Processing of pro-atrial natriuretic peptide by corin in cardiac myocytes

Corin is a type II transmembrane serine protease abundantly expressed in the heart. In a previous study using transfected 293 cells, we showed that corin converted pro-atrial natriuretic peptide (pro-ANP) to atrial natriuretic peptide (ANP), suggesting that corin is likely the pro-ANP convertase. Because other serine proteases such as thrombin and kallikrein had previously also been shown to cleave pro-ANP in vitro, it remained to demonstrate that corin is indeed the endogenous pro-ANP convertase in cardiomyocytes. In this study, we examined pro-ANP processing in a murine cardiac muscle cell line, HL-5. Northern analysis showed that corin mRNA was present in HL-5 cells. In HL-5 cells transfected with a plasmid expressing pro-ANP, recombinant pro-ANP was converted to mature ANP as determined by Western analysis, indicating the presence of the endogenous pro-ANP convertase in these cells. The processed recombinant ANP was shown to be active in an enzyme-linked immunosorbent assay-based cGMP assay in baby hamster kidney cells. The processing of recombinant pro-ANP in HL-5 cells was highly sequence-specific, because mutation R98A, but not mutations R101A and R102A, in pro-ANP prevented the conversion of pro-ANP to ANP. Expression of recombinant wild-type corin enhanced the processing of pro-ANP in HL-5 cells. In contrast, overexpression of active site mutant corin S985A or transfection of oligonucleotide small interfering RNA duplexes directed against the mouse corin gene completely inhibited the processing of recombinant pro-ANP in HL-5 cells. These results indicate that corin is the physiological pro-ANP convertase in cardiac myocytes.     

Chromogranin A: Immunocytochemical localization in secretory granules of frog atrial cardiomyocytes

Chromogranin A (CgA) belongs to the granin family of acidic proteins that are present in the secretory granules of many endocrine, neuroendocrine, and nerve cells. CgA has been shown to be stored in cardiomyocyte secretory granules of the rat heart atrium together with atrial natriuretic peptide (ANP). CgA-derived peptides (vasostatins) are known to produce a cardiosuppressive effect on isolated and working in vitro frog and rat hearts. Recently, CgA-derived vasostatin-containing peptides have been identified in rat hearts, whereas no data are available so far about the presence of CgA in the frog heart. In our work, we have studied the subcellular CgA localization in atrial myocytes of the adult frog R. temporaria heart by using an ultraimmunocytochemical method. Immunocytochemical staining of the frog atrial tissue for CgA and ANP showed the presence of the CgA-immunoreactive material in two types (A and B) of large specific atrial secretory granules, whereas no gold particles were revealed over the small granules (D) with a high electron density core. Similar results were obtained during the immunocytochemical staining by an antibody to ANP of the drog atrial cardiomyocytes. The data of the present work allow for the suggestion that CgA revealed in frog atrial cardiomyocytes, like CgA in rat cardiomyocytes, can be considered to be a precursor of intracardial vasostatins that, together with ANP, can play an important cardioprotector role under conditions of stress.     

Atrial natriuretic peptide detected by immunocytochemistry in peripheral organs of Tupaia belangeri

ANP (atrial natriuretic peptide), a peptide found in granules of mammalian atrial cardiac myocytes, has been shown to be active in regulation of blood pressure and body water homeostasis. The existence of ANP in atrium, pituitary, adrenal gland, and kidney of the rat had been immunocytochemically demonstrated with an antibody against rat ANP (102-126). We used the same antibody in immunocytochemical studies for the detection of ANP in peripheral organs of the tree shrew (Tupaia belangeri). The antibody stained granules in myocytes of cardiac atria which indicated that it reacted with tree shrew ANP. In contrast to the rat, no immunoreactive cells were found in pituitaries and adrenal glands. However, in the kidneys distal tubules in outer medulla and cortex were labeled. Ascending limbs of distal tubules were intensely stained when either the peroxidase-antiperoxidase (PAP) or the indirect immunofluorescence method were used. Collecting ducts and convoluted distal tubules in the outer cortex showed a granular type of staining when the immunofluorescence method was used. These data indicate that ANP is present in epithelial cells of distal tubules and collecting ducts, where it may be involved in the regulation of renal salt excretion.     

Atrial natriuretic peptide detected by immunocytochemistry in peripheral organs of Tupaia belangeri

Summary ANP (atrial natriuretic peptide), a peptide found in granules of mammalian atrial cardiac myocytes, has been shown to be active in regulation of blood pressure and body water homeostasis. The existence of ANP in atrium, pituitary, adrenal gland, and kidney of the rat had been immunocytochemically demonstrated with an antibody against rat ANP (102–126). We used the same antibody in immunocytochemical studies for the detection of ANP in peripheral organs of the tree shrew (Tupaia belangeri). The antibody stained granules in myocytes of cardiac atria which indicated that it reacted with tree shrew ANP. In contrast to the rat, no immunoreactive cells were found in pituitaries and adrenal glands. However, in the kidneys distal tubules in outer medulla and cortex were labeled Ascending limbs of distal tubules were intensely stained when either the peroxidase-antiperoxidase (PAP) or the indirect immunofluorescence method were used. Collecting ducts and convoluted distal tubules in the outer cortex showed a granular type of staining when the immunofluorescence method was used. These data indicate that ANP is present in epithelial cells of distal tubules and collecting ducts, where it may be involved in the regulation of renal salt excretion.     

Functional analysis of the transmembrane domain and activation cleavage of human corin: design and characterization of a soluble corin

Corin is a cardiac transmembrane serine protease. In cell-based studies, corin converted pro-atrial natriuretic peptide (pro-ANP) to mature ANP, suggesting that corin is potentially the pro-ANP convertase. In this study, we evaluated the importance of the transmembrane domain and activation cleavage in human corin. We showed that a soluble corin that consists of only the extracellular domain was capable of processing recombinant human pro-ANP in cell-based assays. In contrast, a mutation at the conserved activation cleavage site, R801A, abolished the function of corin, demonstrating that the activation cleavage is essential for corin activity. These results allowed us to design, express, and purify a mutant soluble corin, EKsolCorin, that contains an enterokinase recognition sequence at the activation cleavage site. Purified EKsolCorin was activated by enterokinase in a dose-dependent manner. Activated EK-solCorin had hydrolytic activity toward peptide substrates with a preference for Arg and Lys residues in the P-1 position. This activity of EKsolCorin was inhibited by trypsin-like serine protease inhibitors but not inhibitors of chymotrypsin-like, cysteine-, or metallo-proteases. In pro-ANP processing assays, purified active EKsolCorin converted recombinant human pro-ANP to biologically active ANP in a highly sequence-specific manner. The pro-ANP processing activity of EKsolCorin was not inhibited by human plasma. Together, our data indicate that the transmembrane domain is not necessary for the biological activity of corin but may be a mechanism to localize corin at specific sites, whereas the proteolytic cleavage at the activation site is an essential step in controlling the activity of corin.     

自发性高血压大鼠右心耳肌细胞特殊颗粒的形态分析和ANP免疫反应强度的观察

本研究运用透射电镜及形态计量学方法结合免疫组织化学技术对成年自发性高血压大鼠（ＳＨＲ）的右心耳肌细胞心房特殊颗粒（ＡＳＧ）和心房利钠肽（ＡＮＰ）的免疫反应强度进行了观察和定量研究。成年自发性高血压大鼠的心肌细胞内,ＡＳＧ数目增加,直径增大,高尔基复合体发达;线粒体轻度肿胀,部分嵴溶解断裂,部分内质网扩张,糖原颗粒增多。ＡＮＰ免疫反应增强与ＡＳＧ数目的增加一致。提示自发性高血压大鼠ＡＮＰ的合成和释放均增加,以维持机体在高血压状态下血压的平衡和内环境的稳定。     

Regional appearance of atrial natriuretic peptide in the ventricles of infarcted rat hearts

The appearance of atrial natriuretic peptide (ANP) in the ventricular myocardium was investigated in rat hearts subjected to severe left ventricular infarction. The left coronary artery was ligated for 1, 2, 3, 4 and 6 days and for 3 weeks, and the tissue was prepared for microscopic examination of immunoreactive ANP and for electron microscopy. In the normal and sham-operated hearts, and in hearts subjected to 1 day of coronary ligation, ANP immunoreactivity was restricted to a few ventricular myocytes of the conduction system. Following 2–3 days of coronary ligation, ANP immunoreactivity was detected in the viable myocardium of the lateral border of the infarct and in a few layers of viable cardiac myocytes located in the subendocardial areas of the ischemic left free ventricular wall. Further, during the following days and after 3 weeks of coronary ligation, a gradient of specific labeling was commonly seen across the lateral border area of the infarct. Thus, the strongest immunoreactivities were present in the cardiac myocytes located adjacent to the non-contracting myocardium. Electron microscopic examination of the immunoreactive cardiac myocytes confirmed the presence of electron-dense specific granules within these cells. The present findings suggest that the increased regional production of ANP within the ventricular myocardium is induced by increased mechanical stretch of the cardiac myocytes, and that this might contribute to the increased release of ANP in myocardial infarction.     

Chromogranin A: immunocytochemical localization in secretory granules of frog atrial cardiomyocytes

Chromogranin A (CgA) is a member of the granin family of acidic proteins that present in the secretory granules (SGs) of many endocrine, neuroendocrine and neuronal cells. Atrial natriuretic peptide (ANP)-storing SGs in atrial cardiomyocytes of rat heart also contain CgA. Cardiosuppressive effect of CgA-derived peptides (vasostatins) on in vitro isolated and perfused working frog and rat hearts has been shown under both basal conditions and beta-adrenergic stimulation. More recently it has been revealed that rat heart produces and processes CgA-derived vasostatin-containing peptides. Until now nothing has been known about the presence of CgA in an amphibian heart. We have investigated the subcellular localization of CgA in atrial myocytes of adult frog Rana temporaria heart using ultraimmunocytochemical method. Immunocytochemical staining of the frog atrial tissue for CgA and ANP has shown that out of three morphologically different types (A, B and D) of specific cytoplasmic granules (SCGs) present in myocytes only two (A and B)--large (120-200 nm in diameter) granules with more and with less electron dense core--exhibit immunoreactivity (IR) to these two antigens. The third type (D) of granules (80-100 nm in diameter) are small membrane bound granules characterized by highly electron dense core surrounded with a thin halo. These granules revealed negative reaction on immunostaining for both CgA and ANP. The presence of CgA- and ANP-IR in the same SCGs in frog atrial myocytes is consistent with the endocrine nature of these granules. Taking into account our and literature data we propose that CgA present in frog atrial cardiomyocite SCGs might be a precursor of vasostatin-containing peptides, as it takes place in rat heart. It is possible that these CgA-derived peptides together with ANP exert their regulatory function through the autocrine and/or paracrine mechanisms and play important cardioprotective role in frog heart under stress condition.     

Upregulation of corin gene expression in hypertrophic cardiomyocytes and failing myocardium

High levels of plasma atrial natriuretic peptides (ANP) are associated with pathological conditions such as congestive heart failure (CHF). Recently, we have identified a cardiac serine protease, corin, that is the pro-ANP convertase. In this study, we examined the regulation of corin gene expression in cultured hypertrophic cardiomyocytes and in the left ventricular (LV) myocardium of a rat model of heart failure. Quantitative RT-PCR analysis showed that both corin and ANP mRNA levels were significantly increased in phenylephrine (PE)-stimulated rat neonatal cardiomyocytes in culture. The increase in corin mRNA correlated closely with the increase in cell size and ANP mRNA expression in the PE-treated cells (r = 0.95, P < 0.01; r = 0.92, P < 0.01, respectively). The PE-treated cardiomyocytes had an increased activity in converting recombinant human pro-ANP to biologically active ANP, as determined by a pro-ANP processing assay and a cell-based cGMP assay. In a rat model of heart failure induced by ligation of the left coronary artery, corin mRNA expression in the noninfarcted LV myocardium was significantly higher than that of control heart tissues from sham-operated animals, when examined by Northern blot analysis and RT-PCR at 8 wk. These results indicate that the corin gene is upregulated in hypertrophic cardiomyocytes and failing myocardium. Increased corin expression may contribute to elevation of ANP in the setting of cardiac hypertrophy and heart failure.     

Evaluation of atrial natriuretic peptide and brain natriuretic peptide in atrial granules of rats with experimental congestive heart failure.

The natriuretic peptides are believed to play an important role in the pathophysiology of congestive heart failure (CHF). We utilized a quantitative cytomorphometric method, using double immunocytochemical labeling, to assess the characteristics of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in atrial granules in an experimental model of rats with CHF induced by aortocaval fistula. Rats with CHF were further divided into decompensated (sodium-retaining) and compensated (sodium-excreting) subgroups and compared with a sham-operated control group. A total of 947 granules in myocytes in the right atrium were analyzed, using electron microscopy and a computerized analysis system. Decompensated CHF was associated with alterations in the modal nature of granule content packing, as depicted by moving bin analysis, and in the granule density of both peptides. In control rats, the mean density of gold particles attached to both peptides was 347.0 +/- 103.6 and 306.3 +/- 89.9 gold particles/microm2 for ANP and BNP, respectively. Similar mean density was revealed in the compensated rats (390.6 +/- 81.0 and 351.3 +/- 62.1 gold particles/microm2 for ANP and BNP, respectively). However, in rats with decompensated CHF, a significant decrease in the mean density of gold particles was observed (141.6 +/- 67.3 and 158.0 +/- 71.2 gold particles/microm2 for ANP and BNP, respectively; p<0.05 compared with compensated rats, for both ANP and BNP). The ANP:BNP ratio did not differ between groups. These findings indicate that the development of decompensated CHF in rats with aortocaval fistula is associated with a marked decrease in the density of both peptides in atrial granules, as well as in alterations in the quantal nature of granule formation. The data further suggest that both peptides, ANP and BNP, may be regulated in the atrium by a common secretory mechanism in CHF.     

Effects of expansion of blood volume and bilateral vagotomy on specific heart granules and release of atrial natriuretic peptide in the rat

Summary There was no statistically significant difference in basal concentrations of immunoreactive atrial natriuretic peptide (ANP), as assessed by radioimmunoassay, between right and left atrial muscle of control rats; similarly, stereological analysis showed no statistically significant difference in the fractional volume of myocytes occupied by specific heart granules, or in numerical density of granules, between right and left atria. Nevertheless, correlated radioimmunoassay and ultrastructural investigations showed that the major source of elevated plasma levels of ANP after expansion of blood volume was the right atrium. Substantial expansion of blood volume caused an increase in the proportion of peripherally located granules in myocytes of both atria, but reduction in the number of granules and in the concentration and total content of ANP occurred in the right atrium only. Bilateral cervical vagotomy also caused a statistically significant elevation of plasma ANP concentration, accompanied by a statistically significant reciprocal reduction in right atrial ANP content; no statistically significant change occurred in left atrial ANP. When blood volume was expanded after bilateral vagotomy, there was a further statistically significant increase in plasma ANP concentration; this was accompanied by further reduction in right atrial ANP and, moreover, the combined manoeuvre also elicited a statistically significant reduction of ANP in the left atrium. Ultrastructural studies confirmed that, under these conditions, myocytes in both atria showed a marked depletion of specific heart granules.     

An immunocytochemical study on co-localization of cathepsin B and atrial natriuretic peptides in secretory granules of atrial myoendocrine cells of rat heart

To examine localization of cathepsin B, a representative lysosomal cysteine protease, in atrial myoendocrine cells of the rat heart, immunohistochemistry at the light and electron microscopic level was applied to the atrial tissue, using a monospecific antibody for rat liver cathepsin B. In serial semi-thin sections, immunoreactivity for cathepsin B and atrial natriuretic peptides (ANP) was detected in the para-nuclear region of atrial myoendocrine cells. Several large granules and many fine granules in the region of the cells were positively stained by the cathepsin B antibody. Gold particles indicating cathepsin B antigenicity labeled secretory granules in the cells, which were also labeled by those indicating ANP, using thin sections of the Lowicryl K4M-embedded material. Moreover, some granules labeled densely by immunogold particles for cathepsin B seemed to be lysosomes. By double immunostaining using thin sections of the Epon-embedded material, gold particles indicating cathepsin B and ANP antigenicities were co-localized in secretory granules of the cells. By enzyme assay, activity of cathepsin B was three times higher in atrial tissue than ventricular tissue. The results suggest that co-localization of cathepsin B and ANP in secretory granules is compatible with the possibility that cathepsin B participates in the maturation process of ANP.     

Proteomic analysis of rat atrial secretory granules: a platform for testable hypotheses

The recent development of powerful proteomic tools has enabled investigators to directly examine the population of proteins present in defined biological systems. We report here the first proteomic analysis of atrial secretory granules. Approximately 100 distinct protein components of the atrial secretory granule proteome were detected using subcellular fractionation and one-dimensional SDS-PAGE in conjunction with peptide mass fingerprinting by MALDI-TOF mass spectrometry. Of this number, 61 proteins were clearly identified by high probability data matches and repeated observation. The majority of the proteome was found to be membrane-associated with the most prominent proteins being peptidylglycine alpha-amidating monooxygenase (PAM) and pro-atrial natriuretic peptide (pro-ANP). This proteomic analysis of the rat atrium secretory granule produced an assembly of proteins with a diverse array of reported functions. The identified proteins fall into seven functional categories: (1) granular transport, docking and fusion; (2) signal transduction; (3) calcium-binding/calcium-dependent; (4) cellular architecture/chaperoning; (5) peptide/protein processing; (6) hormone; (7) proton transport. The novel finding of several protein processing enzymes and signal transduction proteins offer new perspectives on how pro-ANP is stored and processed to ANP during release. Accordingly, defining the proteome of the atrial secretory granule provides a framework for the development of new hypotheses that address key mechanisms governing granule function and ANP secretion.     

Stretch-activated non-selective cation channel: a causal link between mechanical stretch and atrial natriuretic peptide secretion

The polypeptide hormone atrial natriuretic peptide (ANP) plays vital roles in maintaining blood volume and arterial blood pressure. The recognition of clinical benefits of ANP both in healthy and diseased heart identifies ANP as a potential candidate for therapeutic strategy in the treatment of heart disease. ANP is synthesized and stored in cardiac myocytes and it is released through the exocytosis of ANP granules both constitutively and in response to stimuli. It is well known that mechanical stretch is the predominant stimulus for ANP secretion. However, the mechanistic link between mechanical stimuli and exocytosis of ANP vesicles in single atrial myocyte has not yet been demonstrated. Over the last decade, compelling evidence suggested that stretch-activated ion channels might function as mechanosensors. We showed previously that direct stretch of single atrial myocyte using two micro-electrodes activated a non-selective cation channel (SAC). So far it is not known whether activation of SAC is involved in stretch-induced ANP secretion. The present article aims to give an overview of the mechanism of mechanical stretch-stimulated ANP secretion and describes an innovative technique to detect ANP secretion from isolated rat atrial myocytes with high time-resolution. Combined with capacitance measurement and patch-clamp technique in conjunction with in situ ANP bioassay, we were able to demonstrate that SAC in rat atrial myocytes acts as a mechanosensor to transduce stretch signals into the ANP secretion pathway.     

Immunohistochemical identification of Purkinje fibers and transitional cells in a terminal portion of the impulse-conducting system of porcine heart

Summary The ultrastructure of porcine ventricular tissue was studied by electron microscopy and immunocytochemical techniques. Electron-dense specific granules were found in both Purkinje fibers and transitional cells in the ventricular walls, and were positively stained by the immunogold staining method using an antiserum against atrial natriuretic polypeptide (ANP). This suggests that both the Purkinje fibers and transitional cells display the same specific granules as atrial cardiocytes containing ANP. These results demonstrate that Purkinje fibers and two types of transitional cells, in addition to the ordinary ventricular cardiocytes, can be identified in porcine ventricular wall tissue.     

C-type natriuretic peptide inhibits ANP secretion and atrial dynamics in perfused atria: NPR-B-cGMP signaling

The purpose of the present experiments was to define the role of C-type natriuretic peptide (CNP) in the regulation of atrial secretion of atrial natriuretic peptide (ANP) and atrial stroke volume. Experiments were performed in perfused beating and nonbeating quiescent atria, single atrial myocytes, and atrial membranes. CNP suppressed in a dose-related fashion the increase in atrial stroke volume and ANP secretion induced by atrial pacing. CNP caused a right shift in the positive relationships between changes in the secretion of ANP and atrial stroke volume or translocation of the extracellular fluid (ECF), which indicates the suppression of atrial myocytic release of ANP into the paracellular space. The effects of CNP on the secretion and contraction were mimicked by 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP). CNP increased cGMP production in the perfused atria, and the effects of CNP on the secretion of ANP and atrial dynamics were accentuated by pretreatment with an inhibitor of cGMP phosphodiesterase, zaprinast. An inhibitor of the biological natriuretic peptide receptor (NPR), HS-142-1, attenuated the effects of CNP. The suppression of ANP secretion by CNP and 8-BrcGMP was abolished by a depletion of extracellular Ca(2+) in nonbeating atria. Natriuretic peptides increased cGMP production in atrial membranes with a rank order of potency of CNP > BNP > ANP, and the effect was inhibited by HS-142-1. CNP and 8-BrcGMP increased intracellular Ca(2+) concentration transients in single atrial myocytes, and mRNAs for CNP and NPR-B were expressed in the rabbit atrium. From these results we conclude that atrial ANP release and stroke volume are controlled by CNP via NPR-B-cGMP mediated signaling, which may in turn act via regulation of intracellular Ca(2+).     

Localization of the mosaic transmembrane serine protease corin to heart myocytes.

Corin cDNA encodes an unusual mosaic type II transmembrane serine protease, which possesses, in addition to a trypsin-like serine protease domain, two frizzled domains, eight low-density lipoprotein (LDL) receptor domains, a scavenger receptor domain, as well as an intracellular cytoplasmic domain. In in vitro experiments, recombinant human corin has recently been shown to activate pro-atrial natriuretic peptide (ANP), a cardiac hormone essential for the regulation of blood pressure. Here we report the first characterization of corin protein expression in heart tissue. We generated antibodies to two different peptides derived from unique regions of the corin polypeptide, which detected immunoreactive corin protein of approximately 125-135 kDa in lysates from human heart tissues. Immunostaining of sections of human heart showed corin expression was specifically localized to the cross striations of cardiac myocytes, with a pattern of expression consistent with an integral membrane localization. Corin was not detected in sections of skeletal or smooth muscle. Corin has been suggested to be a candidate gene for the rare congenital heart disease, total anomalous pulmonary venous return (TAPVR) as the corin gene colocalizes to the TAPVR locus on human chromosome 4. However examination of corin protein expression in TAPVR heart tissue did not show evidence of abnormal corin expression. The demonstrated corin protein expression by heart myocytes supports its proposed role as the pro-ANP convertase, and thus a potentially critical mediator of major cardiovascular diseases including hypertension and congestive heart failure.     

胰岛素诱导低血糖大鼠心房肌细胞特殊颗粒的形态计量和心房利钠肽免疫组织化学研究

通过形态计量学和免疫组织化学方法发现胰岛素诱导低血糖大鼠心房肌细胞核周区特殊颗粒（ＡＳＧ）的体密度、面数密度和数密度及平均直径均高于对照组（Ｐ＜０．０５）,但高尔基复合体各参数与对照组比较没有差别（Ｐ＞０．０５）。实验组的心房利钠肽（ＡＮＰ）的免疫反应强度比对照组强（Ｐ＜０．００１）。提示胰岛素诱导低血糖对心房利钠肽的释放具有抑制作用,表明ＡＮＰ作为生理和病理调节递质与代谢刺激相拮抗。     

Fine structural and immunohistochemical localization of cardiac hormones (ANP) in the right atrium and hypothalamus of the white rat

Atrial natriuretic peptide (ANP) is a polypeptide hormone secreted primarily by atrial myoendocrine cells. It has diuretic, natriuretic and vasorelaxant effects. ANP has been characterized by non-morphological methods in a number of extra-atrial tissues, particularly the hypothalamus, but little is known of the immunohistochemistry of hypothalamic ANP cells in comparison to atrial ones. Although the presence of ANP-producing cells has previously been confirmed in the right atrium of the rat and other vertebrate species, to our knowledge, this is the first study to demonstrate the presence of these cells in the hypothalamus using a purely morphological method such as electron microscopy. The fine structural and immunohistochemical characteristics of right atrial and hypothalamic ANP positive cells were investigated using immunogold labeling with goat anti-alpha-human ANP (1-28) as primary antibody. Atrial ANP cells were characterized by the presence of membrane-bound electrondense spherical or oval granules with a diameter of about 250 nm. The opaque content of the granules is separated from the limiting membrane by a thin electron translucent band about 20 nm wide. Electron dense crystalloid inclusions were evident within the granule matrix of some atrial ANP granules. Hypothalamic ANP granules were membrane-bound larger in diameter (320 nm), and less electron dense, and lacked crystalloid inclusions. Statistical analyses revealed a significant larger diameter and a significant smaller number of hypothalamic ANP granules compared to atrial ones. The significantly greater number of atrial ANP positive granules suggests a greater volume capacity for the atrial ANP positive granules as compared to the hypothalamic ones. This may indicate that ANP is secreted primarily from the right atrium and to a lesser extent from the hypothalamus; and that both atrial and hypothalamic ANP are closely related in chemical nature and immunohistochemical characteristics. This supports the suggestion that ANP may play the dual role of peripheral hormone and a neurotransmitter or neuromediator.