Circulating fragments of N-Terminal Pro-B-Type Natriuretic Peptides in plasma of heart failure patients

BACKGROUND: The use of nonstandardized N-terminal pro-B-type natriuretic peptide (NT-proBNP) assays can contribute to the misdiagnosis of heart failure (HF). Moreover, there is yet to be established a common consensus regarding the circulating forms of NT-proBNP being used in current assays. We aimed to characterize and quantify the various forms of NT-proBNP in the circulation of HF patients. METHODS: Plasma samples were collected from HF patients (n = 20) at rest and stored at -80 degrees C. NT-proBNP was enriched from HF patient plasma by use of immunoprecipitation followed by mass spectrometric analysis. Customized homogeneous sandwich AlphaLISA (R) immunoassays were developed and validated to quantify 6 fragments of NT-proBNP. RESULTS: Mass spectrometry identified the presence of several N- and C-terminally processed forms of circulating NT-proBNP, with physiological proteolysis between Pro2-Leu3, Leu3-Gly4, Pro6-Gly7, and Pro75-Arg76. Consistent with this result, AlphaLISA immunoassays demonstrated that antibodies targeting the extreme N or C termini measured a low apparent concentration of circulating NT-proBNP. The apparent circulating NT-proBNP concentration was increased with antibodies targeting nonglycosylated and nonterminal epitopes (P < 0.05). CONCLUSIONS: In plasma collected from HF patients, immunoreactive NT-proBNP was present as multiple N- and C-terminally truncated fragments of the full length NT-proBNP molecule. Immunodetection of NT-proBNP was significantly improved with the use of antibodies that did not target these terminal regions. These findings support the development of a next generation NT-proBNP assay targeting nonterminal epitopes as well as avoiding the central glycosylated region of this molecule. (c) 2013 American Association for Clinical Chemistry

Cardiac function before and after treatment for various types of loading conditions and in myocardial restriction : A prospective study on pediatric patients with two- and three-dimensional echocardiography and measurement of serum natriuretic peptides

Background: The incidence of all forms of congenital heart defects is 0.75%. For patients with congenital heart defects, life-expectancy has improved with new treatment modalities. Structural heart defects may require surgical or catheter treatment which may be corrective or palliative. Even those with corrective therapy need regular follow-up due to residual lesions, late sequelae, and possible complications after interventions. Aims: The aim of this thesis was to evaluate cardiac function before and after treatment for volume overload of the right ventricle (RV) caused by atrial septal defect (ASD), volume overload of the left ventricle (LV) caused by patent ductus arteriosus (PDA), and pressure overload of the LV caused by coarctation of the aorta (CoA), and to evaluate cardiac function in patients with Mulibrey nanism. Methods: In Study I, of the 24 children with ASD, 7 underwent surgical correction and 17 percutaneous occlusion of ASD. Study II had 33 patients with PDA undergoing percutaneous occlusion. In Study III, 28 patients with CoA underwent either surgical correction or percutaneous balloon dilatation of CoA. Study IV comprised 26 children with Mulibrey nanism. A total of 76 healthy voluntary children were examined as a control group. In each study, controls were matched to patients. All patients and controls underwent clinical cardiovascular examinations, two-dimensional (2D) and three-dimensional (3D) echocardiographic examinations, and blood sampling for measurement of natriuretic peptides prior to the intervention and twice or three times thereafter. Control children were examined once by 2D and 3D echocardiography. M-mode echocardiography was performed from the parasternal long axis view directed by 2D echocardiography. The left atrium-to-aorta (LA/Ao) ratio was calculated as an index of LA size. The end-diastolic and end-systolic dimensions of LV as well as the end-diastolic thicknesses of the interventricular septum and LV posterior wall were measured. LV volumes, and the fractional shortening (FS) and ejection fraction (EF) as indices of contractility were then calculated, and the z scores of LV dimensions determined. Diastolic function of LV was estimated from the mitral inflow signal obtained by Doppler echocardiography. In three-dimensional echocardiography, time-volume curves were used to determine end-diastolic and end-systolic volumes, stroke volume, and EF. Diastolic and systolic function of LV was estimated from the calculated first derivatives of these curves. Results: (I): In all children with ASD, during the one-year follow-up, the z score of the RV end-diastolic diameter decreased and that of LV increased. However, dilatation of RV did not resolve entirely during the follow-up in either treatment group. In addition, the size of LV increased more slowly in the surgical subgroup but reached control levels in both groups. Concentrations of natriuretic peptides in patients treated percutaneously increased during the first month after ASD closure and normalized thereafter, but in patients treated surgically, they remained higher than in controls. (II): In the PDA group, at baseline, the end-diastolic diameter of LV measured over 2SD in 5 of 33 patients. The median N-terminal pro-brain natriuretic peptide (proBNP) concentration before closure measured 72 ng/l in the control group and 141 ng/l in the PDA group (P = 0.001) and 6 months after closure measured 78.5 ng/l (P = NS). Patients differed from control subjects in indices of LV diastolic and systolic function at baseline, but by the end of follow-up, all these differences had disappeared. Even in the subgroup of patients with normal-sized LV at baseline, the LV end-diastolic volume decreased significantly during follow-up. (III): Before repair, the size and wall thickness of LV were higher in patients with CoA than in controls. Systolic blood pressure measured a median 123 mm Hg in patients before repair (P < 0.001) and 103 mm Hg one year thereafter, and 101 mm Hg in controls. The diameter of the coarctation segment measured a median 3.0 mm at baseline, and 7.9 at the 12-month (P = 0.006) follow-up. Thicknesses of the interventricular septum and posterior wall of the LV decreased after repair but increased to the initial level one year thereafter. The velocity time integrals of mitral inflow increased, but no changes were evident in LV dimensions or contractility. During follow-up, serum levels of natriuretic peptides decreased correlating with diastolic and systolic indices of LV function in 2D and 3D echocardiography. (IV): In 2D echocardiography, the interventricular septum and LV posterior wall were thicker, and velocity time integrals of mitral inflow shorter in patients with Mulibrey nanism than in controls. In 3D echocardiography, LV end-diastolic volume measured a median 51.9 (range 33.3 to 73.4) ml/m² in patients and 59.7 (range 37.6 to 87.6) ml/m² in controls (P = 0.040), and serum levels of ANPN and proBNP a median 0.54 (range 0.04 to 4.7) nmol/l and 289 (range 18 to 9170) ng/l, in patients and 0.28 (range 0.09 to 0.72) nmol/l (P < 0.001) and 54 (range 26 to 139) ng/l (P < 0.001) in controls. They correlated with several indices of diastolic LV function. Conclusions (I): During the one-year follow-up after the ASD closure, RV size decreased but did not normalize in all patients. The size of the LV normalized after ASD closure but the increase in LV size was slower in patients treated surgically than in those treated with the percutaneous technique. Serum levels of ANPN and proBNP were elevated prior to ASD closure but decreased thereafter to control levels in patients treated with the percutaneous technique but not in those treated surgically. (II): Changes in LV volume and function caused by PDA disappeared by 6 months after percutaneous closure. Even the children with normal-sized LV benefited from the procedure. (III): After repair of CoA, the RV size and the velocity time integrals of mitral inflow increased, and serum levels of natriuretic peptides decreased. Patients need close follow-up, despite cessation of LV pressure overload, since LV hypertrophy persisted even in normotensive patients with normal growth of the coarctation segment. (IV): In children with Mulibrey nanism, the LV wall was hypertrophied, with myocardial restriction and impairment of LV function. Significant correlations appeared between indices of LV function, size of the left atrium, and levels of natriuretic peptides, indicating that measurement of serum levels of natriuretic peptides can be used in the clinical follow-up of this patient group despite its dependence on loading conditions.

Association of common variants in NPPA and NPPB with circulating natriuretic peptides and blood pressure

We examined the association of common variants at the NPPA-NPPB locus with circulating concentrations of the natriuretic peptides, which have blood pressure-lowering properties. We genotyped SNPs at the NPPA-NPPB locus in 14,743 individuals of European ancestry, and identified associations of plasma atrial natriuretic peptide with rs5068 (P = 8 × 10 -70), rs198358 (P = 8 × 10 -30) and rs632793 (P = 2 × 10 -10), and of plasma B-type natriuretic peptide with rs5068 (P = 3 × 10 -12), rs198358 (P = 1 × 10 -25) and rs632793 (P = 2 × 10 -68). In 29,717 individuals, the alleles of rs5068 and rs198358 that showed association with increased circulating natriuretic peptide concentrations were also found to be associated with lower systolic (P = 2 × 10 -6 and 6 × 10 -5, respectively) and diastolic blood pressure (P = 1 × 10 -6 and 5 × 10 -5), as well as reduced odds of hypertension (OR = 0.85, 95% CI = 0.79-0.92, P = 4 × 10 -5; OR = 0.90, 95% CI = 0.85-0.95, P = 2 × 10 -4, respectively). Common genetic variants at the NPPA-NPPB locus found to be associated with circulating natriuretic peptide concentrations contribute to interindividual variation in blood pressure and hypertension.

Atrial natriuretic peptides: reproducibility of renal effects and response of liver blood flow.

To assess the variability of the response to exogenous atrial natriuretic peptide (ANP), it was infused at the rate of 1 microgram/min for 2 h in 6 salt-loaded normal volunteers under controlled conditions on 2 occasions at an interval of 1 week. The effect on solute excretion and the haemodynamic and endocrine actions were highly reproducible. The constant ANP infusion caused a delayed and prolonged excretion of sodium, chloride and calcium, no change in potassium or phosphate excretion or in glomerular filtration rate but a marked decrease in renal plasma flow. Blood pressure, heart rate and the plasma levels of angiotensin II, aldosterone, arginine vasopressin and plasma renin activity were unaltered. The effect of a 2-h infusion of ANP 0.5 microgram/min or its vehicle on apparent hepatic blood flow (HBF) was also studied in 14 normal volunteers by measuring the indocyanine green clearance. A 21% decrease in HBF was observed in subjects who received the ANP infusion (p less than 0.01 vs vehicle). Thus, ANP infused at a dose that did not lower blood pressure decreased both renal and liver blood flow in normotensive volunteers. The renal and endocrine responses to ANP were reproducible over a 1-week interval.

Natriuretic peptides and immunoreactants modify osmoticum-dependent volume changes in Solanum tuberosum L. mesophyll cell protoplasts

In plants, as in vertebrates, natriuretic peptide (NP) hormones can influence water and solute homeostasis. Here we demonstrate that a synthetic peptide identical to the C-terminus (amino acids 99–126) of the rat atrial natriuretic peptide (rANP) modulates osmotically induced swelling of mesophyll cell protoplasts (MCPs) in a concentration and time-dependent manner. Osmotically-induced volume changes in MCPs are enhanced by plant extracts with NP immunoreactivity and this effect is concentration-dependent. In contrast, pre-treatment of the plant extracts with rabbit anti-human ANP (99–126) antiserum suppresses enhanced osmoticum-induced swelling. Isolated plant peptides (irPNP) that have been immunoaffinity purified with rabbit anti-human ANP (99–126) antiserum also enhance osmotically-induced swelling. While rANP and irPNP cause increases in cGMP levels in MCPs, elevated cGMP levels do not cause increases in osmoticum-dependent swelling but exert an inhibitory effect. These findings are consistent with a NP-dependent, cGMP-independent effect on plant cell volume regulation and a role in homeostasis for peptides that are recognized by antibodies directed against the C-terminus of vertebrate ANPs.

Guanylyl cyclase stimulation by homologous and heterologous natriuretic peptides in salmonids

A range of homologous (trout ANP, trout CNP, trout VNP) and heterologous (eel ANP, eel ANP-NH₂, rat ANP, porcine CNP) NPs were tested for their effect on guanylyl cyclase in gill and kidney membrane preparations from freshwater and seawater-acclimated rainbow trout and Atlantic salmon. All NPs stimulated guanylyl cyclase at 1 µmol l^-1in all preparations. ANP was the most potent stimulator of kidney guanylyl cyclase and CNP the most potent stimulator of guanylyl cyclase in gills. Some differences were apparent between the potencies of homologous and heterologous peptides at 1 µmol l^-1: tANP was more potent than rANP in the SW trout kidney and tCNP was more potent than pCNP in FW salmon tissues. While eANP was more potent than tANP in trout gills, it was less potent than tANP in FW salmon gills. However, there was no significant difference between the potencies of eANP and eANP-NH₂ in trout or salmon gills. Salinity did not affect guanylyl cyclase activity with the exception that trout ANP at 1 µmol l^-1was more potent in SW trout kidneys than in FW trout kidneys. These results suggest a predomination of NPR-A in the kidney and NPR-B in the gill. It appears that salmonid NPR-A and NPR-B are relatively promiscuous in their ligand affinity, with few differences in the potencies of trout and mammalian NPs and only small differences in cGMP production where these differences do occur.

Natriuretic peptides inhibit adenylyl cyclase activity in dispersed eel gill cells

The effect of natriuretic peptides on forskolin-evoked adenylyl cyclase activity was investigated in dispersed gill cells from the Australian short-finned eel (Anguilla australis). Molecular cloning techniques were employed to identify the putative G-protein-activating motif within the intracellular domain of the eel natriuretic peptide C receptor. Eel ANP, eel CNP and the NPR-C-specific C-ANF inhibited the forskolin-stimulated production of cyclic AMP. This effect was abolished by pretreatment of cells with pertussis toxin. Eel VNP was without effect on adenylyl cyclase activity. PCR and molecular cloning indicated that the intracellular domain of A. australis NPR-C has the same amino acid sequence as Anguilla japonica. Alignment of these sequences with Rattus norvegicus NPR-C indicated conservation of the putative G-protein-activating motif BB...BBXXB (B=basic, X=nonbasic residues). These data suggest that branchially-expressed NPR-C may play a physiological role additional to that of ligand clearance.

A perspective on the role of natriuretic peptides in amphibian osmoregulation

The natriuretic peptide (NP) system is a complex family of peptides and receptors that is primarily linked to the maintenance of osmotic and cardiovascular homeostasis. In amphibians, the potential role(s) of NPs is complicated by the range of osmoregulatory strategies found in amphibians, and the different tissues that participate in osmoregulation. Atrial NP, brain NP, and C-type NP have been isolated or cloned from a number of species, which has enabled physiological studies to be performed with homologous peptides. In addition, three types of NP receptors have been cloned and partially characterised. Natriuretic peptides are always potent vasodilators in amphibian blood vessels, and ANP has been shown to increase the permeability of the microcirculation. In the perfused kidney, ANP causes vasodilation, diuresis and natriuresis that are caused by an increased GFR rather than effects in the renal tubules. These data are supported by the presence of ANP receptors only on the glomeruli and renal blood vessels. In the bladder and skin, the function of NPs is enigmatic because physiological analysis of the effects of ANP on bladder and skin function has yielded conflicting data with no clear role for NPs being revealed. Overall, NPs often have no direct effect, but in some studies they have been shown to inhibit the function of AVT. In addition, there is evidence that ANP can inhibit salt retention in amphibians since it can inhibit the ability of adrenocorticotrophic hormone or angiotensin II to stimulate corticosteroid secretion. It is proposed that an important role for cardiac NPs could be in the control of hypervolaemia during periods of rapid rehydration, which occurs in terrestrial amphibians.

Cyclic GMP modulates stomatal opening induced by natriuretic peptides and immunoreactive analogues

In this paper we demonstrate that compounds that promote stomatal opening such as kinetin, atrial natriuretic peptide (ANP) and plant natriuretic peptide immunoanalogues (irPNP) significantly elevate cGMP in guard cell protoplasts. Stomata opened by irPNP are induced to close in the presence of the guanylate cyclase inhibitor, LY 83583. The effect of cGMP on stomatal opening appears to be linked with Ca2+ levels. ANP, irPNP and 8-Br-cGMP all induce stomatal opening and this is inhibited by compounds that lower intracellular Ca2+ levels such as ethylene glycol bis(β-aminoethyl ether) N,N,N’,N’-tetraacetic acid (EGTA), ruthenium red and procaine.

Molecular cloning of natriuretic peptides from the heart of reptiles : loss of ANP in diapsid reptiles and birds

Atrial natriuretic peptide (ANP) and B-type NP (BNP) are hormones involved in homeostatic control of body fluid and cardiovascular regulation. Both ANP and BNP have been cloned from the heart of mammals, amphibians, and teleost fishes, while an additional cardiac peptide, ventricular NP, has been found in selected species of teleost fish. However, in chicken, BNP is the primary cardiac peptide identified thus far. In contrast, the types of NP/s present in the reptilian heart are unknown, representing a considerable gap in our understanding of NP evolution. In the present study, we cloned and sequenced a BNP cDNA from the atria of representative species of reptile, including crocodile, lizard, snake, and tortoise. In addition, we cloned BNP from the pigeon atria. The reptilian and pigeon BNP cDNAs had ATTTA repeats in the 3′ untranslated region, as observed in all vertebrate BNP mRNAs. A high sequence homology was evident when comparing reptile and pigeon preproBNP with the previously identified chicken preproBNP. In particular, the predicted mature BNP-29 was identical between crocodile, tortoise, and chicken, with pigeon having a single amino acid substitution; lizard and snake BNP had seven and nine substitutions, respectively. Furthermore, an ANP cDNA could only be cloned from the tortoise atria. Since ANP was not isolated from the heart of any non-chelonian reptile and appears to be absent in birds, we propose that the ANP gene has been lost after branching of the turtles in the amniote line. This data provides new avenues for research on NP function in reptiles.

Natriuretic peptides and their receptors in the brain of the amphibian, Bufo marinus

The natriuretic peptides, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) are members of a family of hormones that play an important role in mammalian fluid and electrolyte balance. In the periphery, natriuretic peptides reduce blood volume and subsequently blood pressure by increasing renal natriuresis and diuresis and relaxation of vascular smooth muscle. The actions of natriuretic peptides are mediated via two membrane-linked guanylate cyclase receptors (NPR-GC); natriuretic peptide receptor-A (NPR-A) which has a high affinity for ANP and BNP; and natriuretic peptide receptor-B (NPR-B)which has the greatest affinity for CNP. A third receptor not linked to guanylate cyclase, natriuretic peptide receptor-C (NPR-C) also exists, which binds to ANP, BNP and CNP with a relatively equal affinity, and is involved with clearance of the peptides from the circulation and tissues. The natriuretic peptides are present in the brain and are particularly predominant in cardiovascular and fluid and electrolyte regulating areas such as the anteroventral third ventricle (AV3V) region. This distribution has led to the suggestion natriuretic peptides play a neuromodulatory role in the central control of fluid homeostasis. Natriuretic peptides in the brain have been observed to inhibit the release of other fluid and electrolyte regulating hormones such as arginine vasopressin (AVP) and angiotensin II (AII). Natriuretic peptides have also been identified in the non-mammalian vertebrates although information regarding the distribution of the peptides and their receptors in the non-mammalian brain is limited. In amphibians, immunohistochemical studies have shown that natriuretic peptides are highly concentrated in the preoptic region of the brain, an area believed to be analogous to the A\T3\ region in mammals, which suggests that natriuretic peptides may also be involved in central fluid and electrolyte regulation in amphibians. To date, CNP is the only natriuretic peptide that has been isolated and cloned from the lower vertebrate brain, although studies on the distribution of CNP binding sites in the brain have only been performed in one fish species. Studies on the distribution of ANP binding sites in the lower vertebrate brain are similarly limited and have only been performed in one fish and two amphibian species. Moreover, the nature and distribution of the natriuretic peptide receptors has not been characterised. The current study therefore, used several approaches to investigate the distribution of natriuretic peptides and their receptors in the brain of the amphibian Bufo marinus. The topographical relationship of natriuretic peptides and the fluid and electrolyte regulating hormone arginine vasotocin was also investigated, in order to gain a greater understanding of the role of the natriuretic peptide system in the lower vertebrate brain. Immunohistochemical studies showed natriuretic peptides were distributed throughout the brain and were highly concentrated in the preoptic region and interpeduncular nucleus. No natriuretic peptide-like immunoreactivity (NP-IR) was observed in the pituitary gland. Arginine vasotocin-like immunoreactivity (AvT-IR) was confined to distinct regions, particularly in the preoptic/hypothalamic region and pituitary gland. Double labelling studies of NP-JR and AvT-IR showed the peptides are not colocalised in the same neural pathways. The distribution of natriuretic peptide binding sites using the ligands 125I-rat ANP (125I-rANP) and 125I-porcine CNP (125I-pCNP) showed different distributions in the brain of B. marinus. The specificity of binding was determined by displacement with unlabelled rat ANP, porcine CNP and C-ANF, an NPR-C specific ligand. 125I-rANP binding sites were broadly distributed throughout the brain with the highest concentration in pituitary gland, habenular, medial pallium and olfactory region. Minimal 125I-rANP binding was observed in the preoptic region. Residual 125I-rANP binding in the presence of C-ANF was observed in the olfactory region, habenular and pituitary gland indicating the presence of both NPR-GC and NPR-C in these regions. 125I-pCNP binding was limited to the olfactory region, pallium and posterior pituitary gland. All 125I-pCNP binding was displaced by C-ANF which suggests that CNP in the brain of B. marinus binds only to NPR-C. Affinity cross-linking and SDS-PAGB demonstrated two binding sites at 136 kDa and 65 kDa under reducing conditions. Guanylate cyclase assays showed 0.1 µM ANP increased cGMP levels 50% above basal whilst a 10-fold higher concentration of CNP was required to produce the same result. Molecular cloning studies revealed a 669 base pair fragment showing 91% homology with human and rat NPR-A and 89% homology with human, rat and eel NPR-B. A 432 base pair fragment showing 67% homology to the mammalian NPR-C and 58% homology with eel NPR-D was also obtained. The results show natriuretic peptides and their receptors are distributed throughout the brain of B. marinus which indicates that natriuretic peptides may participate in a range of regulatory functions throughout the brain. The potential for natriuretic peptides to regulate the release of the fluid and electrolyte regulating hormone AVT also exists due to the high number of natriuretic peptide binding sites in the posterior pituitary gland. At least two populations of natriuretic peptide receptors are present in the brain of B. marinus, one linked to guanylate cyclase and one resembling the mammalian clearance receptor. Furthermore, autoradiography and guanylate cyclase studies suggest ANP may be the major ligand in the brain of B. marinus, even though CNP is the only natriuretic peptide that has been isolated from the lower vertebrate brain to date.

Characterisation and physiological functions of plant natriuretic peptides

Natriuretic peptides are bioactive proteins. In plants, biochemical and physiological studies on these molecules has now revealed that they influence stomatal opening, cell volume and the activity of membrane pumps and their localisation within vascular tissues. Thus they have major roles in maintaining water and solute homeostasis.

Natriuretic peptides : do they regulate water balance in desert-adapted rodents?

This research determined how the natriuretic peptide system, which controls water and salt balance, responds to water limitation in a desert rodent. It was found that a uniform down-regulation of the system is not part of the physiological response to minimise water loss during periods of low water availability.