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Results for “Secretory Vesicles”

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PubMed1997

Secretory vesicle pools and rate and kinetics of single vesicle exocytosis in neurosecretory cells.

Secretory vesicles are localized in specific compartments within neurosecretory cells. Morphometric, cytochemical and electrophysiological techniques have allowed the definition of secretory vesicle compartments. These are different pools in which vesicles are in various states of releasability. The transit of vesicles between compartments is not random, but an event controlled and regulated by Ca2+ and the cortical F-actin network. Cortical F-actin disassembly, a Ca(2+)-dependent event, controls the transit of secretory vesicles from the reserve compartment to the release-ready vesicle pool. Furthermore, the recent development of new technical approaches (patch-clamp membrane capacitance, electrochemical detection of amines with carbon-fibre microelectrodes) has now permitted us to understand the kinetics of single vesicle exocytosis.

Animals↗

PubMed1978

Fusion of secretory vesicles isolated from rat liver.

Secretory vesicles isolated from rat liver were found to fuse after exposure to Ca2+. Vesicle fusion is characterized by the occurrence of twinned vesicles with a continuous cleavage plane between two vesicles in freeze-fracture electron microscopy. The number of fused vesicles increases with increasing Ca2+-concentrations and is half maximal around 10(-6)M. Other divalent cations (Ba2+, Sr2+, and Mg2+) were ineffective. Mg2+ inhibits Ca2+-induced fusion. Therefore, the fusion of secretory vesicles in vitro is Ca2+ specific and exhibits properties similar to the exocytotic process of various secretory cells. Various substances affecting secretion in vivo (microtubular inhibitors, local anesthetics, ionophores) were tested for their effect on membrane fusion in our system. The fusion of isolated secretory vesicles from liver was found to differ from that of pure phospholipid membranes in its temperature dependence, in its much lower requirement for Ca2+, and in its Ca2+-specificity. Chemical and enzymatic modifcations of the vesicle membrane indicate that glycoproteins may account for these differences.

Animals↗

PubMed2002

Protein trafficking to chromaffin granules and proteolytic processing within regulated secretory vesicles of neuroendocrine chromaffin cells.

Proteolytic processing within secretory vesicles is required for the production and secretion of biologically active peptide neurotransmitters and hormones, known collectively as neuropeptides. This chapter addresses several new aspects of proteolysis in secretory vesicles, chromaffin granules, with respect to sorting proneuropeptides or prohormones into such regulated secretory vesicles that use specific prohormone sorting signals. Concomitant with prohormone sorting, evidence for the role of chromogranin A in secretory granule biogenesis is presented. Secretory vesicle function involves endogenous serpin protease inhibitors for the regulation of proteolysis. The novel serpins endopin 1 and endopin 2 possess high homology to a(1)-antichymotrypsin, yet they possess distinct target protease specificities. The serpins PAI-1 and neuroserpin are also localized to chromaffin granules. In addition, regulation of secretory vesicle function involves cytochrome b561 that regulates reducing equivalents to maintain the intravesicular redox state. These studies demonstrate multiple components as regulatory factors in the control of secretory vesicle function for the biosynthesis and secretion of neuropeptides and catecholamines.

Animals↗

PubMed1987

Isolation of secretory vesicles from Saccharomyces cerevisiae.

Purification of secretory vesicles from Saccharomyces cerevisiae has been hindered because these organelles normally represent a small proportion of cellular membranes. In the yeast secretory mutant sec1, secretory vesicles accumulate intracellularly in large quantities. Using a sec1 strain we have devised a procedure for the partial purification of these vesicles. The purification employs differential and density gradient centrifugations and an electrophoretic separation of membranes. The fractions obtained from this procedure are enriched for secretory vesicles at least fivefold over other cellular membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized membrane fractions reveals a distinct set of polypeptides associated with secretory vesicles.

Acid Phosphatase↗

PubMed2003

In vitro fusion between Saccharomyces cerevisiae secretory vesicles and cytoplasmic-side-out plasma membrane vesicles.

The final step in the secretory pathway, which is the fusion event between secretory vesicles and the plasma membrane, was reconstructed using highly purified secretory vesicles and cytoplasmic-side-out plasma membrane vesicles from the yeast Saccharomyces cerevisiae. Both organelle preparations were obtained from a sec 6-4 temperature-sensitive mutant. Fusion was monitored by means of a fluorescence assay based on the dequenching of the lipophilic fluorescent probe octadecylrhodamine B-chloride (R18). The probe was incorporated into the membrane of secretory vesicles, and it diluted in unlabelled cytoplasmic-side-out plasma membrane vesicles as the fusion process took place. The obtained experimental dequenching curves were found by mathematical analysis to consist of two independent but simultaneous processes. Whereas one of them reflected the fusion process between both vesicle populations as confirmed by its dependence on the assay conditions, the other represented a non-specific transfer of the probe. The fusion process may now be examined in detail using the preparation, validation and analytical methods developed in this study.

Cell Membrane↗

PubMed1991

Expression of the yeast plasma membrane [H+]ATPase in secretory vesicles. A new strategy for directed mutagenesis.

Secretory vesicles that accumulate in the temperature-sensitive sec6-4 strain of yeast have been shown to contain a vanadate-sensitive ATPase, presumably en route to the plasma membrane (Walworth, N. C., and Novick, P. J. (1987) J. Cell Biol. 105, 163-174). We have now established this enzyme to be a fully functional form of the PMA1 [H+]ATPase, identical in its catalytic properties to that found in the plasma membrane. In addition, the secretory vesicles are sealed tightly enough to permit the measurement of ATP-dependent proton pumping with fluorescent probes. We have gone on to exploit the vesicles as an expression system for site-directed mutants of the ATPase. For this purpose, a sec6-4 strain has been constructed in which the chromosomal PMA1 gene is under control of the GAL1 promoter; the mutant pma1 allele to be studied is introduced on a centromeric plasmid under the control of a novel heat shock promoter. In galactose medium at 23 degrees C, the wild-type ATPase is produced and supports normal vegetative growth. When the cells are switched to glucose medium at 37 degrees C, however, the wild-type gene turns off, the mutant gene turns on, and secretory vesicles accumulate. The vesicles contain a substantial amount of newly synthesized, plasmid-encoded ATPase (5-10% of total vesicle protein), but only traces of residual wild-type PMA1 ATPase and no detectable mitochondrial ATPase, vacuolar ATPase, or acid or alkaline phosphatase. To test the expression strategy, we have made use of pma1-105 (Ser368----Phe), a vanadate-resistant mutant previously characterized by standard methods (Perlin, D. S., Harris, S. L., Seto-Young, D., and Haber, J. E. (1989) J. Biol. Chem. 264, 21857-21864). In secretory vesicles, as expected, the plasmid-borne pma1-105 allele gives rise to a mutant enzyme with a reduced rate of ATP hydrolysis and a 100-fold increase in Ki for vanadate. Proton pumping is similarly resistant to vanadate. Thus, the vesicles appear well suited for the production and characterization of mutant forms of the PMA1 [H+]ATPase. They should also aid the study of other yeast membrane proteins that are essential for growth as well as heterologous proteins whose appearance in the plasma membrane may be toxic to the cell.

Catalysis↗

PubMed1994

ADP ribosylation factor and a 14-kD polypeptide are associated with heparan sulfate-carrying post-trans-Golgi network secretory vesicles in rat hepatocytes.

Constitutive secretory vesicles carrying heparan sulfate proteoglycan (HSPG) were identified in isolated rat hepatocytes by pulse-chase experiments with [35S]sulfate and purified by velocity-controlled sucrose gradient centrifugation followed by equilibrium density centrifugation in Nycodenz. Using this procedure, the vesicles were separated from plasma membranes, Golgi, trans-Golgi network (TGN), ER, endosomes, lysosomes, transcytotic vesicles, and mitochondria. The diameter of these vesicles was approximately 100-200 nm as determined by electron microscopy. A typical coat structure as described for intra-Golgi transport vesicles or clathrin-coated vesicles could not be seen, and the vesicles were not associated with the coat protein beta-COP. Furthermore, the vesicles appear to represent a low density compartment (1.05-1.06 g/ml). Other constitutively secreted proteins (rat serum albumin, apolipoprotein E, and fibrinogen) could not be detected in purified HSPG-carrying vesicles, but banded in the denser fractions of the Nycodenz gradient. Moreover, during pulse-chase labeling with [35S]methionine, labeled albumin did not appear in the post-TGN vesicle fraction carrying HSPGs. These findings indicate sorting of HSPGs and albumin into different types of constitutive secretory vesicles in hepatocytes. Two proteins were found to be tightly associated with the membranes of the HSPG carrying vesicles: a member of the ADP ribosylation factor family of small guanine nucleotide-binding proteins and an unknown 14-kD peripheral membrane protein (VAPP14). Concerning the secretory pathway, we conclude from these results that ADP ribosylation factor proteins are not only involved in vesicular transport from the ER via the Golgi to the TGN, but also in vesicular transport from the TGN to the plasma membrane.

ADP-Ribosylation Factors↗

PubMed1986

Processing and secretion in the neurohypophysis. Stability of isolated secretory vesicles and role of internal pH.

A possible role of low pH in secretory vesicles for processing and secretion in the neurohypophysis was investigated. Subcellular fractionation of guinea-pig neural lobes revealed that a proton present in the membranes from this tissue could not be ascribed to secretory vesicles. However, a proton pump was found in coated microvesicles. Secretory vesicles isolated from rats and guinea pigs were stable under conditions known to lyse secretory vesicles from the adrenal medulla owing to the generation of a proton gradient. These results suggest that the internal pH of secretory vesicles from the neurohypophysis is closer to neutral than is the pH in chromaffin secretory vesicles. Processing of a neurophysin-glycopeptide intermediate from the biosynthesis of vasopressin in intact secretory vesicles incubated in vitro was activated by the addition of NH4Cl, known to increase the intravesicular pH. This activation of neurohormone processing was also apparent in isolated nerve endings incubated in the presence of NH4Cl, suggesting that NH4Cl can also be used to increase the intravesicular pH in intact nerve endings. However, NH4Cl did not affect the secretion of neurohormones, indicating that a low intravesicular pH is not important for exocytosis in the neurohypophysis. Our results indicate that a low pH generated during processing by mechanisms other than ATP-dependent proton transport may inhibit the processing enzymes, thereby preventing extensive breakdown of neurohormone precursors.

Acid Phosphatase↗

PubMed1983

Calcium/sodium exchange in purified secretory vesicles from bovine neurohypophyses.

Purified secretory vesicles isolated from bovine neurohypophyses take up Na+ under the same circumstances where an efflux of Ca2+ takes place, suggesting a Na+/Ca2+ exchange. Potassium cannot substitute for Na+ in this process. Also, a Ca2+/Ca2+ exchange can occur. Inhibiting the latter process by Mg2+ allowed to estimate an apparent KM of 0.7 microM free Ca2+ and a maximal uptake of 1.5 nmol X mg protein-1 X min-1 Ca2+ in exchange for Na+. The vesicles did not contain plasma membrane marker (Na+/K+ ATPase) as shown by distribution analyses on the density gradients on which they were purified. Similarly, distribution studies also showed that no other ATPase activity could be detected in the purified vesicle fraction. It is concluded that a Na+/Ca2+ exchange is operating across the secretory vesicle membrane and that it is not directly dependent on ATP hydrolysis.

Adenosine Triphosphatases↗

PubMed1980

Isolation and characterization of secretory vesicles from bovine neurohypophyses.

A procedure is described for the isolation of secretory vesicles from bovine neurohypophyses by differential centrifugation followed by density gradient centrifugation on iso-osmolal gradients of percoll/sucrose. Only negligible contamination of the secretory vesicle fraction with markers for mitochondria, microsomes and plasma membranes could be detected. The amount of Ca2-ATPase in the isolated neurohypophysial secretory vesicles was of the same low order of magnitude as that of (Na, K)-ATPase. Thin-section electromicrographs confirmed the high purity of the isolated secretory vesicle fractions, In freeze-fracture electronmicrographs, vesicle fusion was demonstrated after incubation with Ca2. As shown in dodecyl sulfate-gel electrophoresis and subsequent autoradiography secretory vesicles exhibited an endogenous phosphorylation activity. The secretory vesicles contained an average of 23.1 microgram vasopressin/mg of protein. On incubation in media differing in ionic strength, pH and Ca2 concentration the vesicles were stable for at least 1 h.

Animals↗

PubMed1983

Ca2+ uptake to purified secretory vesicles from bovine neurohypophyses.

Purified secretory vesicles isolated from bovine neurohypophyses were found to take up Ca2+ when incubated at 30 degrees C in media containing 10(-7) to 10(-4) M free Ca2+. At 10(-4) free Ca2+ 19 nmol/mg protein were taken up within 30 min. The initial uptake at this Ca2+ concentration was about 2 nmol/mg protein per min. The uptake of Ca2+ to secretory vesicles was not affected by ATP, oligomycin, ruthenium red, trifluoperazine, Mg2+ or K+, but was inhibited by Na+ and Sr2+. From these characteristics it can be concluded that the uptake system does not utilize directly ATP (as the Ca2+-ATPases known to be present in the cell membrane and the endoplasmic reticulum) and is different from the mitochondrial Ca2+ uptake system driven by respiration and/or ATP hydrolysis. However, Ca2+-Na+ exchange may well operate: In experiments using different concentrations of Na+ we found half-maximal inhibition of Ca2+ uptake with 33.3 mM Na+. An analysis of the data in a Hill plot indicated that at least 2 Na+ would be exchanged for 1 Ca2+. Also, it was found that Ca2+ previously taken up could be released again by external Na+ but not by K+.

Animals↗

PubMed1982

Uptake of Ca2+ by isolated secretory vesicles from adrenal medulla.

Intact secretory vesicles isolated from bovine adrenal medulla contain 94 nmol Na+ per mg of protein, and Ca2+ influx into the vesicles is inhibited by increasing concentrations of extravesicular Na+ (but not of K+, Li+ or choline+) or by addition of the Na+ ionophore monensin. Thus Ca2+ influx in determined by the Na+ gradient across the vesicular membrane. Half maximal inhibition of Ca2+ influx occurs with 34 nM Na+ extravesicularly. The fact that Ca2+ can also be released from the vesicles by inversion of the Na+ gradient provides direct evidence that an Na+-Ca2+ exchange may operate. According to an analysis of the inhibition of Ca2+ uptake by Na+ in a Hill plot 2 Na+ would be exchanged for 1 Ca2+. Ca2+ influx into the vesicles increases with temperature (energy of activation; 16 kcal/mol), can be observed already with 10(-7) M free Ca2+ and increases up to 10(-4) M Ca2+. Ca2+ influx is not affected by Mg2+ but Sr2+ is inhibitory. Since the process is only slightly influenced by the pH of the incubation medium and is insensitive to Mg2+-ATP or inhibitors of the proton translocating Mg2+-ATPase the electrochemical proton gradient across the vesicular membrane does not affect directly the Ca2+ influx into the secretory vesicles. Ca2+ uptake is insensitive to ruthenium red and oligomycin.

Adrenal Medulla↗

PubMedDate not supplied

Novel secretory vesicle serpins, endopin 1 and endopin 2: endogenous protease inhibitors with distinct target protease specificities.

Secretory vesicles of neuroendocrine cells possess multiple proteases for proteolytic processing of proteins into biologically active peptide components, such as peptide hormones and neurotransmitters. The importance of proteases within secretory vesicles predicts the presence of endogenous protease inhibitors in this subcellular compartment. Notably, serpins represent a diverse class of endogenous protease inhibitors that possess selective target protease specificities, defined by the reactive site loop domains (RSL). In the search for endogenous serpins in model secretory vesicles of neuroendocrine chromaffin cells, the presence of serpins related to alpha1-antichymotrypsin (ACT) was detected by Western blots with anti-ACT. Molecular cloning revealed the primary structures of two unique serpins, endopin 1 and endopin 2, that possess homology to ACT. Of particular interest was the observation that distinct RSL domains of these new serpins predicted that endopin 1 would inhibit trypsin-like serine proteases cleaving at basic residues, and endopin 2 would inhibit both elastase and papain that represent serine and cysteine proteases, respectively. Endopin 1 showed selective inhibition of trypsin, but did not inhibit chymotrypsin, elastase, or subtilisin. Endopin 2 demonstrated cross-class inhibition of the cysteine protease papain and the serine protease elastase. Endopin 2 did not inhibit chymotrypsin, trypsin, plasmin, thrombin, furin, or cathepsin B. Endopin 1 and endopin 2 each formed SDS-stable complexes with target proteases, a characteristic property of serpins. In neuroendocrine chromaffin cells from adrenal medulla, endopin 1 and endopin 2 were both localized to secretory vesicles. Moreover, the inhibitory activity of endopin 2 was optimized under reducing conditions, which required reduced Cys-374; this property is consistent with the presence of endogenous reducing agents in secretory vesicles in vivo. These new findings demonstrate the presence of unique secretory vesicle serpins, endopin 1 and endopin 2, which possess distinct target protease selectivities. Endopin 1 inhibits trypsin-like proteases; endopin 2 possesses cross-class inhibition for inhibition of papain-like cysteine proteases and elastase-like serine proteases. It will be of interest in future studies to define the endogenous protease targets of these two novel secretory vesicle serpins.

Chromaffin Cells↗

PubMed1999

Neuropeptide release by efficient recruitment of diffusing cytoplasmic secretory vesicles.

Neuropeptides are slowly released from a limited pool of secretory vesicles. Despite decades of research, the composition of this pool has remained unknown. Endocrine cell studies support the hypothesis that a population of docked vesicles supports the first minutes of hormone release. However, it has been proposed that mobile cytoplasmic vesicles dominate the releasable neuropeptide pool. Here, to determine the cellular basis of the releasable pool, single green fluorescent protein-labeled secretory vesicles were visualized in neuronal growth cones with the use of an inducible construct or total internal reflection fluorescence microscopy. We report that vesicle movement follows the diffusion equation. Furthermore, rapidly moving secretory vesicles are used more efficiently than stationary vesicles near the plasma membrane to support stimulated release. Thus, randomly moving cytoplasmic vesicles participate in the first minutes of neuropeptide release. Importantly, the preferential recruitment of diffusing cytoplasmic secretory vesicles contributes to the characteristic slow kinetics and limited extent of sustained neuropeptide release.

Animals↗

PubMed1991

Inositol 1,3,4,5-tetrakisphosphate-induced Ca2+ sequestration into bovine adrenal-medullary secretory vesicles.

Ins(1,3,4,5)P4 induced a rapid sequestration of Ca2+ into both secretory vesicles and microsomes of bovine adrenal medulla. The Ca(2+)-sequestering role of Ins(1,3,4,5)P4 contrasts with the Ca(2+)-releasing role of Ins(1,4,5)P3 in adrenal-medullary secretory vesicles and microsomes. The Ins(1,3,4,5)P4-induced Ca2+ sequestration into secretory vesicles was not inhibited by heparin (50 micrograms/ml), whereas Ins(1,4,5)P3-induced Ca2+ release was completely inhibited, indicating two different receptors for Ins(1,4,5)P3 and Ins(1,3,4,5)P4. Furthermore, Ins(1,3,4,5)P4 was as effective at 4 degrees C as at 24 degrees C in sequestering Ca2+ into secretory vesicles, implying Ca2+ sequestration through receptor-operated Ca2+ channels or activation of the Ca(2+)-exchange mechanism by Ins(1,3,4,5)P4. The Ca(2+)-sequestering activity of Ins(1,3,4,5)P4 has also been demonstrated with 45Ca2+; 10 microM-Ins(1,3,4,5)P4 induced rapid uptake of 45Ca2+ into secretory vesicles optimized for Ca2+ uptake, whereas 10 microM-Ins(1,4,5)P3 induced 45Ca2+ release from secretory vesicles in similar experiments.

Adrenal Medulla↗

PubMed1999

The COOH-terminal domain of Myo2p, a yeast myosin V, has a direct role in secretory vesicle targeting.

MYO2 encodes a type V myosin heavy chain needed for the targeting of vacuoles and secretory vesicles to the growing bud of yeast. Here we describe new myo2 alleles containing conditional lethal mutations in the COOH-terminal tail domain. Within 5 min of shifting to the restrictive temperature, the polarized distribution of secretory vesicles is abolished without affecting the distribution of actin or the mutant Myo2p, showing that the tail has a direct role in vesicle targeting. We also show that the actin cable-dependent translocation of Myo2p to growth sites does not require secretory vesicle cargo. Although a fusion protein containing the Myo2p tail also concentrates at growth sites, this accumulation depends on the polarized delivery of secretory vesicles, implying that the Myo2p tail binds to secretory vesicles. Most of the new mutations alter a region of the Myo2p tail conserved with vertebrate myosin Vs but divergent from Myo4p, the myosin V involved in mRNA transport, and genetic data suggest that the tail interacts with Smy1p, a kinesin homologue, and Sec4p, a vesicle-associated Rab protein. The data support a model in which the Myo2p tail tethers secretory vesicles, and the motor transports them down polarized actin cables to the site of exocytosis.

Alleles↗

PubMed2006

Nearly neutral secretory vesicles in Drosophila nerve terminals.

The acidity of mammalian secretory vesicles drives concentration and processing of their contents. Here, pH-sensitive green fluorescent protein (GFP) variants show that the > or =30-fold (H+) difference between secretory vesicles (pH < or = 5.7) and the cytoplasm (pH = 7.2) in mammalian cells is not present in peptidergic and small synaptic vesicles of the Drosophila neuromuscular junction. First, we find that fluorescence from Topaz-tagged atrial natriuretic factor, a peptidergic vesicle pH indicator, is only modestly affected by collapsing the H+ gradient in type III synaptic boutons. Quantitation shows that peptidergic vesicles are nearly neutral (pH = 6.74 +/- 0.05), even when temperature is elevated. Furthermore, small synaptic vesicles in glutamatergic synaptic boutons, studied with synaptophluorin, are as alkaline as peptidergic vesicles. Finally, yellow fluorescent protein measurements show that cytoplasmic pH is only slightly different than in mammals (pH = 7.4). Thus, the marked acidity of mammalian secretory vesicles is not conserved in evolution, and a modest vesicular H+ gradient is sufficient for supporting neurotransmission.

Animals↗

PubMed2001

Dense core secretory vesicles revealed as a dynamic Ca(2+) store in neuroendocrine cells with a vesicle-associated membrane protein aequorin chimaera.

The role of dense core secretory vesicles in the control of cytosolic-free Ca(2+) concentrations ([Ca(2+)](c)) in neuronal and neuroendocrine cells is enigmatic. By constructing a vesicle-associated membrane protein 2-synaptobrevin.aequorin chimera, we show that in clonal pancreatic islet beta-cells: (a) increases in [Ca(2+)](c) cause a prompt increase in intravesicular-free Ca(2+) concentration ([Ca(2+)]SV), which is mediated by a P-type Ca(2+)-ATPase distinct from the sarco(endo) plasmic reticulum Ca(2+)-ATPase, but which may be related to the PMR1/ATP2C1 family of Ca(2+) pumps; (b) steady state Ca(2+) concentrations are 3-5-fold lower in secretory vesicles than in the endoplasmic reticulum (ER) or Golgi apparatus, suggesting the existence of tightly bound and more rapidly exchanging pools of Ca(2+); (c) inositol (1,4,5) trisphosphate has no impact on [Ca(2+)](SV) in intact or permeabilized cells; and (d) ryanodine receptor (RyR) activation with caffeine or 4-chloro-3-ethylphenol in intact cells, or cyclic ADPribose in permeabilized cells, causes a dramatic fall in [Ca(2+)](SV). Thus, secretory vesicles represent a dynamic Ca(2+) store in neuroendocrine cells, whose characteristics are in part distinct from the ER/Golgi apparatus. The presence of RyRs on secretory vesicles suggests that local Ca(2+)-induced Ca(2+) release from vesicles docked at the plasma membrane could participate in triggering exocytosis.

Adenosine Triphosphate↗