Combining the lack of chromogranins with chronic L-DOPA treatment affects motor activity in mice.

We have tested whether the lack of chromogranins (Cgs) A and B could provoke CNS disorders when combined with an excess of dopamine. We chronically treated (over 6 months) mice lacking both chromogranins A and B (Cgs-KO) with a low oral dosage of L-DOPA/benserazide (10/2.5 mg/kg). Motor performance in the rota-rod test, open field activity, and metabolic cages indicated a progressive impairment in motor coordination in these mice, and an increase in rearing behavior, which was accompanied by an increase in DA within the substantia nigra. We conclude that mild chronic L-DOPA treatment does not produce nigro-striatal toxicity that could be associated with parkinsonism, neither in control nor Cgs-KO mice. Rather, Cgs-KO mice exhibit behaviors compatible with an amphetamine-like effect, probably caused by the excess of catecholamines in the CNS.

Electrochemical Investigation of the Interaction between Catecholamines and ATP.

The study of the colligative properties of adenosine 5'-triphosphate (ATP) and catecholamines has received the attention of scientists for decades, as they could explain the capabilities of secretory vesicles (SVs) to accumulate neurotransmitters. In this Article, we have applied electrochemical methods to detect such interactions in vitro, at the acidic pH of SVs (pH 5.5) and examined the effect of compounds having structural similarities that correlate with functional groups of ATP (adenosine, phosphoric acid and sodium phosphate salts) and catecholamines (catechol). Chronoamperometry and fast scan cyclic voltammetry (FSCV) provide evidence compatible with an interaction of the catechol and adenine rings. This interaction is also reinforced by an electrostatic interaction between the phosphate group of ATP and the protonated ammonium group of catecholamines. Furthermore, chronoamperometry data suggest that the presence of ATP subtlety reduces the apparent diffusion coefficient of epinephrine in aqueous media that adds an additional factor leading to a slower rate of catecholamine exocytosis. This adds another plausible mechanism to regulate individual exocytosis events to alter communication.

The intravesicular cocktail and its role in the regulation of exocytosis.

The accumulation of neurotransmitters within secretory vesicles (SVs) far exceeds the theoretical tonic concentrations in the cytosol, a phenomenon that has captivated the attention of scientists for decades. For instance, chromaffin granules can accumulate close to molar concentrations of catecholamines, along with many other products like ATP, calcium, peptides, chromogranins, ascorbate, and other nucleotides. In this short review, we will summarize the interactions that are currently believed to occur between the elements that make up the vesicular cocktail in the acidic environment of SVs, and how they permit the accumulation of such high concentrations of certain components. In addition, we will examine how the vesicular cocktail regulates the exocytosis of neurotransmitters. In this review, we have highlighted the mechanisms that permit the storage of neurotransmitters and hormones inside secretory vesicles. We also have proposed a novel model based in the intravesicular interactions of the main components of this inner cocktail - catecholamines, ATP, and chromogranins - to allow the accumulation of near molar concentrations of transmitters in secretory vesicles. This article is part of a mini review series on Chromaffin cells (ISCCB Meeting, 2015).

The interaction between chromogranin A and catecholamines governs exocytosis.

Chromogranins (Cgs) are acidic proteins that have been described in the large, dense core vesicles (LDCVs) of adrenal chromaffin cells and that have been shown to promote LDCV formation, even in nonsecretory cells. Catecholamines (CAs) are adsorbed by Cgs in vitro, and the absence of Cgs modifies the storage and exocytosis of CAs in chromaffin cells. In this study, we set out to assess the role of CgA in the accumulation and exocytosis of CAs in cells when the levels of CgA and CA are manipulated. We overexpressed CgA in nonsecretory HEK293 cells and in secretory PC12 cells, to study the formation, movement, and exocytosis of newly formed granules by evanescent wave microscopy. We analyzed the association of Cgs/CA by HPLC and amperometry and their role in the accumulation and exocytosis of amines, both under resting conditions and after l-DOPA overloading. To our knowledge, this is the first demonstration that CgA expression in a nonsecretory cell line facilitates the storage and exocytosis of CA. In addition, CgA overexpression causes a doubling of the accumulation of CA, although it slows down exocytosis in PC12 cells. We propose a model to explain how the CgA/CA complex governs the accumulation and exocytosis of secreted amines.

The role of chromogranins in the secretory pathway.

Chromogranins (Cgs) are acidic proteins implicated in several physiological processes, including the biogenesis and sorting of secretory vesicles, the generation of bioactive peptides, and the accumulation of soluble species inside large dense core vesicles (LDCV). Indeed, Cgs are the main protein component of the vesicular matrix in LDCV, and they are involved in the concentration of soluble species like neurotransmitters and calcium. Experiments using electrochemical techniques such amperometry, patch amperometry, and intracellular electrochemistry have clarified the functional roles of Cgs in the accumulation and release of catecholamines. We have focused this review at a single event of exocytosis of chromaffin cells from three mouse strains lacking Cgs. Accordingly, in this brief review, we will focus on the role of Cgs in maintaining the intravesicular environment of secretory vesicles and in exocytosis, bringing together the most recent findings from studies on adrenal chromaffin cells.

Fluorescent ß-Blockers as Tools to Study Presynaptic Mechanisms of Neurosecretion.

Several, if not all adrenergic ß-blockers (ß-Bs), accumulate progressively inside secretory vesicles in a time- and concentration-dependent manner, and could be considered to be false neurotransmitters. This transmitter effect is most likely unrelated to their ability to block adrenergic receptors, but it could explain the delay in lowering arterial pressure in hypertensive patients. We have developed a new drug to monitor the accumulation of ß-Bs inside living cells, RCTM-3, which fluoresces in the visible spectrum. Here we describe the procedure to synthesize this new compound, as well as its fluorescent properties, pharmacological profile and its accumulation inside the secretory vesicles of PC12 cells.

Intravesicular factors controlling exocytosis in chromaffin cells.

Chromaffin granules are similar organelles to the large dense core vesicles (LDCV) present in many secretory cell types including neurons. LDCV accumulate solutes at high concentrations (catecholamines, 0.5-1 M; ATP, 120-300 mM; or Ca(2+), 40 mM (Bulenda and Gratzl Biochemistry 24:7760-7765, 1985). Solutes seem to aggregate to a condensed matrix to elude osmotic lysis. The affinity of solutes for LDCV matrix is responsible for the delayed release of catecholamines during exocytosis. The aggregation of solutes occurs due to a specific H(+) pump denominated V-ATPase that maintains an inner acidic media (pH ≈5.5). This pH gradient against cytosol is also responsible for the vesicular accumulation of amines and Ca(2+). When this gradient is reduced by modulation of the V-ATPase activity, catecholamines and Ca(2+) are moved toward the cytosol. In addition, some drugs largely accumulate inside LDCV and not only impair the accumulation of natural solutes, but also act as false neurotransmitters when they are co-released with catecholamines. There is much experimental evidence to conclude that the physiological modulation of vesicle pH and the manipulation of intravesicular media with drugs affect the LDCV cargo and change the kinetics of exocytosis. Here, we will present some experimental data demonstrating the participation of drugs in the kinetics of exocytosis through changes in the composition of vesicular media. We also offer a model to explain the regulation of exocytosis by the intravesicular media that conciliate the experimentally obtained data.

Chromogranins A and B as regulators of vesicle cargo and exocytosis.

Chromogranins (Cgs) are acidic proteins that have been implicated in several physiological processes such as vesicle sorting, the production of bioactive peptides and the accumulation of soluble species inside large dense core vesicles (LDCV). They constitute the main protein component in the vesicular matrix of LDCV. This latter characteristic of Cgs accounts for the ability of vesicles to concentrate catecholamines and Ca(2+). It is likely that Cgs are behind the delay in the neurotransmitter exit towards the extracellular milieu after vesicle fusion, due to their low affinity and high capacity to bind solutes present inside LDCV. The recent availability of mouse strains lacking Cgs, combined with the arrival of several techniques for the direct monitoring of exocytosis, have helped to expand our knowledge about the mechanisms used by granins to concentrate catecholamines and Ca(2+) in LDCV, and how they affect the kinetics of exocytosis. We will discuss the roles of Cgs A and B in maintaining the intravesicular environment of secretory vesicles and in exocytosis, bringing together the most recent findings from adrenal chromaffin cells.

Chromogranins as regulators of exocytosis.

Chromogranins (Cgs) constitute the main protein component in the vesicular matrix of large dense core vesicles (LDCV). These acidic proteins have been implicated in several physiological processes such as vesicle sorting, the generation of bioactive peptides and the accumulation of soluble species inside LDCV. This latter feature of Cgs accounts for the ability of vesicles to concentrate catecholamines and Ca(2+). Indeed, the low affinity and high capacity of Cgs to bind solutes at the low pH of the LDCV lumen seems to be behind the delay in the neurotransmitter exit towards the extracellular milieu after vesicle fusion. The availability of new mouse strains lacking Cgs in combination with the arrival of several techniques for the direct monitoring of exocytosis (like amperometry, patch-amperometry and intracellular electrochemistry), have helped advance our understanding of how these granins concentrate catecholamines and Ca(2+) in LDCV, and how they influence the kinetics of exocytosis. In this review, we will discuss the roles of Cgs A and B in maintaining the intravesicular environment of secretory vesicles and in exocytosis, bringing together the most recent findings from adrenal chromaffin cells.

Chromogranin B gene ablation reduces the catecholamine cargo and decelerates exocytosis in chromaffin secretory vesicles.

Chromogranins/secretogranins (Cgs) are the major soluble proteins of large dense-core secretory vesicles (LDCVs). We have recently reported that the absence of chromogranin A (CgA) caused important changes in the accumulation and in the exocytosis of catecholamines (CAs) using a CgA-knock-out (CgA-KO) mouse. Here, we have analyzed a CgB-KO mouse strain that can be maintained in homozygosis. These mice have 36% less adrenomedullary epinephrine when compared to Chgb(+/+) [wild type (WT)], whereas the norepinephrine content was similar. The total evoked release of CA was 33% lower than WT mice. This decrease was not due to a lower frequency of exocytotic events but to less secretion per quantum (approximately 30%) measured by amperometry; amperometric spikes exhibited a slower ascending but a normal decaying phase. Cell incubation with L-DOPA increased the vesicle CA content of WT but not of the CgB-KO cells. Intracellular electrochemistry, using patch amperometry, showed that L-DOPA overload produced a significantly larger increase in cytosolic CAs in cells from the KO animals than chromaffin cells from the WT. These data indicate that the mechanisms for vesicular accumulation of CAs in the CgB-KO cells were saturated, while there was ample capacity for further accumulation in WT cells. Protein analysis of LDCVs showed the overexpression of CgA as well as other proteins apparently unrelated to the secretory process. We conclude that CgB, like CgA, is a highly efficient system directly involved in monoamine accumulation and in the kinetics of exocytosis from LDCVs.

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

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 Ca(2+), but the free intragranular [Ca(2+)], the mechanisms for Ca(2+) 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 Ca(2+) from freshly isolated vesicles. Accordingly, vesicle alkalinization released Ca(2+) from the granules to the cytosol, measured with fura-2 in intact chromaffin cells. Using TIRFM in cells overexpressing the EGFP-labeled synaptobrevin (VAMP2-EGFP) protein, we have then shown that the Ca(2+) 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 Ca(2+) around the vesicles and in two of the major functions of secretory cells, vesicle motion and exocytosis.1.

Intravesicular calcium release mediates the motion and exocytosis of secretory organelles: a study with adrenal chromaffin cells.

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 Ca2+ 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 Ca2+, measured with fura-2. Fluorimetric measurements, using the dye Oregon green BAPTA-2, showed that bafilomycin directly released Ca2+ from freshly isolated vesicles. The Ca2+ 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 Ca2+ and in two of the major functions of secretory cells, vesicle motion and exocytosis.

Intragranular pH rapidly modulates exocytosis in adrenal chromaffin cells.

Several drugs produce rapid changes in the kinetics of exocytosis of catecholamines, as measured at the single event level with amperometry. This study is intended to unveil whether the mechanism(s) responsible for these effects involve changes in the intravesicular pH. Cell incubation with bafilomycin A1, a blocker of the vesicular proton pump, caused both a deceleration in the kinetics of exocytosis and a reduction in the catecholamine content of vesicle. These effects were also observed upon reduction of proton gradient by nigericin or NH4Cl. pH measurements using fluorescent probes (acridine orange, quinacrine or enhanced green fluorescent protein-synaptobrevin) showed a strong correlation between vesicular pH and the kinetics of exocytosis. Hence, all maneuvers tested that decelerated exocytosis also alkalinized secretory vesicles and vice versa. On the other hand, calcium entry caused a transient acidification of granules. We therefore propose that the regulation of vesicular pH is, at least partially, a necessary step in the modulation of the kinetics of exocytosis and quantal size operated by some cell signals.

A simple way to build a grinder for carbon-fibre electrodes for amperometry or voltammetry.

Carbon-fibre electrodes are used widely for studying exocytosis by amperometry. Currently, there are two major methods for insulating fibres so as to leave the tip as the only conductive surface: encapsulation with plastic or glass. The latter offers advantages such as better insulation and a known electro-active surface. In addition, such electrodes are suitable for in vivo electrochemistry because they can penetrate brain tissues. However, the construction of glass-encapsulated electrodes requires a grinder to polish the electrode surface with precision. This apparatus is expensive because it needs a very stable motor, a diamond surface and a micromanipulator. We describe the construction of a cheap precision grinder using a computer drive and an old microscope.

Hydralazine reduces the quantal size of secretory events by displacement of catecholamines from adrenomedullary chromaffin secretory vesicles.

The effects of the antihypertensive agent hydralazine (1 to 100 nmol/L) on the exocytotic process of single adrenal chromaffin cells have been studied using amperometry. Hydralazine does not reduce the frequency of exocytotic spikes but rapidly slows the rate of catecholamine release from individual exocytotic events by reducing the quantal size of catecholamine exocytosis. Confocal and standard epifluorescence microscopy studies show that hydralazine rapidly accumulates within secretory vesicles. The blockade of the vesicular H+ pump with bafilomycin A1 inhibits hydralazine uptake. Experiments with permeabilized cells show that hydralazine displaces catecholamines from secretory vesicles. The drug also displaces vesicular Ca2+, as shown by fura-2 microfluorimetry. These data suggest that hydralazine acts, at least partially, by interfering with the storage of catecholamines. These effects of hydralazine occurred within seconds, and at the tissue concentrations presumably reached in antihypertensive therapy; these concentrations are a thousand times lower than those described for relaxing vascular tissues in vitro. We proposed that these novel effects could explain many of the therapeutic and side effects of this drug that are likely exerted in sympathetic nerve terminals.

Nongenomic regulation of the kinetics of exocytosis by estrogens.

The role of nongenomic action of estrogens on elicited catecholamine secretion and exocytosis kinetics was studied in perfused rat adrenals and in cultured bovine chromaffin cells. 17beta-Estradiol as well as the estrogen receptor modulators raloxifene and LY117018, but not 17alpha-estradiol, inhibited at the micromolar range the catecholamine output elicited by acetylcholine or high potassium. However, these agents failed to modify the secretion elicited by high Ca(2+) in glands treated with the ionophore A-23187 (calcimycin), suggesting that estrogens did not directly act on the secretory machinery. At the single cell level, estrogens modified the kinetics of exocytosis at nanomolar range. All of the drugs tested except 17alpha-estradiol produced a profound slowing down of the exocytosis as measured by amperometry. LY117018 also reduced the granule content of catecholamines. 17beta-Estradiol reduced the intracellular free Ca(2+) but only at micromolar concentrations, whereas nanomolar concentrations increased the cAMP levels. These effects were reproduced with the nonpermeable drug 17beta-estradiol-horseradish peroxidase and antagonized with nanomolar concentrations of the antiestrogen ICI 182,780 (fulvestrant). Our data suggest the presence of membrane sites that regulate both the exocytotic phenomenon and the total catecholamine release with high and low affinity, respectively.