Atrial Specific Granules Research Articles

Cardiac Aldosterone Synthesis?

Recognition that a variety of hormones affect the heart and blood vessels dates back well over a century, and about 25 years ago, it was firmly established that the heart is also an endocrine organ. This era began with de Bold’s1 discovery that the quantity of electron-dense “atrial specific granules” previously described by Jamieson and Palade2 varied with maneuvers that altered intravascular volume status. By 1982, it was recognized that these granules contained and released a potent peptide called atrial natriuretic peptide.

Determinants of natriuretic peptide gene expression

The cardiac natriuretic peptides (NP) atrial natriuretic factor or peptide (ANF or ANP) and brain natriuretic peptide (BNP) are polypeptide hormones synthesized, stored and secreted mainly by cardiac muscle cells (cardiocytes) of the atria of the heart. Both ANF and BNP are co-stored in storage granules referred to as specific atrial granules. The biological properties of NP include modulation of intrinsic renal mechanisms, the sympathetic nervous system, the rennin–angiotensin–aldosterone system (RAAS) and other determinants, of fluid volume, vascular tone and renal function. Studies on the control of baseline and stimulated ANF synthesis and secretion indicate at least two types of regulated secretory processes in atrial cardiocytes: one is stretch-stimulated and pertussis toxin (PTX) sensitive and the other is Gq-mediated and is PTX insensitive. Baseline ANF secretion is also PTX insensitive. In vivo, it is conceivable that the first process mediates stimulated ANF secretion brought about by changes in central venous return and subsequent atrial muscle stretch as observed in acute extracellular fluid volume expansion. The second type of stimulation is brought about by sustained hemodynamic and neuroendocrine stimuli such as those observed in congestive heart failure.

Monitoring of ANP secretion from single atrial myocytes using densitometry.

Atrial myocytes secrete atrial natriuretic peptide (ANP) in response to mechanical stretch and can serve as a challenging model for studying stretch-secretion coupling. We have developed a technique for monitoring ANP secretion from single atrial myocytes, using neutral red and a CCD video camera. Atrial-specific granules (ASGs) containing ANP were stained with neutral red. The cells were illuminated with monochromatic light (550 mm) and the grey value monitored within the region of interest (ROI) surrounding the region in which ASGs were densely located. Assuming that neutral red is evenly distributed in ASGs, the change in optical density (OD) was considered to represent the total amount of secretion. Under control, non-stimulated conditions, the OD decreased spontaneously (19.7+/-1.4%/10 min, n=14). Direct mechanical stretch (cell length increased by 20%) with two micropipettes or hypotonic swelling (200 mOsm) accelerated the decrease in OD significantly (48.7+/-7.4%/10 min; n=3, 47.2+/-2.4%/10 min; n=7, respectively). In conclusion, this method allows monitoring of ANP secretion with a relatively high time resolution while mechanical stress is applied. Furthermore, patch-clamp or intracellular perfusion techniques can be combined with the present technique for studying cellular mechanisms of stretch-secretion coupling.

Past and current perspectives on the natriuretic peptides.

It is now some 15 years since de Bold and colleagues observed the profound hypotension, diuresis, and natriuresis that occurred following injection of acid extracts of cardiac atria into anesthetized rats (1). As is now well documented, these effects were due to a peptide hormone, atrial natri-uretic factor or peptide (ANF or ANP), which is stored in precursor form in atrial specific granules (for review of discovery and early research on ANP see Ref. 2). ANP is a 28-amino acid disulfide bonded peptide that forms the C terminus of a larger precursor, pro-ANP, and which is released into the circulation in response to local wall stretch induced by increased intraatrial volume (3).Following the initial experiments of Sonnenberg and de Bold (l), several atrial natriuretic peptides were purified, sequenced, and chemically synthesized. Administration of pharmacological doses of synthetic ANP confirmed that the peptide was a potent natriuretic substance with mild hypotensive effects (4-7). Infusion of pharmacolo...

The pathogenesis of so-called cardiac rhabdomyoma in swine: a histological, immunohistochemical and ultrastructural study.

To determine whether cardiac rhabdomyoma (CR) is a hamartoma of fetal cardiac myocyte, we investigated five cases of CRs that spontaneously developed in five 6-month-old hybrid swine with histological, immunohistochemical, and ultrastructural techniques. The cases were four multiple and one solitary neoplasms, which appeared as intraventricular nodules of various sizes without any congenital malformations. Histologically, the large ovoid CR cells with an occasional spiderweb appearance showed a transition from normal-looking cardiac myocytes or rarely from Purkinje cells, but no mitotic figures. Besides large amounts of glycogen, the CR cells contained many PAS-negative, large cytoplasmic vacuoles filled with eosinophilic or fibrillar substance. Immunohistochemically, the CR cells showed intense positivity for desmin and variable positivities for vimentin, alpha-atrial naturiuretic peptide, and proliferating cell nuclear antigen. These positivities were not seen in adjacent cardiac myocytes. Cytokeratin was negative in the CR cells but was positive in fetal cardiac myocytes of early gestation. Rod-like or granular positivity for alpha-actinin in the CR cells was similar to that in nemaline myopathy. Ultrastructurally, the CR cells contained myofibrils that frequently showed myofibrillar degeneration and produced large intracytoplasmic vacuoles. These myofibrils often mingled with nemaline bodies and leptofibrils that continued to the Z bands. T-systems, sarcoplasmic reticulum, and intercalated discs, which are specific features of postnatal cardiac myocytes, were sometimes observed in the CR cells. Increase of glycogen and mitochondria and appearance of atrial-specific granules associated with the Golgi apparatus were other features noted. The present findings have not been reported, even in human CR. From these new observations with the recent report on the occurrence of CR in neonatal piglets, swine CR does not belong to the entity of hamartoma but may be a congenital dysplasia of the perinatal cardiac tissues with myofibrillar degeneration, affecting mainly cardiac myocytes and rarely Purkinje cells. The various immunophenotypic changes including proliferating cell nuclear antigen and the increase and appearance of cytoplasmic elements compared with mature cardiac myocytes can be interpreted as reactive or regenerative changes due to myofibrillar degeneration.

Changes in endocrine atrial rat cardiocytes during growth and aging: an ultrastructural, morphometric and endocrinological study.

The effects of age on atrial natriuretic factor (ANF) metabolism were studied in male Sprague Dawley rats by standard radioimmunological procedures, transmission electron microscopy, and ultrastructural morphometry. Plasma atrial natriuretic peptide (ANP) levels increased during growth (1-month-old rats: 64 +/- 8.7 pg/mL; 2-month-old rats: 105 +/- 8.1 pg/mL) and did not change thereafter. Immunoreactive (ir)-ANP concentration increased significantly in senescent rats, both in the right (12-month-old rats: 254 +/- 33 ng/mg; 24 month-old: 415 +/- 77 ng/mg) and left atrium (226 +/- 30 and 498 +/- 60 respectively). The ultrastructural morphological features of the endocrine cardiocytes of senescent rats were the presence of a few lysosomal structures and atrial specific granules of higher electron-density. Ultrastructural morphometry studies did not reveal any significant increase in the number or in the mean individual volume of atrial specific granules, compared with young adult rats. In conclusion, unlike younger rats, morphometric data in older and senescent rats show that atrial granularity may not necessarily change together with atrial ir-ANP contents; ir-hormone assay findings show that senescent rats have ANP plasma levels within the norm, and a much larger hormone store in atrial tissue.

Atrial natriuretic polypeptide (ANP)-immunoreactivity and specific atrial granules in cardiac myocytes of stroke-prone spontaneously hypertensive rat (SHRSP).

The distribution of atrial natriuretic polypeptide (ANP) and atrial specific granules in the myocytes of the atria and ventricles of an experimental animal model, stroke-prone spontaneously hypertensive rats (SHRSP) and a control, Wistar Kyoto rats (WKY), was examined using immunocytochemical and electron microscopic techniques. In the atria of both SHRSP and WKY, ANP-immunoreactivity was recognized in the perinuclear regions of essentially all cardiac myocytes. In the ventricles of WKY, ANP-immunoreactivity was hardly seen except for the impulse-conducting system. However, in the ventricles of SHRSP, almost all cardiac myocytes possessed immunoreaction products which were scattered evenly throughout the cytoplasm; this distribution pattern differed from that of the atrial wall of this strain.

Cytochemical localization and calcium quantitation of intracellular calcium store in rat atrial cardiocytes.

Ethanolic phosphotungstic acid (EPTA) method and G-6-Pase method were used to localize cytochemically the atrial specific granules (ASG) and sarcoplasmic reticulum (SR) of rat atrial cardiocytes under the electron microscope, and at the same time, the calcium (Ca) in ASG and SR was analyzed quantitatively by analytical electron microscopy (AEM). In these methods, the procedure of osmium tetroxide fixation was shortened or even abolished to keep more combined Ca. The Ca concentration in ASG and in the terminal cisterns of SR (TSR) was 64±16 and 99±21mM/kg dry wt respectively. These values are approximate to those reported in earlier works that were done on cryosections. It is suggested that these cytochemical methods could reserve a large part of combined Ca in the intracellular Ca store, and they are much simpler than the low temperature techniques of biological specimen preparation. Therefore, these methods are useful for AEM microanalysis of elements in organelles.

Atrial natriuretic factor in cats subjected to acute myocardial ischaemia

Eighteen anaesthetized open chest cats were subjected to 10, 30, or 50 min occlusion of the left anterior descending coronary artery (LAD). Heart rate, left ventricular end-diastolic (LVEDP) and systolic pressure (LVSP), and dp/dt were continuously recorded during the experiments. Prior to LAD-occlusion, and just before termination of the experiments, blood samples were collected from the left femoral artery for measurements of atrial natriuretic factor (ANF), catecholamines, electrolytes, urea, and creatinine. Simultaneously, biopsies were collected from the right auricular wall. The tissue was embedded in Lowicryl K4M, and ultrathin sections were incubated with anti-ANF antibodies and secondary antibodies conjugated to gold particles. The density of ANF-containing atrial-specific granules labelled with gold particles was morphometrically calculated. LVEDP increased significantly in all three time groups, and when pooling the pre- and postocclusion values, there was an increase from 5.1±0.4 to 10.3+1.2 mmHg (p<0.05). The noradrenaline level increased from 0.93±0.18 to 2.34±0.75 nmol l−1 (p<0.05) after LAD-occlusion. Similarly, the mean plasma level of ANF in the 18 cats increased from 57.6±11.9 to 98.9±22.6 pmol l−1 (p<0.05). Atrial granular density appeared to decline after 10 min of occlusion (from 0.141 ±0.017 to 0.127±0.022 granules−1 p.m2 sarcoplasm), and after 30 min there was a significant decrease (0.080±0.012 granules/μm2, p<0.05). However, after 50 min occlusion the granular density was almost restored (0.133±0.017 granules/μm2). Plasma ANF showed a positive linear correlation to LVEDP and to the noradrenaline level. The present study demonstrates that the plasma ANF level is increased in cats subjected to acute LAD-occlusion, and it is suggested that elevated LVEDP and plasma noradrenaline contribute to the ANF secretion from the heart.

Atrial natriuretic factor in experimental acute Chagas' disease.

The atrial myocyte of the mammalian heart produces atrial natriuretic factor (ANF) (de Bold et al. 1981), a polypeptide hormone that participates in the regulation of extracellular fluid volume and blood pressure (de Bold 1985). Changes in ANF synthesis and storage have been observed in a wide spectrum of myocardial pathologies (Morii et al. 1989; Tsunoda et al. 1986) and have been related to the development of congestive heart failure. The heart, particularly the atrial muscle, is the main target of Trypanosoma cruzi during the acute period of infection (Tafuri 1979). We have previously shown a drastic decrease in the number of specific atrial granules, the site of ANF storage, and in the natriuretic activity of atrial extracts in acute Chagas' disease in rats (Piazza et al. 1992). In the present work, we characterized changes in cardiac and plasma immunoreactive ANF levels in acute Chagas' infection together with parasitological, serological, electrocardiographic and histopathological changes. Wistar rats (28+3 days of age) were injected intraperitoneally with 6 x 105 trypomastigotes of the T. cruzi X-1 strain obtained from infected mice (Cano and Rubiolo 1985). A control group was inoculated with the same volume of blood from healthy mice. The acute phase of Chagas' disease was studied until 40 days postinfection (p.i.). The parasitaemia course was followed using the direct method of Hoff (1974) in blood drawn by cardiac puncture twice a week p.i. The serological response was evaluated using three standard methods, indirect haemagglutination (Rubiolo and Vottero-Cima 1979), indirect immunofluorescence (Camargo 1966)

Ultrastructural observations on autolytic changes of atrial specific granules in pig heart

The progressive autolytic changes of atrial specific granules ( ASGS) up to 24 hours after death were studied electron microscopically. Tissue samples taken from the left auricular myocardium of four pig hearts at 0, 0·5, 1·5, 3·5, 6, 12 and 24 hours post mortem were fixed for 24 hours by immersion in 2·5 per cent cacodylate buffered glutaraldehyde (pH 7·4). Two pig hearts were kept at 20°C and two at 4°C. Although typical autolytic changes were seen in many organelles, no remarkable modifications in aspect, distribution, content and diameter of ASGS were observed in all samples. The results of this study indicate that ASGS are not morphologically affected by the autolytic processes, at least during the first 24 hours. Hence, the electron microscopic evaluation of ASGS does not need a particular fixation method such as perfusion. This may eliminate technical problems when slaughterhouse material is used.

Atrial natriuretic factor in cats subjected to acute myocardial ischaemia.

Eighteen anaesthetized open chest cats were subjected to 10, 30, or 50 min occlusion of the left anterior descending coronary artery (LAD). Heart rate, left ventricular end-diastolic (LVEDP) and systolic pressure (LVSP), and dp/dt were continuously recorded during the experiments. Prior to LAD-occlusion, and just before termination of the experiments, blood samples were collected from the left femoral artery for measurements of atrial natriuretic factor (ANF), catecholamines, electrolytes, urea, and creatinine. Simultaneously, biopsies were collected from the right auricular wall. The tissue was embedded in Lowicryl K4M, and ultrathin sections were incubated with anti-ANF antibodies and secondary antibodies conjugated to gold particles. The density of ANF-containing atrial-specific granules labelled with gold particles was morphometrically calculated. LVEDP increased significantly in all three time groups, and when pooling the pre- and postocclusion values, there was an increase from 5.1 +/- 0.4 to 10.3 +/- 1.2 mmHg (p < 0.05). The noradrenaline level increased from 0.93 +/- 0.18 to 2.34 +/- 0.75 nmol l-1 (p < 0.05) after LAD-occlusion. Similarly, the mean plasma level of ANF in the 18 cats increased from 57.6 +/- 11.9 to 98.9 +/- 22.6 pmol l-1 (p < 0.05). Atrial granular density appeared to decline after 10 min of occlusion (from 0.141 +/- 0.017 to 0.127 +/- 0.022 granules-1 microns 2 sarcoplasm), and after 30 min there was a significant decrease (0.080 +/- 0.012 granules/microns 2, p < 0.05). However, after 50 min occlusion the granular density was almost restored (0.133 +/- 0.017 granules/microns 2). Plasma ANF showed a positive linear correlation to LVEDP and to the noradrenaline level.(ABSTRACT TRUNCATED AT 250 WORDS)

Annexins V and VI: major calcium-dependent atrial secretory granule-binding proteins.

Atrial natriuretic peptide is stored by atrial myocytes in secretory granules, known as atrial specific granules, and is released from these granules by exocytosis. We have isolated a group of atrial proteins by affinity chromatography that bind to atrial specific granules in a calcium-dependent manner. The two major proteins isolated (32.5 kd and 67 kd) are calcium-binding proteins and have been identified as annexins V and VI by immunoblotting with specific antisera. The calcium dependence of their binding to atrial specific granules has been characterized in vitro and indicates that this interaction takes place at micromolar levels of calcium. In addition, the group of proteins isolated includes another calcium-binding protein of 20 kd, as well as GTP-binding proteins of 22 to 26 kd. Membrane interactions during exocytosis are presumably mediated by the interaction of specific proteins with the granule membrane. The properties of the proteins described here, and their ability to bind to atrial specific granules in a calcium-dependent manner, make them likely candidates in the search for regulatory proteins mediating atrial natriuretic peptide secretion.

Effect of diabetes mellitus on levels of atrial natriuretic hormone in plasma and the right atrium in the non-obese diabetic mouse

Atrial natriuretic hormone (ANH) levels in plasma and the right atrium of non-obese diabetic mice in the decompensated diabetic state were investigated by radioimmunoassay and histological examination. Severely diabetic mice, with blood glucose levels over 33.6 mmol/l showed significantly higher hematocrit, plasma sodium and calculated plasma osmolarity than the age-matched normoglycemic mice. The plasma ANH levels in diabetic mice were significantly lower (17.5 pmol/l) than those in normoglycemic mice (43.6 pmol/l). The ANH concentrations in the right atrium were 26.6 mg/g protein in the diabetic and 11.2 mg/g protein in the normoglycemic mice. The right atrium in the diabetic mice showed much wider immunohistochemical staining by anti-human ANH antiserum compared with the normoglycemic mice. An increase in the number of atrial specific granules in the diabetic mice was observed by transmission and scanning electron microscopy. Morphometrical analysis indicated that the number of granules increased to more than twice that in the normoglycemic mice. These findings indicate that plasma ANH decreases in diabetic mice. The store of right atrial ANH may be increased to compensate for the marked dehydration in severe diabetes.

The morphology and location of atrial specific granules and the demonstration of atrial natriuretic factor in porcine, lapine and bovine heart by immunoelectronmicroscopy

The atrial specific granules (ASGs) were studied in samples collected from the right and left auricles of conventionally slaughtered cows (10), pigs (16) and rabbits (8). In addition, the presence of atrial natriuretic factor (ANF) was detected by immunocytochemistry. Mature ASGs, characterized by the presence of highly osmiophilic and electron-dense material surrounded by a membrane, were present in all atrial myoendocrine cells and their diameters ranged from 100 to 470 nm in pigs, from 100 to 235 nm in cattle, and from 125 to 275 nm in rabbits. Immunoelectronmicroscopical studies revealed the presence of ANF in the ASGs of pigs and cattle, whereas anti-ANF polyclonal serum failed to detect any significative reaction in lapine ASGs. The ultrastructural features of the ASGs of pigs, cattle and rabbits described may be useful in comparing the morphological picture of several cardiac endocrine pathological conditions.

Distribution of atrial natriuretic peptide in the conduction system and ventricular muscles of the human heart

Atrial natriuretic peptide (ANP), a cardiac hormone, is known to be located in the atrial specific granules, but its presence and localization in the ventricular muscle of the human heart has not been examined fully. Using a specific antibody to human ANP, we studied the conduction system and ventricular muscle with immunohistochemical and ultrastructural methods in 30 hearts obtained at autopsy. These included 12 normal and 18 diseased hearts. In the normal hearts, ANP-positive granules, which were regularly observed in the atrial myocytes, were found in small quantities in the cells of the penetrating and branching bundles in 4 of 12, and in the cells of the ventricular free walls in 2 of the 12 hearts. In the diseased hearts, the positivity increased significantly (P less than 0.05), being found in 13 of 18 (72.2%) conduction systems and 10 of 18 (55.6%) ventricular muscles. The granules were confirmed to be immunoreactive with ANP by ultrastructural examination. Furthermore, the presence of ANP mRNA in the conduction system as well as in the ventricular myocytes was demonstrated by Northern blot hybridization for which we used the complementary DNA of human ANP. Thus, a small quantity of ANP appears to be synthesized and stored in the conduction system and ventricles of some normal hearts. However, ANP was shown to be present in a larger percentage of the diseased hearts.

Changes of specific atrial granules induced by doxorubicin in dog's heart

Changes of specific atrial granules induced by doxorubicin in dog's heart

Atrial specific granules: The intracellular calcium store

It is well known that certain organells in cytosol contain high calcium concentration and they are named intracellular Ca stores. These stores regulate the intracellular Ca concentration. The present project aims at exploring whether the release of atrial natriuretic peptide (ANP) from the atrial specific granules(ASG) is dependent on the release of calcium from the ASG. For this purpose, we have to know first whether the ASG contain high Ca concentration. In this work, positive results were obtained by quantitative electron probe x ray microanalysis (EPMA) and Ca-ATPase cytochemical localization.Adult female Sprague Dawley rats were used. The auricle samples were prepared in five different procedures: 1 )preparation of routine ultrathin sections; 2)ethanolic phosphotungstic acid(EPTA) staining ultrathin sections for ASG showing; [1] 3) cytochemical localization of Ca-ATPase ;[2] 4) preparation of ultrathin cryosections of fresh rapid frozen auricles; [3] 5) quantitative EPMA with Link AN10000 and Link Quantem/FLS quantitative analysis software.

The atrial myocardial cells of mouse heart: A structural and stereological study

Structural and stereological studies of mouse atrial myocardial cells, carried out in the same fashion as our previous investigations on mouse ventricle, demonstrate an extremely well-developed sarcoplasmic reticulum (SR) in atrial cells. The volume fraction ( V v) of the SR exceeds 12% in mouse atrial cells; perimyofibrillar network SR constitutes the major portion. We have confirmed the findings of Bossen et al. (1981, Tissue Cell 13, 71–77) of a difference between atria in terms of coupling density, the right atrium having a significantly lower incidence of interior junctional SR than the left. The SR of mouse atrium comprises a rich variety of specialized segments, including the IJSR, peripheral junctional SR, corbular SR, cisternal SR (including regions similar to fenestrated collars of striated skeletal muscle SR), as well as a peculiar form of extended junctional SR (EJSR). Although less frequent in occurrence than corbular SR, the EJSR seems closely related, since it occurs in multiple clusters at or near the Z-line regions, contains internal granular densities, and bears surface-connected structures resembling junctional processes. Seen in thin sections, mouse atrial EJSR elements are more complex than corbular SR, being larger in diameter and frequently circular in profile. Thick-section and serial-section analyses reveal that bodies of EJSR are in fact hollow spheroids. The transverse-axial tubular system of mouse atrium is rather poorly developed in comparison to its ventricular counterpart. The Golgi apparatus and associated specific atrial granules are prominent cell components. “Focal ellipsoidal deposits” (FEDs) previously described by Page and co-workers (1986, Amer. J. Physiol.) are consistently located adjacent to the Golgi region, but immunocytochemical staining for two different segments of atrial natriuretic peptide reveals no specific reaction in FEDs, whereas the SAGS are densely labeled for both antibodies.

Changes in atrial specific granules and atrial natriuretic factor release in experimental congestive cardiac failure

Changes in atrial specific granules and atrial natriuretic factor release in experimental congestive cardiac failure