Synaptophysin Regulates Fusion Pores and Exocytosis Mode in Chromaffin Cells

In chromaffin cells, the kinetics of fusion pore expansion vary depending on which synaptotagmin isoform (Syt-1 or Syt-7) drives release. Our recent studies have shown that fusion pores of granules harboring Syt-1 expand more rapidly than those harboring Syt-7. Here we sought to define the structural specificity of synaptotagmin action at the fusion pore by manipulating the Ca2+-binding C2B module. We generated a chimeric Syt-1 in which its C2B Ca2+-binding loops had been exchanged for those of Syt-7. Fusion pores of granules harboring a Syt-1 C2B chimera with all three Ca2+-binding loops of Syt-7 (Syt-1:7C2B123) exhibited slower rates of fusion pore expansion and neuropeptide cargo release relative to WT Syt-1. After fusion, this chimera also dispersed more slowly from fusion sites than WT protein. We speculate that the Syt-1:7 C2B123 and WT Syt-1 are likely to differ in their interactions with Ca2+ and membranes. Subsequent in vitro and in silico data demonstrated that the chimera exhibits a higher affinity for phospholipids than WT Syt-1. We conclude that the affinity of synaptotagmin for the plasma membrane, and the rate at which it releases the membrane, contribute in important ways to the rate of fusion pore expansion.

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Morphological Observations on the Granule Types in Rabbit Extra-Adrenal Chromaffin Cells.

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100115213 ◽

1975 ◽

Vol 33 ◽

pp. 318-319

Author(s):

Joe A. Mascorro ◽

Robert D. Yates

Keyword(s):

Chromaffin Cells ◽

Sodium Phosphate ◽

Ganglion Cells ◽

Ultrastructural Level ◽

Osmic Acid ◽

Adrenal Chromaffin Cells ◽

Potassium Iodate ◽

Perfusion Fluid ◽

New Zealand White Rabbits ◽

Adrenal Chromaffin

Extra-adrenal chromaffin organs (abdominal paraganglia) constitute rich sources of catecholamines. It is believed that these bodies contain norepinephrine exclusively. However, the present workers recently observed epinephrine type granules in para- ganglion cells. This report investigates catecholamine containing granules in rabbit paraganglia at the ultrastructural level.New Zealand white rabbits (150-170 grams) were anesthetized with 50 mg/kg Nembutal (IP) and perfused with 3% glutaraldehyde buffered with 0.2M sodium phosphate, pH 7.3. The retroperitoneal tissue blocks were removed and placed in perfusion fluid for 4 hours. The abdominal paraganglia were dissected from the blocks, diced, washed in phosphate buffer and fixed in 1% osmic acid buffered with phosphate. In other animals, the glutaraldehyde perfused tissue blocks were immersed for 1 hour in 3% glutaraldehyde/2.5% potassium iodate buffered as before. The paraganglia were then diced, separated into two vials and washed in the buffer. A portion of this tissue received osmic acid fixation.

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Alleviation of pain and depression by specific neural transplants: Fine structural correlates

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100129966 ◽

1992 ◽

Vol 50 (2) ◽

pp. 1066-1067

Author(s):

George D. Pappas ◽

Jacqueline Sagen

Keyword(s):

Opioid Peptides ◽

Chromaffin Cells ◽

Pain Sensitivity ◽

Opioid Peptide ◽

Local Source ◽

Pain Models ◽

Neural Transplants ◽

Adrenergic Antagonists ◽

Csf Levels ◽

Donor Source

We have been interested in the use of neural transplants mainly as a local source of neuroactive substances, rather than as a replacement for damaged neural circuities. In particular, we have been exploring the possibilities of reducing pain by transplants of opioid peptide producing cells, and reducing depression by transplants of monoamine-producing cells. For the past several years, work in our laboratory has demonstrated in both acute and chronic pain models that transplantation of adrenal medullary tissue or isolated chromaffin cells into CNS pain modulatory regions can reduce pain sensitivity in rodents. Chromaffin cells were chosen as donor source since they produce high levels of both opioid peptides and catecholamines, substances which independently, and probably synergistically, reduce pain sensitivity when injected locally into the spinal cord. The analgesia produced by these transplants most likely results from the release of both opioid peptides and catecholamines, since it can be blocked or attenuated by opiate or adrenergic antagonists, respectively. Furthermore, CSF levels of met-enkephalin and catecholamines are increased by the transplants.

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Immunogold labeling of the calcium binding protein synexin in isolated adrenal chromaffin granules and chromaffin cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100162326 ◽

1990 ◽

Vol 48 (3) ◽

pp. 952-953

Author(s):

Gemma A.J. Kuijpers ◽

Harvey B. Pollard

Keyword(s):

Adrenal Medulla ◽

Calcium Binding ◽

Chromaffin Cells ◽

Cell Activation ◽

Structural Information ◽

Dependent Manner ◽

Chromaffin Granules ◽

Immuno Electron Microscopy ◽

Ultrathin Sections ◽

Adrenal Chromaffin

Exocytotic fusion of granules in the adrenal medulla chromaffin cell is triggered by a rise in the concentration of cytosolic Ca2+ upon cell activation. The protein synexin, annexin VII, was originally found in the adrenal medulla and has been shown to cause aggregation and to support fusion of chromaffin granules in a Ca2+-dependent manner. We have previously suggested that synexin may there fore play a role in the exocytotic fusion process. In order to obtain more structural information on synexin, we performed immuno-electron microscopy on frozen ultrathin sections of both isolated chromaffin granules and chromaffin cells.Chromaffin granules were isolated from bovine adrenal medulla, and synexin was isolated from bovine lung. Granules were incubated in the presence or absence of synexin (24 μg per mg granule protein) and Ca2+ (1 mM), which induces maximal granule aggregation, in 0.3M sucrose-40m MMES buffer(pH 6.0). Granules were pelleted, washed twice in buffer without synexin and fixed with 2% glutaraldehyde- 2% para formaldehyde in 0.1 M phosphate buffer (GA/PFA) for 30 min. Chromaffin cells were isolated and cultured for 3-5 days, and washed and incubated in Krebs solution with or without 20 uM nicotine. Cells were fixed 90 sec after on set of stimulation with GA/PFA for 30 min. Fixed granule or cell pellets were washed, infiltrated with 2.3 M sucrose in PBS, mounted and frozen in liquid N2.

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Fusion and Fission Processes in Exocytosis: Possible Roles for Synexin and Osmotic Lysis in the Two Events

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s1431927600002634 ◽

1980 ◽

Vol 38 ◽

pp. 594-597

Author(s):

H.B. Pollard ◽

C.E. Creutz ◽

C.J. Pazoles ◽

J.H. Scott

Keyword(s):

Small Molecules ◽

Chromaffin Cells ◽

Adrenal Glands ◽

General Concept ◽

Extracellular Calcium ◽

Large Protein ◽

Granule Membrane ◽

Osmotic Lysis ◽

Per Se ◽

Chemical Evidence

Exocytosis is a general concept describing secretion of enzymes, hormones and transmitters that are otherwise sequestered in intracellular granules. Chemical evidence for this concept was first gathered from studies on chromaffin cells in perfused adrenal glands, in which it was found that granule contents, including both large protein and small molecules such as adrenaline and ATP, were released together while the granule membrane was retained in the cell. A number of exhaustive reviews of this early work have been published and are summarized in Reference 1. The critical experiments demonstrating the importance of extracellular calcium for exocytosis per se were also first performed in this system (2,3), further indicating the substantial service given by chromaffin cells to those interested in secretory phenomena over the years.

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