related to reduced binding to the metabolic clearance receptor NPRC. These studies suggest that C53 may be a novel therapeutic strategy for the prevention and treatment of cardiac fibrosis in HF and further investigations are warranted.

Modulation of natriuretic peptide receptors in human adipose tissue: molecular mechanisms behind the “natriuretic handicap” in morbidly obese patients

The B-type natriuretic peptide (BNP) hormone plays a crucial role in the regulation of cardiovascular and energy homeostasis. Obesity is associated with lowcirculating levels of BNP, a condition known as "natriuretic handicap." Recent evidences suggest an altered expression of BNP receptors-both the signaling natriuretic peptide receptors (NPR)-A and the clearance NPR-C receptor-in adipose tissue (AT) as one of the putative causes of natriuretic handicap. The current study aims at clarifying the molecular mechanisms behind the natriuretic handicap, focusing on NPR modulation in the AT of obese and control subjects. The study enrolled 34 obese and 20 control subjects undergoing bariatric or abdominal surgery, respectively. The main clinical and biochemical parameters, including circulating BNP, were assessed. In visceral (VAT) and subcutaneous AT (SAT) samples, collected during surgery, the adipocytes and stromal vascular fraction (SVF) expression of NPR-A and NPR-C and the SVF secretion of interleukin 6 (IL-6) were determined. Both VAT and SAT from obese patients expressed a lower NPR-A/NPR-C ratio in adipocytes and the SVF secreted a higher level of IL-6, compared with the controls. Moreover, NPR-A/NPR-C ratio expressed by VAT and SAT adipocytes negatively correlated with body mass index, insulin, the Homeostasis Model Assessment of Insulin resistance, and IL-6 secreted by SVF, and the expression of the clearance receptor NPR-C, in both the VAT and SAT adipocytes, showed a negative correlation with circulating BNP. Overall, insulin resistance/hyperinsulinemia and AT inflammation (ie, high level of IL-6) are the major determinants of the lower NPR-A/NPR-C ratio in adipocytes, thus contributing to the natriuretic handicap in obese subjects.

PS 16-01 INSULIN AND GLUCOSE INDUCE NATRIURETIC PEPTIDE CLEARANCE RECEPTOR IN HUMAN ADIPOCYTES

Objective: Cardiac natriuretic peptides (NP) are involved in cardio-renal regulation but also in lipolysis and lipid metabolism. The signaling receptor NPRA and the clearance receptor NPRC mediate the response to NP that is largely dependent on the ratio between the two receptors. Lipolysis increases when NPRC is selectively downregulated by starving or very-low calorie diet. Insulin is an anti-lipolytic hormone and also induces sodium-retention, suggesting a possible functional link with NP. Therefore we examined the insulin-mediated regulation of NP receptors in differentiated human adipocytes and tested the association between NP receptors expression in visceral adipose tissue (VAT) and insulin, HOMA index and metabolic profiles in patients undergoing renal surgery. Design and method: Differentiated human adipocytes from VAT and SGBS cell line were treated with insulin in the presence of glucose. Further experiments in low-glucose media were performed to simulate in vivo starving conditions and to study glucose-insulin mediated genes involved in lipogenesis. Lipolysis was assessed by glycerol release assay. Fasting blood samples and VAT samples were taken from patients in the day of renal surgery. Results: We observed a potent insulin-mediated and glucose-dependent up-regulation of NPRC, through the PI3K pathway, associated with lower lipolysis in differentiated adipocytes. No effect was observed on NPRA. Low-glucose medium hampered the insulin effect, likely through the modulation of glucose flux genes. An expression ratio in favor of NPRC in adipose tissue was associated with higher fasting insulinemia, higher HOMA index and higher total, LDL, and non-HDL cholesterol. Conclusions: Insulin and glucose-dependent NPRC induction in adipocytes, especially in the contest of insulin resistance and increased insulin levels, might be a key factor linking hyperinsulinemia, metabolic syndrome and higher blood pressure.

Insulin/glucose induces natriuretic peptide clearance receptor in human adipocytes: a metabolic link with the cardiac natriuretic pathway.

Cardiac natriuretic peptides (NP) are involved in cardiorenal regulation and in lipolysis. The NP activity is largely dependent on the ratio between the signaling receptor NPRA and the clearance receptor NPRC. Lipolysis increases when NPRC is reduced by starving or very-low-calorie diet. On the contrary, insulin is an antilipolytic hormone that increases sodium retention, suggesting a possible functional link with NP. We examined the insulin-mediated regulation of NP receptors in differentiated human adipocytes and tested the association of NP receptor expression in visceral adipose tissue (VAT) with metabolic profiles of patients undergoing renal surgery. Differentiated human adipocytes from VAT and Simpson-Golabi-Behmel Syndrome (SGBS) adipocyte cell line were treated with insulin in the presence of high-glucose or low-glucose media to study NP receptors and insulin/glucose-regulated pathways. Fasting blood samples and VAT samples were taken from patients on the day of renal surgery. We observed a potent insulin-mediated and glucose-dependent upregulation of NPRC, through the phosphatidylinositol 3-kinase pathway, associated with lower lipolysis in differentiated adipocytes. No effect was observed on NPRA. Low-glucose medium, used to simulate in vivo starving conditions, hampered the insulin effect on NPRC through modulation of insulin/glucose-regulated pathways, allowing atrial natriuretic peptide to induce lipolysis and thermogenic genes. An expression ratio in favor of NPRC in adipose tissue was associated with higher fasting insulinemia, HOMA-IR, and atherogenic lipid levels. Insulin/glucose-dependent NPRC induction in adipocytes might be a key factor linking hyperinsulinemia, metabolic syndrome, and higher blood pressure by reducing NP effects on adipocytes.

Impaired atrial natriuretic peptide-mediated lipolysis in obesity.

Catecholamines and natriuretic peptides (NPs) are the only hormones with pronounced lipolytic effects in human white adipose tissue. Although catecholamine-induced lipolysis is well known to be impaired in obesity and insulin resistance, it is not known whether the effect of NPs is also altered. Catecholamine- and atrial NP (ANP)-induced lipolysis was investigated in abdominal subcutaneous adipocytes in vitro and in situ by microdialysis. In a cohort of 122 women, both catecholamine- and ANP-induced lipolysis in vitro was markedly attenuated in obesity (n=87), but normalized after substantial body weight loss (n=52). The impairment of lipolysis differed between the two hormones when expressing lipolysis per lipid weight, the ratio of stimulated over basal (spontaneous) lipolysis rate or per number of adipocytes. Thus, while the response to catecholamines was lower when expressed as the former two measures, it was higher when expressed per cell number, a consequence of the significantly larger fat cell size in obesity. In contrast, although ANP-induced lipolysis was also attenuated when expressed per lipid weight or the ratio stimulated/basal, it was similar between non-obese and obese subjects when expressed per cell number suggesting that the lipolytic effect of ANP may be even more sensitive to the effects of obesity than catecholamines. Obesity was characterized by a decrease in the protein expression of the signaling NP A receptor (NPRA) and a trend toward increased levels of the clearance receptor NPRC. The impairment in ANP-induced lipolysis observed in vitro was corroborated by microdialysis experiments in situ in a smaller cohort of lean and overweight men. ANP- and catecholamine-induced lipolysis is reversibly attenuated in obesity. The pro-lipolytic effects of ANP are relatively more impaired compared with that of catecholamines, which may in part be due to specific changes in NP receptor expression.

Clinical Aspects of C-Type Natriuretic Peptide on the Cardiovascular System

C-type natriuretic peptide (CNP) is one of the three most important members of the human natriuretic peptide family, sharing with them a highly conserved 17-aminoacid ring structure, essential to elicit its biological actions by binding to its specific receptor NPR-B. CNP acts in a paracrine or autocrine way and it is cleared from plasma circulation by the NPR-C receptor. This is one of the reasons why its plasma levels are usually low.

Pseudomonas aeruginosa Expresses a Functional Human Natriuretic Peptide Receptor Ortholog: Involvement in Biofilm Formation.

ABSTRACTConsiderable evidence exists that bacteria detect eukaryotic communication molecules and modify their virulence accordingly. In previous studies, it has been demonstrated that the increasingly antibiotic-resistant pathogen Pseudomonas aeruginosa can detect the human hormones brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) at micromolar concentrations. In response, the bacterium modifies its behavior to adapt to the host physiology, increasing its overall virulence. The possibility of identifying the bacterial sensor for these hormones and interfering with this sensing mechanism offers an exciting opportunity to directly affect the infection process. Here, we show that BNP and CNP strongly decrease P. aeruginosa biofilm formation. Isatin, an antagonist of human natriuretic peptide receptors (NPR), prevents this effect. Furthermore, the human NPR-C receptor agonist cANF4-23 mimics the effects of natriuretic peptides on P. aeruginosa, while sANP, the NPR-A receptor agonist, appears to be weakly active. We show in silico that NPR-C, a preferential CNP receptor, and the P. aeruginosa protein AmiC have similar three-dimensional (3D) structures and that both CNP and isatin bind to AmiC. We demonstrate that CNP acts as an AmiC agonist, enhancing the expression of the ami operon in P. aeruginosa. Binding of CNP and NPR-C agonists to AmiC was confirmed by microscale thermophoresis. Finally, using an amiC mutant strain, we demonstrated that AmiC is essential for CNP effects on biofilm formation. In conclusion, the AmiC bacterial sensor possesses structural and pharmacological profiles similar to those of the human NPR-C receptor and appears to be a bacterial receptor for human hormones that enables P. aeruginosa to modulate biofilm expression.

PP.19.26

Objective: The natriuretic peptide (NP) system is involved in systemic cardio-renal regulation but also in lipolysis and “fat burning” in adipocytes. The signaling receptor NPRA and the clearance receptor NPRC mediate the response to NP that is largely dependent on the ratio between of the expression of the two receptors. Adipocytes are distributed through the body and, by NPRC binding, affect also NP levels and their cardio-renal effects. NPRC is selectively downregulated by starving or very low calorie diet favoring lipolysis whereas insulin, stimulated by food ingestion, is an antilipolytic hormone with sodium retaining effects. Design and method: Therefore we examined the insulin- mediated regulation of NP receptors in differentiated human adipocytes and tested the association between NP receptors expression in visceral adipose tissue, insulin, insulin resistance index and metabolic profiles in patients undergoing renal surgery. Differentiated adipocytes (SGBS cell line and primary visceral human adipocytes) were treated with insulin with or without wortmannin, a specific PI3K inhibitor of the main insulin signaling pathway that also inhibits lipolysis in adipocytes. Results: We observed a potent insulin -mediated and glucose dependent up-regulation of NPRC through the PI3-K pathway in differentiated adipocytes. No effect was observed on NPRA. In the patients, an expression ratio in favor of NPRC in adipose tissue was associated with higher fasting insulinemia, higher HOMA index and higher total, LDL and non-HDL cholesterol. Conclusions: In conclusion, insulin and glucose dependent NPRC induction in adipocytes, especially in the contest of insulin resistance and increased insulin levels, might be a key factor linking hyperinsulinemia, metabolic syndrome and higher blood pressure.

Abstract 469: Influences of Dietary Weight Reduction on the Atrial Natriuretic Peptide System in Obese Subjects

In obese individuals, reduced atrial natriuretic peptide (ANP) bioavailability appears to promote arterial hypertension and type 2 diabetes likely through direct metabolic actions. Mid-regional pro-ANP is cleaved off during ANP processing, thus, serving as ANP release marker. We hypothesized that dietary weight reduction improves ANP release. In 75 overweight and obese women and men who had lost &gt;5% body weight during six months hypocaloric dieting (-9.1±3.8 kg), we analyzed venous mid-regional pro-ANP and natriuretic peptide receptor (NPR) expression in abdominal subcutaneous adipose tissue before and after the intervention. Participants underwent ambulatory blood pressure (ABPM) monitoring and cardiac magnetic resonance imaging. ABPM decreased from 118±9/73±7 to 115±9/71±6 mm Hg and left ventricular mass from 92±20 to 86±19 g (p&lt;0.05 for all). End-diastolic and end-systolic volumes remained unchanged. Mid-regional pro-ANP plasma concentrations did not respond to weight loss (before: 51±24 pmol/; after 53± 24 pmol/l) and increased similarly during exercise testing. Adipose NPR-A mRNA expression remained unchanged whereas NPR-C receptor mRNA decreased from 1.2±0.7 to 1.0±0.5 (p&lt;0.05). Our data suggest that dietary weight loss does not augment ANP release in overweight and obese subjects. However, weight loss could improve ANP bioavailability through decreased clearance. Possible mechanisms are reduced fat mass and reduced expression of adipose tissue ANP scavenger receptor NPR-C.

Vascular relaxation induced by C-type natriuretic peptide involves the ca2+/NO-synthase/NO pathway.

AimsC-type natriuretic peptide (CNP) and nitric oxide (NO) are endothelium-derived factors that play important roles in the regulation of vascular tone and arterial blood pressure. We hypothesized that NO produced by the endothelial NO-synthase (NOS-3) contributes to the relaxation induced by CNP in isolated rat aorta via activation of endothelial NPR-C receptor. Therefore, the aim of this study was to investigate the putative contribution of NO through NPR-C activation in the CNP induced relaxation in isolated conductance artery.Main MethodsConcentration-effect curves for CNP were constructed in aortic rings isolated from rats. Confocal microscopy was used to analyze the cytosolic calcium mobilization induced by CNP. The phosphorylation of the residue Ser1177 of NOS was analyzed by Western blot and the expression and localization of NPR-C receptors was analyzed by immunohistochemistry.Key FindingsCNP was less potent in inducing relaxation in denuded endothelium aortic rings than in intact ones. L-NAME attenuated the potency of CNP and similar results were obtained in the presence of hydroxocobalamin, an intracellular NO0 scavenger. CNP did not change the phosphorylation of Ser1177, the activation site of NOS-3, when compared with control. The addition of CNP produced an increase in [Ca2+]c in endothelial cells and a decrease in [Ca2+]c in vascular smooth muscle cells. The NPR-C-receptors are expressed in endothelial and adventitial rat aortas.SignificanceThese results suggest that CNP-induced relaxation in intact aorta isolated from rats involves NO production due to [Ca2+]c increase in endothelial cells possibly through NPR-C activation expressed in these cells. The present study provides a breakthrough in the understanding of the close relationship between the vascular actions of nitric oxide and CNP.

Effects of natriuretic peptides on electrical conduction in the sinoatrial node and atrial myocardium of the heart

Natriuretic peptides, including B-type and C-type natriuretic peptide (BNP and CNP), are powerful regulators of the cardiovascular system; however, their electrophysiological effects in the heart, particularly in the sinoatrial node (SAN), are incompletely understood. We have used high-resolution optical mapping to measure the effects of BNP and CNP, and the roles of natriuretic peptide receptors (NPR-A, NPR-B and NPR-C), on electrical conduction within the SAN and atrial myocardium. In basal conditions BNP and CNP (50-500nm) increased conduction velocity (CV) within the SAN by ∼30% at the high dose and shifted the initial exit site superiorly. These effects sped conduction from the SAN to the surrounding atrial myocardium and were mediated by the NPR-A and NPR-B receptors. In the presence of isoproterenol (1μm) the NPR-C receptor made a major contribution to the effects of BNP and CNP in the heart. In these conditions BNP, CNP and the NPR-C agonist cANF each decreased SAN CV and shifted the initial exit site inferiorly. The effects of cANF (30% reduction) were larger than BNP or CNP (∼15% reduction), indicating that BNP and CNP activate multiple natriuretic peptide receptors. In support of this, the inhibitory effects of BNP were absent in NPR-C knockout mice, where BNP instead elicited a further increase (∼25%) in CV. Measurements in externally paced atrial preparations demonstrate that the effects of natriuretic peptides on CV are partially independent of changes in cycle length. These data provide detailed novel insight into the complex effects of natriuretic peptides and their receptors on electrical conduction in the heart.

Natriuretic peptides regulate heart rate and sinoatrial node function by activating multiple natriuretic peptide receptors

Natriuretic peptides, including BNP and CNP, elicit their effects via two guanylyl cyclase-linked receptors denoted NPR-A and NPR-B as well as a third receptor, NPR-C. The relative contributions of these receptors to the overall effects of NPs on heart rate (HR) and sinoatrial node (SAN) function are very poorly understood. The effects of BNP and CNP (10–500nM) on HR and SAN myocyte spontaneous action potential (AP) firing were studied using wildtype mice and mice lacking functional NPR-C receptors (NPR-C−/−). In basal conditions and 10nM doses of the β-adrenergic receptor (β-AR) agonist isoproterenol (ISO) BNP and CNP increased HR and AP firing in SAN myocytes. The NPR-C selective agonist cANF (10–500nM) had no effects in basal conditions, but decreased HR and SAN AP frequency in the presence of ISO. These effects of cANF were completely absent in NPR-C−/− mice. Strikingly, in the presence of 1μM doses of ISO, BNP and CNP switched to causing decreases in HR and SAN AP frequency. These decreases were not as large as those elicited by cANF and were absent in NPR-C−/− hearts, where BNP instead elicited a further increase in HR. Inhibition of NPR-A with A71915, in the presence of 1μM ISO, enabled BNP to signal exclusively through NPR-C and to decrease HR as effectively as cANF. Together these data demonstrate that BNP and CNP affect HR and SAN function by activating multiple receptor subtypes. NPR-A/B mediate increases in HR and SAN function, but these effects are opposed by NPR-C, which plays an increasingly important signaling role in the presence of β-AR stimulation.

The natriuretic peptides BNP and CNP increase heart rate and electrical conduction by stimulating ionic currents in the sinoatrial node and atrial myocardium following activation of guanylyl cyclase-linked natriuretic peptide receptors

Natriuretic peptides (NPs) are best known for their ability to regulate blood vessel tone and kidney function whereas their electrophysiological effects on the heart are less clear. Here, we measured the effects of BNP and CNP on sinoatrial node (SAN) and atrial electrophysiology in isolated hearts as well as isolated SAN and right atrial myocytes from mice. BNP and CNP dose-dependently increased heart rate and conduction through the heart as indicated by reductions in R-R interval, P wave duration and P-R interval on ECGs. In conjunction with these ECG changes BNP and CNP (100 nM) increased spontaneous action potential frequency in isolated SAN myocytes by increasing L-type Ca(2+) current (I(Ca,L)) and the hyperpolarization-activated current (I(f)). BNP had no effect on right atrial myocyte APs in basal conditions; however, in the presence of isoproterenol (10nM), BNP increased atrial AP duration and I(Ca,L). Quantitative gene expression and immunocytochemistry data show that all three NP receptors (NPR-A, NPR-B and NPR-C) are expressed in the SAN and atrium. The effects of BNP and CNP on SAN and right atrial myocytes were maintained in mutant mice lacking functional NPR-C receptors and blocked by the NPR-A antagonist A71915 indicating that BNP and CNP function through their guanylyl cyclase-linked receptors. Our data also show that the effects of BNP and CNP are completely absent in the presence of the phosphodiesterase 3 inhibitor milrinone. Based on these data we conclude that NPs can increase heart rate and electrical conduction by activating the guanylyl cyclase-linked NPR-A and NPR-B receptors and inhibiting PDE3 activity.

Opposite effects of ANP receptors in attenuation of LPS-induced endothelial permeability and lung injury

Atrial natriuretic peptide (ANP) has been recently identified as a modulator of acute lung injury (ALI) induced by pro-inflammatory agonists. While previous studies tested effects of exogenous ANP administration, the role of endogenous ANP in the course of ALI remains unexplored. This study examined regulation of ANP and its receptors NPR-A, NPR-B and NPR-C by LPS and involvement of ANP receptors in the modulation of LPS-induced lung injury. Primary cultures of human pulmonary endothelial cells (EC) were used in the in vitro tests. Expression of ANP and its receptors was determined by quantitative RT-PCR analysis. Agonist-induced cytoskeletal remodeling was evaluated by immunofluorescence staining, and EC barrier function was characterized by measurements of transendothelial electrical resistance. In the murine model of ALI, LPS-induced lung injury was assessed by measurements of protein concentration and cell count in bronchoalveolar lavage fluid (BAL). LPS stimulation significantly increased mRNA expression levels of ANP and NPR-A in pulmonary EC. Pharmacological inhibition of NPR-A augmented LPS-induced EC permeability and blocked barrier protective effects of exogenous ANP on LPS-induced intercellular gap formation. In contrast, pharmacological inhibition of ANP clearance receptor NPR-C significantly attenuated LPS-induced barrier disruptive effects. Administration of NPR-A inhibitor in vivo exacerbated LPS-induced lung injury, whereas inhibition of NPR-C suppressed LPS-induced increases in BAL cell count and protein content. These results demonstrate for the first time opposite effects of NPR-A and NPR-C in the modulation of ALI and suggest a compensatory protective mechanism of endogenous ANP in the maintenance of lung vascular permeability in ALI.

Natriuretic peptides in brain physiology

Abstract Natriuretic peptides (NPs) regulate salt and water homeostasis by inducing natriuresis and diuresis in the kidney. These actions in addition to those via the heart and vascular system play important roles in the regulation of blood pressure. In the central nervous system NPs play a significant role in neuronal development, synaptic transmission and neuroprotection. Currently, six different human NPs have been described: atrial natriuretic peptide (ANP), urodilatin (URO, renal natriuretic peptide), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) as well as guanylin and uroguanylin. ANP, URO and BNP activate the natriuretic peptide receptor A (NPR-A or guanylate cyclase A (GC-A)) while CNP activates natriuretic peptide receptor B (NPR-B or guanylate cyclase B (GC-B)). Guanylin and uroguanylin are known to activate guanylate cyclase C (GC-C). The receptors GC-A, GC-B, and GC-C are widely expressed in the human body. Currently, GC-B and CNP seems to have the highest expression in central nervous system compared to other NPs and their receptors. All known NPs generate intracellular cyclic GMP (cGMP) by activating their specific guanylate cyclase receptors. Subsequently, cGMP is able to activate protein kinase I or II (PKG I or II) and/or directly regulate transmembrane proteins such as ion channels, transporters and pumps. NPs also bind to the natriuretic peptide receptor C (also called clearance receptor NPR-C) which is a major pathway for the degradation of NPs and has no guanylate cyclase activity. In this review we will focus on new insights regarding the physiological effects of NPs in the brain, especially specific areas of their signaling pathways in neurons and glial cells.