Recycling of a secretory granule membrane protein after stimulated secretion

1993 ◽  
Vol 106 (2) ◽  
pp. 649-655 ◽  
Author(s):  
S.M. Hurtley
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Secretory Granule ◽  
Chromaffin Cells ◽  
Secretory Granules ◽  
Primary Cultures ◽  
Dopamine Beta Hydroxylase ◽  
Granule Membrane ◽  
Antibody Complex ◽  
Adrenal Chromaffin

Recycling of a secretory granule membrane protein, dopamine-beta-hydroxylase, was examined in primary cultures of bovine adrenal chromaffin cells. Cells were stimulated to secrete in the presence of antibodies directed against the luminal domain of dopamine-beta-hydroxylase. The location of the antibodies after various times of reincubation and after a second secretory stimulus was assessed using immunofluorescence microscopy. Stimulation led to the exposure of dopamine-beta-hydroxylase at the plasma membrane, which could be detected by a polyclonal antibody in living and fixed cells. The plasma membrane dopamine-beta-hydroxylase, either alone or complexed with antibody, was rapidly internalized after removal of the secretagogue. Internalized protein-antibody complex remained stable for at least 24 hours of reculture. Twenty four hours after stimulation the cells with internalized antibody could respond to further stimulation and some of the antibody was re-exposed at the plasma membrane. These findings were confirmed using FACS analysis. This suggests that the antibody-protein complex had returned to secretory granules that could respond to further secretagogue stimulation.

scholarly journals Bovine chromaffin granule membranes undergo Ca(2+)-regulated exocytosis in frog oocytes.

1992 ◽  
Vol 116 (2) ◽  
pp. 359-365 ◽  
Author(s):  
D Scheuner ◽  
C D Logsdon ◽  
R W Holz
Keyword(s):  
Chromaffin Cells ◽  
Secretory Vesicle ◽  
Xenopus Laevis Oocytes ◽  
Chromaffin Granules ◽  
Chromaffin Granule ◽  
Vesicle Membrane ◽  
Sequence Of Events ◽  
Dopamine Beta Hydroxylase ◽  
Granule Membrane ◽  
Adrenal Chromaffin

We have devised a new method that permits the investigation of exogenous secretory vesicle function using frog oocytes and bovine chromaffin granules, the secretory vesicles from adrenal chromaffin cells. Highly purified chromaffin granule membranes were injected into Xenopus laevis oocytes. Exocytosis was detected by the appearance of dopamine-beta-hydroxylase of the chromaffin granule membrane in the oocyte plasma membrane. The appearance of dopamine-beta-hydroxylase on the oocyte surface was strongly Ca(2+)-dependent and was stimulated by coinjection of the chromaffin granule membranes with InsP3 or Ca2+/EGTA buffer (18 microM free Ca2+) or by incubation of the injected oocytes in medium containing the Ca2+ ionophore ionomycin. Similar experiments were performed with a subcellular fraction from cultured chromaffin cells enriched with [3H]norepinephrine-containing chromaffin granules. Because the release of [3H]norepinephrine was strongly correlated with the appearance of dopamine-beta-hydroxylase on the oocyte surface, it is likely that intact chromaffin granules and chromaffin granule membranes undergo exocytosis in the oocyte. Thus, the secretory vesicle membrane without normal vesicle contents is competent to undergo the sequence of events leading to exocytosis. Furthermore, the interchangeability of mammalian and amphibian components suggests substantial biochemical conservation of the regulated exocytotic pathway during the evolutionary progression from amphibians to mammals.

scholarly journals Distinct fusion properties of synaptotagmin-1 and synaptotagmin-7 bearing dense core granules

2014 ◽  
Vol 25 (16) ◽  
pp. 2416-2427 ◽  
Author(s):  
Tejeshwar C. Rao ◽  
Daniel R. Passmore ◽  
Andrew R. Peleman ◽  
Madhurima Das ◽  
Edwin R. Chapman ◽  
...  
Keyword(s):  
Chromaffin Cells ◽  
Secretory Granules ◽  
Fusion Pore ◽  
Cargo Proteins ◽  
Granule Membrane ◽  
Synaptotagmin 1 ◽  
Physiological Homeostasis ◽  
Dense Core Granules ◽  
Adrenal Chromaffin ◽  
Granule Membrane Proteins

Adrenal chromaffin cells release hormones and neuropeptides that are essential for physiological homeostasis. During this process, secretory granules fuse with the plasma membrane and deliver their cargo to the extracellular space. It was once believed that fusion was the final regulated step in exocytosis, resulting in uniform and total release of granule cargo. Recent evidence argues for nonuniform outcomes after fusion, in which cargo is released with variable kinetics and selectivity. The goal of this study was to identify factors that contribute to the different outcomes, with a focus on the Ca2+-sensing synaptotagmin (Syt) proteins. Two Syt isoforms are expressed in chromaffin cells: Syt-1 and Syt-7. We find that overexpressed and endogenous Syt isoforms are usually sorted to separate secretory granules and are differentially activated by depolarizing stimuli. In addition, overexpressed Syt-1 and Syt-7 impose distinct effects on fusion pore expansion and granule cargo release. Syt-7 pores usually fail to expand (or reseal), slowing the dispersal of lumenal cargo proteins and granule membrane proteins. On the other hand, Syt-1 diffuses from fusion sites and promotes the release of lumenal cargo proteins. These findings suggest one way in which chromaffin cells may regulate cargo release is via differential activation of synaptotagmin isoforms.

scholarly journals Localized topological changes of the plasma membrane upon exocytosis visualized by polarized TIRFM

2010 ◽  
Vol 188 (3) ◽  
pp. 415-428 ◽  
Author(s):  
Arun Anantharam ◽  
Bibiana Onoa ◽  
Robert H. Edwards ◽  
Ronald W. Holz ◽  
Daniel Axelrod
Keyword(s):  
Plasma Membrane ◽  
Total Internal Reflection ◽  
Secretory Granules ◽  
Membrane Topology ◽  
Membrane Complex ◽  
Polarization Technique ◽  
Granule Membrane ◽  
Guanosine Triphosphatase ◽  
Adrenal Chromaffin ◽  
Image Simulations

Total internal reflection fluorescence microscopy (TIRFM) images the plasma membrane–cytosol interface and has allowed insights into the behavior of individual secretory granules before and during exocytosis. Much less is known about the dynamics of the other partner in exocytosis, the plasma membrane. In this study, we report the implementation of a TIRFM-based polarization technique to detect rapid submicrometer changes in plasma membrane topology as a result of exocytosis. A theoretical analysis of the technique is presented together with image simulations of predicted topologies of the postfusion granule membrane–plasma membrane complex. Experiments on diI-stained bovine adrenal chromaffin cells using polarized TIRFM demonstrate rapid and varied submicrometer changes in plasma membrane topology at sites of exocytosis that occur immediately upon fusion. We provide direct evidence for a persistent curvature in the exocytotic region that is altered by inhibition of dynamin guanosine triphosphatase activity and is temporally distinct from endocytosis measured by VMAT2-pHluorin.

scholarly journals Visualization of the exocytosis/endocytosis secretory cycle in cultured adrenal chromaffin cells.

1983 ◽  
Vol 97 (6) ◽  
pp. 1906-1917 ◽  
Author(s):  
J H Phillips ◽  
K Burridge ◽  
S P Wilson ◽  
N Kirshner
Keyword(s):  
Chromaffin Cells ◽  
Similar Rate ◽  
Chromaffin Granule ◽  
Secretory Cycle ◽  
Surface Patches ◽  
Dopamine Beta Hydroxylase ◽  
Antigen Complex ◽  
Granule Membrane ◽  
Adrenal Chromaffin ◽  
Concanavalin A Receptors

Cultured bovine adrenal medullary chromaffin cells were stimulated to secrete catecholamines by addition of veratridine or nicotine. The formation of an exocytotic pit exposes a major secretory granule membrane antigen, the enzyme dopamine beta-hydroxylase, to the external medium. By including antiserum to this enzyme in the medium, we were able to visualize sites of exocytosis by decoration of bound antibody using a fluorescent second antibody. Internalization of this antibody-antigen complex was then followed in chase experiments: approximately half the surface complex was internalized in 15-30 min. In other experiments, secretion was triggered in the absence of antiserum, and surface enzyme was revealed by binding antibodies at various times after secretion had been halted by an antagonist. Surface patches of antigen remained discrete from the bulk of the plasma membrane for at least 30 min, although a substantial proportion of the antigen was internalized within this time. Cell surface concanavalin A receptors were internalized at a roughly similar rate, suggesting that mechanisms may be similar. After internalization, chromaffin granule membranes fused to larger structures, possibly lysosomes, and were transported over a few hours to the perinuclear region of the cell.

Dissection of stages in exocytosis in the adrenal chromaffin cell with use of trifluoperazine

1982 ◽  
Vol 216 (1202) ◽  
pp. 111-115 ◽  
Keyword(s):  
Plasma Membrane ◽  
Chromaffin Cells ◽  
Chromaffin Cell ◽  
Morphological Changes ◽  
Secretory Granules ◽  
Adrenal Chromaffin Cell ◽  
Sensitive Stage ◽  
Adrenal Chromaffin ◽  
Two Stages ◽  
Stimulation Of

Stimulation of isolated chromaffin cells with carbamylcholine led to a number of morphological changes, indicative of exocytosis, apparently resulting from translocation of secretory granules to the plasma membrane and their subsequent fusion with the plasma membrane to release their contents. However, stimulation in the presence of trifluoperazine resulted only in the accumulation of secretory granules close to the plasma membrane. Thus exocytosis could be divided into two stages: a trifluoperazine-insensitive stage involving translocation of secretory granules to the plasma membrane and a second trifluoperazine-sensitive stage resulting in granule-plasma membrane fusion.

scholarly journals CONCOMITANT SYNTHESIS OF MEMBRANE PROTEIN AND EXPORTABLE PROTEIN OF THE SECRETORY GRANULE IN RAT PAROTID GLAND

1971 ◽  
Vol 50 (1) ◽  
pp. 187-200 ◽  
Author(s):  
Abraham Amsterdam ◽  
Michael Schramm ◽  
Itzhak Ohad ◽  
Yoram Salomon ◽  
Zvi Selinger
Keyword(s):  
Amino Acids ◽  
Secretory Granule ◽  
De Novo ◽  
Secretory Granules ◽  
Specific Radioactivity ◽  
Staining Intensity ◽  
Granule Membrane ◽  
Rat Parotid ◽  
Cell Fractions

After enzyme secretion the membrane of the secretory granule, which had been fused to the cell membrane, was resorbed into the cell. Experiments were therefore carried out to test whether formation of new secretory granules involves reutilization of the resorbed membrane or synthesis of a new membrane, de novo, from amino acids. Incorporation of amino acids-14C into proteins of various cell fractions was measured in vivo, 30, 120, and. 300 min after labeling. At all times the specific radioactivity of the secretory granule membrane was about equal to that of the granule's exportable content. At 120 and 300 min the specific radioactivity of the granule membrane and of the granule content was much higher than that of any other subcellular fraction. It is therefore concluded that the protein of the membrane is synthesized de novo concomitantly with the exportable protein. The proteins of the granule membrane could be distinguished from those of the granule content by gel electrophoresis. All major bands were labeled proportionately to their staining intensity. The amino acid composition of the secretory granule membrane was markedly different from that of the granule's content and also from that of the mitochondrial membrane. The granule membrane showed a high proline content, 30 moles/100 moles amino acids. The analyses show that the radioactivity of the granule membrane is indeed inherent in its proteins and is not due to contamination by other fractions. The possibility is considered that the exportable protein leaves the endoplasmic reticulum already enveloped by the newly synthesized membrane.

scholarly journals Secretion-related uptake of horseradish peroxidase in neurohypophysial axons.

1976 ◽  
Vol 70 (2) ◽  
pp. 294-303 ◽  
Author(s):  
D T Theodosis ◽  
J Dreifuss ◽  
M C Harris ◽  
L Orci
Keyword(s):  
Plasma Membrane ◽  
Electrical Stimulation ◽  
Horseradish Peroxidase ◽  
Secretory Granule ◽  
Morphometric Analysis ◽  
Neurosecretory Granules ◽  
Neurohypophysial Hormones ◽  
Axon Terminals ◽  
Granule Membrane ◽  
Stimulation Of

During secretion of the neurohypophysial hormones, oxytocin and vasopressin, secretory granule membrane is added to the plasma membrane of the axon terminals. It is generally assumed that subsequent internalization of this additional membrane occurs by endocytosis. In order to study this process, we have traced the uptake of intravenously injected horseradish peroxidase by neurohypophysial axons in rats and golden hamsters. Peroxidase reaction product within the secretory axons was found mainly in vacuolar and C-shaped structures of a size comparable with or larger than the neurosecretory granules. Our observations suggest that these large horseradish peroxidase (HRP)-impregnated vacuoles arise directly by a form of macropinocytosis. Morphometric analysis indicated that this form of membrane retrieval increased significantly after the two types of stimuli used, reversible hemorrhage and electrical stimulation of the pituitary stalk. Microvesicular uptake of HRP was found to be comparatively less.

scholarly journals Targeted Ablation of the Chromogranin A (Chga) Gene: Normal Neuroendocrine Dense-Core Secretory Granules and Increased Expression of Other Granins

2006 ◽  
Vol 20 (8) ◽  
pp. 1935-1947 ◽  
Author(s):  
Geoffrey N. Hendy ◽  
Tong Li ◽  
Martine Girard ◽  
Richard C. Feldstein ◽  
Shree Mulay ◽  
...  
Keyword(s):  
Secretory Granule ◽  
Chromogranin A ◽  
Chromaffin Cells ◽  
Secretory Granules ◽  
Hormone Secretion ◽  
Proteolytic Processing ◽  
Dense Core ◽  
Secretory Process ◽  
Chromogranin B ◽  
Developmental Abnormalities

Abstract Chromogranin A (CgA), originally identified in adrenal chromaffin cells, is a member of the granin family of acidic secretory glycoproteins that are expressed in endocrine cells and neurons. CgA has been proposed to play multiple roles in the secretory process. Intracellularly, CgA may control secretory granule biogenesis and target neurotransmitters and peptide hormones to granules of the regulated pathway. Extracellularly, peptides formed as a result of proteolytic processing of CgA may regulate hormone secretion. To investigate the role of CgA in the whole animal, we created a mouse mutant null for the Chga gene. These mice are viable and fertile and have no obvious developmental abnormalities, and their neural and endocrine functions are not grossly impaired. Their adrenal glands were structurally unremarkable, and morphometric analyses of chromaffin cells showed vesicle size and number to be normal. However, the excretion of epinephrine, norepinephrine, and dopamine was significantly elevated in the Chga null mutants. Adrenal medullary mRNA and protein levels of other dense-core secretory granule proteins including chromogranin B, and secretogranins II to VI were up-regulated 2- to 3-fold in the Chga null mutant mice. Hence, the increased expression of the other granin family members is likely to compensate for the Chga deficiency.

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