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

ARTICLE IN PRESS Ann Anat 189 (2007) 457—464

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Atrial natriuretic peptide (ANP)-granules in the guinea pig atrial and auricular cardiocytes: an immunocytochemical and ultrastructural morphometric comparative study Eliane Florencio Gamaa,, Claudio Antonio Ferraz de Carvalhob, Edson Aparecido Libertib, Romeu Rodrigues de Souzaa a

Universidade Sa˜o Judas Tadeu, Departamento de Anatomia Humana, Rua Taquari, 546, Mooca-Sa˜o Paulo-SP 03166 000, Brazil b Universidade de Sa˜o Paulo, Departamento de Anatomia, Instituto de Cie ˆncias Biome ´dicas, Sa˜o Paulo, Brasil Received 18 September 2006; accepted 9 November 2006

KEYWORDS ANP-granules; Atrial cardiocytes; Immunocytochemistry; Morphometry; Guinea pig

Summary 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. & 2007 Elsevier GmbH. All rights reserved.

Introduction Corresponding author. Tel.: +55 11 60991677.

E-mail address: [email protected] (E.F. Gama).

Atrial natriuretic peptide (ANP) is a peptide hormone that is primarily synthesized and stored

0940-9602/$ - see front matter & 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.aanat.2006.11.006

ARTICLE IN PRESS 458 in the atrial cardiocytes, and rapidly released into the blood stream (Sata et al., 2003). It causes diuresis, natriuresis, vasodilatation and depression of blood pressure. It is also involved in the modification of the water-electrolyte balance (De Bold et al., 1981; Forssmann et al., 1984; Skepper and Navaratman, 1988; Jiao et al., 1993; Toyoshima et al., 1996; Yoshihara et al., 1998). The atrial and auricle wall distension under conditions of hypervolemia (Veress and Sonnerberg, 1984) or blood pressure increase would promote an increase in the circulating ANP (Lang et al., 1985; Itoh et al., 1987). The granules were identified in the four regions of the heart: the right and left atria and the right and left auricles, but ANP was not. Quantitative studies on the granules have been performed in various mammalian species (Jamieson and Palade, 1964; Cantin et al., 1979; Mifune et al., 1996), but authors do not agree on the number and diameter of the granules in the various regions of the atrial–auricular complex. In the rat, the largest number of granules was found in the right atrium, whereas in the guinea pig, in the hamster, in the rabbit and in the cat, the number of granules was largest in the left atrium (Cantin et al., 1979). Chapeau et al. (1985) assert that there are a larger number of granules in the right atrium. According to Mifune et al. (1992), imunohistochemically, the most intensely peptide-reactive cardiocytes were localized in the right auricle. The aim of this study has been to determine whether ANP can be detected in the granules from the four regions of the atrial–auricular complex of the guinea pig, and if so, whether or not there are significant differences in the number and diameter of the granules between these regions.

Material and methods Animals Six guinea pigs (Cavia porcellus), of both sexes, weighing 500–700 g were used. The heart atrial– auricular complexes were removed after anesthesia with sodium pentobarbital (60 mg/kg, i.p.) and then washed out with saline solution. Fragments were obtained from the right atrium, from the left atrium, from the right auricle and from the left auricle.

Ultrastructural immunocytochemistry Fragments from each region and from each animal were immersed in a fixative solution of

E.F. Gama et al. paraformaldehyde, 6%, glutaraldehyde at 0.5% and picric acid at 0.2% in phosphate buffer (0.1 M, pH 7.3), for 4 h at 4 1C. The fragments were then dehydrated in alcohol 701 and 951. All fragments were embedded in LR White resin (Ladd Research Industries, Inc.), without being previously stained with osmium. The resin polymerization was obtained in a sterilizer at 50 1C during 48 h. Ultrathin sections from each region were obtained with a diamond knife and placed on formvar coated nickel grids. The unspecified binding sites were blocked using a Tris–HCl solution (0.02 M, pH 7.2) containing cattle serum 2%, Tween 20 0.2% and Triton X-100 0.1%, over 90 min. In the sequence, the grids were exposed to several solutions prepared with demineralized water. Initially, they were incubated for 18 h at 4 1C in a humid chamber with the primary antibody (Human ANP – KIH, Sigma Immuno Chemicals), developed in goats, diluted in Tris HCl (0.02 M, pH 7.2), cattle serum 2%, Tween 20 0.2% and Triton X-100 0.1% (dilution 1:50). Subsequently, they were washed out in Tris–HCl (0.02 M, pH 7.2) containing cattle serum 0.1%, Tween 20 0.2% and Triton X-100 0.1% for 1 min; Tris–HCl buffer, 1 min and finally they underwent 10 washings with the Tris–HCl buffer for 1 h. The material was once again put through the blockage process at the unspecified binding sites by using the Tris–HCl solution (0.02 M, pH 7.2) containing cattle serum 2%, Tween 20 0.2% and Triton X-100 0.1% for 10 min. The grids were incubated for 1 h in Protein-A Gold (10 nm, Sigma Chemical Co.) diluted in Tris–HCl (0.02 M, pH 7.2) containing cattle serum 2%, Tween 20 0.2% and Triton X-100 0.1%. The material was washed out in Tris–HCl (0.02 M, pH 7.2) containing cattle serum 0.1%, Tween 20 0.2% and Triton X-100 0.1% for 1 min; Tris–HCl buffer, 1 min and 10 washings in Tris–HCl buffer for 1 h. Finally, the grids were washed out in aqua bi-destilled water. The control group was obtained by omitting the primary antibody. The grids were contrasted with uranyl acetate for 5 min and analyzed on the transmission electron microscope, Philips EM 400.

Electron microscopy Three animals were anaesthetized with sodium pentobarbital, and their ventricles perfused with heparinized saline solution (1 mg/300 ml of solution). The atria and the auricles were reduced to fragments of about 1 mm3. Fragments were placed in a glutaraldehyde fixative solution at 5% in cacodylate buffered solution (0.2 M, pH 7.3) for

ARTICLE IN PRESS ANP-granules in rats: a morphometric study 3 h. The fragments were washed 3 times with cacodylate buffer (0.1 M, pH 7.3) for 5 min each time, and subsequently placed in a fixative solution of osmium tetroxide at 1% in a cacodylate buffered solution (0.1 M, pH 7.3) for 2 h. The fragments were left overnight in uranyl acetate at 0.5% with sucrose (540 mg/100 ml) and after being washed with cacodylate buffer they were also dehydrated in an increasing series of alcohols (alcohol 701, alcohol 701+uranyl at 1%, alcohol 951, alcohol 1001 and propylene oxide) and embedded in Epon resin (Polisciences, Inc.), with previous embedment in an 1:1 resin plus propylene oxide solution for 8 h under rotation. Following that, they were embedded in resin alone for 5 h and finally left in the same resin at 60 1C for 5 days. Ultrathin sections were obtained with a diamond knife, on an ultramicrotome (Sorvall MT-2), and after contrasting with uranyl acetate and lead citrate they were analyzed

459 on a Philips EM 400-transmission electron microscope.

Ultrastructural morphometry Sets of 10 electron micrographs/region, and animal, chosen by systematic random sampling of squares were taken at a final magnification of 7500  and the number of granules/field and the size (diameter) were determined, using a computerized program (Axion Plus, Zeiss). A total of one hundred fields were analyzed.

Statistics The means (7SEM) of counts were calculated with the measurements of at least 673 granules in total and statistically analyzed by ANOVA. Differences were considered significant when po0.05.

Figure 1. Immunocytochemical staining of ANP in the atrial–auricular complex in the guinea pig heart. The immunoreaction for ANP is most intense in the right atrium (A, B), and in the left atrium (C) and moderate in the right auricle (D) and in the left auricle (E). No immunocytochemical reaction was observed outside the granules. Observe the presence of numerous Protein-A Gold (10 nm), particles inside the granules. In C, intensely stained granules can be seen.

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Results

Ultrastructure

Immunocytochemistry

The cardiocytes contained electron dense granules with sparse granular and homogeneous content (Fig. 2). The distribution pattern of granules in the cardiocytes was common to the atria and auricles in that most of the granules were localized near the nuclear poles. Sometimes, granular content released into the extra-cellular space (extrusion) can be observed. The plasma membranes of the granules were fused and the amorphous material, apparently derived from the

The ultra thin sections obtained by omitting the primary antibody showed no detection of immunoreactions. ANP-immunoreactivity was detectable in all cardiocytes from the four regions studied. A large number of gold particles related to the granules have been observed, as well as less electron dense material in their vicinity (Fig. 1).

Figure 2. Electron micrographs of atrial (A, B, E) and auricular (C, D, F) cardiocytes in the guinea pig heart. N: nucleus. In the right atrium (B) granules are higher in number than those in the left atrium (A) and auricles (C, D, F) and there are large (arrows), medium (arrowhead) and small (double arrows) granules. In A and D, the granules are found at the poles of the nucleus, but in some cardiocytes there are granules widely dispersed in the cell (C, F). In E, the cardiocyte contains several electron dense granules, with homogeneous content. Observe an empty granule (arrowhead) and a partially empty one (double arrow). Amorphous material, apparently derived from the granules, can be observed in the extra-cellular space (arrow).

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granule, was observed in the intercellular space (Fig. 2E).

Fig. 5. In the right atrium, compared to the other regions there is a shift in the peak of the distribution towards large neurons (Fig 5).

Ultrastructural morphometry The number and diameter of ANP-granules are shown in Figs. 3 and 4. The number of granules was greater (not significant) in the right atrium than in the left atrium and auricles (Fig. 3). The average diameter of granules was significantly larger in the right atrium than in the left atrium and auricles ðpo0:05Þ (Fig. 4). The diameter of the granules varied from 63 to 211 nm. The distribution of granules according to their diameters in the four regions of the atrial–auricular complex is shown in the histograms in 11 10

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Figure 3. Comparison of the number of ANP-granules in the guinea pig atrial and auricular cardiocytes (means7SEM). RAt – right atrium; LAt – left atrium; LAu – left auricle; RAu – right auricle. The right atrium contain the higher number of granules and the left auricle contain the lower.

Discussion This is the first investigation comparing the presence and quantitative aspects of the ANPgranules among the atria and auricles. Thus far, five molecules comprise the natriuretic peptide family: ANP, urodilatin, brain natriuretic peptide (BNP), Ctype natriuretic peptide (CNP) and dendroaspis natriuretic peptide (DNP) (Cea, 2005). This study immunocytochemically demonstrated the presence of immunoreactive ANP in all examined granules in the cardiocytes from the guinea pig heart. Although the ultrastructural evaluation of atrial and auricular cardiocytes showed granules with similar localization and morphology in the four respective regions examined in this study, the number and diameter of granules differed from region to region. Although in this study we have only analyzed the granules in the atrial–auricular complex, in many species, including in the guinea pig, they are also present in the ventricles (Mifune et al., 1992). The ultrastructural morphometry showed that there were numerical differences in atrial and auricular regions. In the present study, we observed that, although not significant, the number of granules in the right atrium was greater than in the other regions of the atrial–auricular complex. These results are in accordance with those of other authors. According to Rinne et al. (1986) and Chiu et al. (1994) the right atrium is a greater source of

115 110 105 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0

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Figure 4. Diameter of ANP-granules in the guinea pig atrial and auricular cardiocytes. Each value is the mean 7SEM. Rat – right atrium; LAt – left atrium; LAu – left auricle; RAu – right auricle. The mean diameter of granules in the right atrium is significantly higher than in other regions.

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Figure 5. Histogram showing the distribution of the ANP-granules according to their diameter (nm) in the four regions studied: right atrium (RAt), left atrium (LAt), right auricle (RAu), left auricle (LAu). In the right atrium there is a clear shift in the peak of the distribution to the right. Observe that while in the right atrium about 50% of the granules measure between 80 and 140 nm (1000  ), with a peak at 120 nm (1000  ), in the other regions the same proportion of granules range between 80 and 100 nm (1000  ).

ANP than the left atrium, probably making it possible to respond with greater intensity to mechanical and nervous stimuli. We observed that the amount of ANP-granules decrease from the right to the left auricle and from the right to the left atrium. However, a larger amount of granules in the left atrium as compared to the right one has been reported in guinea pig hearts (Cantin et al., 1984). The results obtained by Skepper et al. (1989) and Avramovitch et al. (1995) indicate that there is no statistical difference from one side (right) to the other (left) regarding the amount of granules in the atrial–auricular complex. Results showing that the right auricle is functionally more active than the left one have been reported (Veress and Sonnerberg, 1984; Skepper et al., 1989; Stewart et al., 1992). This has also been verified in dogs that the bilateral removal of the auricles eliminates ANP release (Stewart et al., 1992). It is possible, that these divergent results are due to different methods used. Numerical changes in ANP-granules were observed under several physiological conditions: temperature, dehydration and nutritional state (Mifune et al., 1993; Gall et al., 1990; Toyoshima et al., 1996; Penner et al., 1990; Nakayama et al., 1984; Takayanagi et al., 1985). The numerical

differences in NP-granules in the regions of the atrial–auricular complex are suggested to be associated with differences in the synthetic and secretory ability in these regions. It is possible that the synthetic and secretory ability is enhanced in the regions with fewer granules, i.e., in the left atrium and auricles. Relatively low production of the ANP and/or a rapid secretion, with the consequent lower amount of storage of the peptide in these regions, may be the reason. In the present work, there were significant differences in granule diameter among the regions of the atrial–auricular complex. The average diameter of the granules in the right atrium was larger than in the other regions. The distribution of the granular diameter from the right atrium was displaced towards larger values compared to data from the left atrium and auricles. The diameters of ANP-granules are also influenced by several factors: e.g. water depletion (Gall et al., 1990), or hypertension (Takayanagi et al., 1985). It has been reported that the enhancement of ANP synthesis in the cells reduced the granular diameter, or that NP-granules became smaller with a concomitant decrease in the levels of atrial ANP mRNA and plasma ANP in the course of the down regulation (Mifune et al., 1991; Mifune et al., 1992). According to these authors,

ARTICLE IN PRESS ANP-granules in rats: a morphometric study these findings suggest no relationship between the granular size and the ability of ANP synthesis and secretion. In the present study, the lower the number of granules in a cardiocyte, the smaller the granule diameter became, suggesting that the number of granules may be related to the granule size. The granule diameter is therefore possibly determined by the number of granules in the cardiocytes in the species (Mifune et al., 1996). The ratio of peptide content in the right atrium was larger than in the auricles and may indicate a more important endocrine role in right atrium than in left atrium and auricles. Since the discovery of ANP more than 20 years ago, numerous studies have focused on the mechanisms regulating ANP secretion. According to Katoh et al. (1990), Schiebinger and Greening (1992), Seul et al. (1992), De Bold et al. (2001) and Dietz (2005) the main physiological stimulus for increased ANP release is the atrial muscle stretch, which normally occurs when extracellular fluid volume or blood volume is elevated. Our observation of atrial granule proximity to the sarcolemma complement previous descriptions (Imada et al., 1988; Needleman et al., 1989), demonstrating that the ANP is eliminated through the atrial myocytes, via exocytosis. The clinical applications of natriuretic peptides are expanding rapidly. Recent basic science and clinical research findings continue to improve our understanding of this peptide system and guide use of ANP as a biomarker and as a therapeutic agent (Silver, 2006).

Acknowledgments We would like to express our appreciation to Cleide Rosana Duarte Prisco and to Claudia Borin da Silva for their cooperation in the statistical analyses.

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