On the role of intravesicular calcium in the motion and exocytosis of secretory organelles

Abstract

Secretory vesicles of sympathetic neurons and chromaffin granules maintain a pH gradient towards the cytosol (5.5 vs. 7.2) promoted by the V-ATPase activity. This gradient of pH is mainly responsible for the accumulation of amines. The secretory vesicles contain large amounts of total Ca²⁺, but the free intragranular [Ca²⁺], the mechanisms for Ca2+ uptake and release from the granules and their physiological relevance regarding exocytosis are still matters of debate.

We have recently shown that disruption of the pH gradient of secretory vesicles slowed down exocytosis. Fluorimetric measurements, using the dye Oregon green BAPTA-2, showed that the V-ATPase inhibitor bafilomycin A1 directly released Ca²⁺ from freshly isolated vesicles. Accordingly, vesicle alkalinization released Ca²⁺ from the granules to the cytosol, measured with fura-2 in intact chromaffin cells. Using TIRFM in cells over-expressing the EGFP-labeled synaptobrevin (VAMP2-EGFP) protein, we have then shown that the Ca²⁺ released from the vesicles to the cytosol in the presence of bafilomycin, 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 the local cytosolic Ca²⁺ around the vesicles and in two of the major functions of secretory cells, vesicle motion and exocytosis.¹

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Most neurotransmitters and hormones are stored in secretory vesicles that release their contents to the extracellular media after a stimulus.

As exocytosis and vesicle motions are well-established Ca²⁺-dependent mechanisms and large concentrations of Ca²⁺ are stored in secretory vesicles, a considerable effort has been placed to address a physiological role(s) of vesicular Ca²⁺ in their own motion and exocytosis.

In this brief we will discuss recent data about the homeostasis of intravesicular Ca²⁺, which have provided strong evidence that it may represent a crucial source able to create a specific microdomain of Ca²⁺ in the vicinity of granule membrane, the exact location to control both the granule motion and exocytosis.

The Secretory Organelle

Secretory granules from chromaffin cells are large dense core vesicles similar to those present in many other neuroendocrine cells and in sympathetic neurons.2 Chromaffin granules are extremely efficient concentrating soluble transmitters such as catecholamines 500–1000 mM3,4 together with other components as ATP 125–300 mM,5 ascorbate 10–30 mM,6,7 peptides and chromogranins, forming a condensed protein matrix (∼180 mg/ml).8 In addition they concentrate H⁺ to create an acid media and, the main reason of this report, large amounts of Ca²⁺. The mechanisms followed to get these large concentrations of solutes in spite of the large osmotic forces created have intrigued researchers for decades.

Chromaffin granules maintain a pH gradient across their membranes of about two orders of magnitude, ≈5.5 inside and ≈7.3 in the cytosol. This gradient is held stable by the activity of a specific H⁺-ATPase (V-ATPase). This vesicular H⁺ gradient is used as antiporter to accumulate catecholamines, by the vesicular monoamine transporter VMAT2,9 and Ca²⁺ through the H⁺/Ca²⁺ antiport, although most of Ca²⁺ accumulation in the vesicles appears to occur via a SERCA-type Ca²⁺ ATPase10,11 (Fig. 1). The presence of a vesicular matrix composed by the aggregation of the components of the vesicular cocktail with proteins has been proposed as the chelating method to reduce the osmotic forces12 that permit the accumulation of solutes at high concentrations.13 Therefore, most of the intravesicular solutes are not free but associated to the matrix, where the main proteic components are chromogranins, whose pK_a is also around 5.5.12,14 Therefore, it is plausible that intravesicular pH can regulate the ability of chromogranin A to form aggregates15 and that the regulation of vesicular pH could play an important role in the dynamics of vesicular Ca²⁺ and catechols.11,16,17

Bi-compartmental Storage of Ca²⁺

The idea that intravesicular Ca²⁺ could be involved in the exocytotic process was first postulated by Borowitz in 1967.18 Nevertheless, this idea has not received fully acceptance by the scientific community. Endoplasmic reticulum has been classically considered as the main source of Ca²⁺, mainly because the mobilization of Ca²⁺ from stores by InsP₃ was first discovered in this organelle. More recently, the involvement of other cell structures like mitochondria, nucleus and Golgi in the uptake, release and cytosolic redistribution of Ca²⁺ have also been proven.19–21 Therefore secretory vesicles are still frequently ignored and considered as a simply non-functional sink for Ca²⁺. The main argument, with little experimental support, has been that vesicular Ca²⁺ is sequestered into the vesicular matrix from where it experiences little turnover. In spite of the new data that contradicts this assumption let us to show here some numbers.

About 30% of the total a chromaffin cell volume is occupied by around 20,000 granules.22 The recent development of targeted aequorins to the inner side of secretory vesicles has directly confirmed that Ca²⁺ is distributed in two fractions; the chelated Ca²⁺ which is estimated to be about 40 mM,23 and the free fraction which was calculated to be around 50–100 µM.11,23,24 The free fraction is in equilibrium with the Ca²⁺ bound allowing a rapid recovery after an acute depletion. Chromaffin granules contain far more Ca²⁺ than any other organelle, accounting for about 60% of the total in chromaffin cells.23,25 Even considering that this cation is crucial for processes that take place ‘just across their membrane’ like vesicle movement or exocytosis, the old hypothesis of Borowitz is still receiving little attention.

Mobilization of Vesicular Ca²⁺

The disruption of pH gradient using protonophores26 or weak bases27–29 has been used to induce the alkalinization of granules that causes the release of Ca²⁺ and catecholamines towards the cytosol.29 This effect is shared by clinically relevant drugs like the hypotensive agent hydralazine,30 amphetamines31 or β adrenergic blockers.

Other stimuli like histamine, caffeine or depolarization mobilize the free Ca²⁺ fraction.11,24 Targeted aequorine data suggest that intravesicular Ca²⁺ kinetics follows a bi-compartmental model where the total amount of free [Ca²⁺] is nearly three orders of magnitude smaller than bound calcium. This explains the rapid recovery of free Ca²⁺ after the depletion of the free compartment with SERCA inhibitors (BHQ, cyclopiazonic acid) or pH-disrupting agents.11,24 In addition, both InsP₃-induced and Ca²⁺ induced Ca²⁺ release (CICR) are present and functional in chromaffin and PC12 secretory vesicles. The main problem to demonstrate whether the intravesicular Ca²⁺ is actively participating in granule motion and exocytosis, under physiological conditions, is the difficulty in differentiating this Ca²⁺ from the Ca²⁺ arriving from other sources. All known secretagogues increase free cellular Ca²⁺ by activating its entry from external media and/or promoting its release from internal stores. Nevertheless, the vesicular alkalinization observed upon the activation of several second messenger routes will contribute also to the mobilization of vesicular Ca²⁺ and catecholamines; this latter effect was recently demonstrated using single cell amperometry. It seems plausible that the pH gradient across the vesicular membrane could be the necessary link between physiological stimuli and the regulation of Ca²⁺ and catecholamines release from the secretory vesicles.

Besides the role of other known organelles, in future, cell stimulation by different mechanisms, either mediated by InsP₃ receptors, ryanodine receptors or plasma membrane Ca²⁺ channels should take into account that they also induce vesicular Ca²⁺ release. In addition, other stimuli that activate guanylate cyclase or adenylate cyclase, which alkalinize the vesicular lumen, might also mimic these mechanisms.

Taking into account the poor diffusion of Ca²⁺ through the cytosol,32 we consider it highly plausible that vesicular Ca²⁺ could be playing a relevant physiological role in the granule's approach to the membrane33,34 and in their own exocytosis. The physiological relevance of the Ca²⁺ release from secretory vesicles will require further investigation.

An additional support to this argument was found using bafilomycin A1, a potent and highly specific inhibitor of the H⁺-ATPase, to study the effects of vesicle alkalinization and the release of vesicular Ca²⁺ to cytosol. This Ca²⁺ increases the lateral motion of chromaffin granules and triggered exocytosis. Although bafilomycin is not a physiological stimulus, these results revealed a novel mechanism for releasing vesicular Ca²⁺, which is controlled by the pH gradient.

In summary, the recent data from our laboratories and other have demonstrated that: (i) secretory vesicles from PC12,24 and chromaffin cells11 accumulate Ca²⁺ under two different and exchangeable conditions: free (≈50–100 µM) and bound Ca²⁺ (≈40 mM), (ii) the vesicular pH is closely associated with the modulation of the kinetics and quantal characteristics of the exocytosis of catecholamines,29 (iii) secretory granules possess mechanisms for fast uptake and release of Ca²⁺ and (iv) Ca²⁺ release from the granule can participate in their own movement and exocytosis.1

Figures and Tables

Figure 1 Mechanism used for Ca²⁺ (and catecholamines, CA) turnover in chromaffin secretory organelles. The relative sizes for the granule matrix (1) and the free compartment (2) have been change for clarity. The H⁺ are pumped towards the vesicle lumen by an ATP dependent (V-ATPase, 3). Protons maintain the pH and the potential gradients with the help of Cl⁻ channels which acts as counter ions (4) to keep the Ψ ≈ −80 mV. Catecholamines (5) and Ca²⁺ (6) use H⁺ as antiporters to be accumulated inside vesicles, both carriers can work also in the reverse mode. The IP₃ receptors (7) release Ca²⁺ as a response to intracellular IP₃ whereas CICR (8) amplifies the Ca²⁺ signaling by releasing Ca²⁺ a response that is modulated by ryanodine and caffeine. The SERCA (9), not described yet in chromaffin granules will be the Ca²⁺ pump; this pump is blocked by thapsigargin. In these studies the luminal terminal of VAMP (10) (synaptobrevin) has been modified to place a Ca²⁺ sensor (low Ca²⁺-affinity aequorine) or pH sensor (EGFP).

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