Brain natriuretic peptide-like immunoreactivity is present in human plasma

A highly specific and sensitive radioimmunoassay (RIA) for a novel porcine brain natriuretic peptide (BNP) has been established to elucidate whether BNP-like immunoreactivity (LI) is present in human plasma. The antibody used was specific for BNP without any crossreactivities with known human atrial natriuretic peptides (hANP). After extraction of human plasma with Sep-Pak cartridge, this assay allowed for detection of BNP-LI as low as 0.1 fmol/tube. In 12 healthy subjects, the mean concentrations of plasma BNP-LI were 1.5 fmol/ml. Reverse-phase HPLC coupled with BNP RIA revealed that the single major component with BNP-LI, corresponding to porcine BNP(1–26), was apparently distinct from that of -hANP. These data indicate that a small molecular mass form with BNP-LI circulates in human blood.     

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.     

Brain natriuretic peptide is cosecreted with atrial natriuretic peptide from porcine cardiocytes

Using primary cultures of atrial cardiocytes from neonatal pig, the secretion brain natriuretic peptide (BNP) and atrial natriuretic peptide (ANP)-like immunoreactivities (LI) was studied in vitro. Porcine cardiocytes time-dependently secreted both BNP-LI and ANP-LI into medium under a serum-free condition, although the amount of BNP-LI secreted was about one-third that of ANP-LI. Phorbol ester and Ca2+ ionophore had less stimulatory effects on secretion of BNP-LI than that of ANP-LI. Reverse-phase HPLC of the conditioned medium revealed a single major BNP-LI component corresponding to synthetic porcine BNP(1-26). These data suggest that a small molecular weight form BNP, possibly BNP(1-26), is cosecreted with ANP from porcine cardiocytes.     

Biosynthesis, secretion, and receptor selectivity of human brain natriuretic peptide.

Biosynthesis, secretion and receptor selectivity of human brain natriuretic peptide (hBNP) were studied. The BNP mRNA level in the ventricle was approximately 40% of that in the atrium and, taking tissue weight into account, the total amount of BNP mRNA in the ventricle was about twofold greater than the total amount in the atrium. The plasma BNP-like immuno-reactivity (-LI) level in normal subjects was 0.90 +/- 0.07 fmol/mL, which was 16% of the ANP-LI level. In contrast, the plasma BNP-LI level markedly increased in patients with congestive heart failure, with a progressive rise in proportion to its severity. There was a significant step-up of the plasma BNP-LI level in the coronary sinus (CS) compared with that in the aortic root, and the difference in the plasma BNP-LI level between the CS and the aorta (Ao), delta (CS-Ao)BNP, increased with the severity of congestive heart failure. In addition, the difference in the BNP-LI level between the anterior inverventricular vein (AIV) draining the ventricle and the aorta (delta (AIV-Ao)BNP) was comparable to delta (CS-Ao) BNP, indicating that BNP is secreted predominantly from the ventricle. Binding ability to human clearance receptors (C receptors) and cyclic GMP (cGMP) production of hBNP were investigated and compared with those of ANP. hBNP bound to human C receptors very weakly (about 7%), but exerted cGMP production similar to ANP in cultured human mesangial cells and bovine endothelial cells. In conclusion, hBNP is a novel cardiac hormone mainly synthesized in and secreted from the ventricle and plays physiological and pathophysiological roles in the dual cardiac natriuretic peptide system.     

Biological characterization of human brain natriuretic peptide (BNP) and rat BNP: species-specific actions of BNP

We examined the diuretic-natriuretic activities of rat BNP and human BNP in anesthetized rats in vivo and their vasorelaxant activities for rat thoracic aorta and porcine coronary artery in vitro. Rat BNP was almost equipotent to rat ANP in diuresis and natriuresis with relative potencies of 1.6 and 2.5, respectively, while human BNP exerted no significant activity. Rat ANP, rat BNP and human BNP relaxed PGF2 alpha-contracted rat aortic strips with IC50 values of 0.62, 0.64 and 12.1 nM, respectively, while they relaxed PGF2 alpha-contracted porcine coronary arteries with IC50 values of 0.04, 1.10 and 0.02 nM, respectively. These results strongly suggest that the biological action of BNP is species-specific.     

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

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

Peptide YY receptors in the brain

Radiolabelled ligand binding studies demonstrated that specific receptors for peptide YY are present in the porcine as well as the canine brains. Peptide YY was bound to brain tissue membranes via high-affinity (dissociation constant, 1.39 X 10(-10)M) and low-affinity (dissociation constant, 3.72 X 10(-8)M) components. The binding sites showed a high specificity for peptide YY and neuropeptide Y, but not for pancreatic polypeptide or structurally unrelated peptides. The specific activity of peptide YY binding was highest in the hippocampus, followed by the pituitary gland, the hypothalamus, and the amygdala of the porcine brain, this pattern being similarly observed in the canine brain. The results suggest that peptide YY and neuropeptide Y may regulate the function of these regions of the brain through interaction with a common receptor site.     

Isolation and identification of human brain natriuretic peptides in cardiac atrium

By using a radioimmunoassay (RIA) system newly established for human brain natriuretic peptide (BNP), a high concentration of immunoreactive (ir-) human BNP (hBNP) has been found in cardiac atrium (1). Two molecular forms of ir-hBNP of 4K and 13-15K were isolated from atrial extracts by using anti-hBNP IgG immunoaffinity chromatography and reverse phase high performance liquid chromatography (HPLC). By microsequencing, the peptides were determined to be a pro-hBNP (gamma-hBNP) and its C-terminal 32-amino acid peptide (hBNP-32). Based on these results, in cardiac atrium, hBNP is found to be processed in a pathway similar to that of porcine BNP (pBNP) but distinct from that of rat BNP, although low MW hBNP-32 is a major form in contrast to pBNP which exists as a high MW gamma-pBNP.     

The presence of brain natriuretic peptide of 12,000 daltons in porcine heart

Brain natriuretic peptide (BNP) and its N-terminally six amino acid extended form (BNP-32) have been identified in porcine brain. These peptides exert diuretic-natriuretic and hypotensive effects, and have remarkably high sequence homology to atrial natriuretic peptide (ANP). We have set up a radioimmunoassay system specific to BNP and surveyed immunoreactive (ir-) BNP in peripheral tissue. In porcine cardiac atrium, we found the highest concentration of ir-BNP. By using gel filtration and reverse phase high performance liquid chromatography, ir-BNP was characterized. Most of ir-BNP in the atrium was found to exist as a high molecular weight form of 12,000 daltons; less than 15% of the total ir-BNP exist as low molecular weight forms such as BNP and BNP-32. These results suggest that BNP functions as a circulating hormone in addition to the neuropeptide function in brain.     

Radioimmunoassay with heterologous antibody (hetero-antibody RIA): utilization of highly cross-reactive antibody present in polyclonal antiserum.

To develop a homologous radioimmunoassay (RIA) for a hormone of a small or rare animal often meets difficulty in collecting a large amount of purified antigen required for antibody production. On the other hand, to employ a heterologous RIA to estimate the hormone often gives poor sensitivity. To overcome this difficulty, a "hetero-antibody" RIA was studied. In a hetero-antibody RIA system, a purified preparation of a hormone is used for radioiodination and standardization and a heterologous antibody to the hormone is used for the first antibody. Canine motilin and rat LH were selected as examples, and anti-porcine motilin and anti-hCG, anti-hCG beta or anti-ovine LH beta was used as the heterologous antibody. The sensitivities of the hetero-antibody RIAs were much higher than those of heterologous RIAs in any case, showing that these hetero-antibody RIA systems were suitable for practical use. To clarify the principle of hetero-antibody RIA, antiserum to porcine motilin was fractionated on an affinity column where canine motilin was immobilized. The fraction bound had greater constants of affinity with both porcine and canine motilins than the rest of the antibody fractions. This fraction also reacted with a synthetic peptide corresponding to the C-terminal sequence common to porcine and canine motilins in a competitive binding test with labeled canine motilin. These results suggest that an antibody population having high affinity and cross-reactivity is present in polyclonal antiserum and indicate that the population can be used in hetero-antibody RIA at an appropriate concentration.     

Distribution and molecular forms of brain natriuretic peptide in porcine heart and blood

Although brain natriuretic peptide (BNP) is a novel natriuretic peptide originally identified in porcine brain, recent investigation has verified the presence of BNP in porcine heart. In order to identify BNP as a circulating hormone, we analyzed the regional distribution and molecular form of immunoreactive (ir-) BNP in heart and blood. Tissue concentration of ir-BNP was high in atrium, but low in ventricle, in a manner similar to that of atrial natriuretic peptide (ANP). However, the concentration of ir-BNP in atrium was only about 1/50 that of ir-ANP. In plasma, ir-BNP was found at a concentration of 1-3 fmol/ml, which was about 1/20 that of ir-ANP. Both ir-BNP and ir-ANP were present as low molecular weight forms. Three forms of ir-BNP of about 3K daltons, including BNP-26, BNP-29 and BNP-32, are thought to circulate in blood.     

Immunoreactive forms of natriuretic peptides in ovine brain: response to heart failure

In order to elucidate how brain natriuretic peptides (NPs) are affected by experimentally induced heart failure, we have measured the immunoreactive (IR) levels of the NP in extracts from 10 regions of ovine brain, including pituitary, and clarified their molecular forms using high performance liquid chromatography (HPLC). Using species-specific radioimmunoassay (RIA), atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) were all detected in extracts taken from control animals and sheep that had undergone rapid ventricular pacing for 7 days to induce heart failure. CNP was the most abundant NP as assessed by specific RIA, and the pituitary contained the highest IR levels for all three NP. Compared with control animals, the pituitary content of BNP in animals with heart failure was reduced by 40% (control, 0.26±0.02 pmol/g wet weight versus heart failure 0.16±0.01; P<0.01, n=7). No other significant changes were observed. The molecular forms of ANP and CNP in whole brain extracts as assessed by HPLC were proANP and CNP22, CNP53 and proCNP, respectively. BNP in pituitary extracts was assessed to be primarily proBNP with a minor component of mature BNP26.     

Isolation and identification of rat brain natriuretic peptides in cardiac atrium

The amino acid sequence of a precursor for rat brain natriuretic peptide (BNP) has recently been deduced by the cDNA cloning method. By using a radioimmunoassay (RIA) system newly established for rat BNP, a high concentration of ir-BNP was found to exist in rat cardiac atrium. Two ir-BNPs of different molecular weights (11K and 5K) were isolated from rat cardiac atria by anti-rat BNP IgG immunoaffinity chromatography and reverse phase high performance liquid chromatography (HPLC). By microsequencing, the high molecular weight (MW) BNP was deduced to be a pro-BNP of 95 residues (gamma-BNP). The low MW BNP was demonstrated to be a C-terminal 45-amino acid peptide (BNP-45) of pro-BNP. Based on these results, BNP-45 and gamma-BNP are shown to be two major forms in rat cardiac atrium, indicating a unique processing pathway of rat BNP precursor.     

Distribution and molecular forms of brain natriuretic peptide in the central nervous system, heart and peripheral tissue of rat

The amino acid sequence of rat brain natriuretic peptide (rBNP) precursor has recently been deduced by the cDNA cloning method (1). In the present study, a radioimmunoassay (RIA) system for rBNP was newly established, and regional distribution of rBNP in the central nervous system, heart and other peripheral tissue of rat was investigated. In heart, especially in cardiac atrium, a high concentration of immunoreactive (ir-) rBNP was detected and identified as rBNP-45 and gamma-rBNP. No significant amount of ir-rBNP was found in other tissues examined including the central nervous system. Especially in brain, no ir-rBNP was detected, while ir-rat atrial natriuretic peptide (rANP) was observed at a relatively high concentration. These results demonstrate a sharp contrast between rat and porcine brains in ir-BNP distribution.     

Cloning and sequence analysis of cDNA encoding a precursor for rat brain natriuretic peptide

Brain natriuretic peptide (BNP) is a new type of natriuretic peptide, which has so far been identified only in porcine brain and atrium. Immunological observations suggest that rat and porcine BNP may have structural difference according to species. To identify rat BNP, we constructed a rat atrial cDNA library, and screened for clones encoding rat BNP-precursor by using part of porcine BNP cDNA as a probe. By sequencing a cloned cDNA, the amino acid sequence of rat BNP-precursor comprising 121 residues was deduced as carrying a 26-residue putative signal peptide at the N-terminus and a region homologous to porcine BNP-32 at the C-terminus. In addition, remarkably high homology between rat and porcine BNP-precursors was observed in the 3'-untranslated AT-rich region. Comparing sequences of precursors of ANP and BNP thus far identified, structural and processing features characteristic of the BNP family were discussed.     

Further evidence for non-pancreatic insulin immunoreactivity in guinea pig brain

The existence of large amounts of insulin in rat brain and of a porcine- or rat-like insulin in guinea pig brain have been disputed on the basis of differing results from standard (Method I) and hydrophobic adsorption techniques (Method II) for concentrating insulin from acid ethanol extracts. To try to resolve these differences, acid ethanol extracts of rat and guinea pig brains were divided into equal aliquots and concentrated for insulin radioimmunoassay (RIA) by both techniques. The RIA used guinea pig anti-porcine insulin serum, with 50% B0 for purified pancreatic porcine, rat and guinea pig insulin standards being 1.35, 2.38 and greater than 1,000 ng/ml, respectively. Oral glucose (4 g/kg) produced plasma glucose of 377 mg/dl in a guinea pig by 20 min but was not associated with any porcine- or rat-like immunoreactive insulin. Dilutions of guinea pig and rat brain extracts had parallel cross-reactivity with insulin standard curves. Insulin contents of rat brain (uncorrected for recovery) against porcine and rat insulin standards, respectively, were 1.33 and 1.93 ng/g (Method I) and 5.93 and 11.67 ng/g (Method II). Rat plasma was 0.85 and 1.42 ng/ml, respectively. Guinea pig contained 1.35 and 1.89 ng/g (uncorrected), respectively (Method I), and 2.99 and 5.62 ng/g, respectively (Method II). Guinea pig plasma was below the sensitivity of the RIA (less than 0.15 ng/ml). These results suggest that a porcine- or rat-like insulin may exist in guinea pig brain.     

Purification and sequence determination of canine relaxin

Relaxin immunological activity has been observed in the plasma of pregnant bitches, and preliminary studies in our laboratory indicated that the highest relaxin concentrations were found in placentas. Therefore, canine placentas were collected at term and also from spay and relaxin was purified by methods developed for equine relaxin. Tissue was prepared by homogenization and purification on a C₁₈ column. The preparation was further purified by stepwise elution ion-exchange chromatography, gel filtration, and gradient elution ion-exchange chromatography. One predominant peak in relaxin immunoactivity was collected. Canine relaxin was found to be larger than either porcine or equine relaxin as determined by SDS-PAGE. It migrated faster under reducing conditions, indicating a subunit structure. Purified canine relaxin was used for tracer and standard in a canine radioimmunoassay (RIA) using an antiporcine relaxin antibody. Concentrations of relaxin immunoactivity using the canine assay were up to 300-fold higher in placental preparations than those measured in the porcine relaxin assay. Sequence analysis of canine relaxin revealed a structure similar to other relaxins in the presence and placement of cystine residues.     

大鼠脑内脑钠素mRNA分布的原位杂交

本工作以特异性的放射磷标记的鼠脑钠肽基因片段为探针,利用高灵敏度的原位杂交分析方法,对大鼠脑组织切片中脑钠肽的ｍＲＮＡ分布进行测定。结果表明,大鼠脑中存在脑钠肽基因的表达,其中下丘脑以及丘脑的边缘部分脑肽的ｍＲＮＡ浓度最高,杏仁核簇中浓度较高,嗅区其次,海马回和大脑皮层中浓度较低,小脑和延髓中几乎没有基因表达。     

Cloning of a cDNA encoding porcine brain natriuretic peptide

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