The granin (chromogranin/secretogranin) family

The chromogranins/secretogranins, referred to in abbreviated form as granins, are a family of acidic secretory proteins that are found in the secretory granules of a wide variety of endocrine cells and neurons, being stored together with many different peptide hormones and neuropeptides. The recent elucidation of their primary structure has provided insights into possible functions of these proteins. Moreover, the granins have been successfully used as markers for normal and neoplastic endocrine and neuronal cells, as well as model proteins to understand the sorting mechanism involved in the formation of secretory granules.     

The 1989 Upjohn Award lecture. Cellular and molecular mechanisms in hormone and neurotransmitter secretion

Studies on adrenal medulla have had an important influence on the development of a variety of biological concepts, not only within the area of endocrinology, but also in the areas of chemical neurotransmission and secretion in general. The adrenal medulla chromaffin cells are derived embryologically from the neural crest, sharing a common origin with sympathetic neurons and common subcellular features with many endocrine cells. One such feature is the storage of secretory products in membrane-bound organelles, the secretory granules. Secretory cells with these characteristics have been named paraneurons, a term that embraces cells generally and traditionally not considered as neurons, and yet should be regarded as relatives of neurons on the basis of their structure, function, and metabolism. Many of the studies carried out in the past to understand the secretory process have employed perfused adrenal glands. Although this technique has provided very useful information regarding secretion, it did not allow the study of the cellular events involved in the secretory process. To obtain further information on cell secretion, several laboratories including our own have published methods for the isolation and culture of chromaffin cells. The cultured chromaffin cells have shown themselves to be one of the most useful systems developed for the study of the neuroendocrine functions of paraneurons. Studies on cultured chromaffin cells have provided important information on secretory cell cytoskeleton: a group of proteins, some of them previously known from studies on muscle, which form a cytoplasmic network in all non-muscle cells including secretory cells. Immunohistochemical studies have shown at least three types of filament systems: microfilaments, microtubules, and intermediate filaments. In addition, a large variety of cytoskeleton-associated proteins have been characterized. Chromaffin cells are among those non-muscle cells from which cytoskeleton proteins have been isolated and characterized. Owing to similarities between "stimulus-secretion coupling" and "excitation-contraction coupling" in muscle, it has been proposed that the secretory process might be mediated by contractile elements either associated with secretory vesicles or present elsewhere in the secretory cell. Cytoskeletal proteins (actin, myosin, alpha-actinin, fodrin, tubulin, and neurofilament subunits) and their regulatory proteins (calmodulin, gelsolin) have been isolated from chromaffin cells and characterized. Their physiochemical proteins have been studied and their cellular localizations have been revealed by biochemical, immunocytochemical, and ultrastructural techniques. alpha-Actinin and fodrin are components of chromaffin granule membranes and some of the cell actin co-purified with secretory granules. Actin forms a network of microfilaments in the subplasmalemma region.(ABSTRACT TRUNCATED AT 400 WORDS)     

Molecular dissection of regulated secretory pathways in human gastric enterochromaffin-like cells: an immunohistochemical analysis

Enterochromaffin-like (ECL) cells regulate gastric acid secretion through vesicular release of histamine. Until now, the molecular machinery of human ECL cells involved in the formation and release of vesicles is largely unknown. We analyzed tissue samples obtained from normal human gastric mucosa (n=4) and ECLomas (n=5) immunohistochemically using the APAAP method or double immunofluorescence confocal laser microscopy. Human pheochromocytomas (n=5) were investigated in parallel and compared to ECL cells. Secretory pathways were characterized using antibodies specific for marker proteins of large dense-core vesicles (LDCVs; islet cell antigen 512, chromogranin A, pancreastatin, and vesicular monoamine transporter 2) and small synaptic vesicle (SSV) analogues (synaptophysin). Tissues were also analyzed for expression of the peptide hormone processing enzymes, carboxypeptidase E and prohormone convertase 1, as well as the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, 25-kDa synaptosome-associated protein (SNAP25), syntaxin, and synaptobrevin. Immunoreactivity for markers of LDCVs and SSV analogues were detected in normal ECL cells and ECLomas. Both tissues also showed expression of carboxypeptidase E and prohormone convertase 1. Analysis of vesicular SNARE (v-SNARE) and target membrane SNARE (t-SNARE) proteins revealed the presence of SNAP25, syntaxin, and synaptobrevin in normal and neoplastic ECL cells. Our data suggest that ECL cells possess the two vesicle types of regulated neuroendocrine secretory pathways, LDCVs and SSV analogues. Since ECL cells also contain typical SNARE proteins, the molecular machinery underlying secretory processes in this cell type appears to be identical to the secretory apparatus of neuroendocrine cells and neurons. In addition, our findings suggest that the secretory apparatus of ECL cells is maintained during neoplastic transformation. Accepted: 10 June 1999     

Neuroendocrine lung structures and tumours: immunohistochemical study by specific markers

Out of 360 lungs or lobes surgically removed, 13 non neoplastic specimens and 16 neuroendocrine (NE) tumours are investigated with immunohistochemical methods, in order to evaluate the presence of NE structures in normal and pathological human lungs. The markers used are neuron specific enolase (NSE), chromogranin (CG) and the 80 kd antigen (80 kdAg) of NE secretory granules detected by the new monoclonal Phe-5 antibody. In non-neoplastic lung specimens, clearcut immunoreactivity for all three markers appears in NE cells, neuroepithelial bodies (NEB), NE cell-hyperplasias and dysplasias. In the same specimens 4 tumourlets with analogous clearcut immunoreactivities were also observed. The NE tumours show distinct immunoreactivity for all three antisera in the 8 well differentiated cases. The 8 poorly differentiated tumours are variably immunoreactive for NSE and present low to nil staining with antisera to CG and 80 kdAg. The immunohistochemical data are interpreted according to current views about a possible relationship between NE tumours and parent normal NE lung structures.     

The posttranslational phase of gene expression: new possibilities in molecular diagnosis

Proteins in general and secretory proteins in particular undergo posttranslational processes before they reach the structure in which they can fulfill their functional purpose. The protein precursor may undergo a wide variety of proteolytic cleavages, N- and C-terminal trimmings and amino acid derivatizations in cells that express the protein. Occasionally, the same precursor is differently processed in different cell types and, in addition, diseased cells may process a given precursor abnormally. For instance, the translational process is often either increased or decreased in diseased cells, which render the ensuing modifications of the precursor incomplete. As a result, a variable mixture of precursors and processing-intermediates accumulates. Measurement of a single protein or peptide component of the posttranslational processing cascade may not facilitate the diagnosis of a disease, because the pattern of precursors and processing products vary individually among patients. In order to exploit disturbed posttranslational processing for diagnostic use, and--at the same time--provide an accurate measure of the translational product, a simple analytical principle named "processing-independent analysis" (PIA) has been designed. PIA-methods quantitate the total mRNA product irrespective of the degree of precursor processing. PIA-methods have recently been developed for a number of prohormones and neuroendocrine proteins, and their diagnostic potential appears promising in early diagnosis of tumors and cardiovascular diseases. The present review describes posttranslational processing patterns for some neuroendocrine proteins. Second, PIA-measurements of precursor-products are mentioned with indication of problems and pitfalls. Finally, PIA-results obtained in diagnosis of neoplastic and cardiovascular diseases are highlighted. But first general aspects of the posttranslational processing are reviewed as a necessary basis for the understanding of the new diagnostic possibilities.     

An amphicrine pancreatic cell line: AR42J cells combine exocrine and neuroendocrine properties.

The permanent cell culture line AR42J, derived from a rat pancreatic acinar carcinoma, is widely used for functional studies of exocrine pancreatic acinar cells. We now present evidence that these cells are amphicrine in that they contain zymogen granules as well as small (40-80 nm) neuroendocrine (NE) vesicles and typical neurotransmitters. Using the small NE vesicle-specific markers synaptophysin and "protein S.V.2", including synaptophysin cDNA probes, we have found that AR42J cells synthesize these proteins and contain vesicles harboring these proteins with biophysical properties similar to those of small NE vesicles. NE properties of these cells are further indicated by the presence of considerable amounts of stored amino acids (gamma-aminobutyric acid (GABA), glycine, glutamate) and by the presence of the GABA-synthesizing enzyme, glutamic acid decarboxylase. Finally, intermediate filament (IF) protein typing showed only cytokeratins 8 and 18, indicating that AR42J cells possess an IF protein complement indistinguishable from that of acinar and islet cells. Our results document the unusual case of a permanent cell line with combined exocrine and neuroendocrine properties that may be indicative of a derivation from a cell with multipotential character.     

Coordinate regulation of catecholamine uptake by rab3 and phosphoinositide 3-kinase.

Previously we observed that rab3 GTPases modulate both the secretion of catecholamines from PC12 neuroendocrine cells and the steady-state accumulation of exogenous norepinephrine (NE) into these cells (Weber, E., Jilling, T., and Kirk, K. L. (1996) J. Biol. Chem. 271, 6963-6971). Here we addressed the mechanisms by which these monomeric GTPases stimulate NE uptake by PC12 cells including their effects on uptake kinetics, their sites of action (secretory granule membrane versus plasma membrane), and the involvement of rab3-interacting proteins in this process. We observed that rab3B stimulated the rate and maximal accumulation of radiolabeled NE into large dense core vesicles within intact PC12 cells. rab3A and rab3B also increased NE uptake into large dense core vesicles in digitonin-permeabilized PC12 cells, which indicates that these GTPases stimulate catecholamine uptake at the level of the secretory granule membrane. In an attempt to identify rab3B targets that may mediate this effect on NE uptake, we found that rab3B interacts directly with phosphoinositide 3-kinase (PI3K) in a GTP-dependent fashion and that PI3K activity was elevated in PC12 cells overexpressing rab3B. Furthermore, two structurally distinct inhibitors of PI3K (wortmannin and LY294002) inhibited NE uptake in intact as well as digitonin-permeabilized PC12 cells, but had no effect on calcium-evoked NE secretion. Our results indicate that rab3 and PI3K positively and coordinately regulate NE uptake in PC12 neuroendocrine cells at least in part by stimulating the secretory vesicle uptake step.     

Human neuroblastoma cells acquire regulated secretory properties and different sensitivity to Ca2+ and alpha-latrotoxin after exposure to differentiating agents

IMR-32 human neuroblastoma cells are unable to release [3H]dopamine in response to secretagogues. However, they express a normal complement of membrane receptors and ion channels which are efficiently coupled to second messenger production. In the present study we took advantage of the ability of this cell line to differentiate in vitro in the presence of either dibutyrryl-cAMP or 5-bromodeoxyuridine, to analyze any developmentally regulated changes in its secretory properties. Uptake, storage, and release of [3H]dopamine were studied biochemically and by autoradiography. The calcium ionophore ionomycin, phorbol 12-myristate 13-acetate and the presynaptic acting neurotoxin alpha-latrotoxin were used in both control and differentiated cells as secretagogue agents. The presence of secretory organelles was investigated by electron microscopy; the expression of secretory organelle markers, such as chromogranin/secretogranin proteins (secretory proteins) and synaptophysin (membrane protein), was detected by Western blotting and immunofluorescence. The results obtained indicate that IMR-32 cells acquire regulated secretory properties after in vitro drug-induced differentiation: (a) they assemble "de novo" secretory organelles, as revealed by electron microscopy and detection of secretory organelle markers, and (b) they are able to store [3H]dopamine and to release the neurotransmitter in response to secretagogue stimuli. Furthermore, secretagogue sensitivity was found to be different, depending on the differentiating agent. In fact, dibutyrryl-cAMP treated cells release [3H]dopamine in response to alpha-latrotoxin, but not in response to ionomycin, whereas 5-bromodeoxyuridine treated cells release the neurotransmitter in response to both secretagogues. All together these results suggest that IMR-32 cells represent an adequate model for studying the development of the secretory apparatus in cultured human neurons.     

Sorting of synaptophysin into special vesicles in nonneuroendocrine epithelial cells

Synaptophysin is a major transmembrane glycoprotein of a type of small vesicle with an electron-translucent content (SET vesicles), including the approximately 50-nm presynaptic vesicles in neuronal cells, and of similar, somewhat larger (< or = approximately 90 nm) vesicles (SLMV) in neuroendocrine (NE) cells. When certain epithelial non-NE cells, such as human hepatocellular carcinoma PLC cells, were cDNA transfected to synthesize synaptophysin, the new molecules appeared in specific SET vesicles. As this was in contrast to other reports that only NE cells were able to sort synaptophysin away from other plasma membrane proteins into presynaptic- or SLMV-type vesicles, we have further characterized the vesicles containing synaptophysin in transfected PLC cells. Using fractionation and immunoisolation techniques, we have separated different kinds of vesicles, and we have identified a distinct type of synaptophysin-rich, small (30-90-nm) vesicle that contains little, if any, protein of the constitutive secretory pathway marker hepatitis B surface antigen, of the fluid phase endocytosis marker HRP, and of the plasma membrane recycling endosomal marker transferrin receptor. In addition, we have found variously sized vesicles that contained both synaptophysin and transferrin receptor. A corresponding result was also obtained by direct visualization, using double-label immunofluorescence microscopy for the endocytotic markers and synaptophysin in confocal laser scan microscopy and in double- immunogold label electron microscopy. We conclude that diverse non-NE cells of epithelial nature are able to enrich the "foreign" molecule synaptophysin in a category of SET vesicles that are morphologically indistinguishable from SLMV of NE cells, including one type of vesicle in which synaptophysin is sorted away from endosomal marker proteins. Possible mechanisms of this sorting are discussed.     

Localization of calbindin D28K-like immunoreactivity in fish gill: a light microscopic and immunoelectron histochemical study.

The presence of calbindin D28K in fish (Heteropneustes fossilis) gill was studied by use of specific antibodies raised against chick duodenal 28 kDa calbindin in immunoperoxidase and electron-microscopic labelling experiments. Immunoreactivity for calbindin D28K, which has been observed in the intestine of a number of avian and mammalian species, is reported for the first time in the gill. It was primarily located in neuroendocrine (NE) cells. Some immunoreactivity was also located in the glycocalyx of the non-endocrine cells, i.e., the pavement cells, which have ultrastructural characteristics quite different from those of endocrine cells. The calbindin-immunopositive NE cells were ascertained in both gill filamental and lamellar epithelium. All the NE cells contained secretory granules as the most distinctive feature of these cells. Ultrastructurally, two types of NE cells were distinguished according to the morphology of their secretory granules. The calbindin immunoreactivity in the NE cells was stimulated when the calcium concentration of the ambient water was reduced. The present findings suggest that NE cells exert some as yet unidentified function related to calcium-mediated processes involving the expression of calbindin.     

Tracking down the elusive early endosome

Despite significant progress in understanding protein trafficking and compartmentation in plants, the identification and protein compartmentalization for organelles that belong to both the secretory and endocytic pathways have been difficult because protein trafficking has generally been studied separately in these two pathways. However, recent data indicate that the trans-Golgi network serves as an early endosome merging the secretory and endocytic pathways in plant cells. Here, we discuss the proteins identified as markers for post-Golgi compartments in these two pathways and propose that the trans-Golgi network is a pivotal organelle with multiple sorting domains for post-Golgi protein trafficking in plant cells.     

The neuroendocrine phenotype,cellular plasticity,and the search for genetic switches: redefining the diffuse neuroendocrine system

The term neuroendocrine has been used to define cells that secrete their products in a regulated manner, in response to a specific stimulus. The neuroendocrine system includes neurons and endocrine cells sharing a common phenotypic program characterized by the expression of markers such as neuropeptides, chromogranins, neuropeptide processing enzymes SPC2 and SPC3 (subtilase-like pro-protein convertases) or dense core secretory granules. Various theories such as the APUD (amine precursor uptake decarboxylation) concept, the diffuse neuroendocrine system (DNES) or the paraneuron concept have been put forth to classify neuroendocrine cells as a cohesive group. Neuroendocrine characteristics have been used as evidence of a common embryological origin for normal and neoplastic cells. However, it is now recognized that neuroendocrine characteristics can be observed in various cell types, such as immunocytes, that are not of a common embryological origin with either neurons or endocrine cells. We propose to redefine previous "neuroendocrine" concepts to include the notion that activation of specific genetic switches can lead to the expression of a partial or full neuroendocrine phenotype in a variety of cell types, including immune cells.     

Cellular distribution of secretory pathway markers in the haploid synergid cells of Arabidopsis thaliana

In flowering plants, cell–cell communication plays a key role in reproductive success, as both pollination and fertilization require pathways that regulate interactions between many different cell types. Some of the most critical of these interactions are those between the pollen tube (PT) and the embryo sac, which ensure the delivery of sperm cells required for double fertilization. Synergid cells function to attract the PT through secretion of small peptides and in PT reception via membrane‐bound proteins associated with the endomembrane system and the cell surface. While many synergid‐expressed components regulating PT attraction and reception have been identified, few tools exist to study the localization of membrane‐bound proteins and the components of the endomembrane system in this cell type. In this study, we describe the localization and distribution of seven fluorescent markers that labelled components of the secretory pathway in synergid cells of Arabidopsis thaliana. These markers were used in co‐localization experiments to investigate the subcellular distribution of the two PT reception components LORELEI, a GPI‐anchored surface protein, and NORTIA, a MILDEW RESISTANCE LOCUS O protein, both found within the endomembrane system of the synergid cell. These secretory markers are useful tools for both reproductive and cell biologists, enabling the analysis of membrane‐associated trafficking within a haploid cell actively involved in polar transport.     

Endocrine markers of cellular immunity: defining the endocrine phenotype

The term neuroendocrine has been used to define cells that secrete their products in a regulated manner, in response to a specific stimulus. The neuroendocrine system includes neurons and endocrine cells sharing a common phenotypic program characterized by the expression of markers such as neuropeptides, chromogranins, neuropeptide processing enzymes SPC2 and SPC3 (subtilase-like pro-protein convertases) or dense core secretory granules. Various theories such as the APUD (amine precursor uptake decarboxylation) concept, the diffuse neuroendocrine system (DNES) or the paraneuron concept have been put forth to classify neuroendocrine cells as a cohesive group. Neuroendocrine characteristics have been used as evidence of a common embryological origin for normal and neoplastic cells. However, it is now recognized that neuroendocrine characteristics can be observed in various cell types, such as immunocytes, that do not share a common embryological origin with either neurons or endocrine cells. We propose to redefine previous "neuroendocrine" concepts to include the notion that activation of specific genetic switches can lead to the expression of a partial or full neuroendocrine phenotype in a variety of cell types, including immune cells.     

Proteomic inventory of "anchorless" proteins on the colon adenocarcinoma cell surface

Surface proteins play important pathophysiological roles in health and disease, and accumulating proteomics-based studies suggest that several "non-membrane" proteins are sorted to the cell surface by unconventional mechanisms. Importantly, these proteins may comprise attractive therapeutic targets and novel disease markers for colon cancer. To perform a proteomics-based inventory of these so-called "anchorless" surface proteins, intact colon adenocarcinoma SW480 cells were labeled with membrane-impermeable biotin after which only soluble biotinylated proteins were isolated and identified by nanoLC-MS/MS. Computer-assisted analysis predicted that only 9 of the 97 identified surface-exposed proteins have predicted secretory signal peptides, whereas 2 other proteins have a putative transmembrane segment. Of the 9 proteins with putative signal peptides, 1 was predicted to be retained at the cell surface by a GPI-anchor, whereas 5 other proteins contained an ER-retention motif (KDEL) that should prevent them from being sorted to the cell surface. The remaining 86 soluble "surface" proteins lack known export signals and the possibility that these proteins are candidate substrates of non-classical transporters or exported by unconventional mechanisms is discussed. Alternatively, the large number of "intracellular" and ER-resident proteins may imply that biotinylation approaches are not only specific for surface proteins, but also biased against a certain subset of non-surface proteins. This underscores the importance of post-proteomic verification of proteomics-based inventories on surface-exposed proteins, which eventually should reveal to which extent non-classical export and retention mechanisms contribute to the sorting of "anchorless" proteins to the surface of colon tumor cells.     

Laboratory diagnostics of enterovirus cardiac infections

Diagnostic efficacy of different markers of enterovirus cardiac infection (EVCI) has been evaluated. Testing of clinical samples from patients with myocarditis (n=50), dilatation cardiomyopathy (n=122), ischemic heart disease (n=34) and from healthy donors (n=50) revealed diagnostically significant markers in patients suspected for enterovirus cardiac infection: antienterovirus IgM in the patient's blood serum, the expression of viral proteins by myocardium cells and the presence of genome RNA and replicative intermediate in cardiac cells. The results obtained were used for developing up-to-date scheme of the EVCI diagnosis which included the data from the case history, the preliminary rapid diagnosis and the molecular biological study of the cardiac biopsies.     

Lipoprotein lipase and leptin are accumulated in different secretory compartments in rat adipocytes.

Adipose cells produce and secrete several physiologically important proteins, such as lipoprotein lipase (LPL), leptin, adipsin, Acrp30, etc. However, secretory pathways in adipocytes have not been characterized, and vesicular carriers responsible for the accumulation and transport of secreted proteins have not been identified. We have compared the intracellular localization of two proteins secreted from adipose cells: leptin and LPL. Adipocytes accumulate large amounts of both proteins, suggesting that neither of them is targeted to the constitutive secretory pathway. By means of velocity centrifugation in sucrose gradients, equilibrium density centrifugation in iodixanol gradients, and immunofluorescence confocal microscopy, we determined that LPL and leptin were localized in different membrane structures. LPL was found mainly in the endoplasmic reticulum with a small pool being present in low density membrane vesicles that may represent a secretory compartment in adipose cells. Virtually all intracellular leptin was localized in these low density secretory vesicles. Insulin-sensitive Glut4 vesicles did not contain either LPL or leptin. Thus, secretion from adipose cells is controlled both at the exit from the endoplasmic reticulum as well as at the level of "downstream" secretory vesicles.     

The secretory pathway of protists: spatial and functional organization and evolution.

All cells secrete a diversity of macromolecules to modify their environment or to protect themselves. Eukaryotic cells have evolved a complex secretory pathway consisting of several membrane-bound compartments which contain specific sets of proteins. Experimental work on the secretory pathway has focused mainly on mammalian cell lines or on yeasts. Now, some general principles of the secretory pathway have become clear, and most components of the secretory pathway are conserved between yeast cells and mammalian cells. However, the structure and function of the secretory system in protists have been less extensively studied. In this review, we summarize the current knowledge about the secretory pathway of five different groups of protists: Giardia lamblia, one of the earliest lines of eukaryotic evolution, kinetoplastids, the slime mold Dictyostelium discoideum, and two lineages within the "crown" of eukaryotic cell evolution, the alveolates (ciliates and Plasmodium species) and the green algae. Comparison of these systems with the mammalian and yeast system shows that most elements of the secretory pathway were presumably present in the earliest eukaryotic organisms. However, one element of the secretory pathway shows considerable variation: the presence of a Golgi stack and the number of cisternae within a stack. We suggest that the functional separation of the plasma membrane from the nucleus-endoplasmic reticulum system during evolution required a sorting compartment, which became the Golgi apparatus. Once a Golgi apparatus was established, it was adapted to the various needs of the different organisms.