Secretory Granules In Chromaffin Cells Research Articles

Identification of β-synuclein on secretory granules in chromaffin cells and the effects of α- and β-synuclein on post-fusion BDNF discharge and fusion pore expansion

α-Synuclein is strongly implicated in the pathogenesis of Parkinson’s disease as well as in other neurodegenerative diseases. However, its normal function in cells is not understood. The N-termini of α-, β-, and γ-synuclein contains six to seven 11-amino acid repeats that are predicted to form amphipathic helices. Membrane-binding and membrane-curving abilities of synuclein raise the possibility that synuclein could alter cellular processes that involve highly curved structures. In the present study we examined the localization of endogenous synuclein in bovine chromaffin cells by immunocytochemistry and its possible function to control protein discharge upon fusion of the granule with the plasma membrane by regulating the fusion pore. We found with quantitative immunocytochemistry that endogenous β-synuclein associates with secretory granules. Endogenous α-synuclein only rarely co-localizes with secretory granules. Overexpression of α-synuclein but not β-synuclein quickened the post- fusion discharge of BDNF-pHluorin by approxinately 30%. However, neither α- nor β-synuclein significantly altered curvature dynamics associated with fusion pore expansion that were measured by the combination of polarization and total internal reflection fluorescence microscopy (pTIRFM). Whatever the mechanism, the physiological significance of the small increased rate of post-fusion protein discharge caused by α-synuclein remains to be demonstrated, especially since endogenous β-, but not α-synuclein is the predominant synuclein isoform associated with chromaffin granules.

Abstract P127: Chromogranin A Pathway: From Pathogenic Molecule to Renal Disease

Introduction: Chromogranin A (CHGA) is released into circulation with catecholamine from secretory granules of chromaffin cells and post-ganglionic sympathetic neurons. Several CHGA gene polymorphisms contributing to traits of autonomic blood pressure control, hypertension and hypertensive nephropathy have been identified. Plasma CHGA is elevated in hypertension and renal failure. This study seeks to understand the role of CHGA in the progression of CKD/ ESRD. Hypothesis: Kidney injury results in elevated expression of CHGA which in turn affects biochemical and physiological traits that determine progression and development of CKD/ESRD. Methods: The remnant kidney model was used to investigate the influence of CHGA on kidney function in response to injury. Distinction in response to kidney injury in wild-type (WT) and Chga-/- (KO) mice was evaluated by measuring glomerular function, fibrosis and microarray analysis. Mesangial cells were treated with CHGA to delineate pathway leading to inflammation and fibrosis. Results: Nephrectomy (Npx) exacerbated azotemia in WT mice compared to their KO counterparts. WT-Npx mice displayed far excess fibrosis as equated with KO-Npx. Gene expression profiling revealed greater mitochondrial dysfunction due to Npx in WT mice. Mesangial cells in culture treated with CHGA, triggered NO release, by a signaling mechanism involving SR-A. CHGA treatment augmented NO production by a 2-step mechanism involving upregulation of iNOS and the arginine transporter Slc7a7. CHGA-treated vs. untreated cells exhibited differential involvement of cytokine, chemokine, complement, acute phase pro-inflammatory markers and apoptotic pathway genes. Plasma creatinine and CHGA was measured in a twin population (N=740) and an inverse correlation between GFR and CHGA was observed. Therefore, in both mice and humans an inverse correlation suggested decrease in glomerular function with increasing plasma CHGA concentration. Conclusions: Kidney injury leads to elevated levels of CHGA which serves as an insult to the mesangial cells resulting in reduction of mitochondrial efficiency, concerted transcription of genes involved in NO production, inflammation and fibrosis, progression of CKD and development of ESRD.

A common genetic variant of the chromogranin A-derived peptide catestatin is associated with atherogenesis and hypertension in a Japanese population.

Chromogranin A (CHGA) is a major protein in the secretory granules of chromaffin cells. CHGA also gives rise to cardiovascular/metabolism regulatory peptides, such as catestatin (CST) and pancreastatin (PST). While CST is a potent inhibitor of catecholamine secretion, PST is a potent physiological inhibitor of glucose-induced insulin secretion. Recently, several SNPs were identified in the CST and PST domains of CHGA locus in different populations. Among the discovered SNPs, CST variant allele Ser-364 was associated with blood pressure alteration and PST variant allele Ser-297 was associated with significantly higher plasma glucose level. In this study, we examined whether these CST and PST variant alleles exist and influence cardiovascular and metabolic phenotypes in Japanese population. Our study comprised of 343 Japanese subjects aged 45-85 years (143 men and 200 women, mean age 66 ± 8 years). We determined the genotypes of CST and PST by PCR-direct sequencing method and carried out genotype-phenotype association analysis. In 343 participants, the minor allele frequency of CST variant Ser-364 was 6.10%. On the other hand, we did not detect the PST variant Ser-297 in this entire study population. The presence of Ser-364 allele was associated with increased in baPWV (an index of systemic arterial stiffness) that suggests an initiation and/or progression atherogenesis and hypertension. The Ser-364 allele was also associated with elevated systolic blood pressure and pulse pressure, consistent with increased baPWV. In conclusion, the CST Ser-364 allele may increase the risk for cardiovascular diseases in Japanese population.

Chromogranin Location in the Adrenal Glands of ISIAH Rats

Comparative immunohistochemical and electron microscopic study of the adrenals from hypertensive ISIAH rats and normotensive WAG rats (control) showed a more intense reaction to chromogranin A in the ISIAH adrenal in comparison with the control. Electron microscopy and morphometric analysis showed high volume and numerical densities of the secretory granules in chromaffin cells of hypertensive rats. The results indicate stimulation of the adrenal medullary substance in ISIAH rats. Presumably, intensive accumulation of chromogranin A and secretory granules in chromaffin cells of hypertensive rats reflects a certain imbalance of chromogranin A and catecholamines biogenesis, this, in turn, leading to stable stimulation of the sympathoadrenal component and higher stress sensitivity of these animals.

Commentary: Granins, Secretory Granule Biogenesis, and Transport

Secretory granules are the hallmark of secretory cells that secrete granins, peptide hormones, and enzymes for various physiological functions in endocrine, exocrine, and neurons. The granin family of proteins are highly acidic and are the major components in dense core secretory granules, including those in chromaffin cells. Granins include chromogranin (Cg) A, B, and secretogranin (Sg) II which are processed to various derived peptides. In this 16th International Symposium of Chromaffin Cell Biology, a number of papers focusing on the mechanisms of biogenesis and transport of secretory granules in chromaffin cells, other endocrine cells and astrocytes were presented. As well, new physiological roles of granin-derived peptides were discussed. Maite Montero-Hadjadje (France) demonstrated that via its terminal domains, CgA induced granule biogenesis and generated a regulated secretory pathway in fibroblasts. These granules recruited cytoskeletal proteins, actin/myosin and microtubule motors to their surface and exhibited Ca sensitive transport to the plasma membrane for release. Peng Loh (USA) showed that a C-terminal CgA-derived peptide, serpinin, upon being released after stimulation of the cells, upregulated the transcription of a protease inhibitor, protease nexin-1 (PN-1) in a cAMP-PKA-sp1 dependent pathway. This in turn stabilized and augmented the level of granule proteins in the Golgi complex, leading to enhanced granule biogenesis. Ricardo Borges (Spain) using chromaffin cells from CgA knockout (KO), CgB KO and Cg double KO mice showed that the absence of granins led to changes in the accumulation and secretion of catecholamines. They concluded that these Cgs are highly efficient in facilitatingmonoamine and calcium accumulation and in the kinetics of exocytosis of dense core granules (for more details, see review by Borges group below). Seung Yoo (Korea) demonstrated the presence of inositol 1,4,5, triphosphate (IP3) sensitive Ca stores and three forms of IP3 receptors/channels in secretory granules in astrocytes, that presumably interacts with the Cgs to modulate Ca channels and Ca release as in chromaffin granules. Several papers were presented demonstrating the role of cytoskeletal network/proteins in granule transport to the plasma membrane for exocytosis. Luis Gutierrez (Spain) presented confocal microscopy and 3D reconstructed images revealing the complex F-actin structure at the cortex of chromaffin cells. He showed that this F-actin structure underwent reorganization during secretion to reposition the vesicles for exocytosis, (for more details, see their review below). Heidi de Wit (The Netherlands) recapped her extensive studies on the morphology of synaptic protein knockout cells at the electron microscopy level, suggesting that syntaxin, SNAP25 and synaptogamin form the minimal docking machinery. She also discussed the role of Munc 18 in reorganizing cortical F-actin for vesicle docking at the plasma membrane, prior to secretion, in chromaffin cells of embryonic mice. (See her review below). Frederic Meunier (Australia) described a novel phosphatidylinisitol (4,5) biphosphate pathway that controls actin-mediated mobilization and translocation of secretory vesicles to the plasma membrane. Small GTPases such as the Rho GTPase family are involved in actin remodeling during exocytosis. Stephane Gasman (France) showed in chromaffin cells and PC12 cells that Rho A, which is associated with secretory Y. P. Loh (*) Section on Cellular Neurobiology, Program on Developmental Neuroscience, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA e-mail: lohp@mail.nih.gov

Chromogranin A Regulates Renal Function by Triggering Weibel–Palade Body Exocytosis

Chromogranin A (CHGA), a protein released from secretory granules of chromaffin cells and sympathetic nerves, triggers endothelin-1 release from endothelial cells. CHGA polymorphisms associate with an increased risk for ESRD, but whether altered CHGA-endothelium interactions may explain this association is unknown. Here, CHGA led to the release of endothelin-1 and Weibel-Palade body exocytosis in cultured human umbilical vein endothelial cells. In addition, CHGA triggered secretion of endothelin-1 from glomerular endothelial cells and TGF-beta1 from mesangial cells cocultured with glomerular endothelial cells. In humans, plasma CHGA correlated positively with endothelin-1 and negatively with GFR. GFR was highly heritable in twin pairs, and common promoter haplotypes of CHGA predicted GFR. In patients with progressive hypertensive renal disease, a CHGA haplotype predicted rate of GFR decline. In conclusion, these data suggest that CHGA acts through the glomerular endothelium to regulate renal function.

Glomerular Filtration

Methodologic and analytic advances in molecular genetics identify many disease-predisposing variants in the human genome. One of the challenges presented by these studies is to define the mechanisms by which a given genetic variant affects a pathophysiologic disorder. This inevitable quandary confronts the study of Chen et al. 1 in this issue of JASN . The authors previously reported that a genetic polymorphism of chromatogranin A (CHGA), a soluble protein released from secretory granules of chromaffin cells and sympathetic nerves, predicts hypertensive ESRD in black patients. Using population genetic techniques, they further showed that the CHGA locus is a trans -QTL for endothelin-1 secretion and that CHGA stimulates endothelin release from endothelial cells. In view of the major role for endothelin postulated in the pathogenesis of a variety of cardiovascular and renal diseases, the authors questioned whether endothelin secretion is the mechanistic link between CHGA and renal dysfunction. Using cultured human umbilical vein endothelial cells, Chen et al. 1 now show that CHGA increases release of endothelin along with other peptides typically stored in Weibel-Palade bodies, including von Willebrand factor and angiopoietin 2. CHGA also increases endothelin secretion from mouse glomerular endothelial cells, and co-culture of CHGA-exposed glomerular endothelial cells with mesangial cells potentiates secretion of TGF-β1. In humans, plasma CHGA correlates positively with plasma endothelin and negatively with GFR. A promoter haplotype for CHGA predicts the level of GFR in individuals with GFRs in the normal range, whereas CHGA haplotypes in the 3′ untranslated region associate with GFR in individuals with progressive renal disease. The conclusion that CHGA triggers exocytotic release of endothelin from the glomerular endothelium and that this, in turn, regulates the level of GFR in normal and declining renal function offers some novel implications. These include …

The chromogranin A fragment catestatin: specificity, potency and mechanism to inhibit exocytotic secretion of multiple catecholamine storage vesicle co-transmitters

Secretory granules of chromaffin cells and neurons co-store and release, by exocytosis, the acidic soluble protein chromogranin A (human, CHGA; rodent, Chga) along with catecholamines, neuropeptides and adenosine triphosphate (ATP). CHGA serves as a pro-protein and upon proteolytic cleavage it generates active peptides, including catestatin (human CHGA352-372), first discovered in adrenal medullary chromaffin granules. Studies in our laboratory demonstrated that catestatin acts at the nicotinic acetylcholine receptor to inhibit catecholamine secretion. However, the specificity of catestatin to exert nicotinic-cholinergic antagonism among its co-transmitters is not clearly known, nor is the potential effect of catestatin on multiple vesicle co-transmitters understood. Here we probed the specificity of catestatin's actions among its co-transmitters: catecholamines, ATP, and neuropeptide Y (NPY). We studied the effects of each transmitter on exocytotic secretion of its co-transmitters from PC12 chromaffin cells, stimulating secretion by triggering physiological pathways at multiple sites. We observed that, among chromaffin granule co-transmitters, only catestatin and NPY inhibited catecholamine release induced by nicotinic-cholinergic stimulation; catestatin was more than tenfold more potent than NPY in this setting. We also stimulated norepinephrine secretion by other chromaffin cell agonists: catestatin blocked norepinephrine release induced by nicotine, but not by other agents (such as membrane depolarization) acting at later stages in the secretory pathway, nor by agents acting on other receptor classes. By contrast, NPY acted less specifically, blocking norepinephrine release triggered by either nicotine or membrane depolarization. Catestatin inhibited nicotinic-cholinergic co-release of all classes of chromaffin granule co-transmitters: catecholamines, chromogranins, neuropeptides, and ATP. Naturally occurring variants of human catestatin (Gly364Ser and Pro370Leu) exhibited parallel changes in potency to inhibit secretion of catecholamines and ATP. We conclude that, among the chromaffin granule co-transmitters, catestatin acts as the most specific and potent inhibitor of physiological pathway (nicotinic-cholinergic) stimulated secretion. Furthermore, catestatin generally inhibits nicotinically triggered exocytotic release of multiple co-transmitters from chromaffin granules. The results have physiological and pharmacological implications for co-transmission in the sympathochromaffin system.

Presence of syntaxin 1A in secretory granules of chromaffin cells and interaction with chromogranins A and B

Syntaxin 1A and synaptotagmin I are key participants of fusion complex formation during exocytotic processes, and syntaxin 1A is known to be present in the plasma membrane. Here, we show the presence of not only synaptotagmin I but also syntaxin 1A in secretory granules of bovine adrenal chromaffin cells by immunogold electron microscopy, and further demonstrate the interaction of these proteins with chromogranins A and B (CGA and CGB), two major proteins of secretory granules. Interaction between chromogranins and the components of fusion complex also suggests active participation of CGA and CGB in fusion complex formation and subsequent exocytosis.

The Innate Immunity: Roles for New Antifungal and Antibacterial Peptides Secreted by Chromaffin Granules from the Adrenal Medulla

In our search of a function for the chromogranin-derived peptides present within secretory granules of chromaffin cells we observed that a large number of them displayed potent antibacterial and antifungal activities. These peptides are released by exocytosis into the circulation together with catecholamines and are also found in inflammatory fluids together with a variety of other antimicrobial peptides such as defensins. Vasostatin I, the natural N-terminal domain of chromogranin A (1-76), and the vasostatin-derived peptide chromofungin (chromogranin A47-66) are, in addition to fragments of proenkephalin (enkelytin) and ubiquitin (ubifungin), potent antimicrobial peptides of adrenomedullary origin. These peptides interact with the cell wall of fungi and yeast cells and may within minutes penetrate and accumulate within the cells. Once inside, the peptides may target to enzymes that are crucial for the growth of the microorganisms, inhibiting their activities and thus causing the rapid death of the affected species. These properties lead us to propose that these peptides participate in a primary and early line of host defence, acting as a shield against invading microorganisms during stress and infection, linking the neuroendocrine and nervous tissues to the immune system. On the basis of structure, absence of a specific receptor, ability to cross membranes and to inhibit the calmodulin-dependent enzyme calcineurin in vitro, we propose that the vasostatin-derived chromofungin should be recognised as a cell penetrating peptide.

Real-Time Measurement of Exocytosis and Endocytosis Using Interference of Light

We describe a new approach for making real-time measurements of exocytosis and endocytosis in neurons and neuroendocrine cells. The method utilizes interference reflection microscopy (IRM) to image surface membrane in close contact with a glass coverslip (the “footprint”). At the synaptic terminal of retinal bipolar cells, the footprint expands during exocytosis and retracts during endocytosis, paralleling changes in total surface area measured by capacitance. In chromaffin cells, IRM detects the fusion of individual granules as the appearance of bright spots within the footprint with spatial and temporal resolution similar to total internal reflection fluorescence microscopy. Advantages of IRM over capacitance are that it can monitor changes in surface area while cells are electrically active and it can be applied to mammalian neurons with relatively small synaptic terminals. IRM reveals that vesicles at the synapse of bipolar cells rapidly collapse into the surface membrane while secretory granules in chromaffin cells do not.

The N- and C-terminal fragments of ubiquitin are important for the antimicrobial activities.

Secretory granules of chromaffin cells contain catecholamines and several antimicrobial peptides derived from chromogranins and proenkephalin-A. These peptides are secreted in the extracellular medium following exocytosis. Here, we show that ubiquitin is stored in secretory chromaffin granules and released into the circulation upon stimulation of chromaffin cells. We also show that the C-terminal fragment (residues 65-76) of ubiquitin displays, at the micromolar range, a lytic antifungal activity. Using confocal laser scan microscopy and rhodamine-labeled synthetic peptides, we could demonstrate that the C-terminal peptide (residues 65-76) is able to cross the cell wall and the plasma membrane of fungi and to accumulate in fungi, whereas the N-terminal peptide (residues 1-34) is stopped at the fungal wall level. Furthermore, these two peptides act synergistically to kill filamentous fungi. Because of the interaction of the C-terminal sequence of ubiquitin with calmodulin, the synthetic peptide (residues 65-76) was tested in vitro against calmodulin-dependent calcineurin, an enzyme crucial for fungal growth. This peptide was found to inhibit the phosphatase activity of calcineurin. Our data show a new property of ubiquitin C-terminal-derived peptide (65-76) that could be used with N-terminal peptide (1-34) as a new potent antifungal agent.

Subunit composition and functional properties of G-protein heterotrimers on rat chromaffin granules

Heterotrimeric G-proteins at the plasma membrane serve as switches between heptahelical receptors and intracellular signal cascades. Likewise endomembrane associated G-proteins may transduce signals from intracellular compartments provided they consist of a functional trimer. Using quantitative immunoelectron microscopy we found heterotrimeric G-protein subunits Galpha2, Galpha(q/11), Gbeta2 and Gbeta5 to reside on secretory granules in chromaffin cells of rat adrenal glands. Thus rat chromaffin granules are equipped with functional G-proteins that consist of a specific alpha-, beta- and probably gamma-subunit combination. Serotonin uptake into a crude rat chromaffin granule preparation was inhibited by activated Galphao2 (10 nM) to nearly the same extent as by GMppNp (50 microM) whereas GDPbetaS was ineffective. The data support the idea that vesicular G-proteins directly regulate the transmitter content of secretory vesicles. In this respect Galphao2 appears to be the main regulator of vesicular momoamine transporter activity.

Cell Type-specific Storage of Dopamine β-Monooxygenase

Expression of dopamine beta-monooxygenase (DBM), the enzyme that converts dopamine into norepinephrine, is limited to adrenal chromaffin cells and a small population of neurons. We studied DBM trafficking to regulated granules by stably expressing rat DBM in AtT-20 corticotrope tumor cells, which contain regulated granules, and in Chinese hamster ovary (CHO) cells, which lack regulated granules. The behavior of exogenous DBM in both cell lines was compared with endogenous DBM in adrenal chromaffin cells. CHO cells secreted active DBM, indicating that production of active enzyme does not require features unique to neuroendocrine cells. Pulse-chase experiments indicated that early steps in DBM maturation followed a similar time course in AtT-20, CHO, and adrenal chromaffin cells. Use of a conformation-sensitive DBM antiserum indicated that acquisition of a folded structure occurred with a similar time course in all three cell types. Cell type-specific differences in DBM trafficking became apparent only when storage in granules was examined. As expected, DBM was stored in secretory granules in chromaffin cells; CHO cells failed to store DBM. Despite the fact that AtT-20 cells have regulated granules, exogenous DBM was not stored in these granules. Thus storage of DBM in secretory granules requires cell type specific factors.

Localization, expression, and characterization of guanylin in the rat adrenal medulla.

The peptide guanylin, recently isolated from the intestine, and localized to cells of the gut mucosa, is involved in electrolyte/water transport in the intestinal epithelium by means of a paracrine mode of regulation. Since high amounts of this peptide are present also in the systemic circulation, we investigated the adrenal gland as a potential endocrine source of guanylin. Using a reverse transcriptase-polymerase chain reaction and hybridization with an internal oligonucleotide designed for rat guanylin, 514-bp signals were obtained in intestinal tissue and adrenal gland. Successive analyses of extracts from intestine and adrenal gland by HPLC, western blotting, and radioimmunoassay revealed the presence of the same high-molecular mass (about 12.4 kDa) guanylin that corresponds to the mass of the guanylin prohormone. About 60 fmol/ml of circulating immunoreactive guanylin was determined in plasma. Localization studies with antisera directed against different epitopes of guanylin revealed that, in the adrenal gland, guanylin immunoreactivity is restricted to the medulla, where it is mainly confined to norepinephrine chromogranin A-containing cells. On the ultrastructural level, guanylin immunoreactivity was exclusively located to secretory granules of chromaffin cells. The present data indicate that, in addition to entero-endocrine cells, the adrenal medulla represents a further source of guanylin. Thus, an endocrine mode of function of guanylin may accrue to its hitherto evidenced paracrine action in fluid transport in the intestinal epithelium. Furthermore guanylin may be considered as a neurohormonal peptide.

Processing of Chromogranin B in Bovine Adrenal Medulla. Identification of Secretolytin, the Endogenous C-Terminal Fragment of Residues 614-626 with Antibacterial Activity

Chromogranins constitute a family of acidic soluble proteins widely distributed in endocrine cells and neurons. Chromogranin A, the major soluble component in bovine adrenal medullary secretory granules in chromaffin cells, has been shown to be actively processed to peptide fragments [Metz-Boutigue, M. H., Garcia-Sablone, P., Hogue-Angeletti, R. & Aunis, D. (1993) Eur. J. Biochem. 217, 247–257]. In the present paper, the structural features of the proteolytic degradation mechanism of chromogranin B/secretogranin I have been characterized with regard to the possible function of this protein as a precursor of biologically active peptides. Chromogranin-B-derived fragments present in bovine chromaffin granules were identified by microse-quencing after separation by two-dimensional gel electrophoresis or high-performance liquid chromatography. A similar approach was performed to characterize chromogranin-B-derived fragments released into the extracellular space from depolarized bovine cultured chromaffin cells. In chromogranin B, 18 cleavage sites were identified along the protein chain and chromogranin B/secretogranin I fragments were generated by proteolytic attack at both the N-terminus and C-terminus. A major fragment corresponding to residues 614–626 of the C-terminal sequence, was identified in the extracellular space; this peptide was found to share sequence and structural similarities with the lytic domain of cecropins and, as expected from this similarity, to display potent antibacterial properties. Endogenous and synthetic peptides were active on Micrococcus luteus, killing bacteria in the micromolar concentration range. The synthetic peptide slows the growth of Bacillus megaterium and was inactive towards Escherichia coli. In addition, the synthetic peptide was unable to induce hemolytic activity. This antibacterial function might be of biological significance in the neuroendocrine system of living organisms. We propose to name this peptide secretolytin.

Possible functional role of diadenosine polyphosphates: Negative feedback for excitation in hippocampus

Diadenosine polyphosphates (Ap 4A and Ap 5A) are present in secretory granules of chromaffin cells 8 as well as in the rat brain synaptic terminals. 6 Their contribution to the exocytosis of the total synaptosomal content is considerable, ranging from 7% to 12%. Ap 4A and Ap 5A are released from synaptosomes in a Ca 2+-dependent manner. 6 There are indications on the high affinity of diadenosine polyphosphates to P 2 receptors. 5,7 but their action on P 1 receptors remains unclear. Here we report that both substances induce a blocking action on excitatory synaptic transmission in the rat hippocampus. This action is elicited via the A 1 (subclass of P 1) receptors and differs in some respects from the action of adenosine.

Intracellular and extracellular processing of chromogranin A. Determination of cleavage sites.

Chromogranins are a family of acidic soluble proteins which exhibit widespread distribution in endocrine cells and neurons. Chromogranin A (CGA), the major soluble component of the secretory granules in chromaffin cells of the adrenal medulla, is a single polypeptide chain of 431 residues with an apparent molecular mass of 70-75 kDa and a pI of 4.5-5. In mature bovine chromaffin granules about 50% of the CGA has been processed. In the present paper, the structural features of the proteolytic degradation mechanism have been characterized with regard to the possible function of CGA as a prohormone, as suggested by recent studies. CGA-derived components present in chromaffin granules were subjected to either two-dimensional gel electrophoresis or HPLC and the N-terminal of each fragment was sequenced. Immunoblotting with antisera to specific sequences within the CGA molecule were used to characterize these fragments further at their C-terminal. In addition, a similar approach was performed to characterize CGA-derived fragments released into the extracellular space from directly depolarized bovine cultured chromaffin cells. Our results identified several proteolytic cleavage sites involved in CGA degradation. Intragranular processing occurs at 12 cleavage sites along the peptide chain located in both N- and C-terminal moieties of the protein; a preferential proteolytic attack in the C-terminal part was noted. We found that CGA processing also occurs in the extracellular space after release, generating new shorter fragments. The proteolytic cleavage sites identified in this study were compared with the cleavage points which are thought to be involved in generating CGA fragments with specific biological activity: pancreastatin, chromostatin and N-terminal vasostatin fragments. In addition, a new 12-amino-acid CGA-derived peptide corresponding to the sequence 65-76 was identified in the soluble core of purified chromaffin granules. This short peptide was released, together with catecholamines, after stimulation of cultured chromaffin cells suggesting its presence within the storage complex of chromaffin granules. The specific biological activity of this CGA-derived fragment remains to be determined.

Diadenosine polyphosphate (ApxA) as new neurotransmitters

AbstractThe presence of diadenosine polyphosphates (ApxA), diadenosine tetraphosphate (Ap4A), diadenosine pentaphosphate (Ap6A), and diadenosine hexaphosphate (Ap6A), has been described in secretory granules of chromaffin cells, Torpedo synaptic vesicles, and rat brain synaptosomes. The release of these compounds by the action of secretagogues and depolarizing agents, in the presence of calcium, increases their importance as active neurotransmitters. Two high affinity receptors have been described in the three neural models, with Kd values ranging from 0.08 to 0.40 nM for the first binding site and from 5.6 to 18nM for the second lower affinity binding site. Both binding sites exhibit a P2y‐like profile in chromaffin cells and Torpedo synaptic terminals and a different pattern in rat brain synaptosomes, suggesting the presence of a novel P2‐purinoceptor tentatively named P2d. Studies about the second messenger linked to this receptor, in chromaffin cells, demonstrate the mobilization of calcium from internal stores. ApxA receptors at the extracellular milieu are responsible for the inhibition of catecholamine release stimulated by secretagogues. Finally, all diadenosine polyphosphates are destroyed by the action of an ecto‐phosphodiesterase which, in chromaffin cells, shows Km values ranging from 1 to 4 μM. © 1993 Wiley‐Liss, Inc.

Topology of chromogranins in secretory granules of endocrine cells

Chromogranins A and B are glycoproteins originally detected in the adrenal medulla. These proteins are also present in a variety of neuroendocrine cells. The subcellular distribution of the chromogranins, and particularly their intra-granular topology are of special interest with respect to their putative functions. Endocrine cells of the guinea pig adrenal medulla, pancreas and gastric mucosa were investigated immunoelectron microscopically for the subcellular distribution of both chromogranins. Out of 13 established endocrine cell types in all locations, only two endocrine cell types showed immunoreactivity for both chromogranin A and B, and eight endocrine cell types showed immunoreactivities only for chromogranin A. These immunoreactivities varied inter-cellularly. Three endocrine cell types were unreactive for the chromogranins. Moreover, some hormonally non-identified endocrine cells in the pancreas and the gastric mucosa also contained chromogranin A immunoreactivities. Subcellularly, chromogranin A or B were confined to secretory granules. In most endocrine cells, the secretory granules showed chromogranin immunoreactivities of varying densities. Furthermore, the intra-granular topology of chromogranin A or B in the secretory granules varied considerably: in some endocrine cell types, i.e. chromaffin-, gastrin- and enterochromaffin-like-cells, chromogranin A immunoreactivity was localized in the perigranular and/or dense core region of the secretory granules; in others, i.e. insulin-, pancreatic polypeptide- and bovine adrenal medulla dodecapeptide-cells, it was present preferentially in the electron-opaque centre of the secretory granules; chromogranin B immunoreactivity was localized preferentially in the perigranular region of the secretory granules of chromaffin cells and gastrin-cells. The inter-cellular and inter-granular variations of chromogranin A and B immunoreactivities point to differences in biosynthesis or processing of the chromogranins among endocrine cells and their secretory granules.