BACKGROUND

Kraits (genus Bungarus) and cobras (genus Naja) are two representative toxic genera of elapids in the old world. Although they are closely related genera and both of their venoms are very toxic, the compositions of their venoms are very different. To unveil their detailed venoms and their evolutionary patterns, we constructed venom gland cDNA libraries and genomic bacterial artificial chromosome (BAC) libraries for Bungarus multicinctus and Naja atra, respectively. We sequenced about 1500 cDNA clones for each of the venom cDNA libraries and screened BAC libraries of the two snakes by blot analysis using four kinds of toxin probes; i.e., three-finger toxin (3FTx), phospholipase A2 (PLA2), kunitz-type protease inhibitor (Kunitz), and natriuretic peptide (NP).

RESULTS

In total, 1092 valid expressed sequences tags (ESTs) for B. multicinctus and 1166 ESTs for N. atra were generated. About 70% of these ESTs can be annotated as snake toxin transcripts. 3FTx (64.5%) and β bungarotoxin (25.1%) comprise the main toxin classes in B. multicinctus, while 3FTx (95.8%) is the dominant toxin in N. atra. We also observed several less abundant venom families in B. multicinctus and N. atra, such as PLA2, C-type lectins, and Kunitz. Peculiarly a cluster of NP precursors with tandem NPs was detected in B. multicinctus. A total of 71 positive toxin BAC clones in B. multicinctus and N. atra were identified using four kinds of toxin probes (3FTx, PLA2, Kunitz, and NP), among which 39 3FTx-positive BACs were sequenced to reveal gene structures of 3FTx toxin genes.

CONCLUSIONS

Based on the toxin ESTs and 3FTx gene sequences, the major components of B. multicinctus venom transcriptome are neurotoxins, including long chain alpha neurotoxins (α-ntx) and the recently originated β bungarotoxin, whereas the N. atra venom transcriptome mainly contains 3FTxs with cytotoxicity and neurotoxicity (short chain α-ntx). The data also revealed that tandem duplications contributed the most to the expansion of toxin multigene families. Analysis of nonsynonymous to synonymous nucleotide substitution rate ratios (dN/dS) indicates that not only multigene toxin families but also other less abundant toxins might have been under rapid diversifying evolution.

OBJECTIVE

The purpose of this study was to investigate circulating pro-B-type natriuretic peptide (proBNP(1-108)) in the general community and evaluate its ability to detect left ventricular (LV) dysfunction.

BACKGROUND

The current concept for cardiac endocrine function is that, in response to cardiac stress, the heart secretes B-type natriuretic peptide (BNP(1-32)) and amino-terminal pro-B-type natriuretic peptide (NT-proBNP(1-76)) after intracardiac cleavage of their molecular precursor, proBNP(1-108). We hypothesized that proBNP(1-108) circulates in normal human subjects and that it is a useful biomarker for LV dysfunction.

METHODS

Our population-based study included a cohort of 1,939 adults (age ≥45 years) from Olmsted County, Minnesota, with 672 participants defined as healthy. Subjects underwent in-depth clinical characterization, detailed echocardiography, and measurement of proBNP(1-108). Independent factors associated with proBNP(1-108) and test characteristics for the detection of LV dysfunction were determined.

RESULTS

ProBNP(1-108) in normal humans was strongly influenced by sex, age, heart rate, and body mass index. The median concentration was 20 ng/l with a mean proBNP(1-108) to NT-proBNP(1-76) ratio of 0.366, which decreased with heart failure stage. ProBNP(1-108) was a sensitive (78.8%) and specific (86.1%) biomarker for detecting LV systolic dysfunction, which was comparable to BNP(1-32), but less than NT-proBNP(1-76), in several subsets of the population.

CONCLUSIONS

ProBNP(1-108) circulates in the majority of healthy humans in the general population and is a sensitive and specific biomarker for the detection of systolic dysfunction. The proBNP(1-108) to NT-proBNP(1-76) ratio may provide insights into altered proBNP(1-108) processing during heart failure progression. Thus, this highly specific assay for proBNP(1-108) provides important new insights into the biology of the BNP system.

OBJECTIVE

The importance of B-type natriuretic peptide (BNP) as a diagnostic and therapeutic modality in cardiovascular disease is well known. BNP levels correlate clinical and physiologic characteristics, as well as outcomes. We sought to investigate the influence of BNP pathway genetic variation on BNP levels after adjustment for clinical/physiologic factors.

METHODS

DNA was extracted from 147 patients undergoing elective cardiac catheterization. Patients with elevated troponin were excluded. Detailed clinical data was collected including standard demographic, laboratory, echocardiographic and catheterization data. Genotype was determined at 19 loci in five genes relevant to the BNP pathway. Multivariable linear regression models of logBNP, adjusted for clinical variables, were used to assess the incremental influence of the genetic variants.

RESULTS

Natriuretic peptide precursor B gene (NPPB) variants incrementally improved models of logBNP after inclusion of clinical/physiologic parameters. The NPPB -381 T>> C genotype was significantly associated with logBNP in the model (p = 0.0005), with the model predicting 50% lower BNP levels in otherwise similar T/T vs. C/C subjects. The NPPB 777 G>> A (3' flanking region) genotype was of borderline significance (p = 0.0078). None of the other genotypes examined were significant (all p>> 0.2).

CONCLUSIONS

Genetic variation in NPPB significantly impacts BNP levels after adjustment for clinical/physiologic factors. The full linear regression model predicted up to a 50% relative difference in BNP levels between NPPB -381 T>> C genotype groups. This suggests that NPPB sequence variants affect BNP physiology, possibly via transcriptional regulation. Further studies are needed to define whether these variants impact the clinical interpretation of BNP levels.

Atrial natriuretic polypeptides (ANPs) of varying chain length have been identified recently in human and rat atrial tissue. Their potent natriuretic-diuretic activities indicate their key role in the regulation of extracellular fluid volume and electrolyte balance. Furthermore, human and rat cDNAs encoding their precursor have been cloned and identified. Natriuretic-diuretic activity in human atrial extract comprises three distinct components (alpha, relative molecular mass (Mr) approximately 3,000; beta, Mr approximately 6,000; gamma, Mr approximately 13,000). However, only the 3,000-Mr peptide, alpha-human atrial polypeptide (alpha-hANP), comprising 28 amino acids, has so far been identified. We report here the purification and sequence analysis of two novel hANPs of higher Mr, beta- and gamma-hANP, both of which exhibit natriuretic and hypotensive activity. gamma-hANP, composed of 126 amino acids, carries the alpha-hANP sequence at its carboxy terminus. The identification of gamma-hANP reveals that the peptide, being the largest form of hANP, is processed directly from a 151-residue precursor by removal of a 26-residue signal peptide. In contrast, beta-hANP (56 residues) comprises an anti-parallel dimer of alpha-hANP; such a dimeric peptide possessing bioactivity has never been found in the tissue as an endogenous entity.

Using a synthetic common carboxy-terminal fragment of alpha-human atrial natriuretic polypeptide (alpha-hANP) and alpha-rat atrial natriuretic polypeptide (alpha-rANP), we have produced an antiserum for alpha-ANP(17-28) and established a radioimmunoassay (RIA) for alpha-ANP that recognizes alpha-hANP and alpha-rANP equally. High performance gel permeation chromatography coupled with the RIA revealed that alpha-hANP-like immunoreactivity (alpha-hANP-LI) in the human atrium consists of three major components, gamma-hANP, alpha-hANP and another. On the other hand, alpha-rANP-LI in the rat atrium comprised at least two components, 13K alpha-rANP-LI and 3K-5K alpha-rANP-LI, which were presumably gamma-rANP and beta-rANP, respectively. Thus, considerable amounts of gamma-hANP and gamma-rANP are present in human right auricles and rat atria, respectively. The RIA established in this study provides a useful tool to investigate the pathophysiological significance of alpha-ANP and related peptides in cardiovascular disorders and shows that gamma-ANP is not only a precursor of alpha-ANP but acts as an important hormone.

In the normal heart, the endocrine capacity resides in the atria. Atrial myocytes express and secrete natriuretic hormones that regulate fluid homeostasis and blood pressure. But in ventricular disease, atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) gene expression is also activated in ventricular myocytes. Plasma concentrations of natriuretic peptides and their biosynthetic precursors are accordingly increased in patients with marked ventricular dysfunction. In contrast, atrial peptide secretion in ventricular disease has received less attention, and our present understanding of the endocrine atria during ventricular dysfunction is still scarce. Although ventricular disease and increased circulating concentrations are associated, it does not entail that the ventricle is the sole or even the main source in all types of heart disease. Clearly, the endocrine atria are also active in heart failure. Plasma measurement of cardiac natriuretic peptides and their molecular precursors can perhaps help us to discriminate when, where and how.

BACKGROUND

This study was performed to gain insights into novel therapeutic approaches for the treatment of autoimmune myocarditis.

OBJECTIVE

Chemical stimulation of the efferent arm of the vagus nerve through activation of nicotinic acetylcholine receptor subtype-7α (α7-nAChR) has been shown to be protective in several models of inflammatory diseases. In the present study, we investigated the potentially protective effect of vagus nerve stimulation on myocarditis.

RESULTS

A/J mice were immunized with cardiac troponin I (TnI) to induce autoimmune myocarditis. Mice were exposed to drinking water that contained nicotine in different concentrations and for different time periods (for 3 days at 12.5 mg/L; 3 days at 125 mg/L; 21 days at 12.5 mg/L; and 21 days at 125 mg/L after first immunization). TnI-immunized mice with no pharmacological treatment showed extensive myocardial inflammation and fibrosis and significantly elevated levels of interleukin-6 and tumor necrosis factor-α. Furthermore, elevated levels of mRNA transcripts of proinflammatory chemokines (monocyte chemoattractant protein-1, macrophage inflammatory protein-1β, and RANTES) and chemokine receptors (CCR1, CCR2, and CCR5) were found. Oral nicotine administration reduced inflammation within the myocardium, decreased the production of interleukin-6 and tumor necrosis factor-α, and downregulated the expression of monocyte chemoattractant protein-1, macrophage inflammatory protein-1β, RANTES, CCR1, CCR2, and CCR5. In addition, nicotine treatment resulted in decreased expression of matrix metalloproteinase-14, natriuretic peptide precursor B, tissue inhibitor of metalloproteinase-1, and osteopontin, proteins that are commonly involved in heart failure. Finally, we found that nicotine reduced levels of pSTAT3 (phosphorylated signal transducer and activator of transcription 3) protein expression within the myocardium. Neostigmine treatment did not affect the progression of myocarditis.

CONCLUSIONS

We showed that activation of the cholinergic antiinflammatory pathway with nicotine reduces inflammation in autoimmune myocarditis. Our results may open new possibilities in the therapeutic management of autoimmune myocarditis.

OBJECTIVE

Dyspnea is a major symptom of both parenchymal lung disease and chronic heart failure. Underlying cardiac dysfunction can be assessed by measurement of cardiac-derived B-type natriuretic peptide or its precursor in plasma. However, no specific endocrine marker of the lung parenchyma has so far been identified. We therefore examined whether plasma concentrations of apelin, a novel inotropic hormone, is affected in patients with chronic parenchymal lung disease without cardiac dysfunction.

RESULTS

Patients with severe chronic parenchymal lung disease and normal cardiac function (n=53), idiopathic pulmonary hypertension with increased right ventricular pressure (n=10), and patients with severe left ventricular systolic dysfunction (n=22) were enrolled. Plasma apelin-36 and proBNP concentrations were measured with radioimmunoassays. While proBNP plasma concentrations were unaffected in chronic parenchymal lung disease patients compared to normal subjects, the apelin-36 concentration was reduced 3.3-fold (median 35 pmol/l (0-162 pmol/l) vs. 117 pmol/l (55-232 pmol/l), P<0.001). Moreover, the apelin-36 concentration was decreased in chronic heart failure patients (2.1-fold, P<0.01) and in patients with idiopathic pulmonary hypertension (4.0-fold, P<0.001). In contrast, the proBNP concentration was highly increased in both chronic heart failure and idiopathic pulmonary hypertension patients.

CONCLUSIONS

Plasma concentrations of apelin-36, a novel inotropic peptide, are decreased in patients with chronic parenchymal lung disease and preserved cardiac function. Combined measurement of apelin-36 and proBNP may be a new diagnostic approach in distinguishing pulmonary from cardiovascular causes of dyspnea.

Granules from rat atria were isolated by differential centrifugation and by a 53% (v/v) Percoll gradient after tissue homogenization in 0.25 M-sucrose/50 mM-Na2EDTA. About 40% of the immunoreactive ANF (atrial natriuretic factor) sedimented with the atrial granules during differential centrifugations. On the Percoll gradient, two distinct bands were observed. Cell debris, mitochondria, lysosomes, myofilaments and microsomes were mostly contained in the lightest-density (rho) (1.03-1.07 g/ml) fraction, as demonstrated by electron microscopy and by enzymic markers such as lactate dehydrogenase, monoamine oxidase, cytochrome c reductase, beta-glucuronidase and acid phosphatase. Atrial granules were mostly contained in the denser (rho 1.11-1.15 g/ml) band and were only slightly contaminated by lysosomes, as shown by beta-glucuronidase activity. Analysis of the ANF content in these isolated granules by h.p.l.c., amino acid composition and sequencing demonstrated that it was only the pro-ANF [ANF-(Asn1-Tyr126)-peptide]. The precursor was present in all granules, as demonstrated by immunocytochemistry. Since hormonal propeptides usually undergo intracellular processing, and the matured peptides are subsequently stored in the secretory granules, these results indicate that the processing pathway of ANF may be different from that of other hormonal peptides.

BACKGROUND

The blockade of beta-adrenergic receptors reduces both mortality and morbidity in patients with chronic heart failure, but the cellular mechanism remains unclear. Celiprolol, a selective beta(1)-blocker, was reported to stimulate the expression of endothelial NO synthase (eNOS) in the heart, and NO levels have been demonstrated to be related to myocardial hypertrophy and heart failure. Thus, we aimed to clarify whether celiprolol attenuates both myocardial hypertrophy and heart failure via the NO-signal pathway.

RESULTS

In rat neonatal cardiac myocytes, celiprolol inhibited protein synthesis stimulated by either isoproterenol or phenylephrine, which was partially suppressed by N(G)-nitro-L-arginine methyl ester (L-NAME). Four weeks after transverse aortic constriction (TAC) in C57BL/6 male mice, the ratio of heart weight to body weight (mg/g) (8.70+/-0.42 in TAC, 6.61+/-0.44 with celiprolol 100 mg x kg(-1) x d(-1) PO, P<0.01) and the ratio of lung weight to body weight (mg/g) (10.27+/-1.08 in TAC, 7.11+/-0.70 with celiprolol 100 mg x kg(-1) x d(-1) PO, P<0.05) were lower and LV fractional shortening was higher in the celiprolol-treated groups than in the TAC group. All of these improvements were blunted by L-NAME. Celiprolol treatment significantly increased myocardial eNOS and activated phosphorylation of eNOS. Myocardial mRNA levels of natriuretic peptide precursor type B and protein inhibitor of NO synthase, which were increased in the TAC mice, were decreased in the celiprolol-treated mice.

CONCLUSIONS

These findings indicated that celiprolol attenuates cardiac myocyte hypertrophy both in vitro and in vivo and halts the process leading from hypertrophy to heart failure. These effects are mediated by a selective <em>beta</em>1-adrenergic receptor blockade and NO-dependent pathway.

The natriuretic peptide family consists of three biologically active peptides: atrial natriuretic peptide (ANP), brain (or B-type) natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). Among these, ANP and BNP are secreted by the heart and act as cardiac hormones. Both ANP and BNP preferentially bind to natriuretic peptide receptor-A (NPR-A or guanylyl cyslase-A) and exert similar effects through increases in intracellular cyclic guanosine monophosphate (cGMP) within target tissues. Expression and secretion of ANP and BNP are stimulated by various factors and are regulated via multiple signaling pathways. Human ANP has three molecular forms, α-ANP, β-ANP, and proANP (or γ-ANP), with proANP predominating in healthy atrial tissue. During secretion proANP is proteolytically processed by corin, resulting in secretion of bioactive α-ANP into the peripheral circulation. ProANP and β-ANP are minor forms in the circulation but are increased in patients with heart failure. The human BNP precursor proBNP is proteolytically processed to BNP1-32 and N-terminal proBNP (NT-proBNP) within ventricular myocytes. Uncleaved proBNP as well as mature BNP1-32 and NT-proBNP is secreted from the heart, and its secretion is increased in patients with heart failure. Mature BNP, its metabolites including BNP3-32, BNP4-32, and BNP5-32, and proBNP are all detected as immunoreactive-BNP by the current BNP assay system. We recently developed an assay system that specifically detects human proBNP. Using this assay system, we observed that miR30-GALNTs-dependent O-glycosylation in the N-terminal region of proBNP contributes to regulation of the processing and secretion of proBNP from the heart.

Pyroglutamyl, proline-rich oligopeptides, classically referred to as bradykinin-potentiating peptides (BPPs) are found in Bothrops jararaca venom, and are naturally occurring inhibitors of the somatic angiotensin-converting enzyme (ACE). The chemical and pharmacological properties of these peptides were essential for the development of captopril, the first active site directed inhibitor of ACE, currently used to treat human hypertension. ACE is a complex ectoenzyme of the vascular endothelium, possessing two catalytic sites, performing diverse specific roles. Recent advances concerning novel features of BPPs revealed that they might still contribute to a better understanding of the cardiovascular physiology and pathology. The molecular biology of the BPPs revealed that they are part of two distinct C-type natriuretic peptide precursors found in the venom gland and the brain of B. jararaca, each containing seven BPPs. In situ hybridization studies detected the presence of the corresponding mRNA precursor in snake brain regions correlated with neuroendocrine functions, such as the ventro-medial hypothalamus, the paraventricular nuclei, the paraventricular organ, and the subcommissural organ. In this article we discuss the large variety of homologous BPPs in B. jararaca venom and brain, its significance, and whether the BPPs could represent novel endogenous neuropeptides.

C-type natriuretic peptide (CNP) encoded by the NPPC (Natriuretic Peptide Precursor C) gene expressed in ovarian granulosa cells inhibits oocyte maturation by activating the natriuretic peptide receptor (NPR)B (NPRB) in cumulus cells. RT-PCR analyses indicated increased NPPC and NPRB expression during ovarian development and follicle growth, associated with increases in ovarian CNP peptides in mice. In cultured somatic cells from infantile ovaries and granulosa cells from prepubertal animals, treatment with CNP stimulated cGMP production. Also, treatment of cultured preantral follicles with CNP stimulated follicle growth whereas treatment of cultured ovarian explants from infantile mice with CNP, similar to FSH, increased ovarian weight gain that was associated with the development of primary and early secondary follicles to the late secondary stage. Of interest, treatment with FSH increased levels of NPPC, but not NPRB, transcripts in ovarian explants. In vivo studies further indicated that daily injections of infantile mice with CNP for 4 d promoted ovarian growth, allowing successful ovulation induction by gonadotropins. In prepubertal mice, CNP treatment alone also promoted early antral follicle growth to the preovulatory stage, leading to efficient ovulation induction by LH/human chorionic gonadotropin. Mature oocytes retrieved after CNP treatment could be fertilized in vitro and developed into blastocysts, allowing the delivery of viable offspring. Thus, CNP secreted by growing follicles is capable of stimulating preantral and antral follicle growth. In place of FSH, CNP treatment could provide an alternative therapy for female infertility.

OBJECTIVE

Genetic risk factors for essential hypertension are largely unknown. The aim of the present study was to assess the association of 77 previously characterized gene variants in 52 candidate genes from various biological pathways with blood pressure (BP) progression and incident hypertension.

METHODS

We analyzed data from 18 738 white women who participated in a prospective cohort study and were free of hypertension at baseline. BP progression at 48 months and incident hypertension during the entire follow-up according to the different genotypes were assessed by logistic regression and Cox proportional-hazards models, respectively.

RESULTS

At 48 months of follow-up, 7889 of 16 635 women (47.4%) had BP progression. Only three of 70 polymorphisms with a minor allele frequency of at least 2% had a significant association with BP progression. The odds ratio [95% confidence interval (CI)] for 5,10-methylenetetrahydrofolate reductase (MTHFR) rs1801133 (minor allele T), natriuretic peptide precursor A (NPPA) rs5063 (minor allele A) and NPPA rs5065 (minor allele C) were 1.05 (1.00-1.10), 0.84 (0.76-0.94) and 0.93 (0.88-1.00), respectively. After adjustment for multiple testing using the false discovery rate, only the NPPA rs5063 association remained significant. During a median follow-up of 9.8 years, 5540 of 18 738 women developed incident hypertension. Only five of 70 polymorphisms were significantly associated with incident hypertension. The hazard ratio (95% CI) for interleukin 6 (IL-6) rs1800795 (minor allele C), MTHFR rs1801133, NPPA rs5063, nitric oxide synthase 3 rs1799983 (minor allele T) and transforming growth factor, beta 1 rs1800469 (minor allele T) were 0.96 (0.92-1.00), 1.06 (1.02-1.10), 0.88 (0.80-0.96), 1.05 (1.01-1.09) and 1.05 (1.01-1.10), respectively. After adjustment for multiple testing, none of these associations remained significant.

CONCLUSIONS

NPPA gene polymorphisms may have a role in BP progression and incident hypertension. Our data also provide moderate confirmatory evidence of association between MTHFR rs1801133 and the development of hypertension.

Peptide-hormones and other biologically active peptides are synthesized as higher molecular weight precursor proteins (pro-proteins) which must undergo post-translational modification to yield the bioactive peptide(s). These post-translational enzymatic events include limited endoproteolysis and may include other modifications of the generated peptide such as limited exopeptidase digestion, N-terminal acetylation, C-terminal amidation, and formation of N-terminal pyroglutamyl residues (pyrrolation). The secretory vesicle hypothesis, one of the major hypotheses regarding processing, states that the initial endoproteolytic event occurs upon formation of the secretory vesicle (or granule) or within the secretory vesicle from which the bioactive peptides are released. Two different endoproteinases which are likely to be physiologically relevant processing enzymes of pro-atrial natriuretic factor and pro-gonadotropin releasing hormone precursor protein, respectively, have recently been discovered in our laboratory and are discussed as model enzymes in the context of this hypothesis. The results indicate that the precursor protein and its complement of processing enzymes are co-packaged into the secretory granule. Evidence is presented to support the idea that the specific sequence and conformation (secondary structural features) of the processing recognition site within the precursor protein likely contribute in large part to the basis for limited endoproteolysis. In the pro-hormones studied, the recognition site is an extended sequence of five to seven residues which likely exists as a beta-turn at the surface of the precursor protein. By extending our results to appropriate protein sequences in the National Biomedical Research Foundation database, we are suggesting that in addition to the doublet of basic amino acids, the primary processing recognition site in pro-hormone precursor proteins often contains a monobasic amino acid or a strongly polar residue (Glu or Asp) in close sequence proximity to the doublet of basic residues.

This review focuses on some selected aspects of the endocrine heart and natriuretic peptides. The endocrine heart is composed of specific myoendocrine cells of the cardiac atria. The myoendocrine cells synthesize and secrete the natriuretic peptide hormones which exhibit natriuretic, diuretic, and vasorelaxant properties. Immunohistochemical analyses show that natriuretic peptides of the A-type and B-type are localized not only in the specific granules of these myoendocrine cells but also in many other organs including the brain, adrenal medulla, and kidney. Also, their receptors are detected in many organs showing the multiple functions of these regulatory peptides. Of the members of the natriuretic peptide family, ANP (ANP for atrial natriuretic peptide; also denominated cardiodilatin, CDD), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and the A-type, including its renal form, urodilatin, are emphasized in this review. Urodilatin is localized in the kidney, differentially processed, and secreted into the urine. The intrarenal synthesis and secretion is the basis for a paracrine system regulating water and sodium reabsorption at the level of the collecting duct. CDD/ANP-1-126, cleaved from a precursor of 126 amino acids in the heart to a 28-amino acid-containing circulating molecular form (CDD/ANP-99-126), and urodilatin (CDD/ANP-95-126) share similar biochemical features and biological functions, but urodilatin may be more involved in the regulation of body fluid volume and water-electrolyte excretion, while circulating CDD/ANP-99-126 is responsible for blood pressure regulation. The physiological and pharmacological properties of these peptides have great clinical impact, and as a consequence urodilatin is involved in drug development for the treatment of acute renal failure, cardiomyopathia, and acute asthma.

BACKGROUND

B-type natriuretic peptide (BNP) and N-terminal proBNP (NT-proBNP) are useful biomarkers in the management of heart failure. Both peptides are secreted into the circulation after cleavage of their precursor proBNP and excreted from the kidney in the active form or as metabolites. We investigated effects of kidney function on the levels and association of these peptides.

METHODS

Plasma concentrations of BNP and serum concentrations of NT-proBNP were measured in 229 outpatients of our cardiology department (male/female = 138/91, mean age= 68 years) and 53 hospital outpatients on chronic haemodialysis (30/23, 68 years). Kidney function in nondialysed patients was assessed by estimated glomerular filtration rate (eGFR; mL/min/1.73 m(2)) and categorised across five stages. Effects of kidney function on BNP, NT-proBNP and their relationship were investigated.

RESULTS

Deterioration in kidney function increased BNP and NT-proBNP levels, as well as the NT-proBNP/BNP ratio, and these values were highest in patients on haemodialysis. The eGFR inversely correlated with BNP (r = -0.472), NT-proBNP (r = -0.579), and the NT-proBNP/BNP ratio (r = -0.454, all P < 0.0001). A significant correlation was observed between BNP and NT-proBNP at all stages of kidney function including patients on haemodialysis, but the correlation was significantly affected by kidney function.

CONCLUSIONS

Although circulating levels of both BNP and NT-proBNP increased with deteriorating kidney function, the impact of kidney function on NT-proBNP was much more pronounced than that on BNP. Kidney function should be taken into account when interpreting data on BNP, NT-proBNP and their relationship.

BACKGROUND

Three neuropeptides, gastrin releasing peptide (GRP), natriuritic precursor peptide B (NPPB), and neuromedin B (NMB) have been proposed to play roles in itch sensation. However, the tissues in which these peptides are expressed and their positions in the itch circuit has recently become the subject of debate. Here we used next-gen RNA-Seq to examine the expression of transcripts coding for GRP, NPPB, NMB, and other peptides in DRG, trigeminal ganglion, and the spinal cord as well as expression levels for their cognate receptors in these tissues.

RESULTS

RNA-Seq demonstrates that GRP is not transcribed in mouse, rat, or human sensory ganglia. NPPB, which activates natriuretic peptide receptor 1 (NPR1), is well expressed in mouse DRG and less so in rat and human, whereas NPPA, which also acts on the NPR1 receptor, is expressed in all three species. Analysis of transcripts expressed in the spinal cord of mouse, rat, and human reveals no expression of Nppb, but unambiguously detects expression of Grp and the GRP-receptor (Grpr). The transcripts coding for NMB and tachykinin peptides are among the most highly expressed in DRG. Bioinformatics comparisons using the sequence of the peptides used to produce GRP-antibodies with proteome databases revealed that the C-terminal primary sequence of NMB and Substance P can potentially account for results from previous studies which showed GRP-immunostaining in the DRG.

CONCLUSIONS

RNA-Seq corroborates a primary itch afferent role for NPPB in mouse and potentially NPPB and NPPA in rats and humans, but does not support GRP as a primary itch neurotransmitter in mouse, rat, or humans. As such, our results are at odds with the initial proposal of Sun and Chen (2007) that GRP is expressed in DRG. By contrast, our data strongly support an itch pathway where the itch-inducing actions of GRP are exerted through its release from spinal cord neurons.

OBJECTIVE

We hypothesised that assessment of plasma C-terminal pro-endothelin-1 (CT-proET-1), a stable endothelin-1 precursor fragment, is of prognostic value in patients with chronic heart failure (CHF), beyond other prognosticators, including N-terminal pro-B-type natriuretic peptide (NT-proBNP).

METHODS

We examined 491 patients with systolic CHF (age: 63±11 years, 91% men, New York Heart Association [NYHA] class [I/II/III/IV]: 9%/45%/38%/8%, 69% ischemic etiology). Plasma CT-proET-1 was detected using a chemiluminescence immunoassay.

RESULTS

Increasing CT-proET-1 was a predictor of increased cardiovascular mortality at 12-months of follow-up (standardized hazard ratio 1.42, 95% confidence interval [CI] 1.04-1.95, p = 0.03) after adjusting for NT-proBNP, left ventricular ejection fraction (LVEF), age, creatinine, NYHA class. In receiver operating characteristic curve analysis, areas under curve for 12-month follow-up were similar for CT-proET-1 and NT-proBNP (p = 0.40). Both NT-proBNP and CT-proET-1 added prognostic value to a base model that included LVEF, age, creatinine, and NYHA class. Adding CT-proET-1 to the base model had stronger prognostic power (p<0.01) than adding NT-proBNP (p<0.01). Adding CT-proET-1 to NT-proBNP in this model yielded further prognostic information (p = 0.02).

CONCLUSIONS

Plasma CT-proET-1 constitutes a novel predictor of increased 12-month cardiovascular mortality in patients with CHF. High CT-proET-1 together with high NT-proBNP enable to identify patients with CHF and particularly unfavourable outcomes.

The bradykinin-potentiating peptides from Bothrops jararaca venom are the most potent natural inhibitors of the angiotensin-converting enzyme. The biochemical and biological features of these peptides were crucial to demonstrate the pivotal role of the angiotensin-converting enzyme in blood pressure regulation. In the present study, seven bradykinin-potentiating peptides were identified within the C-type natriuretic peptide precursor cloned from snake brain. The bradykinin-potentiating peptides deduced from the B. jararaca brain precursor are strong in vitro inhibitors of the angiotensin-converting enzyme (nanomolar range), and also potentiate the bradykinin effects in ex vivo and in vivo experiments. Two of these peptides are novel bradykinin-potentiating peptides, one of which displays high specificity toward the N-domain active site of the somatic angiotensin-converting enzyme. In situ hybridization studies revealed the presence of the bradykinin-potentiating peptides precursor mRNAs in distinct regions of the B. jararaca brain, such as the ventromedial hypothalamus, the paraventricular nuclei, the paraventricular organ, and the subcommissural organ. The biochemical and pharmacological properties of the brain bradykinin-potentiating peptides, their presence within the neuroendocrine regulator C-type natriuretic peptide precursor, and their expression in regions of the snake brain correlated to neuroendocrine functions, strongly suggest that these peptides belong to a novel class of endogenous vasoactive peptides.

Over the last 50 years, increasing evidence has documented the ability of cardiac non-neuronal cells to synthesize and release catecholamines (CAs) and the vasorelaxant natriuretic peptides (NPs), which both regulate cardiovascular homeostasis in health and disease. This knowledge has firmly established the concept of the heart as an endocrine organ. The contents of this frame have been richly expanded by the identification of an increasing number of intracardiac endocrine modulators, including Chromogranin-A (CgA) and its derived peptides. In the rat heart, CgA is co-stored and co-released with Atrial NP (ANP) in non-adrenergic myoendocrine atrial cells as well as in atrial and ventricular Purkinje fibres. In the ventricular myocardium of the human hypertrophic and dilated heart, CgA co-localizes with B-type NP (BNP). CgA is the precursor of biologically active peptides produced by proteolytic cleavage. One of them, the human recombinant 1-76 CgA-derived vasostatin-1 (VS-1), is an inhibitor of cardiac contraction and relaxation, a non-competitive counter-regulator of beta-adrenergic stimulation and a protecting agent in ischemic preconditioning. Therefore, it may function as a cardiocirculatory homeostatic stabilizer, particularly in the presence of intense adrenergic stimuli, e. g. under stress responses. Since in patients with chronic heart failure circulating CgA levels increase up to 10-20 nM, depending on the severity of the disease and are independent prognostic indicators of mortality, knowledge on the physio-pathological significance of locally produced and/or circulating CgA-derived peptides, as attemped in this synopsis, may pave the way for clinically-oriented cardiovascular applications.

As a family of hormones with pleiotropic effects, natriuretic peptide (NP) system includes atrial NP (ANP), B-type NP (BNP), C-type NP (CNP), dendroaspis NP and urodilatin, with NP receptor-A (guanylate cyclase-A), NP receptor-B (guanylate cyclase-B) and NP receptor-C (clearance receptor). These peptides are genetically distinct, but structurally and functionally related for regulating circulatory homeostasis in vertebrates. In humans, ANP and BNP are encoded by NP precursor A (NPPA) and NPPB genes on chromosome 1, whereas CNP is encoded by NPPC on chromosome 2. NPs are synthesized and secreted through certain mechanisms by cardiomyocytes, fibroblasts, endotheliocytes, immune cells (neutrophils, T-cells and macrophages) and immature cells (embryonic stem cells, muscle satellite cells and cardiac precursor cells). They are mainly produced by cardiovascular, brain and renal tissues in response to wall stretch and other causes. NPs provide natriuresis, diuresis, vasodilation, antiproliferation, antihypertrophy, antifibrosis and other cardiometabolic protection. NPs represent body's own antihypertensive system, and provide compensatory protection to counterbalance vasoconstrictor-mitogenic-sodium retaining hormones, released by renin-angiotensin-aldosterone system (RAAS) and sympathetic nervous system (SNS). NPs play central roles in regulation of heart failure (HF), and are inactivated through not only NP receptor-C, but also neutral endopeptidase (NEP), dipeptidyl peptidase-4 and insulin degrading enzyme. Both BNP and N-terminal proBNP are useful biomarkers to not only make the diagnosis and assess the severity of HF, but also guide the therapy and predict the prognosis in patients with HF. Current NP-augmenting strategies include the synthesis of NPs or agonists to increase NP bioactivity and inhibition of NEP to reduce NP breakdown. Nesiritide has been established as an available therapy, and angiotensin receptor blocker NEP inhibitor (ARNI, LCZ696) has obtained extremely encouraging results with decreased morbidity and mortality. Novel pharmacological approaches based on NPs may promote a therapeutic shift from suppressing the RAAS and SNS to re-balancing neuroendocrine dysregulation in patients with HF. The current review discussed the synthesis, secretion, function and metabolism of NPs, and their diagnostic, therapeutic and prognostic values in HF.

Luteinizing hormone (LH) regulation of the epidermal growth factor (EGF) network is critical for oocyte maturation and the ovulatory process. Recent studies have indicated that C-type natriuretic peptide (CNP) and its receptor natriuretic peptide receptor B (NPR2) play an important role in the control of meiotic arrest. Here, we investigated the involvement of the EGF network in the LH-dependent regulation of the CNP/NPR2 axis and cGMP accumulation. LH/hCG treatment causes a major decrease in both cGMP and the CNP precursor (natriuretic peptide precursor C [Nppc]) mRNA accumulation in vivo and in vitro. However, the cGMP downregulation precedes the decrease in Nppc mRNA by more than 1 h. Amphiregulin, an EGF-like factor, suppresses Nppc mRNA levels in cultured follicles to the same extent as LH, and this effect is completely prevented by the EGF receptor (EGFR) kinase inhibitor AG1478. However, the LH-dependent suppression of Nppc is insensitive to AG1478. Similarly, Nppc suppression by LH occurs in follicles from EGFR null mice. These findings document that EGFR signaling is sufficient to downregulate CNP, but is not necessary for LH action. When cGMP concentration in the follicle is measured, the short-term, but not long-term, LH effects on cGMP are prevented by AG1478, suggesting that ligand availability may be responsible for the late response. Human CG decreases the CNP-dependent cGMP synthesis in wild-type and EGFR knockdown cumulus-oocyte complexes. These findings demonstrate that redundant pathways are involved in the regulation of cGMP. EGFR-dependent events are involved in the short-term regulation of cGMP, whereas the long-term effects may involve regulation of the CNP.

BACKGROUND

SHOX mutations have been detected in approximately 70% of Léri-Weill dyschondrosteosis (LWD) and approximately 2.5% of idiopathic short stature (ISS) cases, suggesting the implication of other genes or loci. The recent identification of NPR2 mutations in ISS suggested that NPR2 mutations may also be involved in disproportionate short stature.

OBJECTIVE

The objective of the study was to investigate whether NPR2 mutations can account for a proportion of the cases referred for LWD and ISS in whom no SHOX mutation was detected.

METHODS

We undertook NPR2 mutation screening in 173 individuals referred for suspected LWD and 95 for ISS, with no known defect in SHOX or its enhancers. Intracellular localization and natriuretic peptide precursor C-dependent guanylate cyclase activity were determined for the identified NPR2 variants.

RESULTS

Eight NPR2 variants were identified in nine individuals, seven referred for suspected LWD and two for ISS. Six were demonstrated to affect NPR-B cell trafficking and/or its ability to synthesize cyclic GMP (cGMP) under response to natriuretic peptide precursor C/brain natriuretic peptide stimulation. All pathogenic mutations were detected in the suspected LWD referral group (∼3%). Interestingly, one of these patients is currently being treated with recombinant human GH and in contrast to previous reports is showing a positive response to the treatment.

CONCLUSIONS

NPR2 mutations account for approximately 3% of patients with disproportionate short stature and/or clinical or radiographic indicators of SHOX deficiency and in whom no SHOX defect has been identified. However, no patient has yet presented with Madelung deformity. Thus, NPR2 should be screened in the SHOX-negative LWD referrals.