Chromogranins A and B as Regulators of Vesicle Cargo and Exocytosis

Extremely High Concentrations of Solutes are Present in LDCV

Chromaffins are essentially secretory cells that contain around 15,000 LDCV; however, only a small fraction is suitable for immediate release (Trifaro et al. 1997; Heinemann et al. 1994). The sympatho-adrenal response to stress requires the secretion of large amounts of catecholamines and peptides into the bloodstream, which is possible due to the surprisingly high concentration of solutes inside the LDCV.

Vesicular catecholamines are estimated to be 0.5–1 M depending on the animal species studied (Winkler and Westhead 1980; Pihel et al. 1994; Albillos et al. 1997; Montesinos et al. 2008). Vesicles also contain large amounts of ATP (Weber et al. 1983), of Ca²⁺ (Bulenda and Gratzl 1985), of ascorbate (Terland and Flatmark 1975), as well as unquantified amounts of biopeptides, other nucleotides, dopamine-β-hydroxylase, and H⁺, all of which together with Cgs forms a condensed matrix at an acidic pH of ≈5.5 in the vesicles. The theoretical osmolarity of this mixture would be around 1,500 mOsm. The functional aggregation of soluble species is necessary to maintain the homeostasis of LDCV and avoid their disruption due to osmotic stress. As CgA and CgB are quantitatively the most important granins in the LDCV, they are the main candidates to facilitate the condensation of soluble species required to generate the dense core (Helle et al. 1985). This functional matrix is probably the same as that observed in electron microscopy images of LDCV as an electrodense core. Indeed, the vesicular content of Cgs in bovine chromaffin LDCV is estimated to be ≈1.8 mM of CgA, ≈200 μM of CgB, and ≈30 μM of SgII (Yoo 2010), although their relative amounts may also vary depending on the species.

Chromogranins are currently considered as a saturable high capacity and low affinity buffer. For instance, CgA binds 32 mol adrenaline per mol with a Kd of 2.1 mM (Videen et al. 1992), and Cgs also bind Ca²⁺ ≈50 mol per mol of Cg with a Kd of 1.5 to 4 mM depending on the type of granin (Yoo and Albanesi 1991; Yoo 2010). The ability of CgA and CgB to interact with each other to form dimers or hetero-tetramers has been studied to explore the interaction of Cgs with Ca²⁺ (Yoo 1996; Yoo and Albanesi 1991). However, a similar interaction with soluble species such as catecholamines and ATP is likely to occur as the presence of multiple dibasic groups in their structure increases their ability to concentrate solutes (Yoo and Albanesi 1990; Yoo 1996; Park et al. 2002).

The intravesicular compounds can be divided into two major groups depending on their capacity to move across the vesicle’s membrane. Hence, amines, ascorbate, H⁺, Ca²⁺ and ATP are “mobile components” that are able to move across the vesicle membrane, whereas Cgs and other proteins like enzymes are considered as “immobile components” as they cannot easily leave the vesicles. All mobile compounds of the vesicular cocktail are in equilibrium with the cytosol and the matrix, and as such, they are all the main candidates to be involved in the regulation of exocytosis since changes in any one of these species will affect the others.

The Release of Catecholamines from the LDCV During Exocytosis is Unexpectedly Slow

When single LDCV fusion events occur the release of adrenaline is two to three orders of magnitude slower than that predicted by the diffusion coefficient of catecholamines in aqueous media (Gerhardt and Adams 1982; Hafez et al. 2005). Two mechanisms could explain why catecholamines are retained inside the fused vesicle:

• The diameter of the fusion pore that could limit the free escape of soluble species from the vesicle and,

• The slow diffusion of solutes from the LDCV matrix.

Measurements obtained with patch-amperometry, a technique that combines amperometry with cell-attached capacitance, revealed that the arrival of catecholamines to the carbon fiber electrode was still delayed even when the fusion pore was dilated (Albillos et al. 1997; Montesinos et al. 2008). This suggests the direct involvement of the vesicle matrix in the slow release of amines observed once vesicle fusion has taken place.

Some indirect approaches also connect the slow release to the nature of the vesicle’s protein matrix. For instance, exocytosis is largely delayed in the presence of cross-linking agents like glutaraldehyde or formaldehyde that should freeze the dissociation of catecholamines from Cgs (Borges et al. 2000). Moreover, in experiments on chromaffin cells cultured in astrocyte conditioned media, the phenotype of the chromaffin cells switches to a neuronal-like form observed with electron microscopy that exhibited many small vesicles containing little dense material (Ardiles et al. 2006). Using amperometry, these authors observed a drastically accelerated exocytosis suggesting a close relationship between the presence of vesicular matrix and the kinetics of exocytosis.

Catecholamine Exocytosis from CgA-KO Chromaffin Cells

The arrival of a mouse lacking CgA was recently achieved by the targeted ablation of the CgA gene in mice to generate CgA-KO animals that were viable and fertile in homozygosis (Mahapatra et al. 2005; Hendy et al. 2006). This provided the first opportunity to study exocytosis in the absence of one of the “immobile components”. Although the effects that the lack of CgA produced should be taken with care due to the partial compensation by the overexpression of CgB (Fig. 1a, b), the redistribution of Cgs produced drastic effects in the storage and release of catecholamines from the LDCV of adrenal chromaffin cells.

Although CgA was suggested to be essential for granule biogenesis and sorting, chromaffin cells from CgA-KO can still release adrenaline by exocytosis. Using amperometry as the method of quantification we concluded that cells from CgA-KO mice released ≈40% less catecholamines after a depolarizing stimulus (Fig. 2b), and that the number of spikes from the WT and CgA-KO cells was similar over a 2 min recording (Fig. 2a). In addition, the net content of catecholamines per quantum (Q) was reduced by 34% (Fig. 2c) and the kinetic analysis of secretory spikes showed that exocytosis occurred faster in CgA-KO cells, these kinetic changes mainly affect the last part of spikes (Fig. 3). Taken together, these data indicate that the matrix of the LDCV without CgA is less capable of concentrating and retaining catecholamines thereby causing exocytosis to occur faster (Montesinos et al. 2008).

When chromaffin cells were overloaded with the catecholamine precursor l-DOPA, a second important consequence of the lack in CgA was revealed. l-DOPA crosses the chromaffin cell membranes and it is rapidly converted into dopamine, which is usually taken up by the LDCV where the dopamine is converted in noradrenaline by dopamine-β-hydroxylase. The usual effect of l-DOPA incubation is a notable increase in the vesicular content of catecholamines (Colliver et al. 2000; Sombers et al. 2007; Gong et al. 2003) as observed in cells from WT mice. However, the LDCV of chromaffin cells from CgA-KO could not take up more amines.

We performed experiments using intracellular electrochemistry in the presence of the monoamino oxidase inhibitor pargyline to check whether this impediment to storing more catecholamines was derived from a reduction in the availability of cytosolic catecholamines. This technique is a modified version of patch-amperometry using the whole-cell configuration, thereby allowing a carbon fiber electrode to be brought into contact with the cytosolic medium. Chromaffin cells from CgA-KO animals had less free catecholamines than their WT counterparts. However, there was a marked increase of free cytosolic amines in the KO mice after incubation with l-DOPA (100 μM during 90 min) when compared with WT mice. Hence, the newly synthesized amines appear to be unable to enter the LDCV because their saturated matrix left no room for new catecholamines (Montesinos et al. 2008).

Using patch-amperometry in the cell-attached configuration to simultaneously monitor the vesicle size (capacitance) and the release of catecholamines from the same vesicle (amperometry) we studied, in more detail, the storage and release properties of the LDCV lacking CgA Our data revealed that the vesicular concentration of catecholamines drops from 870 mM in WT to 530 mM in the CgA-KO. In addition, the lack of CgA causes a delay in the release of catecholamines from the moment that fusion occurs to the arrival of catecholamines at the amperometric detector, suggesting a direct role of CgA in the swelling of the LDCV upon fusion.

Our data indicate that the LDCV from chromaffin cells lacking CgA have a much weaker capacity to accumulate catecholamines, even when considering the overexpression of CgB. Having demonstrated a role for CgA in exocytosis, we concentrated our efforts on the other major chromogranin, CgB.

Catecholamine Exocytosis from CgB-KO Chromaffin Cells

The first mouse strain lacking CgB has recently become available. These mice developed a phenotype of hypertension (Zhang et al. 2009) and intolerance to glucose (Obermuller et al. 2010). In contrast to the CgA-KO mouse, immuno-histochemistry and western blotting confirmed the absence of CgB and the overexpression of CgA (Fig. 1c, d). We carried out experiments to examine the secretory characteristics of chromaffin cells in these mice in a similar way to the one followed in the CgA-KO strain.

As in the case of CgA, CgB has also been proposed as a critical factor in the genesis and sorting of LDCV (Glombik et al. 1999; Kromer et al. 1998; Natori and Huttner 1996) and if this were true, the lack of this protein should result in the absence of LDCV. However, amperometry shows that chromaffin cells from CgB-KO mice can maintain the release of catecholamines by exocytosis. Moreover, chromaffin cells from WT and CgB-KO mice exhibited similar secretory patterns with no differences in the number of spikes (Fig. 2d). However, the total amount of catecholamines released was 33% lower (Fig. 2e), roughly coinciding with the amount released per quanta (Fig. 2f). A careful analysis of the kinetic properties of secretory spikes revealed the slowing of exocytosis that, contrary to that observed with the CgA-KO mouse, largely affected the first (ascending) part of the spikes (Diaz-Vera et al. 2010) (Fig. 3).

l-DOPA overloading showed that LDCV cannot take up more catecholamines and that the excess of amines that is unable to enter the vesicles remains in the cytosol. From these data, we can conclude that in the absence of CgB, CgA was overexpressed in the adrenomedullary tissues of the CgB-KO mice. Moreover, the cells were capable of releasing catecholamines with a similar frequency of events as the WT cells, although exocytosis exhibited a marked reduction in terms of the quantal size of the secretory packages, which produced an important fall in the total secretory response of chromaffin cells. Finally, the kinetics of exocytosis is affected in the initial part of the spikes (Fig. 3) and the LDCV from CgB-KO cells are unable to take up more catecholamines even though they were abundant in the cytosol, suggesting a saturation of their storage mechanisms.

In order to determine whether other granins, in addition to CgA, could fulfill the role of CgB in forming the dense matrix, our group performed a proteomic analysis of the enriched LDCV fraction from mouse adrenal medullas (Diaz-Vera et al. 2010). This kind of study on the LDCV of mouse chromaffin cells was not easy to perform due to the minute amount of adrenomedullary material available to obtain a purified fraction of vesicles, requiring a minimum of 28–30 adrenal glands for a single 2D gel. There were no significant changes in the amount of SgII expressed in the LDCV fraction and in addition, the expression of all the other granins remained constant. However, the exact quantification of Cgs was difficult because they were usually accompanied by fragments generated by intravesicular proteolysis (Lee and Hook 2009). The lack of CgB produced changes in the expression of other proteins and when we combed the areas of the 2D gels where other granins should be present, none of them were overexpressed. These proteins can be roughly grouped into: typical LDCV proteins; proteins classically associated to mitochondria; those involved in protein folding; cytoskeletal; and “unexpected proteins”. One may speculate that the CgB-KO mouse lacks several CgB-derived peptides, and that these animals could therefore release more CgA-derived ones. An attractive hypothesis is that some of the changes observed in the metabolic glucidic or lipidic profile could be originated by the alteration of granule proteins.

Our study revealed important amounts of fibrinogen, for which the three chains (α, β, and γ) were only present in the LDCV of the CgB-KO. Besides its crucial role in clot formation, fibrinogen has recently been associated with the sorting of constitutive vesicles (Gerdes and Glombik 1999). By weight, no other protein seems to be capable of fulfilling the role of Cgs as a matrix-condenser for soluble intravesicular components (Diaz-Vera et al. 2010).

The new data provide the first direct evidence implicating CgB in vesicular storage and in the exocytotic release of CA. We have also demonstrated that the frequency of secretory events is maintained, even in the complete absence of CgA or CgB. The lack of Cgs impairs vesicular accumulation of CA in CgB-KO cells, suggesting saturation of the capacity of the vesicles to store more catecholamines but not any effect on the biogenesis of LDCV. A protein analysis of the secretory vesicle fraction has shown the compensative overexpression of one Cg when the other is absent. Unexpectedly, other proteins apparently unrelated to the secretory process were only present in the adrenomedullary tissue of KO animals.

Concluding Remarks

Since their discovery, Cgs have captivated the attention of scientists, the functions originally proposed for these proteins have expanded into a much wider range of roles including granule biogenesis and sorting, thereby serving as a source of bioactive peptides or as tumor markers, and as proteins with pathophysiological implications in degenerative diseases of the CNS. This review addresses the functional role of CgA and CgB in regulating exocytosis from the LDCV. The generation of mice strains lacking either CgA or CgB has opened up the possibility of studying the role of immobile components. Granulogenesis is not affected by the lack of either granin, although chromaffin cells release significantly less catecholamines per quantum and the kinetics of their release differ substantially. In conclusion, Cgs are highly efficient systems directly involved in monoamine accumulation and in the kinetics of exocytosis from the LDCV. New data point to a fine regulation of the concentration of the “mobile components” of the LDCV by Cgs, where each protein exhibited clear differences in their ability to bind and release neurotransmitters.