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Results for “Secretory Vesicles”

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PubMed1994

An elevation of cytosolic protein phosphorylation modulates trimeric G-protein regulation of secretory vesicle formation from the trans-Golgi network.

The role of protein phosphorylation in the formation of secretory vesicles from the trans-Golgi network (TGN) and in the regulation of this process by TGN-associated trimeric G-proteins was investigated, using a previously established and a novel cell-free system derived from the neuroendocrine cell line PC12. In the absence of exogenous activators of trimeric G-proteins, okadaic acid, an inhibitor of protein serine/threonine phosphatase types 1, 2A, and PPX, had no significant effect on secretory vesicle formation as reconstituted in a postnuclear supernatant. However, okadaic acid antagonized the inhibition of secretory vesicle formation which occurred upon activation of trimeric G-proteins by either aluminum fluoride or guanosine 5'-3-O-(thio)-triphosphate (GTP gamma S). Microcystin-LR, a protein phosphatase inhibitor structurally distinct from okadaic acid, also antagonized the trimeric G-protein-mediated inhibition of secretory vesicle formation but, in contrast to okadaic acid, alone was sufficient to stimulate this process. The antagonistic effect of the phosphatase inhibitors was abolished by a broad spectrum protein kinase inhibitor, staurosporine, which alone, however, did not affect vesicle formation. The effect of okadaic acid was promoted by activators of protein kinase C (phorbol myristate acetate) and protein kinase A (cyclic AMP). To investigate the subcellular localization of the phosphoprotein that is involved in the antagonistic effect of protein phosphatase inhibitors, a novel cell-free system was established which reconstitutes the formation of secretory vesicles from TGN membranes supplemented with cytosol. Using this cell-free system, the relevant phosphoprotein was found to reside in the cytosol. In conclusion, our results suggest that serine/threonine protein phosphorylation is not required for secretory vesicle formation from the TGN but modulates, via a cytosolic phosphoprotein, the regulation of this process by TGN-associated trimeric G-proteins.

Alkaloids↗

PubMed1992

Thiol and aspartyl proteolytic activities in secretory vesicles of bovine pituitary.

Thiol and aspartyl proteolytic activities in isolated secretory vesicles of neural (NL) and intermediate (IL) lobes of bovine pituitary were characterized with heterologous enkephalin and tachykinin precursor substrates, 35S-(Met)-preproenkephalin and 35S-(Met)-beta-preprotachykinin. IL and NL secretory vesicles contained thiol-dependent proteolytic activity that cleaved the enkephalin precursor with a pH optimum of 4.5; this activity resembled a novel "prohormone thiol protease' previously purified and characterized from adrenal medulla chromaffin granules. IL and NL vesicles also demonstrated aspartyl proteolytic activity with acidic pH optimum, as shown by pepstatin A inhibition of tachykinin and enkephalin precursor cleaving activity. This activity may be related to a previously characterized chromaffin granule aspartyl protease (CGAP) related to cathepsin D (2), as indicated by the presence of immunoreactive CGAP in NL secretory vesicles by anti-CGAP immunoblots. These results show that pituitary secretory vesicles, like chromaffin granules, may contain similar thiol-dependent and aspartyl proteolytic activities.

Animals↗

PubMed1998

Probing single secretory vesicles with capillary electrophoresis.

Secretory vesicles obtained from the atrial gland of the gastropod mollusk Aplysia californica were chemically analyzed individually with a combination of optical trapping, capillary electrophoresis separation, and a laser-induced fluorescence detection. With the use of optical trapping, a single vesicle that had attoliters (10(-18) liters) of volume was introduced into the tapered inlet of a separation capillary. Once the vesicle was injected, it was lysed, and its components were fluorescently labeled with naphthalene-2, 3-dicarboxaldehyde before separation. The resultant electropherograms indicated distinct variations in the contents of single vesicles.

Amines↗

PubMed1997

Gi regulation of secretory vesicle swelling examined by atomic force microscopy.

In the last decade, several monomeric and heterotrimeric guanine nucleotide binding proteins have been identified to associate with secretory vesicles and to be implicated in exocytosis. Vesicle volume also has been proposed to play a regulatory role in secretory vesicle fusion at the plasma membrane. However, the molecular mechanism of function of the guanine nucleotide binding proteins and of the regulation of secretory vesicle volume in the exocytotic process remains unclear. In this study, we report association of the secretory vesicle membrane with the alpha subunit of a heterotrimeric GTP binding protein G(alpha i3) and implicate its involvement in vesicle swelling. Using an atomic force microscope in combination with confocal microscopy, we were able to study the dynamics of isolated zymogen granules, the secretory vesicles in exocrine pancreas. Exposure of zymogen granules to GTP resulted in a 15-25% increase in vesicle height as measured by the atomic force microscope and a similar increase in vesicle diameter as determined by confocal microscopy. Mas7, an active mastoparan analog known to stimulate Gi proteins, was found to stimulate the GTPase activity of isolated zymogen granules and cause swelling. Increase in vesicle size in the presence of GTP, NaF, and Mas7 were irreversible and KCl-sensitive. Ca2+ had no effect on zymogen granule size. Taken together, the results indicate that G(alpha i3) protein localized in the secretory vesicle membrane mediates vesicle swelling, a potentially important prerequisite for vesicle fusion at the cell plasma membrane.

Animals↗

PubMed1987

Secretory vesicle cytochrome b561: a transmembrane electron transporter.

The major function of cytochrome b561 is now clear. This transmembrane protein transports electrons across a secretory vesicle bilayer to supply electrons to monooxygenases inside the secretory vesicle. Cytochrome b561 has been localized not only to adrenergic secretory vesicles, where it supplies electrons to dopamine beta-hydroxylase, but also to peptidergic secretory vesicles that contain peptidyl alpha-amidating monooxygenase. Thus, one would expect to find cytochrome b561 in the membranes of all neuroendocrine cells that contain amidated peptide secretory products. In addition, its wide occurrence as an integral membrane protein of secretory vesicles may make it useful for investigation of vesicle biogenesis and turnover. One of the most important potential roles of cytochrome b561 is that it can be used as a model protein to investigate long-range biological electron transport. This cytochrome is a single polypeptide, which can be purified easily and reconstituted into a functional assembly. It also catalyzes an experimentally unambiguous transmembrane transport of electrons. A full molecular characterization of the structure and function of this cytochrome may provide insights into biological electron transfer which would otherwise be difficult or impossible to obtain.

Animals↗

PubMed1990

Inositol 1,4,5-trisphosphate-triggered Ca2+ release from bovine adrenal medullary secretory vesicles.

The effect of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and calcium ionophore A23187 on Ca2+ release from bovine adrenal medullary secretory vesicles and microsomes was examined. Ins(1,4,5)P3 released 3.5 nmol of Ca2+/mg protein from secretory vesicles and 1.5 nmol of Ca2+/mg protein from microsomes as measured by a Ca2(+)-selective electrode. However, A23187 promoted Ca2+ uptake into vesicles while releasing Ca2+ from microsomes. Ins(1,4,5)P3-induced Ca2+ release from secretory vesicles was rapid, but the released Ca2+ was absorbed within 3 min during which the Ins(1,4,5)P3-releasable pools were refilled. The in situ calcium content of secretory vesicle measured by atomic absorption spectrometry was 112 +/- 6.3 nmol/mg protein indicating the potential importance of secretory vesicles as an intracellular Ca2+ store. The high Ca2(+)-buffering capacity of secretory vesicles is presumed to be due to the high Ca2(+)-binding capacity of chromogranin A, the major intravesicular protein, which has calsequestrin-like properties.

Adrenal Medulla↗

PubMed1994

Secretory vesicles are the intracellular reservoir of complement receptor 1 in human neutrophils.

The subcellular localization of complement receptor 1 (CR1) was investigated in human neutrophils. CR1 was located exclusively in the light membrane fractions containing secretory vesicles and plasma membranes in Percoll density gradients of unperturbed neutrophils. Separation of plasma membranes from secretory vesicles by high-voltage free-flow electrophoresis of the light membranes from the Percoll gradient revealed that more than 80% of the CR1 was located intracellularly in secretory vesicles. After weak stimulation of neutrophils, the increase in CR1 surface expression closely paralleled both surface increase of known secretory vesicle membrane Ags and the release of matrix proteins from secretory vesicles. More potent stimulation of the neutrophils did not enhance CR1 surface expression further, in agreement with the lack of CR1 in granules as demonstrated on Percoll gradient. CR3 (Mac-1), which has been shown to be located both in secretory vesicles and in neutrophil granules, was up-regulated in parallel with CR1 after weak stimuli, whereas a profound increase of CR3 was observed after more potent stimuli in accordance with granule mobilization. These results identify secretory vesicles as the reservoir of CR1, which translocates to the plasma membrane after weak stimulation, and underscore the functional significance of this recently identified organelle.

Cytoplasmic Granules↗

PubMed2004

Peptidyl-glycine alpha-amidating monooxygenase targeting and shaping of atrial secretory vesicles: inhibition by mutated N-terminal ProANP and PBA.

ANP (atrial natriuretic peptide) is widely recognized as an important vasorelaxant, diuretic, and cardioprotective hormone. Little is known, however, about how ANP-secretory vesicles form within the atrial myocytes. Secretory vesicles were visualized by fluorescence microscope imaging in live rat atrial myocytes expressing proANP-enhanced green fluorescent protein (EGFP), or N-terminal-mutated fusion proteins thought to suppress the calcium-dependent aggregation of proANP. Results showed the following: (1) aggregates of proANP and coexpressed proANP-EGFP recruited peptidylglycine alpha-amidating monooxygenase (PAM)-1, an abundant atrial integral vesicle membrane protein; (2) coexpressed N-terminal-mutated (Glu23,24-->Gln23,24) and N-terminal-deleted proANP-EGFP inhibited recruitment of PAM-1 by up to 60%; (3) 4-phenyl-3-butenoic acid (PBA) (10 mumol/L), a pharmacological inhibitor of the lumenal peptidylglycine alpha-hydroxylating monooxygenase domain of PAM proteins, inhibited recruitment of endogenous PAM-1 and of coexpressed pro-EGFP-PAM-1; (4) PBA had no effect on exocytosis of the potassium inward rectifier KIR2.1; (5) PBA induced a deformation of the secretory vesicles but did not inhibit docking. These findings suggest that recruitment of PAM-1 to secretory vesicles depends on intact N-terminal proANP and on the lumenal domain of PAM-1. Conversely, PAM-1 participates in shaping the proANP-secretory vesicles. The full text of this article is available online at http://circres.ahajournals.org.

Animals↗

PubMed2006

Dissecting docking and tethering of secretory vesicles at the target membrane.

Secretory vesicles dock at their target in preparation for fusion. Using single-vesicle total internal reflection fluorescence microscopy in chromaffin cells, we show that most approaching vesicles dock only transiently, but that some are captured by at least two different tethering modes, weak and strong. Both vesicle delivery and tethering depend on Munc18-1, a known docking factor. By decreasing the amount of cortical actin by Latrunculin A application, morphological docking can be restored artificially in docking-deficient munc18-1 null cells, but neither strong tethering nor fusion, demonstrating that morphological docking is not sufficient for secretion. Deletion of the t-SNARE and Munc18-1 binding partner syntaxin, but not the v-SNARE synaptobrevin/VAMP, also reduces strong tethering and fusion. We conclude that docking vesicles either undock immediately or are captured by minimal tethering machinery and converted in a munc18-1/syntaxin-dependent, strongly tethered, fusion-competent state.

Actins↗

PubMed1996

Formation of nascent secretory vesicles from the trans-Golgi network of endocrine cells is inhibited by tyrosine kinase and phosphatase inhibitors.

Recent evidence suggests that secretory vesicle formation from the TGN is regulated by cytosolic signaling pathways involving small GTP-binding proteins, heterotrimeric G proteins, inositol phospholipid metabolism, and protein serine/threonine phosphorylation. At the cell surface, protein phosphorylation and dephosphorylation on tyrosine residues can rapidly modulate cytosolic signaling pathways in response to extracellular stimuli and have been implicated in the internalization and sorting of signaling receptors. to determine if phosphotyrosine metabolism might also regulate secretory vesicle budding from the TGN, we treated permeabilized rat pituitary GH3 cells with inhibitors of either tyrosine phosphatases or tyrosine kinases. We demonstrate that the tyrosine phosphatase inhibitors pervanadate and zinc potently inhibited budding of nascent secretory vesicles. Tyrphostin A25 (TA25) and other tyrosine kinase inhibitors also prevented secretory vesicle release, suggesting that vesicle formation requires both phosphatase and kinase activities. A stimulatory peptide derived from the NH2 terminus of the small GTP-binding protein ADP ribosylation factor 1 (ARF1) antagonized the inhibitory effect of TA25, indicating that both agents influence the same pathway leading to secretory vesicle formation. Antiphosphotyrosine immunoblotting revealed that protein tyrosine phosphorylation was enhanced after treatment with tyrosine phosphatase or kinase inhibitors. Subcellular fractionation identified several tyrosine phosphorylated polypeptides of approximately 175, approximately 130, and 90-110 kD that were enriched in TGN-containing Golgi fractions and tightly membrane associated. The phosphorylation of these polypeptides correlated with inhibition of vesicle budding. Our results suggest that in endocrine cells, protein tyrosine phosphrylation and dephosphorylation are required for secretory vesicle release from the TGN.

ADP-Ribosylation Factor 1↗

PubMed1999

Ca2+-induced deprotonation of peptide hormones inside secretory vesicles in preparation for release.

The acidic environment inside secretory vesicles ensures that neuropeptides and peptide hormones are packaged in a concentrated condensed form. Although this is optimal for storage, decondensation limits release. Thus, it would be advantageous to alter the physical state of peptides in preparation for exocytosis. Here, we report that depolarization of the plasma membrane rapidly increases enhanced green fluorescent protein (EGFP)-tagged hormone fluorescence inside secretory vesicles. This effect requires Ca2+ influx and persists when exocytosis is inhibited by N-ethylmaleimide. Peptide deprotonation appears to produce this response, because it is not seen when the vesicle pH gradient is collapsed or when a pH-insensitive GFP variant is used. These data demonstrate that Ca2+ evokes alkalinization of the inside of secretory vesicles before exocytosis. Thus, Ca2+ influx into the cytoplasm alters the physical state of intravesicular contents in preparation for release.

Animals↗

PubMed1986

Conversion of proinsulin to insulin occurs coordinately with acidification of maturing secretory vesicles.

Proinsulin is a single polypeptide chain composed of the B and A subunits of insulin joined by the C-peptide region. Proinsulin is converted to insulin during the maturation of secretory vesicles by the action of two proteases and conversion is inhibited by ionophores that disrupted intracellular H+ gradients. To determine if conversion of prohormone to hormone actually occurs in an acidic secretory vesicle, cultured rat islet cells were incubated in the presence of 3-(2,4-dinitroanilino)-3' amino-N-methyldipropylamine (DAMP), a basic congener of dinitrophenol that concentrates in acidic compartments and is retained there after aldehyde fixation. The cells were processed for indirect protein A-gold colocalization of DAMP, using a monoclonal antibody to dinitrophenol, and proinsulin, using a monoclonal antibody that exclusively reacts with the prohormone. The average density of DAMP-specific gold particles in immature secretory vesicles that contained proinsulin was 71/micron 2 (18 times cytoplasmic background), which indicated that this compartment was acidic. However, the density of DAMP-specific gold particles in the insulin-rich mature secretory vesicle averaged 433/micron 2. This suggests that although proinsulin conversion occurs in an acidic compartment, the secretory vesicles become more acidic as they mature. Since the concentration of anti-proinsulin IgG binding in secretory vesicles is inversely proportional to the conversion of proinsulin to insulin, we were able to determine that maturing secretory vesicles had to reach a critical pH before proinsulin conversion occurred.

Animals↗

PubMed2002

The number of secretory vesicles remains unchanged following exocytosis.

Earlier studies using electron microscopy demonstrate that there is no loss of secretory vesicles following exocytosis. Depletion however, of vesicular contents resulting in the formation of empty or partially empty vesicles is seen in electron micrographs, post exocytosis, in a variety of cells. Our studies using atomic force microscopy (AFM) reveal that following stimulation of secretion, live pancreatic acinar cells having 100-180 nm in diameter fusion pores located at the apical plasma membrane, dilate only 25-35% during exocytosis. Since secretory vesicles in pancreatic acinar cells range in size from 200 nm to 1200 nm in diameter, their total incorporation at the fusion pore, would distend the structure much more then what is observed. These earlier results prompted the current study to determine secretory vesicle dynamics in live pancreatic acinar cells following exocytosis. AFM studies on live acinar cells reveal no loss of secretory vesicle number following exocytosis. Parallel studies using electron microscopy, further confirmed our AFM results. These studies demonstrate that following stimulation of secretion, membrane-bound secretory vesicles transiently dock and fuse to release vesicular contents.

Animals↗

PubMed1985

Structure of catecholamine secretory vesicles from PC12 cells.

Catecholamine secretory organelles were partially purified from PC12 cells. Measurement of the sedimentation coefficient (540S in 0.32 M sucrose), density in an isoosmotic gradient (1.139 g/cm), and density in an isoosmotic gradient using D2O as a solvent (1.205 g/cm3) have allowed us to calculate the molecular weight (1.17 X 10(9) daltons), radius (74 nm), and water content (62% vol/vol) of the secretory vesicle. The vesicle appears to contain ATP, but the molar ratio of 3,4-dihydroxyphenylethylamine (dopamine) to ATP in the particles is high (16.5) and the ATP was frequently asymmetrically distributed in the vesicle fraction. The particle behaves like a true secretory particle in that the dopamine content of the particle is increased by pargyline, diminished by depolarization, and abolished by reserpine. Sequential purification of PC12 lysates on controlled pore glass columns and isoosmotic Ficoll gradients produced a 20-30-fold purification, but this enrichment is not sufficient to produce a homogeneous population of vesicles. An 82,000-dalton protein copurifies with secretory granules and appears to be the major secreted protein. At this stage of purification this single protein makes up about 30% of the protein in the vesicle-containing fractions and so the vesicles must be approaching homogeneity.

Adrenal Gland Neoplasms↗

PubMed1996

A role for ADP-ribosylation factor 1, but not COP I, in secretory vesicle biogenesis from the trans-Golgi network.

A synthetic N-myristoylated peptide corresponding to the amino-terminal domain of ADP-ribosylation factor 1 (ARF1) markedly increases, in a cell-free system using post-nuclear supernatant from PC12 cells, the biogenesis of constitutive secretory vesicles and immature secretory granules from the trans-Golgi network (TGN). The related N-myristoylated ARF4 peptide only weakly stimulates, and the non-myristoylated ARF1 and ARF4 peptides inhibit, the biogenesis of these secretory vesicles. In a modified cell-free system using TGN membranes, coatomer-depleted cytosol supports the biogenesis of TGN-derived secretory vesicles to the same extent as control cytosol. These results suggest a role for ARF1, but not the COP I coat, in secretory vesicle biogenesis from the TGN, possibly via the activation of phospholipase D.

ADP-Ribosylation Factor 1↗

PubMed2006

Secretory vesicles in live cells are not free-floating but tethered to filamentous structures: a study using photonic force microscopy.

It is well established that actin and microtubule cytoskeletal systems are involved in organelle transport and membrane trafficking in cells. This is also true for the transport of secretory vesicles in neuroendocrine cells and neurons. It was however unclear whether secretory vesicles remain free-floating, only to associate with such cytoskeletal systems when needing transport. This hypothesis was tested using live pancreatic acinar cells in physiological buffer solutions, using the photonic force microscope (PFM). When membrane-bound secretory vesicles (0.2-1.2 microm in diameter) in live pancreatic acinar cells were trapped at the laser focus of the PFM and pulled, they were all found tethered to filamentous structures. Mild exposure of cells to nocodazole and cytochalasin B, disrupts the tether. Immunoblot analysis of isolated secretory vesicles, further demonstrated the association of actin, myosin V, and kinesin. These studies demonstrate for the first time that secretory vesicles in live pancreatic acinar cells are tethered and not free-floating, suggesting that following vesicle biogenesis, they are placed on their own railroad track, ready to be transported to their final destination within the cell when required. This makes sense, since precision and regulation are the hallmarks of all cellular process, and therefore would hold true for the transport and localization of subcellular organelles such as secretory vesicles.

Actins↗

PubMed2001

Measurement of secretory vesicle pH reveals intravesicular alkalinization by vesicular monoamine transporter type 2 resulting in inhibition of prohormone cleavage.

1. The acidic interior of neuroendocrine secretory vesicles provides both an energy gradient for amine-proton exchangers (VMATs) to concentrate small transmitter molecules, for example catecholamines, and an optimal pH for the prohormone convertases which cleave hormone precursors. There is evidence that VMAT activity modulates prohormone cleavage, but in the absence of measurements of pH in secretory vesicles in intact cells, it has not been possible to establish whether these effects are attributable to raised intravesicular pH due to proton transport through VMATs. 2. Clones were generated of the hamster insulinoma cell line HIT-T15 expressing a pH-sensitive form of green fluorescent protein (GFP-F64L/S65T) targeted to secretory vesicles, with and without co-expression of VMAT2. In order to study prohormone cleavage, further clones were generated that expressed preprogastrin with and without co-expression of VMAT2. 3. Confocal microscopy of GFP fluorescence indicated that the pH in the secretory vesicles was 5.6 in control cells, compared with 6.6 in cells expressing VMAT2; the latter was reduced to 5.8 by the VMAT inhibitor reserpine. 4. Using a pulse-chase labelling protocol, cleavage of 34-residue gastrin (G34) was found to be inhibited by co-expression with VMAT2, and this was reversed by reserpine. Similar effects on vesicle pH and G34 cleavage were produced by ammonium chloride. 5. We conclude that VMAT expression confers the linked abilities to store biogenic amines and modulate secretory vesicle pH over a range influencing prohormone cleavage and therefore determining the identity of regulatory peptide secretory products.

Alkalies↗

PubMed1975

Vesicular binesis: glucose effect on insulin secretory vesicles.

Binesis is a process whereby the membrane of the insuline secretory vesicle in the beta cell forms a lingula that indents the vesicular membrane of an adjoining secretory vesicle of the plasma membrane. Vesicular binesis in beta cells increases when islets of Langerhans are incubated at a stimulatory glucose concentration (300 miligrams per 100 milliliters). These vesicular membrane alterations may be the morphological concomitants of activation of the insulin secretory vesicle, and indicate an active role for the vesicle and its membrane in the release mechanism.

Animals↗