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.     

Regulated and constitutive secretion. Differential effects of protein synthesis arrest on transport of glycosaminoglycan chains to the two secretory pathways.

Many neural and endocrine cells possess two pathways of secretion: a regulated pathway and a constitutive pathway. Peptide hormones are stored in granules which undergo regulated release whereas other surface-bound proteins are externalized constitutively via a distinct set of vesicles. An important issue is whether proper function of these pathways requires continuous protein synthesis. Wieland et al. (Wieland, F.T., Gleason, M.L., Serafini, T.A., and Rothman, J.E. (1987) Cell 50, 289-300) have shown that a tripeptide containing the sequence Asn-Tyr-Thr can be glycosylated in intracellular compartments and secreted efficiently from Chinese hamster ovary and HepG2 cells, presumably via the constitutive secretory pathway. Secretion is not affected by cycloheximide, suggesting that operation of this pathway does not require components supplied by new protein synthesis. In this report we determined the effects of protein synthesis inhibitor on membrane traffic to the regulated secretory pathway in the mouse pituitary AtT-20 cells. We examined transport of glycosaminoglycan chains since previous studies have shown that these chains enter the regulated secretory pathways and are packaged along with the hormone adrenocorticotropin (ACTH). We found that cycloheximide treatment severely impairs the cell's ability to store and secrete glycosaminoglycan chains by the regulated secretory pathway. In marked contrast, constitutive secretion of glycosaminoglycan chains remains unhindered in the absence of protein synthesis. The differential requirements for protein synthesis indicate differences in the mechanisms for sorting and/or transport of molecules through the constitutive and the regulated secretory pathways. We discuss the possible mechanisms by which protein synthesis may influence trafficking of glycosaminoglycan chains to the regulated secretory pathway.     

On the shoulders of giants: the discovery of atrial natriuretic factor

Investigations culminating at the beginning of this century clearly established that the cardiac muscle cell (cardiocytes) is differentiated for excitation, conduction, and contraction. All of the physiology and pathophysiology of the heart was developed subsequently based on this concept. However, morphological investigations in the mid 1950s suggested a secretory function for mammalian atrial cardiocytes. These cells contain storage granules, the specific atrial granules, which resemble granules found in polypeptide hormone-producing cells. The development of techniques for the study of these granules using a combined biochemical-morphological approach during the 1970s defined their general chemical nature and their behaviour under different experimental conditions. Because the number of atrial granules change dramatically following upsets of water and electrolyte balance, atrial muscle extracts were tested for effects on kidney function. In 1981, it was reported that atrial extracts contain a natriuretic factor (ANF) capable of inducing massive diuresis, increases in hematocrit, and lowering of blood pressure. It was demonstrated soon thereafter that ANF is stored within specific atrial granules. More recent work has defined ANF as a polypeptide hormone that appears to modulate or antagonize the renin-angiotensin-aldosterone system. Current work attempts to define the physiological and pathophysiological role for ANF as well as possible therapeutic uses.     

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.     

Chicken atrial natriuretic peptide (chANP) and its secretion

Summary An immunohistochemical study using antiserum raised against synthetic chicken natriuretic polypeptide was used to investigate the distribution of this peptide in the chicken heart. Immunoreactive cells, both in the atrial and ventricular walls, were identified by electron microscopy, and electron-dense granules in the atrial and ventricular cardiocytes were revealed to be storage sites of the peptide. The electron-dense material, thought to be the peptide, was found in the sarcoplasmic reticulum, and it is suggested that a secretory pathway of the peptide through the latter to extracellular space, may be present, in addition to an exocytotic one.     

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.     

Expression of atrial natriuretic factor gene in heart ventricular tissue

A novel peptide hormone, atrial natriuretic factor (ANF), was recently isolated and characterized in mammalian atria. This hormone has potent natriuretic, diuretic and vasorelaxant activities. Since ANF bioactivity was initially found in atria but not in ventricles, it was assumed that the ANF gene is specifically expressed in atria. We now report that ANF mRNA is present in ventricular tissue as well as in atria. This is clearly demonstrated by in situ hybridization and by Northern blot analysis. Rat ventricular ANF mRNA concentration is a hundred-fold lower than in atria. As in atria, the 126 amino acids precursor form of ANF is predominant in ventricles and it is present at a thousand-fold lower concentration. The ten-fold discrepancy in the ratio of ANF mRNA to immunoreactivity between atria and ventricles could reflect a higher rate of peptide release in the latter. Thus, ventricular ANF production may be physiologically significant in view of the much larger ventricular mass.     

Capsaicin-sensitive nerves influence the release of atrial natriuretic factor by atrial stretch in the rat.

Although many factors may modulate the release of atrial natriuretic factor (ANF), the primary mechanism has been demonstrated to be atrial stretch. Recent studies have led to the suggestion that the peptidergic innervation of the heart, through the release of peptides, may be involved in the control of ANF secretion. We have examined the influence of chronic capsaicin treatment on three models of atrial stretch that release ANF. This treatment inhibited ANF released through in vivo blood volume expansion and through balloon inflation in the right atrium of in vitro isolated perfused hearts. Immunohistochemical and electron microscopical analysis confirmed the absence of innervation of the heart by calcitonin gene related peptide and substance P immunoreactive nerve fibres and apparent lack of effect on atrial granules in capsaicin treated rats. We conclude that capsaicin-sensitive cardiac innervation is a component modulating the release of ANF, stimulated by atrial stretch in the rat.     

Atrial natriuretic factor (ANF) gene expression in the Brattleboro rat

Atrial natriuretic factor (ANF) is a 28-amino acid peptide hormone of cardiac origin. It has natriuretic, diuretic and vasorelaxant properties and inhibits several cardiovascular modulators. Because of the possible effects of arginine vasopressin (AVP) on ANF secretion, we have investigated ANF gene expression in Brattleboro rats which are genetically deficient in AVP. Our results indicate that cardiac ANF mRNA and ANF content are higher in Brattleboro rats compared to Long-Evans controls, whereas the plasma levels are similar in both groups. Typical secretory granules containing immunoreactive ANF are present in ventricular cardiocytes of Brattleboro but not of Long-Evans rats. These data suggest that ANF release may be uncoupled from its synthesis in the absence of AVP.     

The secretion of atrial natriuretic factor-(99-126) by cultured cardiac myocytes is regulated by glucocorticoids

Atrial natriuretic factor (ANF) is stored in mammalian atria primarily as ANF-(1-126), the precursor to the known circulating form of the hormone ANF-(99-126). When primary cultures of atrial myocytes were maintained in a complete serum-free medium, they contained and secreted an ANF-(1-126)-like peptide. The addition of dexamethasone to the culture medium, however, resulted in the secretion of a molecule with chromatographic characteristics identical to ANF-(99-126), although the intracellular storage form of ANF was unchanged. Radiosequencing and amino acid analysis confirmed that the cultures maintained in dexamethasone secreted authentic ANF-(99-126). Chronic exposure of the cells to dexamethasone also resulted in a significant increase in the quantity of immunoreactive ANF both contained and secreted by the cultures. Dexamethasone stimulated ANF processing and secretion by atrial cultures in a dose-dependent manner, with an approximate EC50 of 10 nM. This stimulation could be reversed by removing the glucocorticoid from the culture medium. ANF processing was also stimulated by the specific glucocorticoid receptor agonist RU 28362, and both DEX- and RU 28362-stimulated ANF processing was inhibited by the specific glucocorticoid receptor antagonist RU 38486. Ventricular cells, which possess few granules and release ANF in a constitutive fashion, were also capable of processing ANF in a glucocorticoid-dependent fashion. Medium freshly removed from atrial cultures did not convert ANF-(1-126) to ANF-(99-126) nor was exogenous ANF-(1-126) efficiently processed when added to the medium of actively processing cultures. These results indicate that the post-translational processing of ANF-(1-126) to ANF-(99-126) likely occurs within or in close association with the cardiac myocytes and is not dependent on the presence of large quantities of secretory granules. Furthermore, it is apparent that both the expression and the post-translational processing of ANF by cultured cardiac myocytes is specifically regulated by glucocorticoids.     

In vitro mutagenesis of trypsinogen: role of the amino terminus in intracellular protein targeting to secretory granules

The mouse anterior pituitary tumor cell line, AtT-20, targets secretory proteins into two distinct intracellular pathways. When the DNA that encodes trypsinogen is introduced into AtT-20 cells, the protein is sorted into the regulated secretory pathway as efficiently as the endogenous peptide hormone ACTH. In this study we have used double-label immunoelectron microscopy to demonstrate that trypsinogen colocalizes in the same secretory granules as ACTH. In vitro mutagenesis was used to test whether the information for targeting trypsinogen to the secretory granules resides at the amino (NH2) terminus of the protein. Mutations were made in the DNA that encodes trypsinogen, and the mutant proteins were expressed in AtT-20 cells to determine whether intracellular targeting could be altered. Replacing the trypsinogen signal peptide with that of the kappa-immunoglobulin light chain, a constitutively secreted protein, does not alter targeting to the regulated secretory pathway. In addition, deletion of the NH2-terminal "pro" sequence of trypsinogen has virtually no effect on protein targeting. However, this deletion does affect the signal peptidase cleavage site, and as a result the enzymatic activity of the truncated trypsin protein is abolished. We conclude that neither the signal peptide nor the 12 NH2-terminal amino acids of trypsinogen are essential for sorting to the regulated secretory pathway of AtT-20 cells.     

Microtubule-associated distribution of specific granules and secretion of atrial natriuretic factor in primary cultures of rat cardiomyocytes

A close spatial relationship between specific granules containing atrial natriuretic factor (ANF) and microtubules was demonstrated in primary cultures of neonatal rat cardiac myocytes. For the detection of specific granules and microtubules, the myocytes were double immunolabelled with antibodies against -ANF and -tubulin and examined by conventional fluorescence or laser scanning confocal microscopy. In addition, the ultrastructural distribution of specific granules was demonstrated by electron microscopy. In the atrial myocytes, ANF was stored in numerous specific granules that were mainly localized in the perinuclear sarcoplasm. In the ventricular myocytes, however, a minority of the cells (10%) exhibited limited ANF immunoreactivity after 4 days in culture. Microtubules were present throughout the sarcoplasm of the myocytes. They were most densely packed in the perinuclear regions. Depolymerization of the microtubules with nocodazole was followed by dispersal of ANF immunostaining both in the atrial myocytes and in the ventricular myocytes exhibiting ANF immunoreactivity. When the microtubules were allowed to recover, the perinuclear distribution of specific granules, as seen in non-treated myocytes, reappeared. Measurements of secreted immunoreactive ANF by radioimmunoassay revealed that the secretion of ANF from atrial myocytes into the medium was significantly reduced following nocodazole treatment, whereas a similar decrease in secretion from ventricular myocytes was not observed. These findings indicate that ANF-containing specific granules are closely associated with microtubules within the myocytes. It is suggested that secretion of ANF from the atrial myocytes, in contrast to the ventricular myocytes, is microtubule-dependent.     

Ventricular and atrial myocytes of newborn rats synthesise and secrete atrial natriuretic peptide in culture: Light-and electron-microscopical localisation and chromatographic examination of stored and secreted molecular forms

Summary We have demonstrated that atrial natriuretic peptide-like immunoreactivity is stored and secreted by ventricular and atrial myocytes in dissociated cell culture preparations from the heart of newborn rat. Culture preparations were maintained in either foetal calf serum-supplemented medium 199 or in hormone-supplemented, serum-free medium 199. The presence of atrial natriuretic peptidelike immunoreactivity in the cultured myocytes was demonstrated at both light-and electron-microscopical levels. Release of atrial natriuretic peptide-like immunoreactivity into the culture medium was measured by radioimmunoassay; molecular forms of the stored and secreted peptide were determined by gel column chromatography. The atrial natriuretic peptide-like immunoreactivity of cultured atrial and ventricular myocytes was concentrated in the perinuclear cytoplasm and was localised to electron-dense secretory granules. The number of immunoreactive ventricular myocytes and the intensity of their immunofluorescence changed with time in culture and was higher in cultures in foetal calf serum-supplemented medium than in serum-free medium. Gamma-atrial natriuretic peptide was stored and released by cultured atrial and ventricular myocytes, but was broken down to alpha-atrial natriuretic peptide in the growth medium. This process was foetal calf serum-independent, since it occurred in both the media used, indicating that cardiac myocytes in culture may release a factor that cleaves gamma-atrial natriuretic peptide to form alphaatrial natriuretic peptide.     

Novel salmon cardiac peptide hormone is released from the ventricle by regulated secretory pathway

We used the secretion of the novel salmon cardiac peptide (sCP) as a model to examine the mechanisms of ventricular hormone release. Mechanical load increased dose dependently the secretion of immunoreactive sCP from isolated perfused salmon ventricle, with 3. 3-fold increase when a load of 13 cmH(2)O was applied. Endothelin-1 (5 nmol/l) was also able to rapidly increase the secretion of sCP. The released peptide corresponded to the biologically active sCP-29, whereas the large ventricular storage consisted of pro-sCP-sized material. With the use of immunoelectron microscopy, a large number of granules containing immunoreactive sCP could be detected in salmon ventricle. As judged by RNA blot analysis, there was very active basal expression of the sCP gene in the ventricle, which was not increased by mechanical load of up to 2-h duration. Our results show that the ventricle actively expresses the gene of sCP, stores the prohormone in secretory granules, and releases the peptide in response to mechanical load and endothelin-1. Thus the salmon ventricle uses the regulated pathway to produce and release a hormone structurally related to the mammalian natriuretic peptides.     

Rat pro-atrial natriuretic factor expression and post-translational processing in mouse corticotropic pituitary tumor cells

Atrial natriuretic factor (ANF) is stored within atrial myocyte secretory granules as pro-ANF (ANF-(1-126] and is proteolytically processed co-secretionally C-terminal to a single basic amino acid to form ANF-(1-98) and the bioactive product ANF-(99-126). Pro-ANF is also expressed in certain non-cardiac neuroendocrine cell types (e.g. brain, adrenal). Although the relatively low levels of the peptide in these cell types have precluded detailed processing and secretion studies using cultured cells, some work with tissue extracts suggests that pro-ANF is pre-secretionally processed between or C-terminal to Arg101-Arg102 in such cells. In order to assess whether cultured non-cardiac endocrine cells process pro-ANF pre- or co-secretionally, and to establish whether both paired and single basic amino acids can serve as cleavage sites, transfection studies were carried out using the adrenocorticotropic hormone (ACTH)-producing pituitary tumor cell line AtT-20/D-16v. These cells normally cleave pro-ACTH/endorphin pre-secretionally at selected, but not all, pairs of basic amino acids to a variety of product peptides. A prepro-ANF expression plasmid was constructed and transfected into the AtT-20 cells. The resulting ANF/AtT-20 cell clone selected for this study expressed ACTH at levels similar to the untransfected wild type cells and secreted immunoreactive ANF-related material at a rate of approximately 1 fmol/min/10(5) cells, which was about 10% the rate of ACTH secretion. The rates of secretion of both ANF and ACTH could be increased 3-5-fold with a variety of known AtT-20 cell secretagogues including phorbol esters and the beta-adrenergic agonist, isoproterenol, thus indicating that both peptides were routed through regulated secretory pathways. Utilizing a combination of specific antisera directed against various regions of pro-ANF, size exclusion and reversed phase high performance liquid chromatography, and peptide mapping, it was shown that the ANF/AtT-20 cells contained and secreted the bioactive peptide ANF-(103-126) and -(1-97). These results indicate that the ANF/AtT-20 cells specifically cleave pro-ANF pre-secretionally at the same single basic site used by cardiac tissue; this single basic cleavage is apparently followed by removal of Arg98 by carboxypeptidase H. It is also apparent that the cells can cleave at the sole paired basic site in pro-ANF, which is the probable cleavage site used by neurons and some other endocrine cells that express low levels of the prohormone.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of native and synthetic atrial natriuretic factor on cyclic GMP

Mammalian atrial cardiocyte granules contain a potent natriuretic and diuretic peptide. Since cGMP appears to be involved in the modulation of cholinergic and toxin-induced sodium transport, we examined the effect of atrial natriuretic factor (ANF) on this nucleotide. Atrial but not ventricular extracts elicited approximately a 28-fold increase of urinary cGMP excretion parallel to the natriuresis and diuresis. The atrial extracts also elevated cGMP levels in kidney slices and primary cultures of renal tubular cells. The effect of ANF on cGMP appeared to be specific since antibodies which were capable of inhibiting the ANF-induced diuresis also suppressed cGMP excretion. Furthermore, during the course of ANF purification, the ANF-induced increase of cGMP production by kidney cells paralleled the heightened specific natriuretic activity of the atrial factor. A synthetic peptide (8-33)-ANF similarly increased urinary plasma and kidney tubular cGMP levels. The exact mechanism of action of ANF on cGMP remains to be elucidated, but indirect inhibition of cGMP phosphodiesterase appears to participate in its effect.     

A large form of secretogranin III functions as a sorting receptor for chromogranin A aggregates in PC12 cells

Granin-family proteins, including chromogranin A and secretogranin III, are sorted to the secretory granules in neuroendocrine cells. We previously demonstrated that secretogranin III binds chromogranin A and targets it to the secretory granules in pituitary corticotrope-derived AtT-20 cells. However, secretogranin III has not been identified in adrenal chromaffin and PC12 cells, where chromogranin A is correctly sorted to the secretory granules. In this study, low levels of a large and noncleaved secretogranin III have been identified in PC12 cells and rat adrenal glands. Although the secretogranin III expression was limited in PC12 cells, when the FLAG-tagged secretogranin III lacking the secretory granule membrane-binding domain was expressed excessively, hemagglutinin-tagged chromogranin A was unable to target to the secretory granules at the tips and shifted to the constitutive secretory pathway. Secretogranin III was able to bind the aggregated form of chromogranin A, suggesting that a small quantity of secretogranin III is enough to carry a large quantity of chromogranin A. Furthermore, secretogranin III bound adrenomedullin, a major peptide hormone in chromaffin cells. Indeed, small interfering RNA-directed secretogranin III depletion impaired intracellular retention of chromogranin A and adrenomedullin, suggesting that they are constitutively released to the medium. We suggest that the sorting function of secretogranin III for chromogranin A is common in PC12 and chromaffin cells as well as in other endocrine cells, and a small amount of secretogranin III is able to sort chromogranin A aggregates together with adrenomedullin to secretory granules.     

Analysis of atrial natriuretic factor biosynthesis and secretion in adult and neonatal rat atrial cardiocytes

Atrial natriuretic factor (ANF) is stored in atrial cardiocytes as the 126 amino acid polypeptide, proANF, which is later cleaved to the 24-28 amino acid carboxyterminal peptides, the major circulating forms. Earlier studies have demonstrated that isolated, cultured neonatal rat cardiocytes both store and secrete proANF, which can be cleaved to the smaller circulating form(s) by a serum protease. Since differences may exist between neonatal and adult cardiocytes with respect to ANF synthesis and processing, we compared the forms of ANF stored and secreted by neonatal rat cardiocytes with those of adult cells. Using four to five day cultures of isolated atrial cardiocytes prepared from the hearts of neonatal and adult rats, pulse-chase studies were performed with 35S-cysteine and 35S-methionine. Analysis of ANF stored and secreted by these cells was performed by immunoprecipitation of cell extracts and culture media using antibodies directed to either the carboxyterminus or aminoterminus of proANF followed by SDS-PAGE and autoradiography. Cell extracts from both adult and neonatal cultures were found to contain only a 17-kDa polypeptide, previously identified as proANF. The predominant form found in the culture media was also the 17-kDa peptide, with smaller quantities of its 3-kDa carboxyterminal and 14-kDa aminoterminal cleavage products. We conclude from these studies that proANF is the major form stored and secreted by both adult and neonatal cardiocytes in culture; the activity of the protease that cleaves proANF to the smaller forms found in the circulation is either attenuated or is overwhelmed by high ANF-secretory rates in these cultures. Alternatively, the ANF processing and secretory pathways may be somehow altered in culture such that proANF escapes protease cleavage. Further studies will elucidate the nature and location of this protease.