Dendroaspis natriuretic peptide binds to the natriuretic peptide clearance receptor

Dendroaspis natriuretic peptide (DNP) is a newly-described natriuretic peptide which lowers blood pressure via vasodilation. The natriuretic peptide clearance receptor (NPR-C) removes natriuretic peptides from the circulation, but whether DNP interacts with human NPR-C directly is unknown. The purpose of this study was to test the hypothesis that DNP binds to NPR-C. ANP, BNP, CNP, and the NPR-C ligands AP-811 and cANP(4-23) displaced [(125)I]-ANP from NPR-C with pM-to-nM K(i) values. DNP displaced [(125)I]-ANP from NPR-C with nM potency, which represents the first direct demonstration of binding of DNP to human NPR-C. DNP showed high pM affinity for the GC-A receptor and no affinity for GC-B (K(i)>1000 nM). DNP was nearly 10-fold more potent than ANP at stimulating cGMP production in GC-A expressing cells. Blockade of NPR-C might represent a novel therapeutic approach in augmenting the known beneficial actions of DNP in cardiovascular diseases such as hypertension and heart failure.     

Comparative aspects of natriuretic peptide physiology in non-mammalian vertebrates: a review

The natriuretic peptide system is a complex family of peptides and receptors that is primarily linked to the maintenance of osmotic and cardiovascular homeostasis. A natriuretic peptide system is present in each vertebrate class but there are varying degrees of complexity in the system. In agnathans and chondrichthyians, only one natriuretic peptide has been identified, while new data has revealed that multiple types of natriuretic peptides are present in bony fish. However, it seems in tetrapods that there has been a reduction in the number of natriuretic peptide genes, such that only three natriuretic peptides are present in mammals. The peptides act via a family of guanylyl cyclase receptors to generate the second messenger cGMP, which mediates a range of physiological effects at key targets such as the gills, kidney and the cardiovascular system. This review summarises the current knowledge of the natriuretic peptide system in non-mammalian vertebrates and discusses the physiological actions of the peptides.Abbreviations ANP atrial natriuretic peptide - AVT arginine vasotocin - BNP brain natriuretic peptide - cDNA complementary deoxyribonucleic acid - CNP C-type natriuretic peptide - cAMP adenosine 3, 5 cyclic monophosphate - cGMP guanosine 3, 5 cyclic monophosphate - GC guanylyl cyclase - GFR glomerular filtration rate - mRNA messenger ribonucleic acid - NPR natriuretic peptide receptor - NPs natriuretic peptides - sCP salmon cardiac peptide - VIP vasoactive intestinal peptide - VNP ventricular natriuretic peptide Communicated by I.D. Hume     

Dendroaspis natriuretic peptide system and its paracrine function in rat colon

Dendroaspis natriuretic peptide (DNP), a 38-amino-acid peptide, was isolated from the venom of Green Mamba. It has structural and functional similarities to other members of the natriuretic peptide family. The purpose of this study was to determine whether DNP system is present in the rat colon and to define its biological functions. The serial dilution curve of extracts of colonic tissues was parallel to the standard curve of DNP and a major peak of molecular profile by HPLC was synthetic DNP. The concentration of DNP was 0.5±0.04 ng/g of colonic tissues. DNP as well as atrial natriuretic peptide and C-type natriuretic peptide caused dose-dependent increases in cGMP production in the purified membrane of colonic tissues. Three types of natriuretic peptide receptor mRNAs were detected using semi-quantitative RT-PCR. Functionally, synthetic DNP inhibited the spontaneous contraction of rat colonic circular muscle in a concentration-dependent manner. The potency appeared to be at least 10 times greater than that of CNP. Furthermore, DNP inhibited carbachol-induced muscle contraction, suggesting that it also can modulate the nerve regulation of colonic motility. This study demonstrates the presence of DNP system in rat colon and its function as a local regulator of colonic motility.     

Expression of C-type natriuretic peptide and of its receptor NPR-B in normal and failing heart

C-type natriuretic peptide (CNP) was recently found in the myocardium, but possible insights into differences between atrium and ventricle production are so far lacking. Our aim was to evaluate, in an experimental model of pacing-induced heart failure (HF), plasma and tissue levels of CNP and mRNA expression of the peptide and of its specific receptor, NPR-B. Cardiac tissue was collected from male adult minipigs without (control, n=5) and with pacing-induced HF (n=5). Blood samples were collected at baseline and after pacing (10 min, 1, 2, 3 weeks). CNP in plasma and in cardiac extracts was determined by a radioimmunoassay, while the expression of mRNA by real time PCR. Compared to control, plasma CNP was increased after 1 week of pacing stress (36.9+/-10.4 pg/ml vs.16.7+/-1.1, p=0.013, mean+/-S.E.M.). As to myocardial extract, at baseline, CNP was found in all cardiac chambers and its content was 10-fold higher in atria than in ventricles (RA: 13.7+/-1.9 pg/mg protein; LA: 8.7+/-3.8; RV: 1.07+/-0.33; LV: 0.93+/-0.17). At 3 weeks of pacing, myocardial levels of CNP in left ventricle were higher than in controls (15.8+/-9.9 pg/mg protein vs. 0.9+/-0.17, p=0.01). CNP gene expression was observed in controls and at 3 weeks of pacing. NPR-B gene expression was found in all cardiac regions analyzed, and a down-regulation was observed in ventricles after HF. The co-localization of the CNP system and NPR-B suggests a possible role of CNP in HF and may prompt novel therapeutical strategies.     

Neutral endopeptidase and natriuretic peptide receptors participate in the regulation of C-type natriuretic peptide expression in renal interstitial fibrosis

The initiation and progression of renal interstitial fibrosis (RIF) is a complicated process in which many factors may play an activate role. Among these factors, C-type natriuretic peptide (CNP) is an endothelium-derived hormone and acts in a local, paracrine fashion to regulate vascular smooth muscle tone and proliferation. In this study, we established a rat model of unilateral ureteral obstruction (UUO). CNP expression tends to be higher immediately after ligation and declined at later time points, occurring predominantly in tubular epithelial cells. A high-level CNP may contribute to the elevated expression of natriuretic peptide receptor (NPR)-B in the early phase of UUO. However, the sustained expression of NPR-C and neutral endopeptidase (NEP) observed throughout the study period (that is up to 3 months) helps to, at least partly, explain the subsequent decline of CNP. Thus, NEP and NPRs participate in the regulation of CNP expression in RIF.     

Identification and expression of natriuretic peptide receptor type-A and -B mRNA in freshwater and seawater rainbow trout

Natriuretic peptide receptors mediate the physiological response of natriuretic peptide hormones. One of the natriuretic peptide receptor types is the particulate guanylyl cyclase receptors, of which there are two identified: NPR-A and NPR-B. In fishes, these have been sequenced and characterized in eels, medaka, and dogfish shark (NPR-B only). The euryhaline rainbow trout provides an opportunity to further pursue examination of the system in teleosts. In this study, partial rainbow trout NPR-A-like and NPR-B-like mRNA sequences were identified via PCR and cloning. The sequence information was used in real-time PCR to examine mRNA expression in a variety of tissues of freshwater rainbow trout and rainbow trout acclimated to 35 parts per thousand seawater for a period of 10 days. In the excretory kidney and posterior intestine, real-time PCR analysis showed greater expression of NPR-B in freshwater fish than in those adapted to seawater; otherwise, there was no difference in the expression of the individual receptors in fresh water or seawater. In general, the expression of the NPR-A and NPR-B type receptors was quite low. These findings indicate that NPR-A and NPR-B mRNA expression is minimally altered under the experimental regime used in this study.     

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     

The anti-infective peptide, innate defense-regulator peptide, stimulates neutrophil chemotaxis via a formyl peptide receptor

The anti-infective peptide, innate defense-regulator peptide (IDR-1), has been selectively reported to modulate the innate immune response. We found that IDR-1 stimulates the chemotactic migration in human neutrophils. Moreover, IDR-1-induced neutrophil chemotaxis was completely blocked by pertussis toxin, suggesting the importance of the G_i protein in this process. The mechanism governing the IDR-1-induced neutrophil chemotaxis was found to be completely inhibited by the formyl peptide receptor (FPR) antagonist; cyclosporin H. IDR-1 was also found to induce chemotactic migration in FPR but not in vector-expressing HCT116 cells. Meanwhile, IDR-1 failed to stimulate superoxide anion generation and intracellular calcium increase in human neutrophils. Furthermore, IDR-1 was found to inhibit fMLF (an FPR agonist)-induced superoxide generation and calcium signaling in human neutrophils and FPR-expressing HCT116 cells. Taken together, the results demonstrate that IDR-1 is a partial agonist for FPR and further, stimulates neutrophil chemotaxis without inducing calcium signaling and superoxide generation.     

Antimicrobial peptide scolopendrasin VII,derived from the centipede Scolopendra subspinipes mutilans,stimulates macrophage chemotaxis via formyl peptide receptor 1

In this study, we report that one of the antimicrobial peptides scolopendrasin VII, derived from Scolopendra subspinipes mutilans, stimulates actin polymerization and the subsequent chemotactic migration of macrophages through the activation of ERK and protein kinase B (Akt) activity. The scolopendrasin VII-induced chemotactic migration of macrophages is inhibited by the formyl peptide receptor 1 (FPR1) antagonist cyclosporine H. We also found that scolopendrasin VII stimulate the chemotactic migration of FPR1-transfected RBL-2H3 cells, but not that of vector-transfected cells; moreover, scolopendrasin VII directly binds to FPR1. Our findings therefore suggest that the antimicrobial peptide scolopendrasin VII, derived from Scolopendra subspinipes mutilans, stimulates macrophages, resulting in chemotactic migration via FPR1 signaling, and the peptide can be useful in the study of FPR1-related biological responses. [BMB Reports 2015; 48(8): 479-484]     

Structural features of the reprolysin atrolysin C and tissue inhibitors of metalloproteinases (TIMPs) interaction

Atrolysin C is a P-I snake venom metalloproteinase (SVMP) from Crotalus atrox venom, which efficiently degrades capillary basement membranes, extracellular matrix, and cell surface proteins to produce hemorrhage. The tissue inhibitors of metalloproteinases (TIMPs) are effective inhibitors of matrix metalloproteinases which share some structural similarity with the SVMPs. In this work, we evaluated the inhibitory profile of TIMP-1, TIMP-2, and the N-terminal domain of TIMP-3 (N-TIMP-3) on the proteolytic activity of atrolysin C and analyzed the structural requirements and molecular basis of inhibitor-enzyme interaction using molecular modeling. While TIMP-1 and TIMP-2 had no inhibitory activity upon atrolysin C, the N-terminal domain of TIMP-3 (N-TIMP-3) was a potent inhibitor with a K(i) value of approximately 150nM. The predicted docking structures of atrolysin C and TIMPs were submitted to molecular dynamics simulations and the complex atrolysin C/N-TIMP-3 was the only one that maintained the inhibitory conformation. This study is the first to shed light on the structural determinants required for the interaction between a SVMP and a TIMP, and suggests a structural basis for TIMP-3 inhibitory action and related proteins such as the ADAMs.     

Immunoreactive forms of natriuretic peptides in ovine brain: response to heart failure

In order to elucidate how brain natriuretic peptides (NPs) are affected by experimentally induced heart failure, we have measured the immunoreactive (IR) levels of the NP in extracts from 10 regions of ovine brain, including pituitary, and clarified their molecular forms using high performance liquid chromatography (HPLC). Using species-specific radioimmunoassay (RIA), atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) were all detected in extracts taken from control animals and sheep that had undergone rapid ventricular pacing for 7 days to induce heart failure. CNP was the most abundant NP as assessed by specific RIA, and the pituitary contained the highest IR levels for all three NP. Compared with control animals, the pituitary content of BNP in animals with heart failure was reduced by 40% (control, 0.26±0.02 pmol/g wet weight versus heart failure 0.16±0.01; P<0.01, n=7). No other significant changes were observed. The molecular forms of ANP and CNP in whole brain extracts as assessed by HPLC were proANP and CNP22, CNP53 and proCNP, respectively. BNP in pituitary extracts was assessed to be primarily proBNP with a minor component of mature BNP26.     

Reciprocal regulation of natriuretic peptide receptors by insulin in adipose cells

Atrial- and brain-type natriuretic peptides (ANP and BNP, respectively) have been shown to exert potent lipolytic action in adipocytes. A family of natriuretic peptide receptors (NPRs), NPR-1, NPR-2, and NPR-3, mediates their physiologic effects. NPR-1 and NPR-2 are receptor guanylyl cyclases, while NPR-3 lacks enzymatic activity and functions primarily as a clearance receptor for natriuretic peptides. ANP has a high affinity for NPR-1 and NPR-3 than other natriuretic peptides. There is a possibility that ANP may exhibit its lipolytic effect through the balance of NPR-1 and NPR-3 expressions in adipocytes. However, the regulation of adipose NPRs has not been fully elucidated. We here examined the regulation of mouse adipose NPRs by insulin, an anti-lipolytic hormone. Among the insulin target organs, NPR-1 mRNA levels were higher in white adipose tissue (WAT) than in liver and skeletal muscle. NPR-3 mRNA was expressed most abundantly in WAT. Fasting condition induced NPR-1 mRNA level while suppressed NPR-3 mRNA level in WAT. Administration of streptozotocin resulted in the increase of NPR-1 mRNA level while the decrease of NPR-3 mRNA level in WAT. In ob/ob mice, hyperinsulinemic model, NPR-1 mRNA level was lower whereas NPR-3 mRNA level was higher compared to lean control mice. In 3T3-L1 adipocytes, insulin significantly reduced NPR-1 mRNA level while increased NPR-3 mRNA levels both through phosphatidylinositol 3-kinase (PI3-kinase) pathway. In summary, NPR-1 and NPR-3 were highly expressed in WAT and adipose NPR-1 and NPR-3 were reciprocally regulated by insulin. This study suggests that insulin may efficiently promote lipogenesis partly by reducing the lipolytic action of ANP through the opposite regulation of NPR-1 and NPR-3.     

Gene expression of C-type natriuretic peptide and of its specific receptor NPR-B in human leukocytes of healthy and heart failure subjects

C-type natriuretic peptide (CNP), a member of the family of natriuretic peptides, is synthesized and secreted from monocytes and macrophages that resulted to be a source of CNP at inflammatory sites. This suggests that special attention should be focused on the possible role of CNP in the immune system, in addition to its effects on the cardiovascular system. The aim of this study was to evaluate the possibility of measuring the mRNA expression of CNP and NPR-B, its specific receptor, in human whole blood samples of healthy (N; n=7) and heart failure (HF; n=7) subjects by Real-Time PCR (RT-PCR). Total RNA was extracted from leukocytes with QIAamp RNA Blood Kit and/or with PAXgene Blood RNA Kit. RT-PCR was performed and optimized for each primer. The experimental results were normalized with the three most stably expressed genes. CNP and NPR-B expression trend was similar in both fresh and frozen human whole blood. Significant higher levels of CNP and NPR-B mRNA expression were found in HF patients with respect to controls (CNP: N=1.23±0.33 vs. HF=6.54±2.09 p=0.027; NPR-B: N=0.85±0.23 vs. HF=5.31±1.98 p=0.04). A significant correlation between CNP and NPR-B (r=0.86, p<0.0001) was observed. Further studies are needed to clarify the pathophysiological properties of this peptide but the possibility to measure CNP and NPR-B mRNA expression in human leukocytes with a fast and easy procedure is a useful starting point for future investigation devoted to better understand the biomolecular processes associated to different diseases.     

Receptor-specific ligands distinguish natriuretic peptide receptors-A and -C in primate tissues

Systemic clearance of atrial natriuretic peptide (ANP) is in part due to neutral endopeptidase (NEP) proteolysis and natriuretic peptide receptor-C (NPR-C) mediated endocytosis. Biological responses to ANP are primarily mediated by the membrane guanylyl cyclase-A/natriuretic peptide receptor-A (NPR-A). Analogs of ANP selective for NPR-A and/or resistant to NEP may have increased activity in those tissues where NPR-C and NEP are coexpressed with NPR-A. The analog of ANP termed vANP; [(R3D, G9T, R11S, M12L, G16R)ANP] is selective for human NPR-A with at least 10,000 fold reduction in affinity for human NPR-C. We report that rat NPR-A is insensitive to 10 nM vANP, demonstrating the limitations of this species in evaluating human therapeutic candidates. As an alternative approach we tested the binding and potency of receptor-selective and NEP-resistant ANP analogs in rhesus monkey tissues. Competition binding studies with a simplified version of vANP, sANP [(G9T, R11S, G16R)rANP], in rhesus monkey kidney and lung membrane preparations shows displacement of ¹²⁵I-ANP from only a fraction of the total ANP receptor population, 30 and 85%, respectively. The remaining ANP binding sites can be occupied with the NPR-C selective ligand cANP(4-23). These data strongly suggest that only two classes of ANP receptor are present in these membrane preparations, NPR-A and NPR-C. The NEP resistant sANP derivative called sANP(TAPR) was 8 fold more potent (ED₅₀ = 0.6 nM) than rANP (ED₅₀ = SnM) in stimulating cGMP production in the lung membrane preparation. Our results demonstrate that the rhesus monkey natriuretic peptide receptors reflect the pharmacology of the human receptors, and that this species may be suitable to determine the role of NPR-C and NEP in peptide clearance and attenuating functional responses.     

Affinity profiling using the peptide microarray technology: a case study

Little about the reliability of measurements obtained using synthetic peptide microarrays is known. We report results from a study on the quantitative reliability of microarrays manufactured by robot-supported immobilization of presynthesized peptides for different microarray platforms. Technological precision is assessed for inter- and intra-device readout comparisons. Correlations between measured signals and known dissociation constants using a phenomenological model derived from the mass action law are discussed. Special emphasis is on discussing the pitfalls of high-throughput affinity measurements. We show that the quantitative determination of binding affinities is prone to be biased toward a mean affinity of around 10(-7)M, while the classification of peptides into either "binders" or "nonbinders" provides very high prediction accuracy. The experimental requirements needed to obtain reliable binding affinity predictions are discussed.     

Molecular and evolutionary analyses of formyl peptide receptors suggest the absence of VNO-specific FPRs in primates

Formyl peptide receptors (FPRs) were observed to expand in rodents and were recently suggested as candidate vomeronasal chemo-sensory receptors. Since vomeronasal chemosensory receptors usually underwent positive selection and evolved concordantiy with the vomeronasal organ (VNO) morphology, we surveyed FPRs in primates in which VNO morphology is greatly diverse and thus it would provide us a clearer view of VNO-FPRs evolution. By screening available primate genome sequences, we obtained the FPR repertoires in representative primate species. As a result, we did not find FPR family size expansion in primates. Further analyses showed no evolutionary force variance between primates with or without VNO structure, which indicated that there was no functional divergence among primates FPRs. Our results suggest that primates lack the VNO-specific FPRs and the FPR expansion is not a common phenomenon in mammals outside rodent lineage, regardless of VNO complexity.     

Inhibition of goby posterior intestinal NaCl absorption by natriuretic peptides and by cardiac extracts

Natriuretic peptides abolish active Na⁺ and Cl^- absorption aross the posterior intestine of the euryhaline gobyGillichthys mirabilis. Inhibition by eel and human natriuretic peptides is dose-dependent with the following sequence of potencies based on experimentally determined ID₅₀ values for inhibition of short-circuit current: eel ventricular natriuretic peptide (78 nmol · l^-1), eel atrial natriuretic peptide (156 nmol · l^-1), human brain natriuretic peptide (326 nmol · l^-1), human α atrial natriuretic peptide (1.05 μmol · l^-1), and eel C-type natriuretic peptide (75 μmol · l^-1). Natriuretic peptides also significantly increase transcellular conductance. The observed sequence of natriuretic peptide potencies is suggestive of cellular mediation by GC-A-type NP-R₁ receptors in this tissue; as expected for guanylyl-cyclase-coupled NP-R₁ receptors, cyclic GMP mimics the action of natriuretic peptides on the goby intestine. Crude aqueous extracts of goby atrium and ventricle inhibited short circuit current and increased tissue conductance in a dose-dependent manner. Ventricular extract was more potent than atrial extract on both a per organ and per milligram basis.     

Involvement of the atrial natriuretic peptide in the reduction of arterial pressure induced by swimming but not by running training in hypertensive rats

The aim of this study was to compare, under resting conditions, the influence of chronic training in swimming or running on mean arterial pressure (MAP) and the involvement of the natriuretic peptide system in this response. Two-month-old male spontaneously hypertensive rats (SHR) were divided into three groups—sedentary (SD), swimming (SW) and running (RN)—and were trained for eight weeks under regimens of similar intensities. Atria tissue and plasma atrial natriuretic peptide (ANP) concentrations were measured by radioimmunoassay. ANP mRNA levels in the right and left atria as well as the natriuretic peptide receptors (NPR), NPR-A and NPR-C, mRNA levels in the kidney were determined by real-time PCR. Autoradiography was used to quantify NPR-A and NPR-C in mesenteric adipose tissue. Both training modalities, swimming and running, reduced the mean arterial pressure (MAP) of SHR. Swimming, but not running, training increased plasma levels of ANP compared to the sedentary group (P < 0.05). Expression of ANP mRNA in the left atrium was reduced in the RN compared to the SD group (P < 0.05). Expression of NPR-A and NPR-C in the kidneys of the SW group decreased significantly (P < 0.05) compared to the SD group. Although swimming increased ¹²⁵I-ANP binding to mesenteric adipose tissue, displacement by c-ANF was reduced, indicating a reduction of NPR-C. These results suggest that the MAP reduction induced by exercise in SHR differs in its mechanisms between the training modalities, as evidenced by the finding that increased levels of ANP were only observed after the swimming regimen.