Chromogranin B (secretogranin I), a secretory protein of the regulated pathway, is also present in a tightly membrane-associated form in PC12 cells.

Chromogranin B (CgB, also called secretogranin I) is a secretory protein sorted to secretory granules in a wide variety of endocrine cells and neurons. Unexpectedly, after stimulation of regulated secretion in the neuroendocrine cell line PC12, a fraction of the exocytosed CgB was not released into the medium but remained associated with the plasma membrane. The addition of exogenous CgB to unstimulated cells did not result in the appearance of cell surface CgB, suggesting that the presence of cell surface CgB could not be accounted for by adsorption of released CgB to the cell surface. Upon further incubation of stimulated PC12 cells, the surface CgB was internalized by the cells and largely degraded. The surface CgB was not released by exposure to pH 11, yet it partitioned in the aqueous phase upon Triton X-114 phase separation. Subcellular fractionation and differential extraction studies showed that the membrane-associated CgB constituted at least 10% of the total cellular CgB. These observations suggest that (a) the appearance of CgB at the cell surface is due to fusion of secretory granules with the plasma membrane and (b) a fraction of CgB is present in tight association with the secretory granule membrane. We propose a model in which membrane-associated CgB, by virtue of its ability to interact in a homophilic manner with soluble CgB, plays a key role in the sorting and targeting of CgB to the regulated pathway.     

Chromogranin B (secretogranin I), a neuroendocrine-regulated secretory protein, is sorted to exocrine secretory granules in transgenic mice.

Chromogranin B (CgB, secretogranin I) is a secretory granule matrix protein expressed in a wide variety of endocrine cells and neurons. Here we generated transgenic mice expressing CgB under the control of the human cytomegalovirus promoter. Northern and immunoblot analyses, in situ hybridization and immunocytochemistry revealed that the exocrine pancreas was the tissue with the highest level of ectopic CgB expression. Upon subcellular fractionation of the exocrine pancreas, the distribution of CgB in the various fractions was indistinguishable from that of amylase, an endogenous constituent of zymogen granules. Immunogold electron microscopy of pancreatic acinar cells showed co-localization of CgB with zymogens in Golgi cisternae, condensing vacuoles/immature granules and mature zymogen granules; the ratio of immunoreactivity of CgB to zymogens being highest in condensing vacuoles/immature granules. CgB isolated from zymogen granules of the pancreas of the transgenic mice aggregated in a mildly acidic (pH 5.5) milieu in vitro, suggesting that low pH-induced aggregation contributed to the observed concentration of CgB in condensing vacuoles. Our results show that a neuroendocrine-regulated secretory protein can be sorted to exocrine secretory granules in vivo, and imply that a key feature of CgB sorting in the trans-Golgi network of neuroendocrine cells, i.e. its aggregation-mediated concentration in the course of immature secretory granule formation, also occurs in exocrine cells although secretory protein sorting in these cells is thought to occur largely in the course of secretory granule maturation.     

Chromogranin B: isolation from pheochromocytoma, N-terminal sequence, tissue distribution and secretory vesicle processing

The chromogranins/secretogranins are a family of neuroendocrine vesicle secretory proteins. Immunohistology and immunoblotting have suggested that a major soluble protein in human chromaffin granules may be chromogranin B (CgB). We purified from pheochromocytoma chromaffin granules an SDS-PAGE 110-120 kDa protein whose N-terminal sequence matched that previously deduced from a human CgB cDNA. An antibody directed against a synthetic human CgB N-terminal region specifically recognized the CgB N-terminus, though not the chromogranin A (CgA) N-terminus or the CgB C-terminus on immunoblots. An antiserum directed against CgB's C-terminus also visualized CgB but not CgA. By immunoblotting, CgB was a quantitatively major protein in human pheochromocytoma chromaffin granules, but a relatively minor in normal bovine adrenal medullary chromaffin granules. In a variety of normal bovine neuroendocrine tissues, the relative abundance of CgB immunoreactivity on immunoblots was: adrenal medulla greater than anterior pituitary greater than pancreas greater than small intestine, hypothalamus. Immunoblotting of neuroendocrine tissues (or their hormone storage vesicle cores) with both anti N-terminal and anti C-terminal CgB antisera suggested bidirectional cleavage or processing of CgB; in the anterior pituitary, a unique 40 kDa C-terminal fragment was observed. Bidirectional CgB cleavage was also suggested on immunoblots of chromaffin tissue from three species (human, bovine, rat). C-terminal processing of CgB was also confirmed by amino acid sequencing of SDS-PAGE-separated, polyvinylidene difluoride membrane-immobilized CgB fragments from pheochromocytoma chromaffin granules. Whether such fragments possess biological activity remains to be investigated.     

The granin family of uniquely acidic proteins of the diffuse neuroendocrine system: comparative and functional aspects

The chromogranins A (CgA) and B (CgB) and secretogranin II (SgII) constitute the main members of a family of uniquely acidic secretory proteins in elements of the diffuse neuroendocrine system. These genetically distinct proteins, CgA, CgB, SgII and the less well known secretogranins III-VII are collectively referred to as 'granins' and characterised by numerous pairs of basic amino acids as potential cleavage sites for processing by the co-stored prohormone converting enzymes PC 1/3 and PC2. This review is directed towards comparative and functional aspects of the granins with emphasis on their phylogenetically conserved sequences. Recent developments provide ample evidence of widely different effects and targets for the intact granins and their derived peptides, intracellularly in the directed trafficking of storage components during granule maturation and extracellularly in autocrine, paracrine and endocrine interactions. Most of the effects assigned to the granin derived peptides fit into patterns of direct or indirect inhibitory modulations of major functions. So far, peptides derived from CgA (vasostatins, chromacin, pancreastatin, WE-14, catestatin and parastatin), CgB (secretolytin) and SgII (secretoneurin) are the most likely candidates for granin-derived regulatory peptides, of postulated relevance not only for homeostatic processes, but also for tissue assembly and repair, inflammatory responses and the first line of defence against invading microorganisms.     

Essential Role of the Disulfide-bonded Loop of Chromogranin B for Sorting to Secretory Granules Is Revealed by Expression of a Deletion Mutant in the Absence of Endogenous Granin Synthesis

Sorting of regulated secretory proteins in the TGN to immature secretory granules (ISG) is thought to involve at least two steps: their selective aggregation and their interaction with membrane components destined to ISG. Here, we have investigated the sorting of chromogranin B (CgB), a member of the granin family present in the secretory granules of many endocrine cells and neurons. Specifically, we have studied the role of a candidate structural motif implicated in the sorting of CgB, the highly conserved NH₂-terminal disulfide– bonded loop. Sorting to ISG of full-length human CgB and a deletion mutant of human CgB (Δcys-hCgB) lacking the 22–amino acid residues comprising the disulfide-bonded loop was compared in the rat neuroendocrine cell line PC12. Upon transfection, i.e., with ongoing synthesis of endogenous granins, the sorting of the deletion mutant was only slightly impaired compared to full-length CgB. To investigate whether this sorting was due to coaggregation of the deletion mutant with endogenous granins, we expressed human CgB using recombinant vaccinia viruses, under conditions in which the synthesis of endogenous granins in the infected PC12 cells was shut off. In these conditions, Δcys-hCgB, in contrast to full-length hCgB, was no longer sorted to ISG, but exited from the TGN in constitutive secretory vesicles. Coexpression of full-length hCgB together with Δcys-hCgB by double infection, using the respective recombinant vaccinia viruses, rescued the sorting of the deletion mutant to ISG. In conclusion, our data show that (a) the disulfide-bonded loop is essential for sorting of CgB to ISG and (b) the lack of this structural motif can be compensated by coexpression of loop-bearing CgB. Furthermore, comparison of the two expression systems, transfection and vaccinia virus–mediated expression, reveals that analyses under conditions in which host cell secretory protein synthesis is blocked greatly facilitate the identification of sequence motifs required for sorting of regulated secretory proteins to secretory granules.     

Topology of chromogranin A and secretogranin II in the rat anterior pituitary: potential marker proteins for distinct secretory pathways in gonadotrophs

Summary Chromogranins (Cg)/secretogranins (Sg) are representative acidic glycoproteins in secretory granules of many endocrine cells where they are co-stored and co-released with resident amines or peptides. The exact distribution of these proteins in the rat anterior pituitary is unknown. Therefore, pituitaries from untreated male rats were investigated by light- and electron-microscopical immunocytochemistry for the cellular and subcellular localization of CgA, CgB, and SgII. Endocrine cells, identified light-microscopically as gonadotrophs in adjacent semithin sections immunostained for follicle-stimulating hormone (FSH) and luteinizing hormone (LH), concomitantly were immunoreactive for CgA, CgB, and SgII. Ultrastructurally, gonadotrophs exhibited two types of secretory granules which varied in their immunoreactivities for gonadotropins and Cg/Sg. Large-sized (500 nm), moderately electron-dense granules showed antigenicities for FSH, LH, and CgA. Smaller-sized (200 nm), electron-dense granules were immunoreactive exclusively for LH and SgII. The distinct localization of CgA and SgII to morphologically and hormonally different secretory granules indicates the existence of two regulated secretory pathways in rat pituitary gonadotrophs. Hence, these proteins are considered as valuable tools to analyze the intracellular trafficking during granule biogenesis and the possible different regulation of FSH and LH secretion.     

The disulfide-bonded loop of chromogranin B mediates membrane binding and directs sorting from the trans-Golgi network to secretory granules

The disulfide-bonded loop of chromogranin B (CgB), a regulated secretory protein with widespread distribution in neuroendocrine cells, is known to be essential for the sorting of CgB from the trans-Golgi network (TGN) to immature secretory granules. Here we show that this loop, when fused to the constitutively secreted protein alpha1-antitrypsin (AT), is sufficient to direct the fusion protein to secretory granules. Importantly, the sorting efficiency of the AT reporter protein bearing two loops (E2/3-AT-E2/3) is much higher compared with that of AT with a single disulfide-bonded loop. In contrast to endogenous CgB, E2/3-AT-E2/3 does not undergo Ca2+/pH-dependent aggregation in the TGN. Furthermore, the disulfide-bonded loop of CgB mediates membrane binding in the TGN and does so with 5-fold higher efficiency if two loops are present on the reporter protein. The latter finding supports the concept that under physiological conditions, aggregates of CgB are the sorted units of cargo which have multiple loops on their surface leading to high membrane binding and sorting efficiency of CgB in the TGN.     

Topology of chromogranin A and secretogranin II in the rat anterior pituitary: potential marker proteins for distinct secretory pathways in gonadotrophs.

Chromogranins (Cg)/secretogranins (Sg) are representative acidic glycoproteins in secretory granules of many endocrine cells where they are co-stored and co-released with resident amines or peptides. The exact distribution of these proteins in the rat anterior pituitary is unknown. Therefore, pituitaries from untreated male rats were investigated by light- and electron-microscopical immunocytochemistry for the cellular and subcellular localization of CgA, CgB, and SgII. Endocrine cells, identified light-microscopically as gonadotrophs in adjacent semithin sections immunostained for follicle-stimulating hormone (FSH) and luteinizing hormone (LH), concomitantly were immunoreactive for CgA, CgB, and SgII. Ultrastructurally, gonadotrophs exhibited two types of secretory granules which varied in their immunoreactivities for gonadotropins and Cg/Sg. Large-sized (500 nm), moderately electron-dense granules showed antigenicities for FSH, LH, and CgA. Smaller-sized (200 nm), electron-dense granules were immunoreactive exclusively for LH and SgII. The distinct localization of CgA and SgII to morphologically and hormonally different secretory granules indicates the existence of two regulated secretory pathways in rat pituitary gonadotrophs. Hence, these proteins are considered as valuable tools to analyze the intracellular trafficking during granule biogenesis and the possible different regulation of FSH and LH secretion.     

Immunolocalization of secretogranin II, chromogranin A, and chromogranin B in differentiating human neuroblastoma cells.

In order to obtain further insights into the expression of the known markers of secretory neuroendocrine dense core organelles, secretogranin II (SgII), chromogranin A (CgA), and chromogranin B (CgB) during neuronal differentiation, the immunolocalization of these proteins was studied by means of double immunofluorescence in both undifferentiated and retinoic acid-differentiated SH-SY5Y human neuroblastoma cells. The majority of undifferentiated cells was not immunolabeled for all three proteins. In the majority of differentiated cells, a clearly punctate SgII immunolabeling indicative of the presence of secretory organelles was present in the Golgi region, at the cell periphery, along the neurites and in growth cones. Only relatively few of the SgII-immunolabeled cells were also immunolabeled for CgA and CgB, and in a single cell the three proteins were not always present in the same organelles. These results, obtained in a cultured cell line, confirm the not necessarily parallel distribution of SgII, CgA, and CgB observed in different neuroendocrine tissues and suggest that SgII may be the best marker of human neuroblastoma cell differentiation.     

Brefeldin A inhibits the formation of constitutive secretory vesicles and immature secretory granules from the trans-Golgi network.

The effects of brefeldin A (BFA) on membrane traffic between the trans-Golgi network (TGN) and the plasma membrane were investigated in intact PC12 cells and in a cell-free system derived from PC12 cells. In intact cells, BFA caused a virtually complete block of constitutive secretion, as indicated by the lack of release from, and accumulation in, the cells of a [35S]sulfate-labeled heparan sulfate proteoglycan (hsPG). Pulse-chase experiments with [35S]sulfate followed by subcellular fractionation showed that this block was due to the inhibition of formation of constitutive secretory vesicles (CSVs) from the TGN. BFA did not block the depolarization-induced release of [35S]sulfate-labeled chromogranin B (CgB) and secretogranin II (SgII) from secretory granules formed prior to the addition of the drug, showing that BFA does not block secretory granule fusion with the plasma membrane. The presence of BFA did, however, prevent the appearance of [35S]sulfate-labeled CgB and SgII in secretory granules, indicating that the drug inhibits the formation of secretory granules from the TGN. Evidence for a direct block of vesicle formation by BFA was obtained using a cell-free system derived from [35S]sulfate-labeled PC12 cells. In this system, low concentrations of BFA (5 micrograms/ml) inhibited the formation of the hsPG-containing CSVs and that of the SgII-containing secretory granules from the TGN to the same extent (50-60%) as, and in a non-additive manner with, the nonhydrolyzable GTP analogue GTP gamma S. Consistent with the inhibitory effects of BFA on vesicle formation from the TGN, BFA treatment of intact PC12 cells led to the hypersialylation of CgB, which presumably was due to the increased residence time of the protein in the TGN. In conclusion, our data are consistent with, and allow the generalization of, the concept that the BFA-induced block of anterograde membrane traffic results from the inhibition of vesicle formation from a donor compartment.     

A panel of 13 region-specific radioimmunoassays for measurements of human chromogranin B

INTRODUCTION: The primary structure of human chromogranin B (CgB) contains 15 pairs of basic amino acids, which are potential cleavage sites for specific endogenous proteases, but also other sites in the molecule can be subjected to cleavage. Several CgB-related peptides have been identified in tissue extracts. MATERIALS AND METHODS: Peptides homologous to defined parts of the human CgB molecule were selected and synthesized. Antibodies were raised and 13 specific radioimmunoassays were developed. Plasma samples from 19 patients with neuroendocrine tumors were collected and measured in all assays. RESULTS: All region-specific assays measured circulating levels of CgB-related peptides. Only five of the assays measured high concentrations of circulating CgB and two of them correlated with that of intact chromogranin A (CgA). CONCLUSION: The assays presented allow measurements of defined regions of CgB and will thus become important tools for further studies of the processing of CgB. One of the assays merit further investigations as a new marker for neuroendocrine tumors.     

Immunocytochemical localization of secretogranin III in the anterior lobe of male rat pituitary glands.

Secretogranin III (SgIII) is one of the acidic secretory proteins, designated as granins, which are specifically expressed in neuronal and endocrine cells. To clarify its precise distribution in the anterior lobe of the rat pituitary gland, we raised a polyclonal antiserum against rat SgIII for immunocytochemical analyses. By immunohistochemistry using semithin sections, positive signals for SgIII were detected intensely in mammotropes and thyrotropes, moderately in gonadotropes and corticotropes, but not in somatotropes. The distribution pattern of SgIII in the pituitary gland was similar to that of chromogranin B (CgB), also of the granin protein family, suggesting that the expressions of these two granins are regulated by common mechanisms. The localization of SgIII in endocrine cells was confirmed by immunoelectron microscopy. In particular, secretory granules of mammotropes and thyrotropes were densely and preferentially co-labeled for SgIII and CgB in their periphery. Moreover, positive signals for SgIII were occasionally found in cells containing both prolactin and TSH in secretory granules. These lines of evidence suggest that SgIII and CgB are closely associated with the secretory granule membrane and that this membrane association might contribute to gathering and anchoring of other soluble constituents to the secretory granule membrane.     

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.     

Disruption of disulfide bonds exhibits differential effects on trafficking of regulated secretory proteins

For several secretory proteins, it has beenhypothesized that disulfide-bonded loop structures are required forsorting to secretory granules. To explore this hypothesis, we employeddithiothreitol (DTT) treatment in live pancreatic islets, as well as inPC-12 andGH₄C₁cells. In islets, disulfide reduction in the distal secretory pathwaydid not increase constitutive or constitutive-like secretion ofproinsulin (or insulin). In PC-12 cells, DTT treatment caused adramatic increase in unstimulated secretion of newly synthesizedchromogranin B (CgB), presumably as a consequence of reducing thesingle conserved chromogranin disulfide bond (E. Chanat, U. Weiss, W. B. Huttner, and S. A. Tooze. EMBO J. 12: 2159-2168, 1993). However, inGH₄C₁cells that also synthesize CgB endogenously, DTT treatment reducednewly synthesized prolactin and blocked its export, whereas newlysynthesized CgB was routed normally to secretory granules. Moreover, ontransient expression inGH₄C₁cells, CgA and a CgA mutant lacking the conserved disulfide bond showedcomparable multimeric aggregation properties and targeting to secretorygranules, as measured by stimulated secretion assays. Thus theconformational perturbation of regulated secretory proteins caused bydisulfide disruption leads to consequences in protein trafficking thatare both protein and cell type dependent.

     

Identification and characterization of novel chromogranin B-derived peptides from porcine chromaffin granules by liquid chromatography/electrospray tandem MS.

Chromogranin B (CgB) is a regulated secretory protein that is stored in endocrine and neuroendocrine cells. It can be processed proteolytically to small peptide fragments. In the present study three proteolytic products of porcine CgB were obtained after size-exclusion, immunoaffinity, and reversed-phase chromatography, and then identified by electrospray tandem MS. One novel peptide was identified as S586-R602 (SR-17) and is phosphorylated at one or two serine residues. Another novel peptide H603-Q636 (HQ-34), with molecular mass 3815.56 Da, was found to be oxidized at the methionine residue. In addition, a secretolytin-like peptide fragment (KR-11), which is two amino acids shorter than the bovine secretolytin, was found. This is the first report that the C-terminal region of CgB, the homologue of human CCB, is proteolytically processed further into three small peptide fragments.     

Presence and release of the chromogranin B-derived secretolytin-like peptide KR-11 from the porcine spleen

Chromogranin B (CgB) is a major matrix protein in secretory granules/large dense-cored vesicles and a precursor for smaller peptides. In an earlier study, we have identified a secretolytin-like peptide (KR-11, pCgB(637-647)) from porcine chromaffin granules. Further evidence is presented here to show the processing of chromogranin B to this peptide during axonal transport in the splenic nerve and its release in the spleen upon various conditions of stimulation. Immunohistochemical staining showed that in the porcine spleen chromogranin B and NPY completely colocalize in nerve fibres and varicosities in blood vessel walls and trabeculae, and along the loose network of smooth muscle cells in the red pulp, as identified by their alpha-smooth muscle cell actin content. No antibacterial activity was found for the porcine secretolytin-like peptide, KR-11. The change of one amino acid residue (Thr-->Asn) in the porcine homologous fragment of secretolytin appears to be responsible for its loss of antibacterial activity.     

Antimicrobial peptides and protease inhibitors in the skin secretions of the crawfish frog, Rana areolata

The dorsal skin of the crawfish frog, Rana areolata, is associated with numerous prominent granular glands. Proteomic analysis of electrically stimulated skin secretions from these glands enabled the identification and characterization of eight peptides with antimicrobial and hemolytic activity belonging to the previously identified brevinin-1, temporin-1, palustrin-2, palustrin-3, esculentin-1 (two peptides), and ranatuerin-2 (two peptides) families. The primary structures of the peptides were consistent with a close phylogenetic relationship between R. areolata and the pickerel frog, Rana palustris. Three structurally related cationic, cysteine-containing peptides were identified that show sequence similarity to peptide Leucine–Arginine, a peptide with immunomodulatory and histamine-releasing properties from the skin of the northern leopard frog, Rana pipiens. The skin secretions contained a 61-amino-acid-residue peptide that inhibited porcine trypsin and possessed a 10-cysteine-residue motif that is characteristic of a protease inhibitor previously isolated from the parasitic nematode, Ascaris suum. A 48-amino-acid-residue protein containing eight cysteine residues in the whey acidic protein (WAP) motif, characteristic of elafin (skin-derived antileukoproteinase) and secretory leukocyte protease inhibitor, was also isolated. The data suggest that protease inhibitors in skin secretions may play a role complementary to cationic, amphipathic α-helical peptides in protecting anurans from invasions by microorganisms.     

Region-specific antibodies to chromogranin B display various immunostaining patterns in human endocrine pancreas.

Chromogranin (Cg) B is an acidic glycoprotein present in neuroendocrine tissue. The sequence shows several dibasic amino acid positions susceptible to proteolytic cleavage. The purpose of this study was to elucidate the expression of CgB epitopes in the human endocrine pancreas. Tissue sections of six human pancreata were immunostained with 16 different region-specific antibodies to the CgB molecule, using double immunofluorescence techniques. The CgB epitope pattern varied in the four major islet cell types. B (insulin)-cells expressed immunoreactivity to all region-specific antibodies. The antibodies to the N-terminal and mid-portions of CgB showed moderate immunoreactivity, the C-terminal antibodies weak. A (glucagon)-cells were reactive only to the N-terminal and mid-portion antibodies but, after microwave pretreatment, to all antibodies, whereas D (somatostatin)-cells expressed only the sequence CgB 244-255 and a subpopulation CgB 580-595. PP (pancreatic polypeptide) cells were immunostained with antibodies between CgB 1-417 and a few with CgB 580-593. The fragment CgB 244-255 was expressed in all four cell types. The cause of these differences may be cell-specific cleavage or masking of the molecule, but varying translation of CgB mRNA is also possible. The extent to which these epitopes reflect fragments having biological functions remains to be evaluated.     

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.