Microenzyme immunoassay for the measurement of brain natriuretic peptide (BNP)-like immunoreactivity in porcine plasma

A sensitive and specific microenzyme immunoassay (EIA) procedure for porcine brain natriuretic peptide (BNP)-like immunoreactivity has been developed. Enzyme-labeled antigen was prepared by conjugation of synthetic BNP with beta-D-galactosidase using N-(epsilon-maleimidocaproyloxy)succinimide method. Using a second antibody-coated immunoplate, the minimum amount of BNP-like immunoreactivity (BNP-LI) detectable by this assay system was 1.6 fmol/well. When porcine BNP-LI in porcine plasma was assayed by the present method levels between 1 and 8 pmol/l were detected. Gel filtration of porcine plasma extracts on Sephadex G-25 revealed the presence of two immunoreactive peaks; one eluted at a position identical with that of BNP-26 and the other eluted earlier, close the position of BNP-32.     

Demonstration of atrial natriuretic peptide/cardiodilatin (ANP/CDD)-immunoreactivity in the salt gland of the pekin duck

Summary A novel peptide hormone, atrial natriuretic factor/cardiodilatin (ANP/CDD), was recently isolated and characterized from mammalian heart. Its presence has been demonstrated in several organs that contribute to water and sodium homeostasis, such as salivary glands. This study demonstrates the presence of ANP/CDD immunoreactivity in the salt gland of Pekin ducks by high performance liquid chromatography, radioimmunoassay and immunocytochemistry, using a specific antibody against atriopeptide I. A small number of distinct, ovoid or cuboid shaped ANP/CDD-immunoreactive cells were localized in the connective tissue surrounding and separating the central secretory tubules, whereas no immunostaining was observed in the peripheral tubules. Salt glands of ducks that were adapted to salt water revealed a significant hypertrophy of their secretory lobules. However, no differences were found between the number or localization of immunoreactive cells in the salt gland of salt water-acclimatized ducks and non-stimulated glands of ducks that were housed with ad libitum access to fresh water. Our results indicate that ANP/CDD may play a role in the regulation of sodium secretion in the salt gland of aquatic birds.Supported by the Walter-Schulz-Stiftung and Deutsche Forschungsgemeinschaft DFG, We 608/8-2Dedicated to Prof. Dr. med. Dr. phil. h.c. D. Starck on the occasion of his 80th birthday     

Atrial natriuratic peptide and experimental vasorenal hypertension in rats

Vasorenal hypertension in rats resulted in increase of arterial pressure, the plasma concentration of creatinine and potassium. By EM immunocytochemistry we have demonstrated that atrial natriuretic peptide (ANP) was kept in the granules of secretory cardiomyocytes of the right atrium. It has shown that cardiomyocytes released ANP by means of diffusion. The increased secretion of atrial natriuretic peptide has been detected in blood. However the physiological effects this peptide probably was impaired due to the reducing of the density of natriuretic peptide receptors.     

Development and application of a urodilatin (CDD/ANP-95#x2013;126)-specific radioimmunoassay

Urodilatin, a renal natriuretic peptide that is an analogue to circulating atrial natriuretic peptide [-ANP (99-126)], is measurable with a highly specific and sensitive radioimmunoassay. While most ANP antibodies cannot distinguish between urodilatin and other ANP analogues, the polyclonal urodilatin antibody specifically measures human urodilatin without any cross-reactivity to other ANP analogues. Urodilatin is not detected in blood from healthy volunteers nor from cardiac patients. Urinary urodilatin accounts for only a part of total urinary ANP immunoreactivity. Urodilatin excretion closely parallels sodium excretion in response to an acute volume load while changes in urinary immunoreactive ANP excretion do not reflect this renal response. We conclude that specific urodilatin assays are required to explore further the physiological role of the renal natriuretic peptide.     

Regional cardiac expression and concentration of natriuretic peptides in patients with severe chronic heart failure

The purpose of this study was to examine the regional cardiac mRNA expression and concentration of brain natriuretic peptide (BNP) and atrial natriuretic peptide (ANP) in relation to the circulating peptide concentrations in patients with chronic heart failure (CHF). The myocardial mRNA levels and peptide concentrations of BNP and ANP were analysed in seven different regions of the heart from patients undergoing cardiac transplantation. Autopsy samples from individuals without known cardiovascular disease were used as controls. The plasma levels of natriuretic peptides and their N‐terminal propeptides, Nt‐proBNP and Nt‐proANP, were measured in the CHF patients and healthy volunteers. In the autopsy specimens, the atrial regions appeared to contain the highest peptide levels for BNP as well as ANP, the atrioventricular ratio being 12–262 and 72–637‐fold, respectively. In the CHF patients there was a relative shift towards the ventricle for BNP, reducing the atrioventricular ratio to 6–16‐fold. The circulating concentrations of BNP/Nt‐proBNP in the CHF patients correlated closely to the BNP mRNA expression in most myocardial regions including the left ventricle (r=0.72, P < 0.001). For circulating concentrations of ANP/Nt‐proANP, such correlation were limited to the left atrium free wall (r=0.66, P < 0.002). Thus, of the two natriuretic peptides, BNP/Nt‐proBNP may be a better reflector of left ventricular overload.     

Atrial natriuretic peptide (ANP)-granules in the guinea pig atrial and auricular cardiocytes: an immunocytochemical and ultrastructural morphometric comparative study.

The atrial natriuretic peptide (ANP) is a peptide hormone that is mainly produced in the cardiac atria, where it is stored within granules. It is known that the four regions of the atrial-auricular complex (two atria and two auricles) produce and store ANP in the granules. However, no report has been presented comparing the presence of ANP, and the number and diameter of atrial granules in the atria and auricles. ANP immunoreactivity was detected in cardiocytes from the four regions of the atrial-auricular complex. No differences were observed among the regions. The number of granules was greatest in the right atrium followed by the left atrium and left auricle and right auricle, in this order. The diameter of granules in the cardiocytes was significantly largest in the right atrium and reduced via the left auricle to the left atrium and right auricle. Both the number and diameter of the granules are larger in the right atrium in comparison with the other regions of the atrial-auricular complex, which leads to the supposition that this region is the one that most synthesizes and stores the ANP.     

Immunohistochemical localization of cardiodilatin in myoendocrine cells of the cardiac atria

Summary Region-specific antibodies against synthetic N-terminal fragments of cardiodilatin (CDD) were raised in rabbits and used for the immunohistochemical detection of this new peptide hormone in the myoendocrine cells within the cardiac atria of several species. The peroxidase-antiperoxidase (PAP) and fluorescein isothiocyanate (FITC) immunohistochemical methods gave identical results of cardiodilatin-immunoreactivity (CDD-IR) within the tissue. In addition to the porcine right atrial appendage, myoendocrine cells with CDD-IR were also detected in the left atrium of porcine heart, as well as in other species such as dog and cat. The exact localization of the immunoreactivity in specific secretory granules was mostly related to the Golgi-area which is located on both nuclear poles of auricular myoendocrine cells. The results confirm that cardiodilatin is stored in secretory granules observed through electron microscopical means. This hormone is most likely synthesized and released in myoendocrine cells, exerting its important cardiovascular effects.Supported by the Deutsche Forschungsgemeinschaft Carvas SFB 90     

利尿钠肽在诊断心力衰竭中的应用价值

探讨利尿钠肽的水平对心力衰竭（心衰）早期诊断的应用价值。采用放免法、ELISA法检测了129例心衰患者血浆中的心房利尿钠肽（ANP）、脑利尿钠肽（BNP）、N末端脑钠肽前体蛋白（NT-proBNP）水平,并与30例健康对照者进行了比较分析。结果显示,心衰患者血浆中的ANP、BNP、NT-proBNP显著高于健康对照组,且均随着NYHA分级的升高而逐渐增加,其含量在NYHA Ⅳ级时到达最高,心衰患者的血浆ANP、NT-proBNP水平与LVEF呈明显负相关。检测血浆中的ANP、BNP、NT-proBNP含量简便、快捷,可用于心衰诊断及NYHA分级判断。     

Ventricular expression of atrial and brain natriuretic peptides in dilated cardiomyopathy. An immunohistocytochemical study of the endomyocardial biopsy specimens using specific monoclonal antibodies.

Although brain natriuretic peptide is expressed in ventricles of failing hearts including dilated cardiomyopathy, its morphological localization is still unclear. In this study, we analyzed the immunohistocytochemical localization of atrial and brain natriuretic peptides in ventricles of dilated cardiomyopathy at both light and electron microscopic levels. Ventricular specimens were obtained by endomyocardial biopsy in 31 patients (26 with dilated cardiomyopathy and 5 controls without any specific cardiac disease). By light microscopic immunohistochemistry using specific monoclonal antibodies, all (26 of 26) of the left ventricular endomyocardial biopsy specimens and 31% (8 of 26) of the right ventricular specimens showed immunoreactivity for both of these natriuretic peptides in dilated cardiomyopathy. In contrast, none of the normal controls showed immunoreactivity for either of these peptides. The percentage of atrial natriuretic peptide-containing or brain natriuretic peptide-containing myocytes in the left ventricular specimens showed an inverse correlation with the left ventricular ejection fraction (r = -0.72 and r = -0.69, respectively). By electron microscopy, we identified specific secretory granules in ventricular myocytes from patients with dilated cardiomyopathy, but not in those from normal controls. Double immunocytochemistry using a two-face immunogold staining method revealed brain natriuretic peptide colocalized with atrial natriuretic peptide in the same ventricular granules. These findings suggest that brain natriuretic peptide is expressed in ventricular myocytes in response to hemodynamic stress in dilated cardiomyopathy. Brain natriuretic peptide may be, at least in part, synthesized simultaneously and secreted together with atrial natriuretic peptide by granules from failing ventricles, although the secretory turnover is different between these two peptides.     

A specific and highly sensitive radioimmunoassay of human brain natriuretic peptide

We established specific and highly sensitive radioimmunoassay (RIA) for the detection of human brain natriuretic peptide (BNP), and confirmed the presence of BNP-like immunoreactivity (BNP-LI) in human circulating blood and the heart. The antibody used in this RIA did not react any known human atrial natriuretic peptide (ANP). Gel permeation chromatography coupled with the RIA revealed that BNP-LI in atrium is composed with high molecular BNP and low molecular BNP. Furthermore, dominant form of BNP-LI in human plasma was thought to be low molecular BNP. Mean concentration of BNP-LI in plasma from normal subjects was 5.9 pg/ml, which was about 5 times lower than that of ANP-LI. Mean concentration of BNP-LI in human cardiac atrium was 45.6 ng/mg protein, which was about 40-fold lower than that of ANP-LI. Furthermore, concentration of BNP-LI in human cardiac ventricle was in a range of 0.1-4.8 ng/mg protein, being about 1/20 that of cardiac atrium. These data suggest that BNP synthesized in human heart and secreted into circulation.     

Presence of immunoreactive atrial natriuretic peptide in nerve fibres and conduction cells in the conduction system of the bovine heart

Previous findings of atrial (A-type) natriuretic peptide (ANP) in nervous tissue, such as the brain and the superior cervical ganglia, led us to investigate the possible occurrence of ANP in nervous tissue in the heart. The distribution of ANP in the bovine heart, particularly its conduction system, was examined by the use of immunohistochemical methods and an antiserum against -hANP. ANP immunoreactivity was frequently detected in atrial myocytes and in the Purkinje fibres of the AV-bundle, and was sometimes seen in the Purkinje fibres of the bundle branches and their ramifications. On the other hand, ANP immunoreactivity was never seen in the conduction cells of SA- and AV-nodes. ANP immunoreactivity was also detected in small nerve-fibre varicosities, mainly in the AV-node and AV-bundle. Most of these varicosities were located in the proximity of the conduction cells, but some occurred close to fine blood vessels or in the walls of arterioles. These observations show for the first time that ANP immunoreactivity is present not only in atrial myocytes and conduction cells but also in nerve-fibre varicosities in the conduction system. The observations suggest that ANP may act as a neuromodulator and/or neurotransmitter in the conduction system.     

Atrial natriuretic polypeptides in the specific atrial granules of the rat heart: immunohistochemical and immunoelectron microscopical localization and radioimmunological quantification

Atria of several mammalian species contain atrial natriuretic polypeptides (ANP) with natriuretic, diuretic, and vasodilating activity. In the present studies ANP were localized and quantitated in different parts of the heart by immunocytochemical and radioimmunological methods. The concentration of immunoreactive ANP as determined by quantitative radioimmunoassay in rat heart atria was a follows (ng/mg, mean +/- SD, n = 5): right auricle (688 +/- 156), left auricle (556 +/- 156), right atrium (334 +/- 60), and left atrium (93 +/- 36). The staining intensities in immunohistochemical localizations were consistent with the quantitative data. The location of the peptides was sarcoplasmic and granular. The highest concentration of ANP was found in the perinuclear area of the atrial myocyte sarcoplasm, but some staining was also seen in the periphery of the cells. The indirect immunoelectron microscopical gold method showed that ANP are located in the specific atrial granules supporting previous findings.     

利钾尿肽与心钠素摩尔比在老年原发性高血压中的作用

目的:探讨利钾尿肽(KP)与心钠素摩尔比变化在老年原发性高血压发病中的意义。方法:用放射免疫分析方法测定老年高血压患者血浆、老年自发性高血压大鼠(SHR)血浆及心房和心室组织的KP和心钠素(ANP)含量。结果:高血压患者血浆KP、ANP水平与对照组比较无显著差异。但是,KP与ANP的摩尔比显著低于对照组(P＜0.05),SHR血浆KP、ANP水平显著高于对照组(WKY)(P＜0.01和P＜0.05),KP/ANP摩尔比则显著低于WKY(P＜0.01)。SHR心房KP含量高于心室(P＜0.05),但与对照组WKY比较无显著差异。结论:KP含量及KP与ANP的比例关系的改变,在老年原发性高血压发病中起着一定的作用。     

CARDIAC RHABDOMYOMA: A Case Report with Reference to Atrial Natriuretic Peptide

An autopsy case of cardiac rhabdomyoma in a male infant is reported. Many nodules of rhabdomyoma were present in all four cardiac chambers and were microscopically composed of ovoid, glycogen-laden cells and typical "spider cells". Atrial natriuretic peptide (ANP) was immunohistochemically demonstrated in both normal myocytes and rhabdomyoma cells of both atria, but not in normal myocytes and rhabdomyoma cells of both ventricles. Ultrastructurally, atrial specific granules were present in atrial rhabdomyoma cells and normal atrial cardiocytes, and these showed ANP immunoreactivity with protein A-gold technique. It could be said that the localization and intracel-Mar distribution of ANP in this cardiac rhabdomyoma were closely similar to those of normal human heart. With regard to the presence of ANP, cardiac rhabdomyoma cells arising in atria seemed to differ from those in ventricles, although many tumor nodules occurred in both atria and ventricles. Furthermore, it seemed that cardiac rhabdomyomas could also be divided into two parts: 1) an atrial part with ANP, and 2) a ventricular part without ANP. Therefore, this study confirms the hypothesis that cardiac rhabdomyoma is a hamartoma rather than a true neoplasm. ACTA PATHOL JPN 38 : 95–104, 1988.     

Ultrastructural and immunocytochemical study of the myoendocrine cells of the fish Hypostomus cordovae

Summary The myoendocrine cells of the heart of Hypostomus cordovae (Günther 1880), a teleost fish from South America, were investigated by electron microscopy and immunohistochemistry. By applying antibodies raised against synthetic cardiodilatin 99–126 (CDD/ ANP 99–126), a specific labeling of this hormone was found in the heart of this fish, mainly in myoendocrine cells of atrial trabeculae, where specific secretory granules are stored. The distribution of secretory granules exhibited striking seasonal variations. In winter there were fewer differentiated myoendocrine cells, which were easily recognized by the presence of specific secretory granules, most of which occur clustered in perinuclear areas of the cells. By contrast, in summer the majority of the myocardic cells of the atrium are active endocrine cells. They contain abundant secretory granules widely scattered in the cytoplasm, many of them polarized toward the subendocardial aspect of the cell. The secretory granules can be easily differentiated from the Weibel-Palade granules of endothelial cells, the shape, size and content of which were typical at electron-microscopic level. In addition, these endothelial granules did not display CDD immunoreactivity. The presence of cardiodilatin in a fish such as Hypostomus cordovae further supports the view that cardiac hormones are present in many Vertebrates and may preserve analogous roles such as those reported in other species throughout the group.Supported by the Program of International Cooperation between the CONICET Argentina and the DAAD, GermanyResearch Professor of the Alexander von Humboldt Foundation at the IPF     

The goat mammary gland as a target organ for atrial natriuretic peptide

Milk secretion represents a major route for electrolyte and water excretion in the dairy goat. The aims of this study were to investigate whether the mammary gland is a target site for atrial natriuretic peptide (ANP) in the goat and whether ANP affects mammary sodium and water secretion. Receptor autoradiography using ¹²⁵I-ANP as radioligand revealed specific binding sites in the secretory tissue of the mammary gland. The radioligand was totally displaced by unlabelled ANP, but not by brain natriuretic peptide or the ANP fragment c-ANP_4–23, indicative of ANP-A receptor preference. To elucidate the role of ANP in milk secretion, ANP (30 ng kg^?1min^?1; 120 min) or 0.15 m NaCl (control) were administered in vivo. The ANP infusions caused haemoconcentration, but did not change milk flow or the concentrations of sodium, potassium, lactose, fat and protein in the milk. The results show that the mammary gland of the goat expresses ANP-specific binding sites, however, a physiological role of ANP in goat mammary gland function remains to be elucidated.     

Origin and characterization of atrial natriuretic peptide in the rat parotid gland

Summary The heart atria represent the major site of synthesis for atrial natriuretic peptide (ANP) which exerts potent natriuretic, diuretic and vasoactive functions. Recently, ANP-immunoreactivity has been detected in extracardial organs involved in water and electrolyte homeostasis, such as the intestine and certain exocrine glands. The present study investigates ANP in the parotid gland. It was found by immunohistochemical techniques that the peptide is localized in ductal cells of the gland. An analysis of the immunoreactive material by high-pressure liquid chromatography and radioimmunoassay revealed the prohormone of ANP (ANP 1-126) and the biologically active fragment (ANP 99-126). Furthermore, Northern blot hybridization disclosed the presence of mRNA coding for ANP. It is suggested that ANP is synthesized and released from the parotid gland and functions in the control of saliva production.     

Increased brain natriuretic peptide and atrial natriuretic peptide plasma concentrations in dialysis-dependent chronic renal failure and in patients with elevated left ventricular filling pressure

Brain natriuretic peptide (BNP) and atrial natriuretic peptide (ANP) plasma concentrations were measured in patients with dialysis-dependent chronic renal failure and in patients with coronary artery disease exhibiting normal or elevated left ventricular end-diastolic pressure (LVEDP) (n = 30 each). Blood samples were obtained from the arterial line of the arteriovenous shunt before, 2 h after the beginning of, and at the end of hemodialysis in patients with chronic renal failure. In patients with coronary artery disease arterial blood samples were collected during cardiac catheterization. BNP and ANP concentrations were determined by radioimmunoassay after Sep Pak C₁₈ extraction. BNP and ANP concentrations decreased significantly (P < 0.001) during hemodialysis (BNP: 192.1 ± 24.9, 178.6 ± 23.0, 167.2 ± 21.8 pg/ml; ANP: 240.2 ± 28.7, 166.7 ± 21.3, 133.0 ± 15.5 pg/ml). The decrease in BNP plasma concentrations, however, was less marked than that in ANP plasma levels (BNP 13.5 ± 1.8%, ANP 40.2 ± 3.5%; P < 0.001). Plasma BNP and ANP concentrations were 10.7 ± 1.0 and 60.3 ± 4. 0 pg/ml in patients with normal LVEDP and 31.7 ± 3.6 and 118.3 ± 9.4 pg/ml in patients with elevated LVEDP. These data demonstrate that BNP and ANP levels are strongly elevated in patients with dialysis-dependent chronic renal failure compared to patients with normal LVEDP (BNP 15.6-fold, ANP 2.2-fold, after hemodialysis; P < 0.001 or elevated LVEDP (BNP 6.1-fold, ANP 2.0-fold, before hemodialysis; P < 0.001), and that the elevation in BNP concentrations was more pronounced than that in ANP plasma concentrations. The present results provide support that other factors than volume overload, for example, decreased renal clearance, are also involved in the elevationin BNP and ANP plasma levels in chronic renal failure. The stronger elevation in BNP concentrations in patients with chronic renal failure and in patients with elevated LVEDP and the less pronounced decrease during hemodialysis suggest a different regulation of BNP and ANP plasma concentrations.[/ p]Abbreviations ANP atrial natriuretic peptide - BNP brain natriuretic peptide - LVEDP left ventricular end-diastolic pressure Correspondence to: C. Haug     

Atrial natriuretic peptide in human urine

Summary A highly sensitive radioimmunoassay to measure atrial natriuretic peptide (ANP) concentration in urine has been established, and its clinical usefulness is presented. ANP in urine was stable at 4° C for several days and was easily measured by our radioimmunoassay. The average ANP excretion in 65 healthy persons was 25.0±1.4 ng/day (mean ± SEM) and the fractional excretion of ANP was 0.7±0.05%. In 14 patients with congestive heart failure, the average ANP excretion was 119.2±29.4 ng/day, which decreased to 53.3±11.0 after successful treatment.Abbreviations ANP atrial natriuretic peptide - hANP human atrial natriuretic peptide - RIA radioimmunoasay - NSB non specific bound - FE_ANP the fractional excretion of atrial natriuretic peptide - FE_Na the fractional excretion of sodium - SIADH the syngrome of inappropriate secretion of antidiuretic hormone     

Case report of a heart transplantation in a 16-year-old male

We examined ultrastructural changes and did immunohistochemical studies of atrial natriuretic peptide (ANP) in biopsied samples obtained from the heart of a 16-year-old Japanese boy with dilated cardiomyopathy and from the transplanted donor heart. In the right ventricle of the diseased heart, a small number of atrial granules containing ANP were found in the perinuclear area of the cardiomyocytes and near areas lacking some myofibrils. Although no evidence of rejection was seen in the right ventricle of the transplanted donor heart under the light microscope, electron microscopy revealed moderate degeneration of cardiomyocytes and injuries to capillary endothelial cells. We did not find atrial granules, although the serum ANP level was elevated. These data suggest that the ultrastructural changes in the transplanted heart were related to a mild rejection, the effects of cyclosporin, or the effects of a domino heart transplantation from a patient with cystic fibrosis. Because of the absence of atrial granules in the right venticle, it is postulated that the high serum ANP level may be attributed to merely an increased secretion of ANP from atrial granules in the atria, without secretion of ANP in the ventricles.