Brain natriuretic peptide is a novel cardiac hormone

Using a radioimmunoassay for brain natriuretic peptide (BNP), we have measured levels of BNP-like immunoreactivity (-LI) in extract of the porcine heart, in perfusate from the isolated porcine heart and in porcine plasma. BNP-LI was detected in the extract of the atrium, though no detectable amount of BNP-LI (more than 1 ng/g) was present in the ventricle. The BNP-LI level in the porcine atrium was 148.7 +/- 23.3 ng/g. BNP-LI was also detected in the perfusate from the heart. Basal secretory rate of BNP was 3.18 +/- 0.76 ng/min. Moreover, BNP-LI was detected in porcine plasma at the concentration of 4.2 +/- 1.3 pg/ml. Gel filtration studies showed that BNP is present in the atrium as a large molecule and is secreted into the circulation as a small molecule. The percentage of BNP-LI to atrial natriuretic peptide (ANP)-LI was almost the same among the extract, the perfusate and the plasma (2-3 percent). These results indicate that BNP is synthesized in and is secreted into the circulation from the heat in a similar fashion as ANP.     

Plasma clearance and tissue binding of rANP[99-126] and iso-rANP[1-45] in the rat.

Plasma clearance and tissue binding of atrial natriuretic peptide (ANP) and iso-ANP were compared in Inactin-anaesthetized rats. It was found that the plasma half-life of iso-ANP was comparable to ANP. Appearance of trichloroacetic acid-soluble radioactivity of iso-ANP in the plasma was considerably slower than that of ANP, suggesting that the metabolic process of these two peptides may be different. Although the binding distribution of these two peptides was similar, the total binding of iso-ANP to organs other than the kidney was much lower. The kidney, lung, heart and adrenal gland appeared to be major target organs for iso-ANP. Autoradiography showed that iso-ANP bound specifically to the renal glomerulus and proximal part of the proximal tubule. This latter binding site in the kidney was not apparent with ANP, suggesting that iso-ANP may exerts its physiological action at different sites in this organ.     

Isolation and sequence determination of rat cardiac natriuretic peptide

We have isolated a cardiac natriuretic peptide of 5K daltons from the rat atrium and determined its amino acid sequence. The 5K cardiac natriuretic peptide was elucidated to be a 45-amino acid peptide with the sequence of S-Q-D-S-A-F-R-I-Q-E-R-L-R-N-S-K-M-A-H-S-S-S-C-F-G-Q-K-I-D-R-I-G-A-V-S-R- L-G-C-D - G-L-R-L-F by sequencing the native peptide and its lysyl endopeptidase digests. The sequence of this peptide was identical to the amino acid sequence [51-95] of the rat brain natriuretic peptide (BNP) precursor deduced from the cDNA sequence. The 5K cardiac natriuretic peptide, or BNP[51-95], was identified as the major storage and secretory form derived from the BNP precursor in the rat heart.     

Rat corin gene: molecular cloning and reduced expression in experimental heart failure

Stored cardiac pro-atrial natriuretic peptide (pro-ANP) is converted to ANP and released upon stretch from the atria into the circulation. Corin is a serin protease with pro-ANP-converting properties and may be the rate-limiting enzyme in ANP release. This study was aimed to clone and sequence corin in the rat and to analyze corin mRNA expression in heart failure when ANP release upon stretch is blunted. Full-length cDNA of rat corin was obtained from atrial RNA by RT-PCR and sequenced. Tissue distribution as well as regulation of corin mRNA expression in the atria were determined by RT-PCR and RNase protection assay. Heart failure was induced by an infrarenal aortocaval shunt. Stretch was applied to the left atrium in a working heart modus, and ANP was measured in the perfusates. The sequence of rat corin cDNA was found to be 93.6% homologous to mouse corin cDNA. Corin mRNA was expressed almost exclusively in the heart with highest concentrations in both atria. The aortocaval shunt led to cardiac hypertrophy and heart failure. Stretch-induced ANP release was blunted in shunt animals (control 1,195 +/- 197 fmol.min(-1).g(-1); shunt: 639 +/- 99 fmol.min(-1).g(-1), P < 0.05). Corin mRNA expression was decreased in both atria in shunt animals [right atrium: control 0.638 +/- 0.004 arbitrary units (AU), shunt 0.566 +/- 0.014 AU, P < 0.001; left atrium: control 0.564 +/- 0.009 AU, shunt 0.464 +/- 0.009 AU, P < 0.001]. Downregulation of atrial corin mRNA expression may be a novel mechanism for the blunted ANP release in heart failure.     

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.     

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.     

Distribution of atrial natriuretic peptide and its effects on contraction and intracellular calcium in ventricular myocytes from streptozotocin-induced diabetic rat

The distribution of atrial natriuretic peptide (ANP) in blood plasma and cardiac muscle and its effects on ventricular myocyte contraction and intracellular free calcium concentration [Ca2+]i in the streptozotocin (STZ)-induced diabetic rat have been investigated. Blood plasma concentration and heart atrial and ventricular contents of ANP were significantly increased in STZ-treated rats compared to age-matched controls. STZ treatment increased the number of ventricular myocytes immunolabeled with antibodies against ANP. In control myocytes the percentage of cells that labeled positively and negatively were 17% versus 83%, respectively. However, in myocytes from STZ-treated rat the percentages were 52% versus 53%. Time to peak (TPK) shortening was significantly and characteristically prolonged in myocytes from STZ-treated rats (360+/-5 ms) compared to controls (305+/-5 ms). Amplitude of the Ca2+ transient was significantly increased in myocytes from STZ-treated rats compared to controls (0.39+/-0.02 versus 0.29+/-0.02 fura-2 RU in controls) and treatment with ANP reduced the amplitude of the Ca2+ transient to control levels. ANP may have a protective role in STZ-induced diabetic rat heart.     

Organization of the gene for iso-rANP, a rat B-type natriuretic peptide

Using the polymerase chain reaction and oligonucleotide primers constructed from knowledge of the cDNA sequence we have sequenced the gene for iso-rANP, a peptide of the B-type of atrial natriuretic peptides. The overall organization of the rat iso-ANP gene is the same as that of ANP and BNP consisting of three exons and two introns at relatively similar positions. Iso-rANP and it's gene are more closely related to BNPs than ANP and yet there are significant differences at both the protein and DNA levels. Our results suggest that iso-rANP and BNP are distinct members of the same sub-family (B-type natriuretic peptides) within the family of natriuretic peptide genes.     

Removal of atrial natriuretic peptide by perfused rabbit lungs in situ

Removal of iodinated 28-amino acid atrial natriuretic peptide ([125I]ANP) by rabbit lungs was measured by indicator-dilution methods. After bolus injection of 6.5 pmoles of [125I]ANP, 66.9 +/- 2.9% was removed in a single pass through the lungs. Removal was unaltered by a kininase II inhibitor but was reversibly decreased by unlabelled ANP. Thus the lungs can remove ANP from the pulmonary circulation by a mechanism that does not involve hydrolysis by kininase II. Lungs therefore may be involved in regulating systemic concentrations and hence renal and other actions of ANP.     

Ischemia stimulates the release of atrial natriuretic peptide from rat cardiac ventricular myocardium in vitro.

The effect of ischemia on atrial natriuretic peptide (ANP) release from heart ventricles was studied by exposing the perfused hearts of Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats to global ischemia after excision of the atria. Ischemia for 2, 5 and 20 min caused an increase of 0.3 +/- 1.1, 12.4 +/- 5.5 and 11.4 +/- 4.2 ng/g dry weight in ANP release of the WKY ventricles, respectively. ANP release increased 3.4 +/- 2.8 ng/g dry weight after 5 minutes' ischemia from the SHR ventricles. The increase was not caused by cell damage, as only processed form of the peptide was detected in the perfusates. The increase in ANP release in the WKY ventricles correlated positively with the tissue lactate/pyruvate ratio (r = 0.85) and adenosine (r = 0.99), and negatively with the phosphorylation potential (r = -0.70). The results indicate that ventricular ischemia increases ANP release, probably due to changes in myocardial energy metabolism.     

Binding of Brain and Atrial Natriuretic Peptides to Cultured Mouse Astrocytes and Effect on Cyclic GMP

125I-Porcine brain natriuretic peptide (125I-pBNP) bound to mouse astrocytes in primary culture in a time-dependent manner (t1/2 = 4.5 min), similar to 125I-human atrial natriuretic peptide (125I-hANP) (t1/2 = 5 min). Binding was saturable and reached equilibrium after 90 min at 22 degrees C for both radioligands. Scatchard analysis suggested a single class of binding sites for pBNP with a binding affinity and capacity (KD = 0.08 nM; Bmax = 78.3 fmol/mg of protein) similar to those of hANP1-28 (KD = 0.1 nM; Bmax = 90.3 fmol/mg of protein). In competition binding studies, pBNP or human/rat atrial natriuretic peptide (ANP) analogues [hANP1-28, rat ANP1-28 (rANP1-28), and rANP5-28] displaced 125I-hANP, 125I-pBNP, and 125I-rANP1-28 completely, all with IC50 values of less than nM (0.14-0.83 nM). All four peptides maximally stimulated cyclic GMP (cGMP) production by 10 min at 22 degrees C at concentrations of 1 microM with EC50 values ranging from 50 to 100 nM. However, maximal cGMP induction by brain natriuretic peptide (BNP) (25.9 +/- 2.1 pmol/mg of protein) was significantly greater than that by hANP1-28 (11.5 +/- 2.2 pmol/mg of protein), rANP1-28 (16.5 +/- 2.0 pmol/mg of protein), and rANP5-28 (15.8 +/- 2.2 pmol/mg of protein). These studies indicate that BNP and ANPs act on the same binding sites and with similar affinities in cultured mouse astrocytes. BNP, however, exerts a greater effect on cGMP production. The difference in both affinity and selectivity between binding and cGMP production may indicate the existence of receptor subtypes that respond differentially to natriuretic peptides despite similar binding characteristics.     

The lung as a possible target organ for atrial natriuretic polypeptide secreted from the heart

Using a specific radioimmunoassay (RIA) for alpha-rat atrial natriuretic polypeptide (alpha-rANP), we have demonstrated the presence of a considerable amount (6.10 +/- 0.38 ng/g) (mean +/- SE) of alpha-rANP-like immunoreactivity (alpha-rANP-LI) in the rat lung, the first organ through which atrial natriuretic polypeptide (ANP) released from the heart passes. High performance gel permeation chromatography coupled with the RIA revealed that most of alpha-rANP-LI eluted at the position of a low molecular weight form corresponding to synthetic alpha-rANP. In 2- or 5-day water-deprived rats, the concentration and content of alpha-rANP-LI in the lung decreased significantly compared with those of control rats. In addition, water-deprivation induced a significant decrease in the plasma concentration of alpha-rANP-LI simultaneously determined. There was a significant positive correlation between the concentrations of alpha-rANP-LI in the lung and plasma (r = 0.591, P less than 0.01). These results indicate the presence of ANP in the lung and suggest physiological roles of ANP in pulmonary function.     

Natriuretic peptides cause relaxation of human and guinea-pig gallbladder muscle through interaction with natriuretic peptide receptor-B

Atrial natriuretic peptide (ANP) binding sites have been demonstrated in the guinea-pig gallbladder muscle with unclear function. To investigate effects of natriuretic peptides in the gallbladder, we measured relaxation of isolated human and guinea-pig gallbladder strips caused by natriuretic peptides, including C-type natriuretic peptide (CNP), brain natriuretic peptide (BNP) and ANP, as well as des[Gln18, Ser19, Gly20, Leu21, Gly22]ANP(4-23) amide (cANP(4-23)), a selective natriuretic peptide receptor-C (NPR-C) agonist. Results in the human gallbladder were similar to those in the guinea-pig gallbladder. CNP, BNP, ANP and cANP(4-23) alone did not cause contraction or relaxation in resting gallbladder strips. However, in carbachol or endothelin-1-contracted strips, CNP caused moderate, sustained and concentration-dependent relaxation. The relaxation was not affected by tetrodotoxin or atropine in endothelin-1-contracted gallbladder strips and not by tetrodotoxin in carbachol-contracted strips. These indicate a direct effect of CNP on the gallbladder muscle. The relative potencies for natriuretic peptides to cause relaxation were CNP>BNP> or = ANP. cANP(4-23) did not cause relaxation. These indicate the existence of the natriuretic peptide receptor-B (NPR-B) mediating the relaxation. Taken together, these results demonstrate that natriuretic peptides cause relaxation of human and guinea-pig gallbladder muscle through interaction with the natriuretic peptide receptor-B.     

Role of natriuretic peptides in regulation of conduit artery distensibility

Arterial distensibility, assessed by the pulse-wave velocity (PWV), is an independent predictor of cardiovascular risk. We investigated whether natriuretic peptides, acting locally, modify conduit artery distensibility in vivo. All studies were conducted in anesthetized sheep (n = 18) by using a validated ovine hindlimb model. In brief, the PWV was calculated, with the use of the foot-to-foot methodology, from two pressure waveforms recorded simultaneously with a high-fidelity dual pressure-sensing catheter placed in the common iliac artery. Drugs were infused either proximally, via the catheter to perfuse the segment of artery under study, or distally, via the sheath to control for any reflex changes in flow or sympathetic activation. First, the effects of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and c-type natriuretic peptide (CNP) were studied. Second, the role of endogenous ANP was investigated by infusing the natriuretic peptide receptor type A (NPRA)-selective receptor antagonist A71915. Third, A71915 was coinfused with ANP. Fourth, the NPRC-selective agonist cANF was infused. Infusion of CNP or des-[Gln18Ser19Gly20Leu21Gly22]-ANF-(4-23)-NH2 (cANF) had no effect on iliac PWV. However, infusion of ANP, and to a lesser degree BNP, resulted in a reduction in PWV (-9%; P < 0.01 and -6%; P < 0.05, respectively). A71915 increased iliac PWV from 2.97 +/- 0.13 to 3.06 +/- 0.13 m/s; P < 0.01. Coinfusion of A71915 with ANP completely abolished the effects of ANP (P < 0.01). Importantly, ANP-BNP infusion via the sheath did not alter PWV. In conclusion, ANP, and to a lesser extent BNP, modify large artery distensibility via the NPRA receptor. Neither CNP nor cANF altered PWV, suggesting that the NPRB and NPRC receptors do not acutely influence distensibility in vivo.     

Role of NPR-C natriuretic receptor in nitric oxide system activation induced by atrial natriuretic peptide

Atrial natriuretic peptide (ANP) exerts its hypotensive, natriuretic and diuretic effects, almost in part, through the activation of nitric oxide synthase (NOS). The aim was to investigate the natriuretic receptor type and the signaling cascade involved in NOS activation induced by ANP. Male Wistar rats were sacrificed and NOS activity was determined in kidney, aorta and heart with L-[U14C]-arginine, as substrate. ANP and cANP (4-23), a selective NPR-C ligand, increased NOS activity in all tissues. ANP induced a more marked activation in aorta and kidney than cANP (4-23), but no difference in atria NOS activation was observed. NOS activity induced by both peptides was blunted by nifedipine (L-type channel blocker) and calmidazolium (calmodulin antagonist) in heart and aorta. In kidney, nifedipine and calmidazolium abolished NOS activity stimulated by cANP (4-23) but only partially inhibited NOS activity elicited by ANP. Gi inhibition with pertussis toxin abolished NOS activity stimulated by ANP and cANP in atria but only partially inhibited the increased NOS activity induced by ANP and cANP in kidney, aorta and ventricle. Our results show that NPR-C receptor would mediate the activation of NOS by ANP in atria. In kidney, aorta and ventricle, NOS activation would also involve NPR-A and/or B. ANP would interact with NPR-C coupled via Gi to activation Ca2+ -dependent NOS.     

Bay K 8644, a voltage-sensitive calcium channel agonist, facilitates secretion of atrial natriuretic polypeptide from isolated perfused rat hearts

Effects of Bay K 8644, a voltage-sensitive calcium channel agonist, on atrial natriuretic polypeptide (ANP) secretion from isolated rat hearts perfused with Krebs-Henseleit solution were investigated. After a ninety-min period for stabilization, coronary sinus effluents were collected every two min and ANP levels were measured by radioimmunoassay. The basal secretory rate of ANP was 1.65 +/- 0.15 ng/min (mean +/- standard error). Bay K 8644 stimulated ANP secretion dose-dependently. This stimulatory action was blocked by simultaneous administration of nifedipine, its competitive antagonist. Heart rate was also increased by Bay K 8644 administration. In the gel filtration study, the major secretory form of ANP corresponded to alpha-rat ANP, a 28-amino acid peptide. These results suggest that voltage-sensitive calcium channels are involved in two principal biological properties, contraction and ANP secretion, of atrial cardiocytes.     

HS-142-1, a novel polysaccharide of microbial origin, specifically recognizes guanylyl cyclase-linked ANP receptor in rat glomeruli.

HS-142-1, a novel polysaccharide, of microbial origin had been characterized as a specific antagonist of guanylyl cyclase-linked atrial natriuretic peptide (ANP) receptors (ANP-GC receptor) in bovine adrenal cortex. The effect of HS-142-1 on ANP receptors of rat glomeruli were examined. HS-142-1 blocked rat ANP (r-ANP)-stimulated cGMP production in a concentration-dependent manner, although it caused only slight inhibition in the specific binding of [125I]-rANP to the glomeruli where only a small portion of the binding sites are coupled to guanylyl cyclase. HS-142-1 recognized the 135K ANP receptor which is thought to be ANP-GC receptors but did not recognized 60K receptor, guanylyl cyclase-free type from affinity cross-linking studies with glomerular membranes. These results indicate that HS-142-1 is a specific antagonist for the ANP-GC receptor in rat glomeruli, and that it will be a powerful tool for understanding the physiological roles of ANP in renal responses.     

Regulation of atrial natriuretic peptide secretion by cholinergic and PACAP neurons of the gastric antrum

Atrial natriuretic peptide (ANP) released from enterochromaffin cells helps regulate antral somatostatin secretion, but the mechanisms regulating ANP secretion are not known. We superfused rat antral segments with selective neural agonists/antagonists to identify the neural pathways regulating ANP secretion. The nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) stimulated ANP secretion; the effect was abolished by hexamethonium but doubled by atropine. Atropine's effect implied that DMPP activated concomitantly cholinergic neurons that inhibit and noncholinergic neurons that stimulate ANP secretion, the latter effect predominating. Methacholine inhibited ANP secretion. Neither bombesin nor vasoactive intestinal polypeptide stimulated ANP secretion, whereas pituitary adenylate cyclase-activating polypeptide (PACAP)-27, PACAP-38, and maxadilan [PACAP type 1 (PAC1) agonist] each stimulated ANP secretion. The PAC1 antagonist M65 1) abolished PACAP-27/38-stimulated ANP secretion; 2) inhibited basal ANP secretion by 28 +/- 5%, implying that endogenous PACAP stimulates ANP secretion; and 3) converted the ANP response to DMPP from 109 +/- 21% above to 40 +/- 5% below basal, unmasking the cholinergic component and indicating that DMPP activated PACAP neurons that stimulate ANP secretion. Combined atropine and M65 restored DMPP-stimulated ANP secretion to basal levels. ANP secretion in the antrum is thus regulated by intramural cholinergic and PACAP neurons; cholinergic neurons inhibit and PACAP neurons stimulate ANP secretion.     

人和大鼠脊髓内的心钠素样物质

应用特异的心钠素免疫金银染色和放射免疫测定法,证明在人和大鼠脊髓内亦存在有心钠素样物质。心钠素免疫金银染色发现在人脊髓各段均有心钠素免疫反应阳性的神经元广泛分布。这些神经元主要位于脊髓腹角,同时脊髓背角和侧角亦有少量分布。应用对照吸收试验,其心钠素免疫反应阳性颗粒便消失或明显减少。心钠素放射免疫测定发现,从大鼠颈髓到胸、腰、骶髓均有心钠素样物质存在,其中以骶髓含量最高,为21.9±4.48ng/g组织;腰髓次之,为3.78±0.74ng/g组织;颈、胸髓含量最低,分别为0.58±0.14和0.46±0.21ng/g组织。应用凝胶过滤和高压液相层析证明,大鼠脊髓中心钠素亦以多分子形式存在,但以28个氨基酸的大鼠心房利纳多肽(rANP)为主。此外,对在体大鼠脊髓蛛网膜下腔灌流研究发现,高钾去极化刺激可使大鼠脊髓心钠素样物质释放。     

Hypoxia reduces atrial natriuretic peptide clearance receptor gene expression in ANP knockout mice

We tested the hypotheses that hypoxic exposure is associated with exacerbated pulmonary hypertension and right ventricular (RV) enlargement, reduced atrial natriuretic peptide (ANP) clearance receptor (NPR-C) expression, and enhanced B-type natriuretic peptide (BNP) expression in the absence of ANP. Male wild-type [ANP(+/+)], heterozygous [ANP(+/-)], and homozygous [ANP(-/-)] mice were studied after a 5-wk hypoxic exposure (10% O(2)). Hypoxia increased RV ANP mRNA and plasma ANP levels only in ANP(+/+) and ANP(+/-) mice. Hypoxia-induced increases in RV pressure were significantly greater in ANP(-/-) than in ANP(+/+) or ANP(+/-) mice (104 +/- 17 vs. 45 +/- 10 and 63 +/- 7%, respectively) as were increases in RV mass (38 +/- 4 vs. 26 +/- 5 and 29 +/- 4%, respectively). NPR-C mRNA levels were greatly reduced in the kidney, lung, and brain by hypoxia in all three genotypes. RV BNP mRNA and lung and kidney cGMP levels were increased in hypoxic mice. These findings indicate that disrupted ANP expression worsens hypoxic pulmonary hypertension and RV enlargement but does not alter hypoxia-induced decreases in NPR-C and suggest that compensatory increases in BNP expression occur in the absence of ANP.