Localization of wheat-germ agglutinin-binding sites in the Golgi complex of cultured rat atrial myocytes

Summary In the Golgi region of cultured rat atrial myocytes, condensed secretory protein was seen in Golgi-associated tubules or cisternae which lay beyond, and often separated from, the remainder of the Golgi stacks. These structures appeared to be involved in packaging of condensed secretory protein into atrial granules. Binding sites of HRP-conjugated wheat-germ agglutinin (WGA) in saponin-treated cultured atrial myocytes were examined by electron microscopy with special reference to atrial granules and the tubular structures associated with the Golgi stacks. HRP reaction products were observed in both trans-cisternae of the Golgi stacks and the associated tubular structures. While the majority of atrial granules were devoid of reaction products, some granules, which were connected to the WGA-positive tubular structures in the vicinity of the Golgi trans-cisternae, showed HRP reaction products at their connected necks. Similar results were obtained when sections of the cells embedded in Lowicryl K4M were labeled with WGA coupled to colloidal gold (G-WGA); the Golgi complex was G-WGA positive, whereas no specific binding of G-WGA to atrial granules was observed. These results suggest that glycoproteins and/or glycolipids with oligosaccharides recognized by WGA in the Golgi transcisternae, may be separated from atrial natriuretic peptides which are packaged into atrial granules.Abbreviations ANP atrial natriuretic peptide - HRP horseradish peroxidase - M199 medium 199 - TGN trans-Golgi network - WGA wheat-germ agglutinin - G-WGA WGA coupled to colloidal gold     

Glucose Transporter (GLUT-4) Is Targeted to Secretory Granules in Rat Atrial Cardiomyocytes

The insulin-responsive glucose transporter GLUT-4 is found in muscle and fat cells in the transGolgi reticulum (TGR) and in an intracellular tubulovesicular compartment, from where it undergoes insulindependent movement to the cell surface. To examine the relationship between these GLUT-4–containing compartments and the regulated secretory pathway we have localized GLUT-4 in atrial cardiomyocytes. This cell type secretes an antihypertensive hormone, referred to as the atrial natriuretic factor (ANF), in response to elevated blood pressure. We show that GLUT-4 is targeted in the atrial cell to the TGR and a tubulo-vesicular compartment, which is morphologically and functionally indistinguishable from the intracellular GLUT-4 compartment found in other types of myocytes and in fat cells, and in addition to the ANF secretory granules. Forming ANF granules are present throughout all Golgi cisternae but only become GLUT4 positive in the TGR. The inability of cyclohexamide treatment to effect the TGR localization of GLUT-4 indicates that GLUT-4 enters the ANF secretory granules at the TGR via the recycling pathway and not via the biosynthetic pathway. These data suggest that a large proportion of GLUT-4 must recycle via the TGR in insulin-sensitive cells. It will be important to determine if this is the pathway by which the insulin-regulatable tubulo-vesicular compartment is formed.     

Age-related structural and functional characteristics of cardiac myoendocrine cells of rats in a normal state and with hereditary hypertension

A qualitative and quantitative ultrastructural study of right atrial cardiomyocytes in WAG (normotensive control) and ISIAH (inherited stress-induced arterial hypertension) rats of different age (on day 18 of embryogenesis, on days 12 and 21 after birth, and at an age of 6 and 13 months) was performed. It was shown that, in embryos with an as yet incomplete atrial morphogenesis, secretory granules containing natriuretic peptides are actively formed, accumulated, and dissolved. In postnatal ontogeny, the secretory product is accumulated in atrial cells. In all ontogeny stages studied, the numerical density of secretory granules in the myoendocrine cells of hypertensive rats is greater and the qualitative composition of these granules is more diverse than in the control. It was established that, in atrial myocytes of ISIAH rats, the morphological signs of natriuretic peptide hypersecretion precede the development of genetically programmed high blood pressure. In adult hypertensive rats, hypertrophic and degenerative changes in myocytes are accompanied by excessive accumulation of secretory granules, some of which undergo intracellular degradation.     

Immuno-electron microscopy of formation,degradation and exocytosis of the ovulation neurohormone in Lymnaea stagnalis

Summary The cerebral caudodorsal cells (CDC) of the pulmonate snail Lymnaea stagnalis are involved in the control of egg laying and associated behaviour by releasing various peptides. One of these is the ovulation hormone (CDCH). The cellular dynamics of this peptide have been studied using an antiserum raised to a synthetic portion of CDCH comprising the 20–36 amino acid sequence. With the secondary antibody-immunogold technique, specific immunoreactivity was found in all CDC. Rough endoplasmic reticulum and Golgi apparatus showed very little reactivity as did secretory granules that were in the process of being budded off from the Golgi apparatus. However, secretory granules that were being discharged from the Golgi apparatus, were strongly reactive. Secretory granules within lysosomal structures revealed various degrees of immunoreactivity, indicating their graded breakdown. Large electrondense granules, formed by the Golgi apparatus and thought to be involved in intracellular degradation of secretory material, were only slightly reactive. In the axon terminals secretory granules released their contents into the haemolymph by the process of exocytosis. The exteriorized contents were in most cases clearly immunopositive.The possibility has been discussed that CDCH is cleaved from its polypeptide precursor within secretory granules during granule discharge from the Golgi apparatus; subsequently, the mature secretory granules would be transported towards the neurohaemal axon terminals where they release CDCH into the haemolymph. Superfluous secretory material would be degraded by the lysosomal system including the large electron-dense granules.     

Functional Golgi units in microtubule-disrupted cultured atrial myocytes.

We examined the effects of disassembly of microtubules (MT) on the structure and the functions of the Golgi apparatus (GA) in cultured atrial myocytes. MT disassembly with nocodazole led to fragmentation of the GA into small units. The fragmented Golgi units retained their cis-trans polarity and post-cisternal elements, including the trans-Golgi network (TGN). Neither endocytosis of lectin-labeled membrane nor its delivery to the fragmented Golgi units was interrupted by fragmentation of the GA after MT disassembly with nocodazole treatment. A fraction of the secretory granules associated with the fragmented Golgi units was also labeled with the internalized tracer. These results suggest that in nocodazole-treated cultured atrial myocytes, the fragmented Golgi units appear to be structurally and functionally intact despite the altered geometric arrangement of the GA in the cells.     

Ultrastructure of atrial and ventricular myocytes of newborn rats: evidence for the existence of specific atrial granule-like organelles in the ventricle

The present study examined the ultrastructure of atrial and ventricular myocytes from the heart of newborn rats. It was found that, despite former reports stating that ventricular myocytes in adults do not contain cytoplasmic granules, specific atrial granule-like organelles are present in the ventricles of rats at birth. The presence of these granules together with the relatively underdeveloped contractile apparatus and extensive Golgi complex suggests that the ventricular, like the atrial, myocytes may have an endocrine function before or at birth. Further study is required to determine whether these ventricular cytoplasmic granules contain the same atrial natriuretic peptide species known to be present in the atrial specific granules.     

Immuno-electron microscopy of atrial natriuretic factor secretory pathways in atria and ventricles of control and cardiomyopathic hamsters with heart failure

Summary The secretory pathways of atrial natriuretic factor have been investigated in atrial and ventricular cardiocytes of control and cardiomyopathic Syrian hamsters in severe congestive heart failure with four antibodies: a monoclonal antibody (2H2) against rat synthetic atrial natriuretic factor (101–126), which is directed against region 101–103 of rat atrial natriuretic factor (99–126), and polyclonal, affinity-purified antibodies produced in rabbits against synthetic C-terminal atrial natriuretic factor (101–126), synthetic N-terminal atrial natriuretic factor (11–37) or the putative cleavage site of atrial natriuretic factor (98–99): atrial natriuretic factor (94–103). Application of the immunogold technique on thin frozen sections (immunocryoultramicrotomy) revealed an identical picture with the four antibodies. In atria of both control and cardiomyopathic hamsters where atrial natriuretic factor secretion is regulated, the atrial natriuretic factor propeptide travels, uncleaved, from the Golgi complex to immature and mature secretory granules. In ventricles of control hamsters, where secretion is constitutive, the atrial natriuretic factor propeptide travels from the Golgi complex to secretory vesicles. In the ventricles of hamsters with severe congestive heart failure, the Golgi complex is larger, secretory vesicles more abundant and a few secretory granules are present in 20% of cardiocytes. Here again, the peptide travels uncleaved in all these pathways. These results reveal the pathways of secretion of atrial natriuretic factor in atrial and ventricular cardiocytes and indicate that the propeptide is not cleaved intracellularly.Supported by a grant from the Medical Research Council of Canada to the Multidisciplinary Research Group on Hypertension, by the Canadian Heart Foundation and the Pfizer Company (England)     

Human fetal ventricular cardiomyocytes in vitro: proliferation and differentiation

In this study, in the primary cell culture of human fetal cardiomyocytes proliferation of myocytes combines with their differentiation. The cells were isolated enzymatically from 19-22 week-old human fetuses and cultured for 14 days. DNA synthesis, ultrastructure and presence of atrial natriuretic peptide (ANP) were examined. In 7 day-old culture, the myocytes make about 60%, in 14 day-old culture--about 50%. Myocytes synthesize DNA and divide mitotically. After a 24 h incubation with 3H-thymidine in 7 day-old culture 1.8 +/- 0.5% of muscle and 25.2 +/- 11.7% of non-muscle cells are labeled, in 14 day-old culture--2.5 +/- 0.5 and 8.1 +/- 1.7% of cells are labeled, respectively. In 7 and 14 day-old cultures the degree of redifferentiation of contractile apparatus in myocytes varies from scattered actin and myosin filaments surrounded by ribosomes to differentiating myofibrils with distinct sarcomeres and Z-discs. Single electron-dense granules, morphologically similar to secretory atrial granules, display ANP-immunoreactivity. Thus, human fetal ventricular cardiomyocytes in cell culture proliferate, differentiate and synthesize ANP for 14 days; this is indicative of vitality of these cells.     

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.     

Quantitative aspects of membrane shifts in rat parathyroid cells initiated by decrease in serum calcium

The secretory activity of parathyroid glands in rats was stimulated by decreasing the serum Ca++ concentration through constant intravenous infusion of EGTA. The morphometric analysis of the nuclear and cytoplasmic volume and of the surface area of the rough endoplasmic reticulum, Golgi complex, secretory granules and plasma membrane revealed a membrane shift from secretory granules and Golgi complex to the plasma membrane within 1 hr of calcium depression. Subsequently, between 1 and 3 hr of calcium depression, the membrane shift was from the plasma membrane to the Golgi complex. It is considered likely that these membrane shifts are related to a rise in release of parathyroid hormone by exocytosis and a subsequent increase in retrieval of plasma membrane by endocytosis—probably through the compartment of coated pits and coated and uncoated vesicles.     

Quantitative analyses of atrial myoendocrine cells and plasma atrial natriuretic peptides (ANP) of the rat with special reference to the twenty-four-hour variations in secretory granules and plasma ANP concentrations

Summary Subcellular structures of atrial myoendocrine cells in the rat heart and plasma concentrations of atrial natriuretic peptides (ANP) were examined at six evenlyspaced time points over 24 h, using morphometric techniques and radioimmunoassay.Myofibrils and mitochondria of the cells occupied 73.3% of the cytoplasm; 2% of the cytoplasm was occupied by secretory granules, rough endoplasmic reticulum and Golgi complexes, structures characteristic of endocrine cells. Plasma ANP concentration was maximal at 08.00 h, when the individual volume of secretory granules was minimal. The numerical density of secretory granules was increased at 12.00 h. The plasma ANP concentration was minimal at 20.00 h, when the numerical density was minimal and the individual volume was maximal. The fluctuation in plasma ANP concentrations over 24 h was thus parallel to that in the numerical densities of secretory granules and inverse to that in individual volumes.These results suggest that in rats the secretory activity of atrial myoendocrine cells increases at the beginning of the resting period, whereas it decreases at the beginning of the active phase.     

Novel structures in secreting the androgenic gland hormone

The secretory granules in the androgenic gland of the terrestrial isopod Armadillidium vulgare, which have been indistinct for long time because of vulnerable structures, were revealed by using the rapid-freezing and freeze-substitution method. The fine structure of the androgenic gland is conspicuous by the distribution of numerous particular organelles in the cytoplasm consisting of the endoplasmic reticulum and the Golgi complex, and by having a number of highly organized structures developed between the androgenic gland cells. The structures connect to the intercellular space, which is seen as intercellular canaliculi for exporting the androgenic gland hormone. The plasma membranes near the particular structure of the intercellular canaliculi in the androgenic gland are often specialized to form cellular junctions. The secretory granules including the electron-dense materials, which are supposed to be peptides of androgenic gland hormone, are distributed beside the particular structure of the intercellular canaliculi. Some of the granules are seen to fuse with the plasma membranes. This observation suggests that, in the Armadillidium vulgare, the secretory granules containing androgenic gland hormone are transferred to the extracellular space through the intercellular canaliculi particularly developed for exporting the peptide hormone. This is the first evidence to show the secretory mechanism of the androgenic gland hormone in the Isopoda.     

Etude histologique,histochimique et ultrastructurale de la pars intercerebralis chez Locusta migratoria L. (Orthoptere)

Summary A histological, histochemical and ultrastrucutral study of the pars intercerebralis (PI) has been made in Locusta migratoria. The acellular neural lamella is made up of an elastic tissue and collagen fibrils. The cells of the perilemma contain numerous lysosome structures and lipid granules.Three different types of neurosecretory cells (NSC A, B and C) have been distinguished in the PI associated with giant neurons.The cells termed A and B seem not to have an activity cycle during the two last larval instars. At the moment of sexual maturity the NSC A show an important accumulation of neurosecretory material and their number increases at the expense of the NSC B. The NSC A, which are characterized by a highly developped endoplasmic reticulum, contain numerous secretory granules which appear to be individualized in the Golgi complex in three different ways. The NSC B, with a reduced endoplasmic reticulum and an almost quiescent Golgi complex, contain abundant lysosome structures and more seldom some neurosecretory granules. In fact, the study of the fine structure shows different intermediate types, linking in a continuous way typical A cells and typical B cells. NSC A and NSC B might correspond to two opposed stages of secretory activity of one single cell type: the A cell representing the activity stage and the B cell the quiescent stage.NSC C show an accumulation of their neurosecretory products in relation to metamorphosis and sexual maturity. Ultrastructural evidence confirms their neurosecretory activity.A mode of regulating neurosecretion in NSC A and B by internal catabolism of the secretion and formation of lysosome like structures is discussed in the present paper.The giant neurons, which are surrounded by a glial envelope (trophospongium), contain several dense granules originated from Golgi complex.     

Atrial natriuretic factor in the vena cava and sinus node

We investigated the localization of atrial natriuretic factor (ANF) mRNA and of immunoreactive ANF in the vena cava and sinus node of rat and, for comparative purposes, in atria and ventricles. In situ hybridization with an ANF cRNA probe revealed that the supradiaphragmatic portion of the inferior vena cava contains almost as much mRNA as the atria, whereas the levels were less in the superior vena cava and higher than in ventricles in the sinus node. Immunoreactive ANF (high Mr form) was found to be 22 times less abundant in the supradiaphragmatic vena cava and 148 times less abundant in the superior vena cava than in atrial cardiocytes. The wall of the supradiaphragmatic portion of the vena cava and the valve (eustachian valve) that separates the atrial cavity from that of the vein are made up of atrial-like cardiocytes containing secretory granules. The subendothelial area of the superior vena cava also contains atrial-like cardiocytes with secretory granules, whereas the outer portion of the vein is made up of "transitional cells" without or with only a few secretory granules. Secretory granules in the vena cava and nodal cells, as well as transitional cells, contain immunoreactive ANF. With immunocryoultramicrotomy, virtually all cells, whether atrial-like, transitional, or nodal, and even those without secretory granules, were found to contain immunoreactive ANF in their Golgi complex and in secretory vesicles in the vena cava and in the sinus node.     

Studies on secretory activity in the pars intermedia of Xenopus laevis 1: Fine structural changes related to the onset of secretory activity in vivo

Changes in the fine structural organization of the pars intermedia related to the onset of secretory activity within the gland have been studied. It is shown that during the first seven days, following the onset of secretory activity, there is an extensive membraneogenesis within the cytoplasm of the parenchymatous cells of the pars intermedia which results in the formation of a well-organized array of endoplasmic reticulum and an increased development of the Golgi complex. Simultaneously the large population of secretory granules present in the cells in the inactive condition is reduced, the granules fusing with the plasma membrane and releasing their contents into the extracellular space. During the process of intracellular reorganization, in addition to the elaboration of those components of the Golgi complex which are believed to be concerned with the formation of the secretory granules, a second distinctive cisternal element develops within the Golgi area. This component which remains confined to the Golgi area for only a short time (days 2-4) appears to be responsible for the production of membrane-bound dense bodies with a finely granular content. The dense bodies, in turn, become transformed into the larger heterogeneous structures which are a prominent feature of actively secreting pars intermedia cells.     

Golgi apparatus, GERL, and secretory granule formation within neurons of the hypothalamo-neurohypophysial system of control and hyperosmotically stressed mice

The vasopressin-producing neurons of the hypothalamo-neurohypophysial system are a particularly good model with which to consider the relationship between the Golgi apparatus nd GERL and their roles in secretory granule production because these neurons increase their synthesis and secretion of vasopressin in response to hyperosmotic stress. Enzyme cytochemical techniques for acid phosphatase (AcPase) and thiamine pyrophosphatase (TPPase) activities were used to distinguish GERL from the Golgi apparatus in cell bodies of the supraoptic nucleus from normal mice, mice hyperosmotically stressed by drinking 2% salt water, and mice allowed to recover for 5-10 d from hyperosmotic stress. In nonincubated preparations of control supraoptic perikarya, immature secretory granules at the trans face of the Golgi apparatus were frequently attached to a narrow, smooth membrane cisterna identified as GERL. Secretory granules were occasionally seen attached to Golgi saccules. TPPase activity was present in one or two of the trans Golgi saccules; AcPase activity appeared in GERL and attached immature secretory granules, rarely in the trans Golgi saccules, and in secondary lysosomes. As a result of hyperosmotic stress, the Golgi apparatus hypertrophied, and secretory granules formed from all Golgi saccules and GERL. Little or no AcPase activity could be demonstrated in GERL, whereas all Golgi saccules and GERL-like cisternae were TPPase positive. During recovery, AcPase activity in GERL returned to normal; however, the elevated TPPase activity and secretory granule formation seen in GERL-like cisternae and all Golgi saccules during hyperosmotic stress persisted. These results suggest that under normal conditions GERL is the predominant site for the secretory granule formation, but during hyperosmotic stress, the Golgi saccules assume increased importance in this function. The observed cytochemical modulations in Golgi saccules and GERL suggest that GERL is structurally and functionally related to the Golgi saccules.     

The formation of glandular secretion in larval Austramphilina elongata (Amphilinidea)

The ultrastructure of three types of gland cells of embryos and free-swimming larvae of Austramphilina elongata is described. Type I gland cells contain large, more or less round electron-dense granules which are formed by numerous Golgi complexes. Type II gland cells contain thread-like, membrane-bound secretory granules with longitudinally arranged microtubules inside the granules; secretory droplets are produced by Golgi complexes and the microtubules apparently condense in the cytoplasm or in the droplets. Type III gland cells contain irregular-ovoid membrane-bound granules with coiled up microtubules which have an electron-dense core; the granules are formed by secretionderived from Golgi complexes and the microtubules aggregate around and migrate into the secretion; microtubules are at first hollow and the early secretory granules have a central electron-dense region.     

Sorting of three secretory proteins to distinct secretory granules in acidophilic cells of cow anterior pituitary

The distribution of three proteins discharged by regulated exocytosis--growth hormone (GH), prolactin (PRL), and secretogranin II (SgII)--was investigated by double immunolabeling of ultrathin frozen sections in the acidophilic cells of the bovine pituitary. In mammotrophs, heavy PRL labeling was observed over secretory granule matrices (including the immature matrices at the trans Golgi surface) and also over Golgi cisternae. In contrast, in somatotrophs heavy GH labeling was restricted to the granule matrices; vesicles and tubules at the trans Golgi region showed some and the Golgi cisternae only sparse labeling. All somatotrophs and mammotrophs were heavily positive for GH and PRL, respectively, and were found to contain small amounts of the other hormone as well, which, however, was almost completely absent from granules, and was more concentrated in the Golgi complex, admixed with the predominant hormone. Mixed somatomammotrophs (approximately 26% of the acidophilic cells) were heavily positive for both GH and PRL. Although admixed within Golgi cisternae, the two hormones were stored separately within distinct granule types. A third type of granule was found to contain SgII. Spillage of small amounts of each of the three secretory proteins into granules containing predominantly another protein was common, but true intermixing (i.e., coexistence within single granules of comparable amounts of two proteins) was very rare. It is concluded that in the regulated pathway of acidophilic pituitary, cell mechanisms exist that cause sorting of the three secretory proteins investigated. Such mechanisms operate beyond the Golgi cisternae, possibly at the sites where condensation of secretion products into granule matrices takes place.     

Delivery of lectin-labeled membrane to the trans-Golgi network and secretory granules in cultured atrial myocytes

To examine whether and how internalized plasma membrane components are routed to the compartment of the biosynthetic-exocytic pathway in cultured atrial myocytes, the plasma membrane labeled with wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) was traced electron microscopically by cytochemical detection of HRP. The WGA-HRP label was internalized via a coated pit-small vesicle pathway and reached vacuoles and endosomes by 3 min. Labeled endosomes comprised vacuoles and tubular elements containing reaction product. By 15 min, similar tubular structures containing reaction product accumulated in the area of the trans-Golgi network (TGN). The labeled TGN consisted of interconnected tubular elements, which often connected to atrial granules containing reaction product. In contrast, neither native HRP nor Lucifer Yellow reached Golgi elements or atrial granules. These results suggest that a proportion of the plasma membrane labeled with WGA-HRP is delivered to endosomes, from which tubules might bud off to transfer the tracer molecules to the TGN, where the lectin conjugate and associated membranes are packaged into atrial granules.     

Clathrin-coated vesicular transport of secretory proteins during the formation of ACTH-containing secretory granules in AtT20 cells

We have studied by electron microscopy and immunocytochemistry the formation of secretory granules containing adrenocorticotropic hormone (ACTH) in murine pituitary cells of the AtT20 line. The first compartment in which condensed secretory protein appears is a complex reticular network at the extreme trans side of the Golgi stacks beyond the TPPase-positive cisternae. Condensed secretory protein accumulates in dilated regions of this trans Golgi network. Examination of en face and serial sections revealed that "condensing vacuoles" are in fact dilations of the trans Golgi network and not detached vacuoles. Only after presumptive secretory granules have reached an advanced stage of morphological maturation do they detach from the trans Golgi network. Frequently both the dilations of the trans Golgi network containing condensing secretory protein and the detached immature granules in the peri-Golgi region have surface coats which were identified as clathrin by immunocytochemistry. Moreover both are the site of budding (or fusion) of coated vesicles, some of which contain condensed secretory protein. The mature granules below the plasma membrane do not, however, have surface coats. Immunoperoxidase labeling with an antiserum specific for ACTH and its precursor polypeptide confirmed that many of the coated vesicles associated with the trans Golgi network contain ACTH. The involvement of the trans Golgi network and coated vesicles in the formation of secretory granules is discussed.