Immunohistochemical localisation of natriuretic peptides in the heart and brain of the gulf toadfish Opsanus beta

Summary The distribution of natriuretic peptide immunoreactivity was determined in the heart and brain of the gulf toadfish Opsanus beta using the avidin-biotin peroxidase technique. Four antisera were used: the first raised against porcine brain natriuretic peptide which cross-reacts with atrial natriuretic and C-type natriuretic peptides (termed natriuretic peptide-like immunoreactivity); the second raised against porcine brain natriuretic peptide which cross-reacts with C-type natriuretic peptide but not with atrial natriuretic peptide (termed porcine brain natriuretic peptide-like immunoreactivity); the third raised against rat atrial natriuretic peptide; and the fourth raised against eel atrial natriuretic peptide. Natriuretic peptide- and porcine brain natriuretic peptide-like immunoreactivity was observed in all cardiac muscle cells of the atrium. In the ventricle, natriuretic peptide-like immunoreactivity was found in all cardiac muscle cells, however porcine brain natriuretic peptidelike immunoreactivity was confined to muscle cells adjacent to the epicardium. There was no discernible difference in the distribution of natriuretic peptide-like immunoreactivity and porcine brain natriuretic peptide-like immunoreactivity in the brain. Immunoreactive perikarya were observed only in the preoptic region of the diencephalon, and many immunoreactive fibres were found in the telencephalon, preoptic area, and rostral hypothalamus, lateral to the thalamic region. There was no immunoreactivity in any region of the hypophysis. A pair of distinct immunoreactive fibre tracts ran caudally from the preoptic area to the thalamic region, from which fibres extended to the posterior commissure, area praetectalis, dorsolateral regions of the midbrain tegmentum, and tectum. Many immunoreactive fibres were present in the rostral regions of the inferior lobes of the hypothalamus and in the dorsolateral and ventrolateral aspects of the rhombencephalon. No immunoreactivity was observed in the heart and brain using rat atrial natriuretic and eel natriuretic peptide antisera. Although the chemical structure of natriuretic peptides in the heart and brain of toadfish is unknown, these observations show that a component of the natriuretic peptide complement is similar to porcine brain natriuretic and/or porcine C-type natriuretic peptides. The presence of natriuretic peptides in the brain suggests that they could be important neuromodulators and/or neurotransmitters.     

Human brain natriuretic peptide-like immunoreactivity in human brain.

The presence of immunoreactive human brain natriuretic peptide in the human brain was studied with a specific radioimmunoassay for human brain natriuretic peptide-32. This assay showed no significant cross-reaction with human alpha atrial natriuretic peptide, porcine brain natriuretic peptide or rat brain natriuretic peptide. Immunoreactive human brain natriuretic peptide was found in all 5 regions of human brain examined (cerebral cortex, thalamus, cerebellum, pons and hypothalamus) (0.6-6.7 pmol/g wet weight, n = 3). These values were comparable to the concentrations of immunoreactive alpha atrial natriuretic peptide in human brain (0.5-10.1 pmol/g wet weight). However, Sephadex G-50 column chromatography showed that the immunoreactive human brain natriuretic peptide in the human brain eluted earlier than synthetic human brain natriuretic peptide-32. These findings suggest that human brain natriuretic peptide is present in the human brain mainly as larger molecular weight forms.     

Immunohistochemical localisation of natriuretic peptides in the brains and hearts of the spiny dogfishSqualus acanthias and the Atlantic hagfishMyxine glutinosa

Summary The avidin-biotin peroxidase technique was used to determine the distribution of natriuretic peptides in the hearts and brains of the dogfishSqualus acanthias and the Atlantic hagfishMyxine glutinosa. Three antisera were used: one raised against porcine brain natriuretic peptide which cross-reacts with atrial natriuretic and C-type natriuretic peptides (termed natriuretic peptide-like immunoreactivity); the second raised against porcine brain natriuretic peptide which cross-reacts with C-type natriuretic peptide, but not with atrial natriuretic peptide (termed porcine brain natriuretic peptide-like immunoreactivity); and the third raised against rat atrial natriuretic peptide (termed rat atrial natriuretic peptide-like immunoreactivity). Only natriuretic peptide-like immunoreactivity was observed in the heart ofS. acanthias which was most likely due to the antiserum cross-reacting with C-type natriuretic peptide. No immunoreactivity was found in theM. glutinosa heart. In the brain ofS. acanthias, natriuretic peptide-like immunoreactive fibres were located in many areas of the telencephalon, diencephalon, mesencephalon, rhombencephalon, and spinal cord. Extensive immunoreactivity was observed in the hypothalamo-hypophyseal tract and the neurointermediate lobe of the hypophysis. Natriuretic peptide-like immunoreactive perikarya were found in ventromedial regions of the telencephalon and in the nucleus preopticus. Most perikarya had short, thick processes which extended toward the ventricle. Another group of perikarya was observed in the rhombencephalon. Porcine brain natriuretic peptide-like immunoreactive fibres were observed in the telencephalon, diencephalon, mesencephalon, and rhombencephalon, but perikarya were only present in the preoptic area. In theM. glutinosa brain, natriuretic peptide-like immunoreactive fibres were present in all regions. Immunoreactive perikarya were observed in the pallium, primordium hippocampi, pars ventralis thalami, pars dorsalis thalami, nucleus diffusus hypothalami, nucleus profundus, nucleus tuberculi posterioris, and nucleus ventralis tegmenti. Procine brain natriuretic peptide-like immunoreactive perikarya and fibres had a similar, but less abundant distribution than natriuretic peptide-like immunoreactive structures. Although the chemical structures of natriuretic peptides in the brains of dogfish and hagfish are unknown, these observations show that a component of the natriuretic peptide complement is similar to porcine brain natriuretic peptide or porcine C-type natriuretic peptide. The presence of natriuretic peptides in the brain suggest they could be important neuromodulators and/or neurotransmitters. Furthermore, there appears to be divergence in the structural forms of natriuretic peptides in the hearts and brains of dogfish and hagfish.     

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.     

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.     

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

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

Atrial and brain natriuretic peptides share binding sites on cultured cells from the rat trachea

Summary We examined the distribution of binding sites for alpha-atrial natriuretic peptide (¹²⁵I-ANP_1–28) and the recently discovered porcine brain natriuretic peptide (¹²⁵I-pBNP) on immunocytochemically identified cells in dissociated culture preparations of the rat trachea. Specific binding sites for both ¹²⁵I-ANP_1–28 and ¹²⁵I-pBNP were evenly distributed over distinet subpopulations of smooth muscle myosin-like immunoreactive muscle cells, fibronectin-like immunoreactive fibroblasts and S-100-like immunoreactive glial cells. Neither keratin-like immunoreactive epithelial cells nor protein gene product 9.5-like immunoreactive paratracheal neurones expressed natriuretic peptide binding sites, although autoradiographically labelled glial cells were seen in close association with both neuronal cell bodies and neurites. The binding of each radiolabelled peptide was abolished by the inclusion of either excess (1 M) unlabelled rat ANP or excess unlabelled porcine BNP, suggesting that ANP and BNP share binding sites in the trachea. Furthermore, the ring-deleted analogue, Des-[Gln¹⁸, Ser¹⁹, Gly²⁰, Leu²¹, Gly²²]-ANF_4–23-NH₂, strongly competed for specific ¹²⁵I-ANP_1–28 and ¹²⁵I-pBNP binding sites in the tracheal cultures; this suggests that virtually all binding sites were of the clearance (ANP-C or ANF-R₂) receptor subtype.     

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.     

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.     

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.     

Cloning and sequence analysis of complementary DNA encoding a precursor for chicken natriuretic peptide

Chicken -natriuretic peptide (-chNP) has been identified in chicken heart, which showed higher homology to brain natriuretic peptide (BNP) than to atrial natriuretic peptide (ANP) [1]. Complementary DNA (cDNA) clone encoding a chNP precursor (pre-chNP) precursor (pre-chNP) was isolated from cardiac cDNA library and sequenced. Pre-chNP was 140-residue signal peptide at the N-terminus and -chNP at the C-terminus, and did not exhibit high homology to poreine BNP except for the C-terminal region. However, a characteristic AT-rich nucleotide sequence commonly found in mammalian BNPs was also present in the 3′-untranslated region. Thus, chNP is concluded to be classified into the BNP-type     

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.     

Isolation and characterization of a glycine-extended form of calcitonin receptor-stimulating peptide-1: another biologically active form of calcitonin receptor-stimulating peptide-1

In this study, we isolated a peptide eliciting a potent stimulatory effect on cAMP production in LLC-PK₁ cells from acid extracts of porcine brain. By structural analysis, this peptide was determined to be a C-terminal glycine-extended form of calcitonin receptor-stimulating peptide-1 (CRSP-1-Gly). Synthetic CRSP-1-Gly enhanced the cAMP production in COS-7 cells expressing calcitonin (CT) receptor as strongly as CRSP-1. Measurement of immunoreactive (IR) CRSP-1-Gly by radioimmunoassay using the specific antisera against CRSP-1-Gly showed that a relatively high level (>1 pmol/g wet weight) of IR-CRSP-1-Gly was detected in the midbrain, hypothalamus, anterior and posterior lobes of pituitary, and thyroid gland, and the ratio of IR-CRSP-1-Gly to total IR-CRSP-1 varies from 0.02 to 0.35 in each tissue. These results suggest that CRSP-1-Gly is actually present in the tissues as one of major endogenous molecular forms of CRSP-1, and can regulate the cells expressing the CT receptor both in the central nervous system and peripheral tissues in a manner similar to that of CRSP-1. IR-CRSP-2 and IR-CRSP-3 are also present in the brain and other tissues, but their tissue concentrations are 33% on average and less than 3% that of total IR-CRSP-1, respectively.     

Dendroaspis natriuretic peptide system and its paracrine function in rat colon

Dendroaspis natriuretic peptide (DNP), a 38-amino-acid peptide, was isolated from the venom of Green Mamba. It has structural and functional similarities to other members of the natriuretic peptide family. The purpose of this study was to determine whether DNP system is present in the rat colon and to define its biological functions. The serial dilution curve of extracts of colonic tissues was parallel to the standard curve of DNP and a major peak of molecular profile by HPLC was synthetic DNP. The concentration of DNP was 0.5±0.04 ng/g of colonic tissues. DNP as well as atrial natriuretic peptide and C-type natriuretic peptide caused dose-dependent increases in cGMP production in the purified membrane of colonic tissues. Three types of natriuretic peptide receptor mRNAs were detected using semi-quantitative RT-PCR. Functionally, synthetic DNP inhibited the spontaneous contraction of rat colonic circular muscle in a concentration-dependent manner. The potency appeared to be at least 10 times greater than that of CNP. Furthermore, DNP inhibited carbachol-induced muscle contraction, suggesting that it also can modulate the nerve regulation of colonic motility. This study demonstrates the presence of DNP system in rat colon and its function as a local regulator of colonic motility.     

大鼠中枢和外周组织中脑钠素样物质的分布、特性和作用

本工作首先应用特异性脑钠素放射免疫测定、放射受体分析和免疫组织化学的方法,研究了大鼠中枢神经系统和一些外周组织中脑钠素免疫活性物质的分布、生化特性、受体结合和生物学作用,提出脑钠素可作为一种新的神经递质或循环激素,广泛分布于体内不同组织内,并参与水电介质和心血管活动的调节。     

Ventricular and atrial myocytes of newborn rats synthesise and secrete atrial natriuretic peptide in culture: Light-and electron-microscopical localisation and chromatographic examination of stored and secreted molecular forms

Summary We have demonstrated that atrial natriuretic peptide-like immunoreactivity is stored and secreted by ventricular and atrial myocytes in dissociated cell culture preparations from the heart of newborn rat. Culture preparations were maintained in either foetal calf serum-supplemented medium 199 or in hormone-supplemented, serum-free medium 199. The presence of atrial natriuretic peptidelike immunoreactivity in the cultured myocytes was demonstrated at both light-and electron-microscopical levels. Release of atrial natriuretic peptide-like immunoreactivity into the culture medium was measured by radioimmunoassay; molecular forms of the stored and secreted peptide were determined by gel column chromatography. The atrial natriuretic peptide-like immunoreactivity of cultured atrial and ventricular myocytes was concentrated in the perinuclear cytoplasm and was localised to electron-dense secretory granules. The number of immunoreactive ventricular myocytes and the intensity of their immunofluorescence changed with time in culture and was higher in cultures in foetal calf serum-supplemented medium than in serum-free medium. Gamma-atrial natriuretic peptide was stored and released by cultured atrial and ventricular myocytes, but was broken down to alpha-atrial natriuretic peptide in the growth medium. This process was foetal calf serum-independent, since it occurred in both the media used, indicating that cardiac myocytes in culture may release a factor that cleaves gamma-atrial natriuretic peptide to form alphaatrial natriuretic peptide.     

Natriuretic peptides in cardiovascular diseases

The natriuretic peptide family comprises atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), dendroaspis natriuretic peptide (DNP), and urodilatin. The activities of natriuretic peptides and endothelins are strictly associated with each other. ANP and BNP inhibit endothelin-1 (ET-1) production. ET-1 stimulates natriuretic peptide synthesis. All natriuretic peptides are synthesized from polypeptide precursors. Changes in natriuretic peptides and endothelin release were observed in many cardiovascular diseases: e.g. chronic heart failure, left ventricular dysfunction and coronary artery disease.     

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

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

A Comparison of the Lipolytic Activity of Different Natriuretic Peptides on Human Adipocytes

Atrial natriuretic peptide (ANP) was recently shown to promote triacylglycerol hydrolysis in human white adipocytes both in vitro and in vivo through a cGMP-dependent pathway. The ANP-stimulated lipolytic effect is known to be specific to primates. In this study, we compared the lipolytic effect of different natriuretic peptides obtained from several species, including ANP from human, rat, chicken, frog, and eel, brain natriuretic peptide (BNP) from porcine and rat, C-type natriuretic peptide (CNP) from human, chicken, and frog, Dendroaspis natriuretic peptide (DNP), urodilatin, and des-[Gln¹⁸, Ser¹⁹, Gly²⁰, Leu²¹, Gly²²]-ANP (C-ANP), on human and rat adipocytes. We also compared the amount of intracellular cGMP produced in both human and rat adipocytes that were treated with natriuretic peptides. Among these NPs, rat ANP, as well as porcine and rat BNP, DNP and urodilatin showed the ability to elevate intracellular cGMP and to stimulate lipolysis as human ANP. No natriuretic peptide showed the ability to stimulate lipolysis in rat adipocytes, though some of them induced significant elevation of intracelluar cGMP concentrations. These results suggest that ANP and BNP from species close to human have the ability to induce lipolysis in human adipocytes. Jiahua Yu and Yeon Jun Jeong contributed equally.