A new member of the natriuretic peptide family is present in the venom of the green mamba (Dendroaspis angusticeps).

This paper describes the purification, sequence, and biological properties of a 38-amino acid residue peptide from the venom of Dendroaspis angusticeps which shared important sequence homologies with natriuretic peptides. Dendroaspis natriuretic peptide (DNP) relaxed aortic strips that had been contracted by 40 mM KCl with a potency (K0.5 = 20 nM) similar to that of atrial natriuretic peptide (ANP) and larger than that of C type natriuretic peptide (CNP). The relaxing actions of ANP and DNP (both at 100 nM) were mutually exclusive. Bovine aortic endothelial cells responded to ANP (K0.5 = 3 nM) and DNP (K0.5 = 3 nM) but not to CNP by a large activation of guanylate cyclase. Rat aortic myocytes showed larger cGMP responses to CNP (K0.5 = 10 nM) than to ANP or DNP (K0.5 = 100 nM). Finally, DNP completely prevented the specific 125I-ANP binding to clearance receptors in cultured aortic myocytes with a potency (Kd = 10 nM) that was less than that of ANP (Kd = 0.3 nM). It is concluded that DNP is a new member of the family of natriuretic peptides and that it recognizes ANPA receptors and clearance, ANPc receptors, but not CNP-specific ANPB receptors.     

Dendroaspis natriuretic peptide and its functions in pig ovarian granulosa cells

Dendroaspis natriuretic peptide (DNP), a 38-amino acid peptide, was isolated from the venom of green mamba. It has structural and functional similarities to the other members of the natriuretic peptide family. The purpose of this study was to determine whether DNP is present in pig ovarian granulosa cells and to define its biological functions. The serial dilution curves of extracts of granulosa cells and follicular fluid were parallel to the standard curve of DNP, and a major peak of molecular profile of both extracts by HPLC was synthetic DNP. The concentration of DNP was 7.51+/-1.46 pg/10(7) cells and 24.81+/-2.38 pg/ml in granulosa cells and follicular fluid, respectively. Natriuretic peptides increased cGMP production in the purified membrane of granulosa cells with a rank order of potency of C-type natriuretic peptide (CNP)>atrial natriuretic peptide (ANP)=DNP. mRNAs for natriuretic peptide receptor-A (NPR-A), NPR-B and NPR-C were detected by RT-PCR. The binding site of (125)I-DNP was also observed in granulosa cell layer by in vitro autoradiography. Synthetic DNP inhibited the secretion of ANP from granulosa cells in a concentration-dependent manner and the potency was similar to CNP. The concentration of DNP and CNP, which inhibited the secretion of ANP by 50%, was about 1 nM. Increases in production of cGMP in granulosa cells were observed by DNP or CNP. Therefore, these results show the existence of DNP system and the cross-talk between natriuretic peptides in pig ovarian granulosa cells.     

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.     

Histamine release induced by dendroaspis natriuretic peptide from rat mast cells

Dendroaspis natriuretic peptide (DNP), recently isolated from the venom of the green Mamba snake Dendroaspis angusticeps, is a 38 amino acid peptide containing a 17 amino acid disulfide ring structure similar to that of the natriuretic peptide family. The natriuretic peptide family is known to induce histamine release from human and rat mast cells, but there are no published data concerning the effects of DNP on histamine release from mast cells. The purpose of this study is to investigate whether DNP induces the histamine release from rat peritoneal mast cells (RMPCs) and to determine the mechanism of DNP-induced histamine release from RPMCs. After treatment of RPMC with DNP, mast cell degranulation was observed, and calcium uptake and histamine release were measured. DNP released the histamine, induced the mast cell degranulation, and increased the calcium uptake of RPMCs, in a dose-dependent manner. The results indicate that DNP can increase Ca-uptake and induce histamine release.     

Renal actions of dendroaspis natriuretic peptide in rabbits

Dendroaspis natriuretic peptide (DNP) is one of four members of the natriuretic peptide family sharing functional and structural properties. The purpose of the present study was to elucidate the physiological role of DNP on renal functions and its cellular mechanism in the rabbit kidney. DNP (5 μg/kg/min) infused intravenously increased urine volume and urinary excretion of electrolytes. These renal actions induced by DNP were more pronounced than those caused by atrial natriuretic peptide (ANP). We compared profiles of (125)I-ANP and (125)I-DNP by reverse-phase HPLC during incubation in rabbit plasma at 37°C for 1, 2, and 4h. While (125)I-ANP was quickly degraded within 1h, (125)I-DNP was still stable in plasma for 4h. DNP induced the greatest cyclic guanosine monophosphate (cGMP) production in the glomeruli in a dose-dependent manner, when compared to other renal structures including cortical tubules, outer medullary tubules, and inner medullary tubules. Affinity cross-linking analysis revealed NPR-A is selective receptor for DNP in glomeruli. Forskolin, a stimulator of adenylyl cyclase, significantly decreased cGMP production in the renal glomeruli but not in the renal medulla. In summary, DNP is a more effective activator of renal functions than ANP, possibly because of the degradation resistance of DNP against the endogenous peptidases in plasma or tissues. These findings suggest that DNP plays a pivotal role as a renal regulating peptide via specific natriuretic peptide receptors with a guanylyl cyclase domain.     

Presence of dendroaspis natriuretic peptide and its binding to NPR-A receptor in rabbit kidney

Natriuretic peptides help to maintain sodium and fluid volume homeostasis in a healthy cardio-renal environment. Since the identification of Dendroaspis natriuretic peptide (DNP) as a new member of the natriuretic peptide family, DNP has been considered as an important regulator of natriuresis and dieresis. The present study was undertaken to investigate the presence of immunoreactive Dendroaspis natriuretic peptide (DNP) and its specific receptor in rabbit. DNP was detected in heart, kidney, liver, brain, and plasma by radioimmunoassay (RIA). DNP contents of cardiac atrium and ventricle, renal cortex and medulla, liver, and brain were 1.42 ± 0.15, 1.0 6 ± 0.08, 2.55 ± 0.21, 1.81 ± 0.16, 1.36 ± 0.22, and 0.69 ± 0.15 pg/mg of wet weight, respectively. The concentration of DNP in plasma was 235.44 ± 15.44 pg/ml. By quantitative in vitro receptor autoradiography, specific 12?I-DNP binding sites were revealed in glomeruli, interlobular artery, acuate artery, vasa recta bundle, and inner medulla of the kidney with an apparent dissociation constant (K(d)) of 0.29 ± 0.05, 0.36 ± 0.03, 0.84 ± 0.19, 1.18 ± 0.23, and 10.91 ± 1.59 nM, respectively. Basal rate of 3', 5'-cyclic guanosine monophosphate (cGMP) production by particulate guanylyl cyclase (GC) activation of glomerular membranes was basally 13.40 ± 1.70 pmol/mg protein/min. DNP caused an increment of cGMP production in similar magnitude to that caused by ANP, BNP, and urodilatin, while the production of cGMP by CNP was significantly lower than that by DNP. Our results show that plasma levels of DNP were higher when compared to other tissues. DNP produces cGMP via the NPR-A receptor subtype in the kidney, similarly to ANP and BNP, suggesting that plasma DNP could have similar functions as ANP and BNP.     

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.     

Dendroaspis natriuretic peptide-like immunoreactivity and its regulation in rat aortic vascular smooth muscle

Dendroaspis natriuretic peptide (DNP) is a recently isolated 38 amino acid peptide that shares structural and functional properties with the other members of the natriuretic peptide family. The present study demonstrates the presence of DNP-like immunoreactivity in sections of rat aorta, carotid artery and renal vasculature and tubules. DNP-like immunoreactivity was detected in culture aortic vascular smooth muscle cells and medium and is regulated by endothelin-1, angiotensin II and sodium nitroprusside but not by transforming growth factor-beta. Our observations indicate that DNP elicits a marked inhibitory effect on DNA synthesis in culture rat aortic vascular smooth muscle cells.     

Novel bifunctional natriuretic peptides as potential therapeutics

Synthetic atrial natriuretic peptide (carperitide) and B-type natriuretic peptide (BNP; nesiritide) are used to treat congestive heart failure. However, despite beneficial cardiac unloading properties, reductions in renal perfusion pressures limit their clinical effectiveness. Recently, CD-NP, a chimeric peptide composed of C-type natriuretic peptide (CNP) fused to the C-terminal tail of Dendroaspis natriuretic peptide (DNP), was shown to be more glomerular filtration rate-enhancing than BNP in dogs. However, the molecular basis for the increased responsiveness was not determined. Here, we show that the DNP tail has a striking effect on CNP, converting it from a non-agonist to a partial agonist of natriuretic peptide receptor (NPR)-A while maintaining the ability to activate NPR-B. This effect is specific for human receptors because CD-NP was only a slightly better activator of rat NPR-A due to the promiscuous nature of CNP in this species. Interesting, the DNP tail alone had no effect on any NPR even though it is effective in vivo. To further increase the potency of CD-NP for NPR-A, we converted two different triplet sequences within the CNP ring to their corresponding residues in BNP. Both variants demonstrated increased affinity and full agonist activity for NPR-A, whereas one was as potent as any NPR-A activator known. In contrast to a previous report, we found that DNP binds the natriuretic peptide clearance receptor (NPR-C). However, none of the chimeric peptides bound NPR-C with significantly higher affinity than endogenous ligands. We suggest that bifunctional chimeric peptides represent a new generation of natriuretic peptide therapeutics.     

Cenderitide-Eluting Film for Potential Cardiac Patch Applications

Cenderitide, also known as CD-NP, is a designer peptide developed by combining native mammalian c-type natriuretic peptide (CNP) and the C-terminus isolated from the dendroapis natriuretic peptide (DNP) of the venom from the green mamba. In early studies, intravenous and subcutaneous infusion of cenderitide was reported to reduce left ventricular (LV) mass and ameliorate cardiac remodelling. In this work, biodegradable polymeric films encapsulating CD-NP were developed and were investigated for their in vitro release and degradation characteristics. Subsequently, the bioactivity of released peptide and its effects on human cardiac fibroblast (HCF) were explored. We achieved sustained release from three films with low, intermediate and high release profiles for 30 days. Moreover, the bioactivity of released peptide was verified from the elevated production of cyclic guanosine monophospate (cGMP). The CD-NP released from films was able to inhibit the proliferation of hypertrophic HCF as well as suppress DNA synthesis in HCF. Furthermore, the sustained delivery from films showed comparable or superior suppressive actions on hypertrophic HCF compared to daily infusion of CD-NP. The results suggest that these films could be used to inhibit fibrosis and reduce cardiac remodelling via local delivery as cardiac patches.     

Actions of a novel synthetic natriuretic peptide on hemodynamics and ventricular function in the dog

Dendroaspis natriuretic peptide (DNP) is a recently discovered peptide with structural similarity to known natriuretic peptides. DNP has been shown to possess potent renal actions. Our objectives were to define the acute hemodynamic actions of DNP in normal anesthetized dogs and the acute effects of DNP on left ventricular (LV) function in conscious chronically instrumented dogs. In anesthetized dogs, DNP, but not placebo, decreased mean arterial pressure (141 +/- 6 to 109 +/- 7 mmHg, P < 0.05) and pulmonary capillary wedge pressure (5.8 +/- 0.3 to 3.4 +/- 0.2 mmHg, P < 0.05). Cardiac output decreased and systemic vascular resistance increased with DNP and placebo. DNP-like immunoreactivity and guanosine 3',5'-cyclic monophosphate concentration increased without changes in other natriuretic peptides. In conscious dogs, DNP decreased LV end-systolic pressure (120 +/- 7 to 102 +/- 6 mmHg, P < 0.05) and volume (32 +/- 6 to 28 +/- 6 ml, P < 0.05) and LV end-diastolic volume (38 +/- 5 to 31 +/- 4 ml, P < 0.05) but not arterial elastance. LV end-systolic elastance increased (6.1 +/- 0.7 to 7.4 +/- 0.6 mmHg/ml, P < 0.05), and Tau decreased (31 +/- 2 to 27 +/- 1 ms, P < 0.05). The effects on hemodynamics, LV function, and second messenger generation suggest synthetic DNP may have a role as a cardiac unloading and lusitropic peptide.     

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.     

Adipocyte-derived factors impair insulin signaling in differentiated human vascular smooth muscle cells via the upregulation of miR-143

Cardiovascular complications are common in patients with type 2 diabetes. Adipokines have been implicated in the induction of proliferative and pro-atherogenic alterations in human vascular smooth muscle cells (hVSMC). Other reports demonstrated the importance of the miRNA cluster miR-143/145 in the regulation of VSMC homeostasis and insulin sensitivity. Here we investigated whether the detrimental effects of adipokines on hVSMC function could be ascribed to alterations in miR-143/145 expression. The exposure of hVSMC to conditioned media (CM) from primary human subcutaneous adipocytes increased the expression of smooth muscle α-actin (SMA), and the miR-143/145 cluster, but markedly impaired the insulin-mediated phosphorylation of Akt and its substrate endothelial nitric oxide synthase (eNOS). Furthermore, CM promoted the phosphorylation of SMAD2 and p38, which have both been linked to miR-143/145 induction. Accordingly, the induction of miR-143/145 as well as the inhibition of insulin-mediated Akt- and eNOS-phosphorylation was prevented when hVSMC were treated with pharmacological inhibitors for Alk-4/5/7 and p38 before the addition of CM. The transfection of hVSMC with precursor miR-143, but not with precursor miR-145, resulted in impaired insulin-mediated phosphorylation of Akt and eNOS. This inhibition of insulin signaling by CM and miR-143 is associated with a reduction in the expression of the oxysterol-binding protein-related protein 8 (ORP8). Finally, the knock-down of ORP8 resulted in impaired insulin-mediated phosphorylation of Akt in hVSMC. Thus, the detrimental effects of adipocyte-derived conditioned media on insulin action in primary hVSMC can be ascribed to the Alk- and p38-dependent induction of miR-143 and subsequent downregulation of ORP8.     

Oleic acid and adipokines synergize in inducing proliferation and inflammatory signalling in human vascular smooth muscle cells

In the context of obesity, perivascular fat produces various adipokines and releases free fatty acids, which may induce inflammation and proliferation in the vascular wall. In this study we investigated how adipokines, oleic acid (OA) and the combined treatment regulate human vascular smooth muscle cell (hVSMC) proliferation and migration and the underlying signalling pathways. Adipocyte‐conditioned media (CM) generated from human adipocytes induces a prominent proliferation and migration of hVSMC. Autocrine action of adiponectin totally abolishes CM‐induced proliferation. Furthermore, OA but not palmitic acid induces proliferation of hVSMC. CM itself does not contain fatty acids, but CM in combination with OA markedly enhances proliferation of hVSMC in a synergistic way. Both the nuclear factor (NF)‐κB and the mammalian target of rapamycin (mTOR) pathway were synergistically activated under these conditions and found to be essential for hVSMC proliferation. Expression of iNOS and production of nitric oxide was only enhanced by combined treatment inducing a marked release of VEGF. Combination of OA and VEGF induces an additive increase of hVSMC proliferation. We could show that the combination of CM and OA led to a synergistic proliferation of hVSMC. Expression of iNOS and production of nitric oxide were only enhanced under these conditions and were paralleled by a marked release of VEGF. These results suggest that the combined elevated release of fatty acids and adipokines by adipose tissue in obesity might be critically related to hVSMC dysfunction, vascular inflammation and the development of atherosclerosis.     

Ectopic expression of active processed form of atrial natriuretic peptide in skeletal myoblasts

Atrial natriuretic peptide (ANP) is a cardiac hormone that elicits a profound diuresis, natriuresis, and hypotension. As a preliminary study toward ANP gene therapy of cardiovascular disorders, we have cloned a cDNA for mouse preproANP and carried out expression studies in muscle cells. The expression cassette, which was flanked by ITRs from AAV-2, consisted of HCMV IE enhancer/promoter, preproANP gene, and polyadenylation signal from bovine growth hormone. We transfected this expression vector into primary skeletal myoblasts and examined the following points: (1) secretion of immunoreactive ANP, (2) biological activity, and (3) nature of secreted ANP(s). The conditioned media from cells transfected with ANP vector had significantly higher levels of irANP in comparison to mock control. The secreted irANP had biological activity as confirmed by the elevated level of intracellular cGMP in human umbilical vein endothelial cells. Reverse-phase HPLC analysis showed that the processed form of ANP was the predominant form. These results demonstrate that preproANP gene could be ectopically expressed and correctly processed in skeletal myoblasts, which has implications for development of muscle-based ANP gene therapy.     

Renal C-type natriuretic peptide and natriuretic peptide receptor B mRNA expression are affected by water deprivation in the Spinifex Hopping mouse

This study investigated the effect of water deprivation on the expression of C-type natriuretic peptide (CNP) and natriuretic peptide receptor B (NPR-B) mRNA, and the ability of NPR-B to generate cGMP in the Spinifex Hopping mouse, Notomys alexis. This rodent is a native of central and western Australia that is well adapted to survive in arid environments. Initially, CNP and NPR-B cDNAs (partial for NPR-B) were cloned and sequenced, and were shown to have high homology with those of rat and mouse. RT-PCR analysis showed CNP mRNA expression in the kidney, proximal and distal colon and small intestine, whilst NPR-B mRNA expression was found in the kidney, proximal and distal colon and the atria. Using a semi-quantitative multiplex PCR technique, the expression of renal CNP and NPR-B mRNA was determined in 7- and 14-day water-deprived hopping mice, in parallel with control hopping mice (access to water). Water deprivation significantly decreased the relative levels of CNP and NPR-B mRNA expression in both the 7- and 14-day water-deprived hopping mice, when compared to control hopping mice. In contrast, the ability of CNP to stimulate cGMP production was significantly increased after 14 days of water deprivation. This study shows that alterations in the renal CNP/NPR-B system may be an important physiological adjustment when water is scarce.     

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.     

Dendroaspis natriuretic peptide is the most potent natriuretic peptide to cause relaxation of lower esophageal sphincter

Atrial natriuretic peptide (ANP) causes relaxation in the opossum lower esophageal sphincter. The effects of dendroaspis natriuretic peptide (DNP) and other natriuretic peptides in the lower esophageal sphincter were not known. We measured the relaxation of transverse strips from the guinea pig lower esophageal sphincter caused by DNP, ANP, brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and a natriuretic peptide receptor-C agonist des[Gln(18), Ser(19), Gly(20), Leu(21), Gly(22)]ANP(4-23) amide (cANF(4-23)) in vitro. In resting strips of the guinea pig lower esophageal sphincter DNP and BNP caused marked relaxations. Furthermore, in both sarafotoxin S6c and carbachol-contracted lower esophageal sphincter strips, DNP caused marked and BNP caused moderate, concentration-dependent relaxations. ANP as well as CNP caused mild relaxations. In contrast, cANF(4-23) did not cause relaxation. The relative potencies for natriuretic peptides to cause relaxation were DNP>BNP>ANP>=CNP in both sarafotoxin S6c and carbachol-contracted lower esophageal sphincter strips. The DNP and BNP-induced relaxations were not affected by tetrodotoxin or atropine, suggesting that the natriuretic peptide-induced response was not neutrally mediated. In conclusion, these results demonstrate that natriuretic peptides cause the relaxation of the guinea pig lower esophageal sphincter. DNP is the most potent natriuretic peptide to cause lower esophageal sphincter relaxation, which might be mediated by natriuretic peptide receptor-A or a novel DNP-selective natriuretic peptide receptor.