Secretory granule biogenesis and chromogranin A: master gene, on/off switch or assembly factor?

Secretory granules are found in specialized cell types, including endocrine cells, suggesting that a coordinated programme of gene expression is involved in their biogenesis. Indeed, it has been proposed that chromogranin A (CgA) acts as an on/off switch for secretory granule biogenesis. However, this proposed function is difficult to reconcile with the large body of evidence suggesting that secretory granules exist in the absence of CgA and that cells can synthesize CgA in the absence of secretory granules. Indeed, recent evidence suggests that, rather than a master gene or universal on/off switch, a series of on/off switches combines to induce expression of subsets of secretory granule-associated genes. The assembly of newly synthesized proteins and the inclusion of existing granule proteins would produce functional secretory granules. CgA and related proteins might act as assembly factors in this process.     

Chromogranin a induces the biogenesis of granules with calcium- and actin-dependent dynamics and exocytosis in constitutively secreting cells

Chromogranins are a family of acidic glycoproteins that play an active role in hormone and neuropeptide secretion through their crucial role in secretory granule biogenesis in neuroendocrine cells. However, the molecular mechanisms underlying their granulogenic activity are still not fully understood. Because we previously demonstrated that the expression of the major component of secretory granules, chromogranin A (CgA), is able to induce the formation of secretory granules in nonendocrine COS-7 cells, we decided to use this model to dissect the mechanisms triggered by CgA leading to the biogenesis and trafficking of such granules. Using quantitative live cell imaging, we first show that CgA-induced organelles exhibit a Ca(2+)-dependent trafficking, in contrast to native vesicle stomatitis virus G protein-containing constitutive vesicles. To identify the proteins that confer such properties to the newly formed granules, we developed CgA-stably-expressing COS-7 cells, purified their CgA-containing granules by subcellular fractionation, and analyzed the granule proteome by liquid chromatography-tandem mass spectrometry. This analysis revealed the association of several cytosolic proteins to the granule membrane, including GTPases, cytoskeleton-based molecular motors, and other proteins with actin- and/or Ca(2+)-binding properties. Furthermore, disruption of cytoskeleton affects not only the distribution and the transport but also the Ca(2+)-evoked exocytosis of the CgA-containing granules, indicating that these granules interact with microtubules and cortical actin for the regulated release of their content. These data demonstrate for the first time that the neuroendocrine factor CgA induces the recruitment of cytoskeleton-, GTP-, and Ca(2+)-binding proteins in constitutively secreting COS-7 cells to generate vesicles endowed with typical dynamics and exocytotic properties of neuroendocrine secretory granules.     

Localization of inositol trisphosphate receptor subtype 3 to insulin and somatostatin secretory granules and regulation of expression in islets and insulinoma cells.

Calcium ions play a central role in stimulus-secretion coupling in pancreatic beta cells, and an elevation of cytosolic Ca2+ levels is necessary for insulin secretion. Inositol 1,4,5-trisphosphate mobilizes intracellular Ca2+ stores in the beta cell by binding to specific receptors that are ligand-activated Ca2+ channels. The inositol trisphosphate receptors comprise a family of structurally related proteins with distinct but overlapping tissue distributions. Previous studies indicated that the predominant inositol trisphosphate receptor subtype expressed in rat pancreatic islets was the protein designated IP3R-3. We have confirmed the expression of IP3R-3 in pancreatic islets by immunohistocytochemistry and localized this protein to the secretory granules of insulin-secreting beta cells and somatostatin-secreting delta cells by immunogold electron microscopy. Secretory granules contain high levels of Ca2+, and the presence of IP3R-3 in the granule provides a mechanism for mobilizing granule Ca2+ stores in response to glucose and/or hormones. The release of Ca2+ from granule stores would increase the Ca2+ concentration in the surrounding cytoplasm and promote rapid exocytosis of granules, especially those granules in close proximity to the plasma membrane. The levels of IP3R-3 were increased in pancreatic islets of diabetic rats and rats that had been refed after a period of fasting. They were also increased in rat insulinoma RINm5F cells cultured in 25 mM glucose compared with cells cultured in 5 mM glucose. The localization of IP3R-3 to secretory granules of insulin-secreting beta cells and somatostatin-secreting delta cells suggests that granule Ca2+ stores actively participate in the secretory process and that their release is regulated by inositol 1,4,5-trisphosphate. The regulation of IP3R-3 levels by glucose, diabetes, and refeeding may allow the beta cell to adjust the insulin secretory response to changing physiological conditions.     

The priming effect of glucose on insulin release does not involve redistribution of secretory granules within the pancreatic B-cell

Short-term stimulation of the pancreatic B-cell with glucose produces a time-dependent potentiation of this cell, which markedly enhances the insulin response to a renewed stimulation with the hexose. To study if a redistribution of the B-cell secretory granules to a location close to the B-cell plasma membrane could underlie the priming effect of glucose, an investigation by ultrastructural morphometry was performed. After exposure of perfused rat pancreas to non-priming or priming concentrations of glucose, pale and dark B-cell secretory granules were distinguished and analysed both within a central and a peripheral zone of the B-cell. The pale secretory granules comprised 30-40% of the total granule population in the B-cell. Whereas no difference in diameter of the granules was observed, there was evidence for a greater numerical density of dark granules in the central than in the peripheral part of the B-cell. This finding may be in line with observations implying that newly synthesized insulin is released preferentially to older insulin. The present experiments did, however, not reveal any significant priming effect of glucose on the intracellular distribution of secretory granules in the pancreatic B-cell. The lack of morphological changes in the B-cell by glucose priming of insulin release should, rather, direct increased attention to the biochemical aspects of the priming phenomenon.     

Monitoring of glucose-regulated single insulin secretory granule movement by selective photoactivation

Aims/hypothesis Fluorescence microscopy opens new perspectives for the analysis of insulin secretory granule movement. In this study, we examined whether recently developed photoactivatable/photoconvertible proteins are a useful tool for studying this process at the single granule level in insulin-secreting cells after glucose stimulation. Methods Plasmids were generated for expression of fusion proteins of the granule membrane phosphatase phogrin or the granule cargo protein neuropeptide Y (NPY) with the photoactivatable green fluorescent protein mutant A206K (PA-GFP-A206K), the photoconvertible protein Dendra2 and the fluorescent protein mCherry. Transfected insulin-secreting MIN6 cells were analysed by fluorescence microscopy. Results Point-resolved 405 nm light exposure during image acquisition of MIN6 cells transiently transfected with Phogrin-PA-GFP-A206K or NPY-PA-GFP-A206K as well as of stable MIN6-Phogrin-Dendra2 cells resulted in selective visualisation of few granules by green or red fluorescence, respectively. Movement of these granules was analysed by an automated tracking method from confocal 3D image series. The high spatiotemporal resolution facilitated an elongated tracking of single granules. Interestingly, the track speed and track displacement of granules after 1 h starvation and subsequent glucose stimulation was lower in cells pre-cultured for 48 h at 3 mmol/l glucose than in cells pre-cultured at 25 mmol/l glucose. Conclusions/interpretation Targeting of the granule membrane or its cargo with a photoactivatable/photoconvertible protein allows in-depth visualisation and tracking of single insulin granules in dependence upon glucose. This technique may also open the way to elucidating the regulation of granule movement velocity within the pancreatic beta cell with respect to secretory defects in type 2 diabetes.     

Effects of glucose and amino acids on insulin, glucagon and zymogen granule size of foetal rat pancreas grown in organ culture.

Foetal rat pancreatic rudiments explanted on day 14 of gestation were grown for 6 days in organ culture in medium containing glucose (5.5(1G) or 16.5(3G)mmol/l) and amino acids at the 'physiological' (1AA) or seven times the 'physiological' (7AA) concentration. Cultures were also performed in medium to which zinc sulphate had been added at 10(-7) to 10(-5) mol/l concentration. At the end of the period of culture the diameters of insulin, glucagon and zymogen granule profiles in the rudiments were compared with those in normal 20-day foetal pancreas by quantitative morphology. The beta cell volume, the number of granules per beta cell, the insulin granular volume fraction and the area of insulin granule core and halo were also measured under selected experimental conditions. Zymogen granule profiles were largest in vivo, intermediate in diameter when grown in 1G x 7AA medium and smallest in 1G x 1AA medium. The mean diameter of glucagon granule profiles remained constant for growth in vivo, in 1G x 7AA medium. Insulin granule profiles were largest in 1G x 1AA medium or in 1G x 7AA medium, smallest in 3G x 1AA mdeium and of intermediate diameter in vivo. Amino-acid enrichment increased the diameter of insulin granules and glucose enrichment decreased it. The addition of zinc to the culture medium had no effect on insulin granule diameter. In 1G x 7AA cultures the beta cells were of similar size to those in vivo, but there were 29% fewer insulin granules per cell. The increased size of the insulin granules in 1G x 7AA cultures resulted in the insulin granule volume fraction in 1G x 7AA being 17.6 compared with 10.8% in vivo. Insulin granule cores were made larger by amino-acid enrichment of the culture medium but they were unaffected by glucose. The haloes were larger in 7AA medium and smaller in 3G medium. Glucose and amino-acid enrichment had a significant interaction on halo area, the mean area in 3G x 7AA medium being less than would have been expected from the summation of the effects of the two conditions.     

Lysosomes and pancreatic islet function: adaptation of beta-cell lysosomes to various metabolic demands

The effect of various functional demands on the lysosomes of pancreatic islet beta cells was studied in vivo. To expose pancreatic islets to different metabolic situations, normal syngeneic mouse islets were transplanted to either lean mice, alloxan-diabetic mice, or obese hyperglycemic mice. Two weeks after transplantation, primary and secondary beta-cell lysosomes of the islet grafts were analyzed by morphometry. The beta-cell lysosomes and secretory granules of the endogenous islets of lean and obese hyperglycemic mice were also measured. The beta cells of the islets transplanted to lean normoglycemic mice showed only a moderately developed synthetic apparatus and a great number of secretory granules. They had mainly secondary lysosomes, frequently containing secretory granule material, indicating a high crinophagic activity. The islet beta cells exposed to the high serum glucose concentration of alloxan-diabetic and obese hyperglycemic mice had an extensive synthetic apparatus, but a very small content of secretory granules. In these beta cells, there was a predominance of small primary lysosomes, indicating a low crinophagic activity. It is concluded that crinophagy may provide a mechanism for the pancreatic beta cell to moderate its content of insulin. When its secretory granule stores are diminished due to increased demands on insulin secretion, the beta cell seems able to drastically decrease the crinophagic activity. The detailed morphometric analysis of the endogenous islets of the lean and obese hyperglycemic mice showed that the beta cells of the obese hyperglycemic mice had a smaller number and size of the secretory granules.(ABSTRACT TRUNCATED AT 250 WORDS)     

Control of insulin granule dynamics by AMPK dependent KLC1 phosphorylation

The movement of insulin granules along microtubules, driven by kinesin-1/Kif5B, is essential for glucose-stimulated insulin secretion from pancreatic β-cells. 5?AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine kinase, which is activated in β-cells at low glucose concentrations, but inhibited as glucose levels increase. AMPK activation blocks glucose-stimulated recruitment of secretory granules to the cell surface and insulin secretion, suggesting motor proteins may be targets for this kinase. Whilst both kinesin-1/Kif5B and kinesin light chain-1 (KLC1) contain consensus AMPK phosphorylation sites only a peptide corresponding to Ser520 in mouse KLC1 and purified recombinant GST-KLC1 were phosphorylated by purified AMPK in vitro. To test the hypothesis that phosphorylation at this site may modulate kinesin-1-mediated granule movement, we developed a novel approach to study the dynamics of the granules within a cell in three-dimensions using Nokigawa spinning disc confocal microscopy. This cell-wide approach revealed that the number of longer excursions (>10 μm) increased significantly in response to elevated glucose concentration (30 vs 3 mM) in control MIN6 cells. However, similar changes were seen in cells over-expressing wild-type KLC1, phosphomimetic (S517/520D) or non-phosphorylatable (S517/520A) mutants of KLC1. Moreover, no evidence for a change in the phosphorylation state of KLC1 at Ser520 after AMPK activation was obtained using an anti-phospho Ser520-specific antibody. Thus, changes in the phosphorylation state of KLC1 at Ser517/520 are unlikely to affect motor function. In conclusion, we describe a new three-dimensional cell wide approach for the analysis of secretory granule dynamics in living β-cells.     

Distribution of cholecystokinin forms in intestinal secretory granule subtypes.

Peptides and proteins destined to be released in response to stimuli are found in the regulated secretory pathway. Substances in this pathway are packaged into secretory granules, wherein they are often rendered osmotically inactive by complexing to an oppositely charged molecule. The complexing mechanism employed by members of the cholecystokinin (CCK) peptide family is unknown, but the heterogenous charges of CCK peptides makes it possible that different CCK peptides have different abilities to form intragranular complexes. If the number of osmotically active intragranular CCK peptides varies, corresponding variations in secretory granule density should result, and when intestinal CCK secretory granules were purified on isotonic density gradients, four granule peaks were observed. Granules containing greater proportions of short CCK forms tended to have the lowest buoyant densities, suggesting that they contain a greater number of osmotically active molecules than granules of lower density and that short or all intragranular CCK forms are osmotically active. CCK secretory granules also contained novel CCK forms; in addition to previously characterized forms, granules contained a CCK form that appears to be CCK-6 and a form that could arise from cleavage of CCK-58 at position 4, 10, or 12. Because intragranular enzymes are responsible for peptide posttranslational processing, the intragranular CCK forms observed in the present study are likely to be authentic CCK-processing products. Finally, CCK sorting in intestine apparently differs from that in a rat medullary thyroid carcinoma cell line, in which CCK-22 and CCK-33 are not found in the regulated secretory pathway.     

Control of insulin granule dynamics by AMPK dependent KLC1 phosphorylation

The movement of insulin granules along microtubules, driven by kinesin-1/Kif5B, is essential for glucose-stimulated insulin secretion from pancreatic β-cells. 5?AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine kinase, which is activated in β-cells at low glucose concentrations, but inhibited as glucose levels increase. AMPK activation blocks glucose-stimulated recruitment of secretory granules to the cell surface and insulin secretion, suggesting motor proteins may be targets for this kinase. Whilst both kinesin-1/Kif5B and kinesin light chain-1 (KLC1) contain consensus AMPK phosphorylation sites only a peptide corresponding to Ser520 in mouse KLC1 and purified recombinant GST-KLC1 were phosphorylated by purified AMPK in vitro. To test the hypothesis that phosphorylation at this site may modulate kinesin-1-mediated granule movement, we developed a novel approach to study the dynamics of the granules within a cell in three-dimensions using Nokigawa spinning disc confocal microscopy. This cell-wide approach revealed that the number of longer excursions (>10 µm) increased significantly in response to elevated glucose concentration (30 vs 3 mM) in control MIN6 cells. However, similar changes were seen in cells over-expressing wild-type KLC1, phosphomimetic (S517/520D) or non-phosphorylatable (S517/520A) mutants of KLC1. Moreover, no evidence for a change in the phosphorylation state of KLC1 at Ser520 after AMPK activation was obtained using an anti-phospho Ser520-specific antibody. Thus, changes in the phosphorylation state of KLC1 at Ser517/520 are unlikely to affect motor function. In conclusion, we describe a new three-dimensional cell wide approach for the analysis of secretory granule dynamics in living β-cells.     

Isolation and characterisation of insulin secretory granules from a rat islet cell tumour

Summary Density gradient centrifugation techniques, using iso-osmotic colloidal silica suspensions (Percoll), were developed for the isolation of insulin secretory granules from a transplantable rat islet cell tumour. These procedures were readily completed within 7 h and from each animal yielded approximately 1 mg of granule protein. The isolated granules were essentially free of other subcellular organelles as evaluated by their contents of marker proteins, electron microscopy and by electrophoretic analyses. Their susceptibilities to lysis at low osmotic strength, at pH values above 7 or in media containing sodium ions were similar to those of granules partially purified from islets. Insulin comprised 50–60% of the total granule protein when determined by immunoassay or by densitometry of electrophoretic profiles. The proinsulin content was marginally higher than that of islets, as was the ratio of insulins I to II. Electrophoretic analyses revealed that the secretory granules contained 150 or more proteins besides insulin-related peptides. The majority of these had acidic isoelectric points and were located both within the granule interior and its enveloping membrane.     

The role of chemiosmotic lysis in the exocytotic release of insulin

The role of chemiosmotic lysis in the exocytotic release of insulin has been studied using perifused rat pancreatic islets of Langerhans. Established criteria for osmotic lysis of secretory granules requires proton translocation across the secretory granule membrane and the influx of a permeant anion. The consequent increase in granule osmolarity induces water entry and granule lysis. A proton gradient has been previously established to exist across the insulin secretory granule membrane. We have examined the sensitivity of insulin release to 1) hyperosmolar solutions, 2) replacement of medium Cl-, 3) replacement of medium Na+, and 4) anion transport inhibitors. The addition of 200-600 mM sucrose resulted in a 32-69% inhibition of insulin release due to 16.7 mM glucose. Replacement of Cl- by isethionate or SO4--reversibly inhibited glucose-induced insulin release by 47% and 78%, respectively. Na+ replacement by choline did not influence the secretory response. 4,4'-Diisothiocyano-2,2'-stilbene disulfonic acid (500 microM) and probenecid (10 mM) inhibited insulin release by 73% and 79%, respectively. These drugs are known to inhibit anion exchange in erythrocytes and may be influencing Cl- entry into the secretory granule fused to the plasma membrane by a similar mechanism. Furosemide inhibits NaKCl2 cotransport in erythrocytes, but had no influence on glucose-induced insulin release, suggesting that Cl- does not enter the secretory granule by this pathway. The primary criteria for the participation of a chemiosmotic mechanism subserving lysis of the insulin secretory granule are fulfilled by these results.     

Secretory granules of an anterior pituitary cell line, AtT-20, contain only mature forms of corticotropin and beta-lipotropin.

The pituitary cell line, AtT-20, synthesizes the precursor to corticotropin (adrenocorticotropic hormone; ACTH) and beta-endorphin and correctly glycosylates and cleaves it to make the mature forms of the hormones before they are secreted. This cell line was used to study the intracellular transport, packaging, and secretion of these hormones. Secretory granules from the cells were isolated by homogenization and differential centrifugation and isopycnic sedimentation on a 2H2O-Ficoll gradient to give a preparation having a specific activity of 90micrograms ACTH per mg of protein, which is 30- to 90-fold greater than that of whole cells. The granules have density characteristics and a sedimentation coefficient that are appropriate for spheres of 1000 A radius. They contain all of the fragments of the initial ACTH/endorphin precursor but almost undetectable amounts of the intact precursor. The fragments constitute about 50% of the protein in the secretory granule fraction and, from density measurements, we estimate that they are present in approximately 60,000 copies per vesicle. The cell line secretory granules appear, therefore, to be similar to mature secretory granules in normal differentiated tissues. ACTH first appears in the secretory granule at 30-45 min after synthesis. Cleavage of the precursor to mature ACTH occurs at about the same time in the whole cell. Therefore, proteolysis of the prohormone to ACTH and to beta-lipotropin is a metabolic event that can be correlated with the packaging of the hormone into a mature secretory granule. Cleavage of beta-lipotropin to beta-endorphin occurs later, probably in the secretory granule.     

Influence of steroids and GnRH on biosynthesis and secretion of secretogranin II and chromogranin A in relation to LH release in LbetaT2 gonadotroph cells

The granin proteins secretogranin II (SgII) and chromogranin A (CgA) are commonly found associated with LH and/or FSH within specialised secretory granules in gonadotroph cells, and it is possible that they play an important role in the differential secretion of the gonadotrophins. In this study we have examined the regulation of the biosynthesis and secretion of SgII and CgA, in relation to LH secretion, in the LbetaT2 mouse pituitary gonadotroph cell line. Three experiments were carried out to investigate the effects of oestradiol (E2) and dexamethasone (Dex) in the presence and absence of GnRH (experiment 1), differing GnRH concentrations (experiment 2) and alterations in GnRH pulse frequency (experiment 3). In experiment 1, exposure to E2, Dex or E2+Dex, either with or without GnRH treatment, resulted in increased LH secretion. Steroids alone had no effect on LHbeta mRNA levels, but in the presence of GnRH LHbeta mRNA levels were increased in Dex- and E2+Dex-treated cells. GnRH receptor (GnRH-R) mRNA levels were up-regulated by Dex and E2+Dex, but were unaffected by GnRH. There were no steroid-induced changes in SgII or CgA mRNA, but increased levels of CgA mRNA were observed after GnRH treatment in cells cultured in the presence of Dex. In experiment 2, increasing concentrations of GnRH resulted in increases in LH secretion that were inversely dose-dependent. No changes in LHbeta, GnRH-R or SgII mRNA levels were observed, but there were dose-dependent increases in CgA mRNA levels. In experiment 3, GnRH was given as either 1 pulse/day or 4 pulses/day for 3 days. Both pulse regimes resulted in increased LH, SgII and CgA secretion compared with controls during the first 15 min pulse on day 3. Exposure to GnRH at 4 pulses/day increased LH and SgII secretion compared with controls during all 4 pulses, but secretion of both proteins was reduced during pulses 2-4 compared with pulse 1. CgA secretion also increased due to GnRH in pulse 1, but was decreased by GnRH treatment during pulse 2, and unchanged by GnRH during pulses 3 and 4. Total daily secretion of LH and SgII from cells given 1 pulse/day of GnRH increased compared with controls on all three treatment days, while total CgA secretion increased in response to GnRH on days 2 and 3 only. Intracellular levels of SgII, but not LH, decreased after GnRH treatment. In contrast, intracellular CgA was increased, but only after 4 pulses/day of GnRH. Levels of LHbeta, but not SgII, mRNA were increased by both pulse regimes, while CgA mRNA levels increased after 1 pulse/day of GnRH. These results indicate that there is a close correlation between the GnRH-stimulated release of LH and SgII from LbetaT2 cells, suggesting that SgII may have an influential role in the regulated secretion of LH, possibly by inducing LH aggregation to facilitate trafficking into secretory granules. CgA secretion does not appear to be closely associated with that of LH, but CgA expression does appear to be regulated by GnRH, which may indicate involvement in the control of LH secretion, possibly by influencing the proportion of LH in the different types of secretory granules.     

Beta cell chromogranin B is partially segregated in distinct granules and can be released separately from insulin in response to stimulation

Aims/hypothesis We investigated, in three beta cell lines (INS-1E, RIN-5AH, betaTC3) and in human and rodent primary beta cells, the storage and release of chromogranin B, a secretory protein expressed in beta cells and postulated to play an autocrine role. We asked whether chromogranin B is stored together with and discharged in constant ratio to insulin upon various stimuli. Methods The intracellular distribution of insulin and chromogranin B was revealed by immunofluorescence followed by three-dimensional image reconstruction and by immunoelectron microscopy; their stimulated discharge was measured by ELISA and immunoblot analysis of homogenates and incubation media. Results Insulin and chromogranin B, co-localised in the Golgi complex/trans-Golgi network, appeared largely segregated from each other in the secretory granule compartment. In INS-1E cells, the percentage of granules positive only for insulin or chromogranin B and of those positive for both was 66, 7 and 27%, respectively. In resting cells, both insulin and chromogranin B were concentrated in the granule cores; upon stimulation, chromogranin B (but not insulin) was largely redistributed to the core periphery and the surrounding halo. Strong stimulation with a secretagogue mixture induced parallel release of insulin and chromogranin B, whereas with 3-isobutyl-1-methylxantine and forskolin ± high glucose release of chromogranin B predominated. Weak, Ca²⁺-dependent stimulation with ionomycin or carbachol induced exclusive release of chromogranin B, suggesting a higher Ca²⁺ sensitivity of the specific granules. Conclusions/interpretation The unexpected complexity of the beta cell granule population in terms of heterogeneity, molecular plasticity and the differential discharge, could play an important role in physiological control of insulin release and possibly also in beta cell pathology. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorised users. T. Giordano and C. Brigatti contributed equally to this work.     

A proposed role for chromogranin A as a glucocorticoid-responsive autocrine inhibitor of proopiomelanocortin secretion

Chromogranin A (CgA) is a 50 kilodalton (kDa) acidic glycoprotein that is costored and cosecreted from secretory granules with endogenous hormone from diverse endocrine cell types. The physiological role(s) of CgA is yet to be defined. In this study we used the AtT-20 mouse corticotropic cell line, which produces both CgA and POMC-derived peptides, to study 1) the regulation of CgA and POMC synthesis and secretion, and 2) the influence of CgA on POMC secretion. To study regulation of CgA and POMC biosynthesis and secretion, cells were treated with dexamethasone (DEX) or CRF for 48 h and CgA and POMC messenger RNAs and proteins were analyzed. Exposure to DEX for 48 h (10 nM) inhibited secretion of the 16 K fragment of POMC by 60% while stimulating CgA secretion 500% of control value. Consonant with these changes in protein, POMC mRNA levels fell to 40% of control levels while CgA mRNA levels increased to 250% of control values with DEX treatment. DEX treatment had no effect on the sizes of the CgA [2.1 kilobase (kb)] and POMC (1.0 kb) mRNAs. CRF (100 nM) stimulated secretion of both CgA (4-fold) and ACTH (2.5-fold) above basal values. By contrast, CRF increased POMC mRNA levels but had no effect on levels of CgA mRNA. Changes in total peptide production paralleled the changes in mRNA levels. Because DEX differentially regulated CgA and POMC synthesis and secretion, we questioned whether CgA could function as an autocrine inhibitor of hormone secretion. CgA inhibited CRF-stimulated secretion of 16 K fragment in a concentration-dependent manner (100% at 100 nM) without affecting basal 16 K fragment secretion. Moreover, anti-CgA antiserum, but not nonimmune serum, increased basal 16 K fragment secretion 2-fold and CRF-stimulated 16 K fragment secretion 1.5-fold. These results suggest that CgA plays an autocrine role as a glucocorticoid responsive inhibitor of POMC-derived peptide secretion.     

Direct imaging shows that insulin granule exocytosis occurs by complete vesicle fusion

Confocal imaging of GFP-tagged secretory granules combined with the use of impermeant extracellular dyes permits direct observation of insulin packaged in secretory granules, trafficking of these granules to the plasma membrane, exocytotic fusion of granules with the plasma membrane, and eventually the retrieval of membranes by endocytosis. Most such studies have been done in tumor cell lines, using either confocal methods or total internal reflectance microscopy. Here we compared these methods by using GFP-syncollin or PC3-GFP plus rhodamine dextrans to study insulin granule dynamics in insulinoma cells, normal mouse islets, and primary pancreatic beta cells. We found that most apparently docked granules did not fuse with the plasma membrane after stimulation. Granules that did fuse typically fused completely, but a few dextran-filled granules lingered at the membrane. Direct recycling of granules occurred only rarely. Similar results were obtained with both confocal and total internal reflection microscopy, although each technique had advantages for particular aspects of the granule life cycle. We conclude that insulin exocytosis involves a prolonged interaction of secretory granules with the plasma membrane, and that the majority of exocytotic events occur by full, not partial, fusion.     

Different mouse mast cell populations express various combinations of at least six distinct mast cell serine proteases.

Mouse serosal mast cells (SMCs) and Kirsten sarcoma virus-immortalized mast cells store large amounts of mast cell carboxypeptidase A and serine proteases in their secretory granules. Secretory granule proteins from 2.6 x 10(6) purified SMCs were separated by NaDodSO4/PAGE, trans-blotted to poly(vinylidine difluoride) membranes, and subjected to amino-terminal amino acid sequencing. Four distinct mast cell serine proteases were identified. With mast cell carboxypeptidase A, these serine proteases comprise the major proteins of mouse SMC secretory granules. Each of the four SMC serine proteases was distinct from the two serine proteases present in mucosal mast cells in the intestines of helminth-infected mice. The secretory granules of a Kirsten sarcoma virus-immortalized mast cell line contained three of the SMC-derived serine proteases and one of the mucosal mast cell-derived serine proteases. Thus, the family of mouse mast cell secretory granule serine proteases has at least six distinct members that can be expressed in different combinations in different mast cell populations.     

Insulin synthesis in a clonal cell line of simian virus 40-transformed hamster pancreatic beta cells.

A clonal hamster beta cell line (HIT) was established by simian virus 40 transformation of Syrian hamster pancreatic islet cells. Cytoplasmic insulin was detected in all cells by indirect fluorescent antibody staining, and membrane-bound secretory granules were observed ultrastructurally. Acidified-ethanol extracts of HIT cell cultures contained hamster insulin as determined by radioimmunoassay, radioreceptor assay, and bioassay. One subclone at passage 39 contained 2.6 micrograms of insulin per mg of cell protein. [3H]Leucine-labeled HIT insulin and proinsulin were identical to islet-derived proteins when compared by NaDodSO4/polyacrylamide gel electrophoresis of immunoprecipitates. HIT cell insulin secretion was stimulated by glucose, glucagon, and 3-isobutyl-1-methylxanthine. Insulin secretion at optimal glucose concentration (7.5 mM) was 2.4 milliunits per 10(6) cells per hr. Somatostatin and dexamethasone markedly inhibited HIT insulin secretion. The HIT cell line represents a unique in vitro system for studying beta cell metabolism and insulin biosynthesis.