The immediately releasable vesicle pool: highly coupled secretion in chromaffin and other neuroendocrine cells (original) (raw)
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The Immediately Releasable Pool of Secretory Vesicles in Adrenal Chromaffin Cells
2018
The exocytosis of hormone-filled vesicles in adrenal chromaffin cells is triggered by localized Ca gradients that develops after the activation of voltage-dependent Ca channels. To reach the fusion competent state the vesicles go through a variety of maturation steps, including the mobilization through the cytoskeleton, the docking to membrane, and the priming of the exocytotic molecular machinery. However, the fusion readiness of vesicles will also depend on their proximity to the Ca source. The immediately releasable pool is a small group of ready–to-fuse vesicles, whose fusion is tightly coupled to Ca entry through channels. Recent work indicates that such a coupling is not produced by a random distribution between parts, but is the result of a specific interaction of secretory vesicles with P/Q-type Ca channels. The immediately releasable pool is able to sustain with high efficiency the secretion triggered by brief depolarizations applied at low frequencies, like action potentia...
Journal of neurochemistry, 2016
The pattern of stimulation defines important characteristics of the secretory process in neurons and neuroendocrine cells, including the pool of secretory vesicles being recruited, the type and amount of transmitters released, the mode of membrane retrieval and the mechanisms associated with vesicle replenishment. This review analyzes the mechanisms that regulate these processes in chromaffin cells, as well as in other neuroendocrine and neuronal models. A common factor in these mechanisms is the spatial and temporal distribution of the Ca(2+) signal generated during cell stimulation. For instance, neurosecretory cells and neurons have pools of vesicles with different locations with respect to Ca(2+) channels, and those pools are therefore differentially recruited following different patterns of stimulation. In this regard, a brief stimulus will induce the exocytosis of a small pool of vesicles that is highly coupled to voltage-dependent Ca(2+) channels, whereas longer or more inten...
Vesicle Pools: Lessons from Adrenal Chromaffin Cells
Frontiers in Synaptic Neuroscience, 2011
The adrenal chromaffin cell serves as a model system to study fast Ca2+-dependent exocytosis. Membrane capacitance measurements in combination with Ca2+ uncaging offers a temporal resolution in the millisecond range and reveals that catecholamine release occurs in three distinct phases. Release of a readily releasable (RRP) and a slowly releasable (SRP) pool are followed by sustained release, due to maturation, and release of vesicles which were not release-ready at the start of the stimulus. Trains of depolarizations, a more physiological stimulus, induce release from a small immediately releasable pool of vesicles residing adjacent to calcium channels, as well as from the RRP. The SRP is poorly activated by depolarization. A sequential model, in which non-releasable docked vesicles are primed to a slowly releasable state, and then further mature to the readily releasable state, has been proposed. The docked state, dependent on membrane proximity, requires SNAP-25, synaptotagmin, and syntaxin. The ablation or modification of SNAP-25 and syntaxin, components of the SNARE complex, as well as of synaptotagmin, the calcium sensor, and modulators such complexins and Snapin alter the properties and/or magnitudes of different phases of release, and in particular can ablate the RRP. These results indicate that the composition of the SNARE complex and its interaction with modulatory molecules drives priming and provides a molecular basis for different pools of releasable vesicles.
Cytosolic calcium facilitates release of secretory products after exocytotic vesicle fusion
FEBS Letters, 1995
We monitored single vesicle exocytosis by simultaneous measurements of cell membrane capacitance as an indicator of fusion and amperometric detection of serotonin release. We show here that vesicle-plasma membrane fusion in rat mast cell granules is followed by a variable, exponentially distributed, delay before bulk release. This delay reflects the time required for the expansion of the exocytotic fusion pore, lasting, on average, 231 ms in resting cytosolic calcium, [Ca2+L (50 nM). In the presence of [Ca2+]i in the low micromollar range, the lag between fusion and release was reduced to 123 ms. The characteristics of the amperometric signals were unchanged by [Ca2+]i. These resuits show a novel site of regulation in the exocytotic process, the fusion pore, which may represent a different mechanism facilitating transmitter release.
1999
Calcium-dependent activator protein for secretion (CAPS) is a neural/endocrine cell-specific protein that has been shown to function at the Ca(2+)-dependent triggering step of dense-core vesicle (DCV) exocytosis in permeabilized PC12 cells. To evaluate the function of CAPS under physiological conditions, we introduced affinity-purified anti-CAPS IgGs into calf adrenal chromaffin (AC) cells via a patch pipette and tested the kinetics of catecholamine secretion using both amperometric and membrane capacitance techniques. The antibodies reacted with a single major approximately 145 kDa protein in AC cells based on immunoblot analysis. AC cells stimulated with sequential trains of action potentials at 7 Hz resulted in successive secretory episodes of equivalent magnitude. When either of two different anti-CAPS IgGs or their Fab fragments were present, a rapid and progressive inhibition of catecholamine release ensued to a maximum of >80%. The effect was specific because preabsorption...
Docked Secretory Vesicles Undergo Ca2+-activated Exocytosis in a Cell-free System
Journal of Biological Chemistry, 1997
The Ca 2؉-activated fusion of secretory vesicles with the plasma membrane responsible for regulated neurotransmitter and hormone secretion has previously been studied in permeable neuroendocrine cells, where requirements for ATP and cytosolic proteins were identified. As reported here, Ca 2؉-activated fusion mechanisms are also preserved following cell homogenization. The release of norepinephrine (NE) and other vesicle constituents from a PC12 cell membrane fraction was activated by micromolar Ca 2؉ (EC 50 ϳ 3 M) and exhibited a dependence upon MgATP and cytosol. Ca 2؉-dependent NE release was inhibited by botulinum neurotoxins and by CAPS (Ca 2؉-dependent activator protein for secretion) antibody implying that syntaxin, synaptobrevin, SNAP-25 (synaptosomal-associated protein of 25 kDa), and CAPS are required for regulated exocytosis in this system. The exocytosis-competent membrane fraction consisted of rapidly sedimenting dense core vesicles associated with plasma membrane fragments. Free vesicles did not release NE either in the absence or presence of plasma membranes, indicating that only docked vesicles were competent for exocytosis under the reconstitution conditions used. A cell-free system for Ca 2؉-activated fusion will facilitate studies on the roles of essential proteins such as syntaxin, synaptobrevin, SNAP-25, and CAPS that act at post-docking steps in the regulated exocytotic pathway.
1998
Secretion from dense-core vesicles is reputedly much slower than that from typical synaptic vesicles, possibly because of noncolocalization of Ca channels and release sites. We reinvestigated this question by measuring the kinetics of catecholamine release in chromaffin cells from calf and adult bovines. Amperometric recording from calf chromaffin cells stimulated by action potentials exhibited two latencies of secretion that depended on both the frequency of stimulation and the pathway of Ca entry. Short-latency responses (Ͻ25 msec delay; "strongly coupled") appeared at low (0.25 and 1 Hz) and high (7 Hz) frequencies and were entirely dependent on recruitment of "facilitation" L-type Ca channels as revealed by nisoldipine blockade. Long-latency responses (Ͼ25 msec delay; "weakly coupled") were more apparent at higher frequencies (7 Hz) and were substantially reduced by toxins that blocked N-and P-type Ca channels. Ca current recordings revealed that adult bovine chromaffin cells lack facilitation channels; virtually all secretion was weakly coupled in these cells. The mean delay of the strongly coupled signal was ϳ3 msec after the peak of the action potential (at 24°C), indicating that dense-core vesicles can exhibit a rate of exocytosis approaching that occurring in neurons. Although other explanations are possible, these results are consistent with the idea that facilitation Ca channels are colocalized with release sites in calf chromaffin cells. Calculations based on a model incorporating this assumption suggest that these channels must be within 13 nm of secretory sites to account for such rapid exocytosis.
PLoS ONE, 2013
It is generally accepted that the immediately releasable pool is a group of readily releasable vesicles that are closely associated with voltage dependent Ca 2+ channels. We have previously shown that exocytosis of this pool is specifically coupled to P/Q Ca 2+ current. Accordingly, in the present work we found that the Ca 2+ current flowing through P/Q-type Ca 2+ channels is 8 times more effective at inducing exocytosis in response to short stimuli than the current carried by L-type channels. To investigate the mechanism that underlies the coupling between the immediately releasable pool and P/Q-type channels we transiently expressed in mouse chromaffin cells peptides corresponding to the synaptic protein interaction site of Cav2.2 to competitively uncouple P/Q-type channels from the secretory vesicle release complex. This treatment reduced the efficiency of Ca 2+ current to induce exocytosis to similar values as direct inhibition of P/Q-type channels via v-agatoxin-IVA. In addition, the same treatment markedly reduced immediately releasable pool exocytosis, but did not affect the exocytosis provoked by sustained electric or high K + stimulation. Together, our results indicate that the synaptic protein interaction site is a crucial factor for the establishment of the functional coupling between immediately releasable pool vesicles and P/Q-type Ca 2+ channels.
Calcium-dependent activator protein for secretion (CAPS) is a neural/endocrine cell-specific protein that has been shown to function at the Ca 2ϩ -dependent triggering step of dense-core vesicle (DCV) exocytosis in permeabilized PC12 cells. To evaluate the function of CAPS under physiological conditions, we introduced affinity-purified anti-CAPS IgGs into calf adrenal chromaffin (AC) cells via a patch pipette and tested the kinetics of catecholamine secretion using both amperometric and membrane capacitance techniques. The antibodies reacted with a single major ϳ145 kDa protein in AC cells based on immunoblot analysis. AC cells stimulated with sequential trains of action potentials at 7 Hz resulted in successive secretory episodes of equivalent magnitude. When either of two different anti-CAPS IgGs or their Fab fragments were present, a rapid and progressive inhibition of catecholamine release ensued to a maximum of Ͼ80%. The effect was specific because preabsorption of IgGs with the respective antigens ablated the inhib-itory effect, and the IgGs had no effect on Ca currents. CAPS immunoneutralization not only reduced the number of amperometric spikes but markedly altered the kinetic characteristics of the residual events. The remaining spikes were much smaller (by 85%) and broader (by ϳ3.5-fold) than those in control cells, suggesting that CAPS plays a role in determining release of vesicle contents via the fusion pore. Anti-CAPS IgGs also slowed the rate of the initial exocytotic capacitance burst, representing the docked-and-primed vesicle pool, by ϳ90% but had no effect on the kinetics of rapid endocytosis. These results suggest that CAPS is a key component regulating the fusion of DCVs to the plasma membrane, and possibly fusion pore dilation, in catecholamine secretion from AC cells.
Brain Research Reviews, 2000
Neuroendocrine cells display a similar calcium dependence of release as synapses but a strongly different organization of channels and vesicles. Biophysical and biochemical properties of large dense core vesicle release in neuroendocrine cells suggest that vesicles and channels are dissociated by a distance of 100-300 nm. This distinctive organization relates to the sensitivity of the release process to mobile calcium buffers, the resulting relationship between calcium influx and release and the modulatory mechanisms regulating the efficiency of excitation-release coupling. At distances of 100-300 nm, calcium buffers determine the calcium concentration close to the vesicle. Notably, the concentration and diffusion rate of mobile buffers affect the efficacy of release, but local saturation of buffers, possibly enhanced by diffusion barriers, may limit their effects. Buffer conditions may result in a linear relationship between calcium influx and exocytosis, in spite of the third or fourth power relation between intracellular calcium concentration and release. Modulation of excitation-secretion coupling not only concerns the calcium channels, but also the secretory process. Transmitter regulation mediated by cAMP and PKA, as well as use-dependent regulation involving calcium, primarily stimulates filling of the releasable pool. In addition, direct effects of cAMP on the probability of release have been reported. One mechanism to achieve increased release probability is to decrease the distance between channels and vesicles. GTP may stimulate release independently from calcium. Thus, while in most cases primary inputs triggering these pathways await identification, it is evident that large dense core vesicle release is a highly controlled and flexible process.