Vesicular Ca 2+ Participates in the Catalysis of Exocytosis (original) (raw)
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Calcium dynamics in catecholamine-containing secretory vesicles
Cell Calcium, 2005
We have used an aequorin chimera targeted to the membrane of the secretory granules to monitor the free [Ca 2+ ] inside them in neurosecretory PC12 cells. More than 95% of the probe was located in a compartment with an homogeneous [Ca 2+ ] around 40 M. Cell stimulation with either ATP, caffeine or high-K + depolarization increased cytosolic [Ca 2+ ] and decreased secretory granule [Ca 2+ ] ([Ca 2+ ] SG ). Inositol-(1,4,5)trisphosphate, cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate were all ineffective to release Ca 2+ from the granules. Changes in cytosolic [Na + ] (0-140 mM) or [Ca 2+ ] (0-10 M) did not modify either ([Ca 2+ ] SG ). Instead, [Ca 2+ ] SG was highly sensitive to changes in the pH gradient between the cytosol and the granules. Both carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) and nigericin, as well as cytosolic acidification, reversibly decreased [Ca 2+ ] SG , while cytosolic alcalinization reversibly increased [Ca 2+ ] SG . These results are consistent with the operation of a H + /Ca 2+ antiporter in the vesicular membrane. This antiporter could also mediate the effects of ATP, caffeine and high-K + on [Ca 2+ ] SG , because all of them induced a transient cytosolic acidification. The FCCP-induced decrease in [Ca 2+ ] SG was reversible in 10-15 min even in the absence of cytosolic Ca 2+ or ATP, suggesting that most of the calcium content of the vesicles is bound to a slowly exchanging Ca 2+ buffer. This large store buffers [Ca 2+ ] SG changes in the long-term but allows highly dynamic free [Ca 2+ ] SG changes to occur in seconds or minutes.
Journal of Biological Chemistry, 2008
Secretory vesicles of sympathetic neurons and chromaffin granules maintain a pH gradient toward the cytosol (pH 5.5 versus 7.2) promoted by the V-ATPase activity. This gradient of pH is also responsible for the accumulation of amines and Ca 2؉ because their transporters use H ؉ as the counter ion. We have recently shown that alkalinization of secretory vesicles slowed down exocytosis, whereas acidification caused the opposite effect. In this paper, we measure the alkalinization of vesicular pH, caused by the V-ATPase inhibitor bafilomycin A1, by total internal reflection fluorescence microscopy in cells overexpressing the enhanced green fluorescent protein-labeled synaptobrevin (VAMP2-EGFP) protein. The disruption of the vesicular gradient of pH caused the leak of Ca 2؉ , measured with fura-2. Fluorimetric measurements, using the dye Oregon green BAPTA-2, showed that bafilomycin directly released Ca 2؉ from freshly isolated vesicles. The Ca 2؉ released from vesicles to the cytosol dramatically increased the granule motion of chromaffin-or PC12-derived granules and triggered exocytosis (measured by amperometry). We conclude that the gradient of pH of secretory vesicles might be involved in the homeostatic regulation of cytosolic Ca 2؉ and in two of the major functions of secretory cells, vesicle motion and exocytosis.
Calcium Entry, Calcium Redistribution, and Exocytosis
Annals of The New York Academy of Sciences, 2002
Abstract: At a given cytosolic domain of a chromaffin cell, the rate and amplitude of the Ca2+ concentration, [Ca2+]c, depend on at least three efficient regulatory mechanisms: (1) the plasmalemmal Ca2+ channels; (2) the endoplasmic reticulum (ER); and (3) the mitochondria. High-voltage activated Ca2+ channels of the L, N, P/Q, and R subtypes are expressed with different densities in various mammalian species; they are regulated by G proteins coupled to purinergic and opiate receptors, as well as by voltage and the local changes of [Ca2+]c. Targeted aequorin and confocal microscopy show that Ca2+ entry through Ca2+ channels can refill the ER to near millimolar concentrations and causes the release of ER Ca2+ (CICR). We have also seen that, depending on its degree of filling, the ER may act as a sink or source of Ca2+ that modulates the release of catecholamine. Targeted aequorins with different Ca2+ affinities show that mitochondria undergo surprisingly rapid millimolar Ca2+ transients ([Ca2+]M) upon stimulation of chromaffin cells with ACh, high K+, or caffeine. Physiological stimuli generate [Ca2+]c microdomains at these functional complexes in which the local subplasmalemmal [Ca2+]c rises abruptly from 0.1 μM to about 50 μM. This triggers CICR, mitochondrial Ca2+ uptake, and exocytosis in nearby secretory active sites. That this is true is shown by the observation that protonophores abolish mitochondrial Ca2+ uptake and drastically increase catecholamine release by 3- to 5-fold. This increase is likely due to acceleration of vesicle transport from a reserve pool to a ready-release vesicle pool; such transport might be controlled by Ca2+ redistribution to the cytoskeleton, through CICR and/or mitochondrial Ca2+ release.
Vesicular Ca2+ mediates granule motion and exocytosis
Cell Calcium, 2012
Secretory vesicles of chromaffin cells are acidic organelles that maintain an increasing pH gradient towards the cytosol (5.5 vs. 7.3) that is mediated by V-ATPase activity. This gradient is primarily responsible for the accumulation of large concentrations of amines and Ca 2+ , although the mechanisms mediating Ca 2+ uptake and release from granules, and the physiological relevance of these processes, remain unclear. The presence of a vesicular matrix appears to create a bi-compartmentalised medium in which the major fractions of solutes, including catecholamines, nucleotides and Ca 2+ , are strongly associated with vesicle proteins, particularly chromogranins. This association appears to be favoured at acidic pH values. It has been demonstrated that disrupting the pH gradient of secretory vesicles reduces their rate of exocytosis and promotes the leakage of vesicular amines and Ca 2+ , dramatically increasing the movement of secretory vesicles and triggering exocytosis. In this short review, we will discuss the data available that highlights the importance of pH in regulating the association between chromogranins, vesicular amines and Ca 2+ . We will also address the potential role of vesicular Ca 2+ in two major processes in secretory cells, vesicle movement and exocytosis.
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-ethylmaleimidesensitive fusion protein Attachment Protein REceptordependent exocytosis of large dense core vesicles, but only in the presence of extracellular Ca 2+ . However, this response to trachynilysin does not rely on Ca 2+ influx through voltage-activated Ca 2+ 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 Ca 2+ from intracellular stores. Moreover, depletion of the latter stores inhibited trachynilysin-induced exocytosis. Thus, the observed requirement of external Ca 2+ for stimulation of large dense core vesicles exocytosis from chromaffin cells implicates plasma membrane channels that signal efflux of Ca 2+ 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.
Cell Calcium, 2014
Classic calcium hypothesis states that depolarization-induced increase in intracellular Ca 2+ concentration ([Ca 2+ ] i) triggers vesicle exocytosis by increasing vesicle release probability in neurons and neuroendocrine cells. The extracellular Ca 2+ , in this calcium hypothesis, serves as a reservoir of Ca 2+ source. Recently we find that extracellular Ca 2+ per se inhibits the [Ca 2+ ] i dependent vesicle exocytosis, but it remains unclear whether quantal size is regulated by extracellular, or intracellular Ca 2+ or both [1]. In this work we showed that, in physiological condition, extracellular Ca 2+ per se specifically inhibited the quantal size of single vesicle release in rat adrenal slice chromaffin cells. The extracellular Ca 2+ in physiological concentration (2.5 mM) directly regulated fusion pore kinetics of spontaneous quantal release of catecholamine. In addition, removal of extracellular Ca 2+ directly triggered vesicle exocytosis without eliciting intracellular Ca 2+. We propose that intracellular Ca 2+ and extracellular Ca 2+ per se cooperately regulate single vesicle exocytosis. The vesicle release probability was jointly modulated by both intracellular and extracellular Ca 2+ , while the vesicle quantal size was mainly determined by extracellular Ca 2+ in chromaffin cells physiologically.
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.
Brain Research, 2002
Since Pb substitutes for Ca in essential steps leading to exocytosis, we have investigated whether Ca and Pb induce exocytosis through similar pathways. Vesicular catecholamine release was measured from dexamethasone-differentiated PC12 cells using carbon fiber microelectrode amperometry. Effects of drugs known to modulate PKC (PMA, staurosporine), calcineurin (cyclosporin A), calmodulin (W7), and CaM kinase II (KN-62) activity were investigated in intact and in ionomycin-permeabilized PC12 cells. Activation 1 of PKC and inhibition of calmodulin decrease the frequency of exocytotic events evoked by high K stimulation in intact cells. In addition, inhibition of calmodulin enhances the frequency of basal exocytosis from intact cells. Activation of PKC and inhibition of calcineurin enhance the frequency of basal exocytosis in intact as well as in ionomycin-permeabilized cells. Inhibition of PKC and of CaM kinase II cause no significant effects. None of the treatments has a significant effect on vesicle contents. The combined results indicate that PKC and calcineurin enhance and inhibit exocytosis through direct effects on the exocytotic machinery, whereas calmodulin 21 and CaM kinase II exert indirect effects only. Conversely, Pb -evoked exocytosis in permeabilized cells is strongly reduced by inhibition of CaM kinase II, but is not sensitive to modulation of PKC and calcineurin activity. Inhibition of calmodulin only reduces the delay to 21 onset of Pb -evoked exocytosis. Synaptotagmin I-and II-deficient PC12-F7 cells exhibit vesicular catecholamine release following 21 depolarization or superfusion with Pb . However, the frequency of exocytosis and the contents of vesicles released are strongly reduced 21 as compared to PC12 cells. It is concluded that Ca -evoked exocytosis is modulated mainly by PKC and calcineurin, whereas 21 Pb -evoked exocytosis is mainly modulated by CaM kinase II. 2002 Elsevier Science B.V. All rights reserved.
Calcium Signaling and Exocytosis in Adrenal Chromaffin Cells
Physiological Reviews, 2006
At a given cytosolic domain of a chromaffin cell, the rate and amplitude of the Ca 2ϩ concentration ([Ca 2ϩ ] c ) depends on at least four efficient regulatory systems: 1) plasmalemmal calcium channels, 2) endoplasmic reticulum, 3) mitochondria, and 4) chromaffin vesicles. Different mammalian species express different levels of the L, N, P/Q, and R subtypes of high-voltage-activated calcium channels; in bovine and humans, P/Q channels predominate, whereas in felines and murine species, L-type channels predominate. The calcium channels in chromaffin cells are regulated by G proteins coupled to purinergic and opiate receptors, as well as by voltage and the local changes of [Ca 2ϩ ] c . Chromaffin cells have been particularly useful in studying calcium channel current autoregulation by materials coreleased with catecholamines, such as ATP and opiates. Depending on the preparation (cultured cells, adrenal slices) and the stimulation pattern (action potentials, depolarizing pulses, high K ϩ , acetylcholine), the role of each calcium channel in controlling catecholamine release can change drastically. Targeted aequorin and confocal microscopy shows that Ca 2ϩ entry through calcium channels can refill the endoplasmic reticulum (ER) to nearly millimolar concentrations, and causes the release of Ca 2ϩ (CICR). Depending on its degree of filling, the ER may act as a sink or source of Ca 2ϩ that modulates catecholamine release. Targeted aequorins with different Ca 2ϩ affinities show that mitochondria undergo surprisingly rapid millimolar Ca 2ϩ transients, upon stimulation of chromaffin cells with ACh, high K ϩ , or caffeine. Physiological stimuli generate [Ca 2ϩ ] c microdomains in which the local subplasmalemmal [Ca 2ϩ ] c rises abruptly from 0.1 to ϳ50 M, triggering CICR, mitochondrial Ca 2ϩ uptake, and exocytosis at nearby secretory active sites. The fact that protonophores abolish mitochondrial Ca 2ϩ uptake, and increase catecholamine release three-to fivefold, support the earlier observation. This increase is probably due to acceleration of vesicle transport from a reserve pool to a ready-release vesicle pool; this transport might be controlled by Ca 2ϩ redistribution to the cytoskeleton, through CICR, and/or mitochondrial Ca 2ϩ release. We propose that chromaffin cells have developed functional triads that are formed by calcium channels, the ER, and the mitochondria and locally control the [Ca 2ϩ ] c that regulate the early and late steps of exocytosis. 1096 GARCÍA ET AL. Physiol Rev • VOL 86 • OCTOBER 2006 • www.prv.org