N-terminally extended form of C-type natriuretic peptide (CNP-53) identified in porcine brain

C-type natriuretic peptide of 22 residues (CNP-22) is very recently identified in porcine brain as a third member of the mammalian natriuretic peptide family (1). Using a radioimmunoassay system newly established for CNP-22, we searched for CNP-related peptides in porcine brain. In addition to CNP-22, one major form of immunoreactive CNP was detected in porcine brain extracts, being isolated by immunoaffinity chromatography and reverse phase high performance liquid chromatography. By microsequence analysis, the peptide was deduced to be a 53-amino acid peptide carrying a CNP-22 sequence at the C-terminus, and was designated C-type natriuretic peptide-53 (CNP-53). CNP-53 was found to be a major molecular form of CNP in porcine brain.     

C-type natriuretic peptide (CNP) signal peptide fragments are present in the human circulation

Background

Signal peptides may be novel biomarkers in cardiovascular diseases.

Methods

We developed a novel immunoassay to the signal peptide of preproCNP (CNPsp) and used this to document circulating venous concentrations of CNPsp in normal healthy volunteers (n = 109), regional plasma CNPsp concentrations in patients undergoing clinically indicated catheterisation (n = 24) and temporal CNPsp concentrations in patients with ST-elevation myocardial infarction (STEMI) <4 h after symptom onset (n = 8). The structure/sequence of circulating CNPsp was confirmed by tandem mass spectrometry (MS/MS).

Results

In normal human plasma, CNPsp was detectable at levels higher than NT-proCNP (74 ± 17 vs. 20 ± 5.5 pmol/L). There was no correlation between NTproCNP and CNPsp, but plasma concentrations of sibling signal peptides – CNPsp and BNPsp – were strongly correlated (r = 0.532, P < 0.001). In patients undergoing catheterisation, there were significant arterio-venous step-ups in CNPsp concentrations across the heart (P < 0.01) and kidney (P < 0.01). Arterial concentrations of CNPsp significantly correlated with heart rate (r = 0.446, P < 0.05). In STEMI patients, plasma concentrations of CNPsp showed a biphasic elevation pattern between 6 and 12 h after symptom onset, with 12 h values significantly elevated (∼3-fold) compared with levels at presentation (P < 0.05). MS/MS verified circulating CNPsp to be preproCNP(14–23) and preproCNP(16–23) peptides.

Conclusions

This is the first report of a circulating preproCNP derived signal peptide. Given the clear cardiac and renal secretion profiles of CNPsp and its response in STEMI patients, further studies on potential biological functions and biomarker applications of CNPsp in cardiovascular disease are warranted.     

Reduced ability of C-type natriuretic peptide (CNP) to activate natriuretic peptide receptor B (NPR-B) causes dwarfism in lbab -/- mice

C-type natriuretic peptide (CNP) stimulates endochondrial ossification by activating the transmembrane guanylyl cyclase, natriuretic peptide receptor-B (NPR-B). Recently, a spontaneous autosomal recessive mutation that causes severe dwarfism in mice was identified. The mutant, called long bone abnormality (lbab), contains a single point mutation that converts an arginine to a glycine in a conserved coding region of the CNP gene, but how this mutation affects CNP activity has not been reported. Here, we determined that 30-fold to greater than 100-fold more CNP(lbab) was required to activate NPR-B as compared to wild-type CNP in whole cell cGMP elevation and membrane guanylyl cyclase assays. The reduced ability of CNP(lbab) to activate NPR-B was explained, at least in part, by decreased binding since 10-fold more CNP(lbab) than wild-type CNP was required to compete with [(125)I][Tyr(0)]CNP for receptor binding. Molecular modeling suggested that the conserved arginine is critical for binding to an equally conserved acidic pocket in NPR-B. These results indicate that reduced binding to and activation of NPR-B causes dwarfism in lbab(-/-) mice.     

Brain natriuretic peptide-32: N-terminal six amino acid extended form of brain natriuretic peptide identified in porcine brain

Brain natriuretic peptide (BNP) is a newly identified peptide of 26 residues, which has a remarkable homology to but is distinct from atrial natriuretic peptide. The peptide exerts natriuretic-diuretic activity as well as potent chick rectum relaxant activity. By using radioimmunoassay specific to BNP and immunoaffinity chromatography, we have isolated from porcine brain a novel peptide of 32 residues carrying a BNP structure at the C-terminus. The amino acid sequence of this peptide was determined to be: Ser-Pro-Lys-Thr-Met- Arg-Asp-Ser-Gly-Cys-Phe-Gly-Arg-Arg-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly- Leu- Gly-Cys-Asn-Val-Leu-Arg-Arg-Tyr. This peptide is an N-terminal six amino acid extended form of BNP and henceforth is designated BNP-32. BNP and BNP-32 are found to be major forms of BNP family in porcine brain.     

Cloning and sequence analysis of a cDNA encoding a precursor for rat C-type natriuretic peptide (CNP).

Recent identification of a C-type natriuretic peptide (CNP) in porcine brain strongly suggested that a third member of the natriuretic peptide family still remains to be identified in other species of mammals. A cDNA encoding a precursor for rat CNP was cloned from a rat brain cDNA library and sequenced. The precursor was a 126-residue peptide, carrying a 23-residue signal sequence at the N-terminus and the known porcine CNP-53 sequence at the C-terminus. By RNA blot analysis, rat CNP mRNA was found to be expressed exclusively in the brain, implying that CNP may function in the central nervous system as a neuropeptide.     

C-type natriuretic peptide: A new cardiac mediator

Natriuretic peptides are endogenous hormones released by the heart in response to myocardial stretch and overload. While atrial and brain natriuretic peptides (ANP, BNP) were immediately considered cardiac hormones and their role was well-characterized and defined in predicting risk in cardiovascular disease, evidence indicating the role of C-type natriuretic peptide (CNP) in cardiovascular regulation was slow to emerge until about 8 years ago. Since then, considerable literature on CNP and the cardiovascular system has been published; the aim of this review is to examine current literature relating to CNP and cardiovascular disease, in particular its role in heart failure (HF) and myocardial infarction (MI). This review retraces the fundamental steps in research that led understanding the role of CNP in HF and MI; from increased CNP mRNA expression and plasmatic concentrations in humans and in animal models, to detection of CNP expression in cardiomyocytes, to its evaluation in human leukocytes. The traditional view of CNP as an endothelial peptide has been surpassed by the results of many studies published in recent years, and while its physiological role is still under investigation, information is now available regarding its contribution to cardiovascular function. Taken together, these observations suggest that CNP and its specific receptor, NPR-B, can play a very important role in regulating cardiac hypertrophy and remodeling, indicating NPR-B as a new potential drug target for the treatment of cardiovascular disease.     

Gene and precursor structure of porcine C-type natriuretic peptide

Recently we isolated from porcine brain two related peptides, a 22-residue peptide (CNP-22) and its N-terminally elongated peptide (CNP-53; 53-residue), which belong to the third type of mammalian natriuretic peptide designated C-type natriuretic peptide family (CNP) (1,2). To elucidate the structure of their precursor form, we have now isolated the gene for this porcine CNP and prepared its cDNA from COS-1 cells transfected with the gene. Nucleotide sequence analyses have revealed that the gene consists of a least two exons and an intron and encodes the 126-residue CNP precursor (porcine prepro-CNP), in which a putative signal peptide and the CNP-53 sequence are located at the N- and C-terminus, respectively. The C-terminal cysteine codon of CNP-53 is directly followed by a termination codon, indicating that the C-terminus of porcine CNP is generated per se.     

Central and peripheral forms of C-type natriuretic peptide (CNP): evidence for differential regulation in plasma and cerebrospinal fluid

Aminoterminal proCNP (NTproCNP), a stable product of CNP gene expression and readily measured in human plasma, provides a new approach to studies of CNP which is rapidly degraded at source. CNP is detectable in human CSF but the presence and proportions of NTproCNP in CSF are unknown. Since CNP is widely expressed throughout the CNS, we hypothesized that the ratio of NTproCNP to CNP in CSF is greatly increased when compared to plasma and that CSF CNP peptides may contribute to their concentrations in the systemic circulation. Concurrent plasma and CSF concentrations of CNP forms were measured in 51 subjects undergoing spinal anesthesia for arranged orthopedic procedures. Elevated concentrations of NTproCNP (1045 ± 359 pmol/L), characterized by HPLC-RIA, were found in CSF and greatly exceeded those of CNP (7.9 ± 3.2 pmol/L). The ratio of NTproCNP to CNP in CSF (145 ± 55) was much higher than in plasma (31 ± 27). A significant inverse relation was found between plasma and CSF CNP concentrations (r = −0.29, p < 0.05). cGMP and neprilysin were unrelated to CNP levels in CSF. We conclude that CNP is differentially regulated across the brain in normal health. Despite markedly elevated levels of NTproCNP in CSF, it is unlikely that these contribute to systemic levels in healthy adults. Identifying NTproCNP as the dominant CNP form in CSF opens up the possibility of its use in future studies exploring CNP regulation within the CNS and possible applications in the diagnosis and monitoring of subjects with central neural disorders.     

Distribution and characterization of immunoreactive porcine C-type natriuretic peptide.

C-type natriuretic peptide (CNP) is a new member of the natriuretic peptide family recently identified in porcine brain (1). We raised an antiserum against porcine CNP and set up a radioimmunoassay (RIA) for CNP. Using this RIA system, distribution of immunoreactive (ir-) CNP in porcine tissue was measured and compared with that of ir-atrial natriuretic peptide (ANP) and ir-brain natriuretic peptide (BNP). Tissue concentration of ir-CNP in brain was the highest of the three natriuretic peptides at about 0.79 pmol/g wet wt. CNP was present in medulla-pons in high concentration, with a significant concentration detected in cerebellum. In contrast, ir-CNP was not detected in peripheral tissue, including heart, in a significant concentration. These data demonstrated sharp contrasts in the distribution of the three natriuretic peptides, suggesting that CNP is a natriuretic peptide functioning in the central nervous system.     

Isolation and identification of C-type natriuretic peptide in chicken brain

C-type natriuretic peptide (CNP) has recently been identified in porcine brain as a third member of the mammalian natriuretic peptide family (1). Using a radioimmunoassay system for porcine CNP, we found a significant concentration of immunoreactive (ir-) CNP in chicken brain, from which a new peptide was isolated. By microsequence analysis, the amino acid sequence of the peptide was determined to be Gly-Leu-Ser-Arg-Ser-Cys-Phe- Gly-Val-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Met-Ser-Gly-Leu-Gly-Cys. Based on its high homology to porcine CNP, the peptide was designated chicken C-type natriuretic peptide. Chicken CNP also elicits pharmacological effects highly similar to porcine CNP, suggesting that CNP functions as a neuropeptide in the chicken central nervous system.     

Cloning of a cDNA encoding porcine brain natriuretic peptide

Complimentary DNA (cDNA) clones encoding porcine brain natriuretic peptide (BNP) were isolated from a porcine atrial cDNA library. The longest of the cDNA clones (1507 nucleotides) apparently originated from an unprocessed messenger RNA, since the nucleotide sequence encoding BNP-26 was interrupted by an intron of 554 nucleotides. A partial cDNA clone representing processed BNP mRNA was prepared by polymerase chain reaction. A comparison of the sequence of these two cDNAs reveals the presence of an additional intron within the sequence encoding the BNP precursor. The identification of these introns suggests that the BNP gene structure differs from the atrial natriuretic peptide gene in the location of intron 2. BNP mRNA encodes a propeptide of 131 amino acids, including a signal peptide domain (25 amino acids) and a prohormone domain (106 amino acids). Like atrial natriuretic peptide, the bioactive BNP sequence is localized at the carboxyl terminus of the prohormone. Although the carboxyl-terminal peptide sequences of porcine atrial natriuretic peptide and BNP are well conserved, there is relatively little homology within their propeptide regions.     

C-type natriuretic peptide: new candidate for endothelium-derived hyperpolarising factor

Endothelium-derived hyperpolarising factor (EDHF) is an important regulator of vascular tone; however, its identity is still unclear. Several different molecules have been suggested, the most recent of which is the 22-amino acid peptide C-type natriuretic peptide (CNP). CNP induces hyperpolarisation and relaxation of rat mesenteric resistance artery vascular smooth muscle through activation of natriuretic peptide receptor subtype C (NPR-C) and the same potassium channels as EDHF. In addition, this peptide is released from endothelial cells of the perfused rat mesenteric bed in response to endothelium-dependent vasodilators. Thus, CNP is likely to play a vital role in regulation of vascular tone. In addition, since there is evidence that up-regulation of EDHF occurs where normal endothelium function has been compromised, modulation of this pathway represents a novel target for therapeutics in the treatment of inflammatory cardiovascular pathologies characterised by endothelial dysfunction.     

mRNA quantification of C-type natriuretic peptide in brain areas of rodents

C-type natriuretic peptide (CNP) is regarded as an endothelium-derived vasodilator and might therefore have an important role in controlling vascular tone and remodeling. Because CNP also is expressed in the brain, it is considered to be a neurotransmitter. The present study compares expression levels of CNP mRNA in distinct areas of the mouse brain with the expression pattern in the rat brain. A distinct expression of CNP was found in all investigated areas with the exception of the mouse striatum. In both rodents, high CNP expression was detected in the tegmentum.     

Characterization of immunoreactive human C-type natriuretic peptide in brain and heart.

Amino acid sequence of human C-type natriuretic peptide (CNP) has recently been deduced to be identical to those of porcine and rat CNPs in the bioactive unit of C-terminal 22 residues (CNP-22) (1). Thus, tissue concentrations and molecular forms of immunoreactive (ir-) CNP in human brain and heart were determined or characterized using a radioimmunoassay established for porcine CNP. In human brain (hypothalamus and medullapons), ir-CNP was detected at a concentration of 1.04 pmol/g, being about 25 times or 70 times higher than ir-atrial (A-type) natriuretic peptide (ANP) or ir-brain (B-type) natriuretic peptide (BNP). CNP was present mainly as CNP-53, with CNP-22 as well as 13K CNP (presumed to be pro-CNP) as minor components. In heart, 1 approximately 5 pmol/g of ir-CNP was detected in both atrium and ventricle, but this ir-CNP was shown to be derived from crossreactivity of ANP. These results demonstrated that human CNP functions exclusively in the central nervous system in contrast to ANP and BNP which mainly function in the circulation system.     

An insect brain peptide as a member of insulin family

Amino acid sequencing of bombyxin (previously called 4K-PTTH) isolated from the heads of the silkmoth Bombyx mori has disclosed sequence homology of this insect neuropeptide with insulin. Immunohistochemistry using an antibody against a synthetic bombyxin fragment detected 4 pairs of immunoreactive neurosecretory cells in the dorso-medial region of the Bombyx brain. The same cells were reactive to bovine insulin antibody.     

Regulation of C-type natriuretic peptide expression

C-type natriuretic peptide (CNP) is a member of the small family of natriuretic peptides that also includes atrial natriuretic peptide (ANP) and brain, or B-type natriuretic peptide (BNP). Unlike them, it performs its major functions in an autocrine or paracrine manner. Those functions, mediated through binding to the membrane guanylyl cyclase natriuretic peptide receptor B (NPR-B), or by signaling through the non-enzyme natriuretic peptide receptor C (NPR-C), include the regulation of endochondral ossification, reproduction, nervous system development, and the maintenance of cardiovascular health. To date, the regulation of CNP gene expression has not received the attention that has been paid to regulation of the ANP and BNP genes. CNP expression in vitro is regulated by TGF-β and receptor tyrosine kinase growth factors in a cell/tissue-specific and sometimes species-specific manner. Expression of CNP in vivo is altered in diseased organs and tissues, including atherosclerotic vessels, and the myocardium of failing hearts. Analysis of the human CNP gene has led to the identification of a number of regulatory sites in the proximal promoter, including a GC-rich region approximately 50 base pairs downstream of the Tata box, and shown to be a binding site for several putative regulatory proteins, including transforming growth factor clone 22 domain 1 (TSC22D1) and a serine threonine kinase (STK16). The purpose of this review is to summarize the current literature on the regulation of CNP expression, emphasizing in particular the putative regulatory elements in the CNP gene and the potential DNA-binding proteins that associate with them.     

C-type natriuretic peptide system in rabbit colon

C-type natriuretic peptide (CNP) is mainly distributed in the brain and vascular endothelium and is considered to act as a local regulator in many tissues. The present study was aimed to determine the presence of CNP system and its biological function in rabbit colon. The serial dilution curves of tissue extracts were parallel to the standard curve of CNP-22. With gel permeation chromatography and reverse-phase HPLC, the major immunoreactive peak of CNP was observed at the same elution time corresponding to the synthetic CNP-53. The concentration of CNP in the mucosal layer of colon was 212.49 ± 30.44 pg/g tissue wet weight (n = 7), which was significantly higher than that in the muscular layer. The presence of CNP mRNA was also detected by RT-PCR and Southern blot analysis. Production of cGMP by the activation of particulate guanylyl cyclase stimulated by BNP and CNP was higher in membranes obtained from the muscular layer than from mucosal layer. More cGMP was produced by CNP than by ANP. Both natriuretic peptide receptor-A and -B mRNAs were detected by RT-PCR and specific binding sites to ¹²⁵I-[Tyr⁰]-CNP-22 were mainly localized to the muscular layer. Synthetic CNP inhibited basal tension, frequency and amplitude of basal motility of taenia coli of the right colon. This study showing the presence of CNP system and its biological function in colon suggests that endogenous CNP synthesized in the mucosal layer may have a paracrine function as a local regulator of colonic motility.     

C-type natriuretic peptide (CNP) effects on intracellular calcium [Ca2+]i in mouse gonadotrope-derived alphaT3-1 cell line.

C-type natriuretic peptide (CNP), the third member of the atrial natriuretic peptide family, acts via guanylyl cyclase containing GC-B receptors to stimulate cyclic guanosine 3',5' monophosphate (cGMP) accumulation in the gonadotrope-derived alphaT3-1 cell line and rat pituitary cells. This effect is inhibited by concomitant activation of the phospholipase C (PLC)-coupled gonadotrophin hormone-releasing hormone (GnRH) receptors in these cells. Since GnRH stimulates gonadotrophin secretion from gonadotropes by increasing the cytosolic Ca2+ concentration ([Ca2+]i) and natriuretic peptides have been found to influence PLC/Ca2+ signalling in other systems, we have investigated whether CNP can alter basal or GnRH-stimulated changes in [Ca2+]i in alphaT3-1 cells. In Ca 2+-containing medium, 10(-7) M CNP modestly, but significantly increased [Ca2+]i over several min, but subsequently inhibited the elevation of [Ca2+]i in response to 10(-7) M GnRH in both Ca2+-containing and Ca2+-free medium. This inhibitory effect was mimicked by 10(-6) M 8-Br-cGMP, but not by ANP, indicating mediation by cyclic GMP and the CNP-specific GC-B receptor. However, basal and GnRH-stimulated inositol (1,4,5) trisphosphate (Ins(1,4,5)P3) generation were not measurably affected by CNP, and CNP failed to affect thapsigargin-induced capacitative Ca2+ entry. Thus, it appears that the cross-talk between CNP and GnRH in these cells is reciprocal in that GnRH modulates CNP effects on cGMP generation, whereas, CNP modulates GnRH effects on Ca2+ mobilisation.     

Human bone marrow endothelial cells: a new identified source of B-type natriuretic peptide

B-type natriuretic peptide (BNP) is a hormone mainly secreted by cardiac ventricle myocytes and which is increased in cardiac diseases. Moreover, BNP expression has been shown in various cell/tissue types. Six different human endothelial cell (EC) culture models arising from macro and microcirculation either primary cultures or cell lines were cultured and screened for BNP presence and secretion. All cell types expressed BNP mRNA while only the ECs arising from bone marrow stromal compartment secreted high amounts of BNP protein. This report is the first to identify ECs as a new source of BNP. However, BNP secretion is limited to a particular EC type.