Stimulus-Dependent Alterations in Quantal Neurotransmitter Release

2006 ◽  
Vol 96 (6) ◽  
pp. 3082-3087 ◽  
Author(s):  
Chad P. Grabner ◽  
Aaron P. Fox
Keyword(s):  
Neurotransmitter Release ◽  
Chromaffin Cells ◽  
Time Course ◽  
Ion Concentration ◽  
Fusion Pore ◽  
Release Process ◽  
Large Dense Core Vesicles ◽  
Intracellular Calcium Ion ◽  
Individual Vesicle ◽  
Digitonin Permeabilization

Neurotransmitter release is a steep function of the intracellular calcium ion concentration ([Ca2+]i) at the release sites. Both the Ca2+ amplitude and the time course appear to be important for specifying neurotransmitter release. Ca2+ influx regulates the number of vesicles exocytosed as well as the amount of neurotransmitter each individual vesicle releases. In our study we stimulated mouse chromaffin cells in two different ways to alter Ca2+ presentation at the release sites. One method, digitonin permeabilization followed by exposure to Ca2+, allows for a large uniform global elevation of [Ca2+]i, whereas the second method, application of nicotine, depolarizes chromaffin cells and activates voltage-dependent Ca2+ channels, thereby producing more phasic and localized changes in [Ca2+]i. Using amperometry to monitor catecholamine release, we show that both kinds of stimuli elicit the exocytosis of similar quantities of neurotransmitter per large dense core vesicles (LDCVs) released. Even so, the release process was quite different for each stimulus; nicotine-elicited events were small and slow, whereas digitonin events were, in comparison, large and fast. In addition, the transient opening of the fusion pore, called the “foot,” was essentially absent in digitonin-stimulated cells, but was quite common in nicotine-stimulated cells. Thus even though both strong stimuli used in this study elicited the release of many vesicles it appears that the differences in the Ca2+ levels at the release sites were key determinants for the fusion and release of individual vesicles.

scholarly journals Neonatal Intermittent Hypoxia Leads to Long-Lasting Facilitation of Acute Hypoxia-Evoked Catecholamine Secretion From Rat Chromaffin Cells

2009 ◽  
Vol 101 (6) ◽  
pp. 2837-2846 ◽  
Author(s):  
Dangjai Souvannakitti ◽  
Ganesh K. Kumar ◽  
Aaron Fox ◽  
Nanduri R. Prabhakar
Keyword(s):  
Intermittent Hypoxia ◽  
Chromaffin Cells ◽  
Acute Hypoxia ◽  
Adult Life ◽  
Underlying Mechanism ◽  
Ion Concentration ◽  
Rat Pups ◽  
Intracellular Ca ◽  
Intracellular Calcium Ion ◽  
Calcium Ion Concentration

The objective of the present study was to examine the effects of intermittent hypoxia (IH) and sustained hypoxia (SH) on hypoxia-evoked catecholamine (CA) secretion from chromaffin cells in neonatal rats and assess the underlying mechanism(s). Experiments were performed on rat pups exposed to either IH (15-s hypoxia/5-min normoxia; 8 h/day) or SH (hypobaric hypoxia, 0.4 atm) or normoxia (controls) from P0 to P5. IH treatment facilitated hypoxia-evoked CA secretion and elevations in the intracellular calcium ion concentration ([Ca2+]i) and these responses were attenuated, but not abolished, by treatments designed to eliminate Ca2+ flux into cells (Ca2+-free medium or Cd2+), indicating that intracellular Ca2+ stores were augmented by IH. Norepinephrine (NE) and epinephrine (E) levels of adrenal medullae were elevated in IH-treated pups. IH treatment increased reactive oxygen species (ROS) production in adrenal medullae and antioxidant treatment prevented IH-induced facilitation of CA secretion, elevations in [Ca2+]i by hypoxia, and the up-regulation of NE and E. The effects of neonatal IH treatment on hypoxia-induced CA secretion and elevation in [Ca2+]i, CA, and ROS levels persisted in rats reared under normoxia for >30 days. In striking contrast, chromaffin cells from SH-treated animals exhibited attenuated hypoxia-evoked CA secretion. In SH-treated cells hypoxia-evoked elevations in [Ca2+]i, NE and E contents, and ROS levels were comparable with controls. These observations demonstrate that: 1) neonatal IH and SH evoke opposite effects on hypoxia-evoked CA secretion from chromaffin cells, 2) ROS signaling mediates the faciltatory effects of IH, and 3) the effects of neonatal IH on chromaffin cells persist into adult life.

scholarly journals Exocytosis from pancreatic β-cells: mathematical modelling of the exit of low-molecular-weight granule content

2010 ◽  
Vol 1 (1) ◽  
pp. 143-152 ◽  
Author(s):  
Juris Galvanovskis ◽  
Matthias Braun ◽  
Patrik Rorsman
Keyword(s):  
Molecular Weight ◽  
Membrane Receptors ◽  
Fusion Pore ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Release Process ◽  
Cell Plasma ◽  
Insulin Granules ◽  
Large Dense Core Vesicles ◽  
Insulin Exocytosis

Pancreatic β-cells use Ca 2+ -dependent exocytosis of large dense core vesicles to release insulin. Exocytosis in β-cells has been studied biochemically, biophysically and optically. We have previously developed a biophysical method to monitor release of endogenous intragranular constituents that are co-released with insulin. This technique involves the expression of ionotropic membrane receptors in the β-cell plasma membrane and enables measurements of exocytosis of individual vesicles with sub-millisecond resolution. Like carbon fibre amperometry, this method allows fine details of the release process, like the expansion of the fusion pore (the narrow connection between the granule lumen and the extracellular space), to be monitored. Here, we discuss experimental data obtained with this method within the framework of a simple mathematical model that describes the release of low-molecular constituents during exocytosis of the insulin granules. Our findings suggest that the fusion pore functions as a molecular sieve, allowing differential release of low- and high-molecular-weight granule constituents.

scholarly journals The V-ATPase membrane domain is a sensor of granular pH that controls the exocytotic machinery

2013 ◽  
Vol 203 (2) ◽  
pp. 283-298 ◽  
Author(s):  
Sandrine Poëa-Guyon ◽  
Mohamed Raafet Ammar ◽  
Marie Erard ◽  
Muriel Amar ◽  
Alexandre W. Moreau ◽  
...  
Keyword(s):  
Neurotransmitter Release ◽  
Chromaffin Cells ◽  
Secretory Granules ◽  
Secretory Vesicle ◽  
Fusion Pore ◽  
Membrane Domain ◽  
Reversible Dissociation ◽  
Open Time ◽  
Vesicle Exocytosis ◽  
Kinetics Of

Several studies have suggested that the V0 domain of the vacuolar-type H+-adenosine triphosphatase (V-ATPase) is directly implicated in secretory vesicle exocytosis through a role in membrane fusion. We report in this paper that there was a rapid decrease in neurotransmitter release after acute photoinactivation of the V0 a1-I subunit in neuronal pairs. Likewise, inactivation of the V0 a1-I subunit in chromaffin cells resulted in a decreased frequency and prolonged kinetics of amperometric spikes induced by depolarization, with shortening of the fusion pore open time. Dissipation of the granular pH gradient was associated with an inhibition of exocytosis and correlated with the V1–V0 association status in secretory granules. We thus conclude that V0 serves as a sensor of intragranular pH that controls exocytosis and synaptic transmission via the reversible dissociation of V1 at acidic pH. Hence, the V-ATPase membrane domain would allow the exocytotic machinery to discriminate fully loaded and acidified vesicles from vesicles undergoing neurotransmitter reloading.

scholarly journals Chromogranin A, the major lumenal protein in chromaffin granules, controls fusion pore expansion

2018 ◽  
Vol 151 (2) ◽  
pp. 118-130 ◽  
Author(s):  
Prabhodh S. Abbineni ◽  
Mary A. Bittner ◽  
Daniel Axelrod ◽  
Ronald W. Holz
Keyword(s):  
Plasma Membrane ◽  
Small Molecules ◽  
Chromogranin A ◽  
Chromaffin Cells ◽  
Fusion Pore ◽  
Chromaffin Granules ◽  
Chromaffin Granule ◽  
Chromogranin B ◽  
Tissue Plasminogen ◽  
Pore Expansion

Upon fusion of the secretory granule with the plasma membrane, small molecules are discharged through the immediately formed narrow fusion pore, but protein discharge awaits pore expansion. Recently, fusion pore expansion was found to be regulated by tissue plasminogen activator (tPA), a protein present within the lumen of chromaffin granules in a subpopulation of chromaffin cells. Here, we further examined the influence of other lumenal proteins on fusion pore expansion, especially chromogranin A (CgA), the major and ubiquitous lumenal protein in chromaffin granules. Polarized TIRF microscopy demonstrated that the fusion pore curvature of granules containing CgA-EGFP was long lived, with curvature lifetimes comparable to those of tPA-EGFP–containing granules. This was surprising because fusion pore curvature durations of granules containing exogenous neuropeptide Y-EGFP (NPY-EGFP) are significantly shorter (80% lasting <1 s) than those containing CgA-EGFP, despite the anticipated expression of endogenous CgA. However, quantitative immunocytochemistry revealed that transiently expressed lumenal proteins, including NPY-EGFP, caused a down-regulation of endogenously expressed proteins, including CgA. Fusion pore curvature durations in nontransfected cells were significantly longer than those of granules containing overexpressed NPY but shorter than those associated with granules containing overexpressed tPA, CgA, or chromogranin B. Introduction of CgA to NPY-EGFP granules by coexpression converted the fusion pore from being transient to being longer lived, comparable to that found in nontransfected cells. These findings demonstrate that several endogenous chromaffin granule lumenal proteins are regulators of fusion pore expansion and that alteration of chromaffin granule contents affects fusion pore lifetimes. Importantly, the results indicate a new role for CgA. In addition to functioning as a prohormone, CgA plays an important role in controlling fusion pore expansion.

scholarly journals Trachynilysin mediates SNARE-dependent release of catecholamines from chromaffin cells via external and stored Ca2+

2000 ◽  
Vol 113 (7) ◽  
pp. 1119-1125 ◽  
Author(s):  
F.A. Meunier ◽  
C. Mattei ◽  
P. Chameau ◽  
G. Lawrence ◽  
C. Colasante ◽  
...  
Keyword(s):  
Chromaffin Cells ◽  
Motor Nerve ◽  
Neuroendocrine Cells ◽  
Dense Core ◽  
Frog Neuromuscular Junction ◽  
Dense Core Vesicles ◽  
Protein Receptor ◽  
Large Dense Core Vesicles ◽  
Quantal Transmitter Release ◽  
Bovine Chromaffin Cells

Trachynilysin, a 159 kDa dimeric protein purified from stonefish (Synanceia trachynis) venom, dramatically increases spontaneous quantal transmitter release at the frog neuromuscular junction, depleting small clear synaptic vesicles, whilst not affecting large dense core vesicles. The basis of this insensitivity of large dense core vesicles exocytosis was examined using a fluorimetric assay to determine whether the toxin could elicit catecholamine release from bovine chromaffin cells. Unlike the case of the motor nerve endings, nanomolar concentrations of trachynilysin evoked sustained Soluble N-ethylmaleimide-sensitive fusion protein Attachment Protein REceptor-dependent exocytosis of large dense core vesicles, but only in the presence of extracellular Ca2+. However, this response to trachynilysin does not rely on Ca2+ influx through voltage-activated Ca2+ channels because the secretion was only slightly affected by blockers of L, N and P/Q types. Instead, trachynilysin elicited a localized increase in intracellular fluorescence monitored with fluo-3/AM, that precisely co-localized with the increase of fluorescence resulting from caffeine-induced release of Ca2+ from intracellular stores. Moreover, depletion of the latter stores inhibited trachynilysin-induced exocytosis. Thus, the observed requirement of external Ca2+ for stimulation of large dense core vesicles exocytosis from chromaffin cells implicates plasma membrane channels that signal efflux of Ca2+ from intracellular stores. This study also suggests that the bases of exocytosis of large dense core vesicles from motor nerve terminals and neuroendocrine cells are distinct.

Synaptic interactions between mammalian central neurons in cell culture. II. Quantal Analysis of EPSPs

1983 ◽  
Vol 49 (6) ◽  
pp. 1442-1458 ◽  
Author(s):  
P. G. Nelson ◽  
K. C. Marshall ◽  
R. Y. Pun ◽  
C. N. Christian ◽  
W. H. Sheriff ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Culture Medium ◽  
Ion Concentration ◽  
Release Mechanism ◽  
Drg Neurons ◽  
Release Process ◽  
Central Neurons ◽  
Synaptic Interactions ◽  
Presynaptic Release ◽  
The Relationship

The presynaptic release mechanism involved in excitatory synaptic connections between neurons in cell cultures of fetal mouse spinal cord were studied by statistical analysis of intracellularly recorded postsynaptic responses. Quantal parameters were determined for the EPSPs evoked in spinal cord (SC) neurons by stimulation of either other SC or dorsal root ganglion (DRG) neurons. Transmitter release was manipulated by varying the Ca2+ and Mg2+ content of the culture medium. The release process was represented better by binomial than by Poisson statistics. A method was derived for obtaining the probability of release and the number of release elements. The quantal content and the number of release elements were substantially higher for the SC-SC connection than for the DRG-SC connection. This was partially compensated for by a larger quantal amplitude for the DRG-SC connection. There was some indication that the probability of release was higher for the SC-SC connection. The relationship between transmitter output and effective external Ca2+ ion concentration was approximately linear.

Influence of the Membrane Potential on the Intracellular Light Induced Ca2+ -Concentration Change of the Limulus Ventral Photoreceptor Monitored by Arsenazo III under Voltage Clamp Conditions

1984 ◽  
Vol 39 (9-10) ◽  
pp. 986-992 ◽  
Author(s):  
I. Ivens ◽  
H. Stieve
Keyword(s):  
Voltage Clamp ◽  
Light Flash ◽  
Membrane Voltage ◽  
Ion Concentration ◽  
Arsenazo Iii ◽  
Substantial Part ◽  
Voltage Range ◽  
Intracellular Ca ◽  
Intracellular Calcium Ion ◽  
Pass Through

Abstract The light induced transmission change (Arsenazo signal) of an Arsenazo III injected ventral photoreceptor cell of Limulus polyphemus was studied under voltage clamp. The transmission change which represents a change of free intracellular calcium ion concentration, [Ca2+]i, was investigated for its dependence upon membrane voltage. The peak amplitude of the Arsenazo signal decreases in a linear fashion with the clamp voltage in the examined voltage range (from -80 to + 40 mV). In low Ca2+ saline ([Ca2+]e = 250 μᴍ) this decrease in the amplitude of the Arsenazo signal was more pronounced, while in saline with increased Ca2+ ([Ca2+]e = 40, 50 and 100 mᴍ), there is almost no change of the Arsenazo signal with varied membrane voltage. The recovery of the Arsenazo signal (i.e. recovery of the transmission back to the value before the light flash) is faster during hyperpolarization, this recovery being slowed down when the cell is depolarized. From these experiments it is concluded that a substantial part of the Arsenazo signal is due to a light induced influx of Ca2+ from the extracellular space across the cell membrane into the cytoplasma. Conceivably the Ca2+ could pass through light activated Na+ channels. Subsequently the increased intracellular Ca2+ is lowered to the preillumination level, by a membrane voltage dependent mechanism possibly an Na+-Ca2+ exchange. The data do not exclude the possibility that a part of the Ca2+ responsible for the Arsenazo signal is released from intracellular stores.

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