©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Long Term Stimulation Changes the Vesicular Monoamine Transporter Content of Chromaffin Granules (*)

Claire Desnos (§) , Marie-Pierre Laran , Keith Langley (1), Dominique Aunis (1), Jean-Pierre Henry (¶)

From the (1)Centre National de la Recherche Scientifique, Unité Associée 1112, Service de Neurobiologie Physico-Chimique, Institut de Biologie Physico-Chimique, Paris 75005, France and the Institut National de la Santé et de la Recherche Médicale, Unité 338 de Biologie de la Communication Cellulaire, Centre de Neurochimie, Strasbourg 67084, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Bovine chromaffin cells cultured for 5 days in the presence of depolarizing concentrations of K ions show a decreased number of secretory (chromaffin) granules per cell. These cells were still capable of exocytosis. Their contents in catecholamine and chromogranin A, components of the granule matrix, and cytochrome b, a major protein of the granule membrane, were decreased to 35, 30, and 50% of control cells, respectively. However, in the same cells, the number of [H]dihydrotetrabenazine binding sites, a specific ligand of the vesicular monoamine transporter, was increased to 180% of controls. In situ uptake of noradrenaline in permeabilized cells indicated that [H]dihydrotetrabenazine binding sites were associated with a functional vesicular monoamine transporter. When analyzed by isopycnic centrifugation, these sites cosedimented with catecholamine, chromogranin A, and cytochrome b, in a peak with a density lighter than that from controls. The composition of this peak suggests that it contains incompletely matured secretory granules, with a 3-5-fold increase in the vesicular monoamine transporter content of this membrane. This increase might indicate that an adaptative process occurs which allows a faster filling of the granules in continuously secreting cells.


INTRODUCTION

Trans-synaptic induction (stimulation-secretion-synthesis coupling) is an important regulatory process, allowing the neuron to adapt to a strong stimulation by increasing the rate of synthesis of proteins required for secretion(1) . Trans-synaptic induction was initially observed for the biosynthetic enzymes of adrenal medulla, tyrosine hydroxylase(2) , and dopamine -hydroxylase(3) , but it has been extended to proteins of the chromaffin granule matrix, such as proenkephalin(4) . Strong stimulation may therefore change the composition of the ``secretory mixture''(5) , presumably for physiological purposes. These adaptative changes can be reproduced in vitro by stimulation with nicotinic agonists or by depolarization of bovine chromaffin cells in culture(6, 7, 8) .

In adrenal medulla, the catecholamine biosynthetic pathway involves the translocation of dopamine from the cytosol to the chromaffin granule matrix. The uptake is catalyzed by the vesicular monoamine transporter, a H/monoamine antiporter utilizing the H-electrochemical gradient generated by an ATP-dependent proton pump of the V-type(9) . The same transporter also operates in monoaminergic neurons, although two genes, VMAT and VMAT (vesicular monoamine transporter 1 and 2)()encoding this activity have been recently described(10, 11) . In rats, VMAT is expressed in adrenal medulla, whereas VMAT is present in the brain. On the other hand, both genes are expressed in bovine adrenal medulla (12, 13) and from analysis of the N-terminal extremity of the chromaffin granule protein VMAT is the major gene(14) . Several ligands are specific for the vesicular monoamine transporter, such as reserpine or dihydrotetrabenazine ([H]TBZOH)(15) . These ligands are useful tools not only for biochemical experiments, but also for physiological studies, including study of the regulation of the expression of the vesicular monoamine transporter. After an insulin shock, the number of [H]TBZOH binding sites of rat adrenal medulla increased for several days and was maximal after 4-6 days(5) . A similar increase was observed in vitro in bovine chromaffin cells in culture(16) . The [H]TBZOH binding site increase was maximal after several days of culture in the presence of carbamylcholine or depolarizing concentrations of potassium ions. The response involved Ca channels, and it was mimicked by forskolin and by phorbol esters. Increase in the number of binding sites seemed likely to involve transcriptional activation, since it was blocked by actinomycin and cycloheximide. The hypothesis of a regulated expression of the vesicular monoamine transporter was strongly supported by the finding that K depolarization of bovine chromaffin cells increased the transcription of the VMAT gene, encoding the tetrabenazine-sensitive vesicular monoamine transporter(12) . This was clearly seen on Northern blots, and competitive polymerase chain reaction indicated a 3-fold increase in VMAT expression after a 6-h depolarization in 55 mM KCl. The concept of trans-synaptic induction might therefore be extended to the vesicular monoamine transporter.

What are the consequences of an increased expression of the gene at the cellular level? Does an increase of the monoamine transporter result in a differential change in the composition of the secretory granule membrane? To answer these questions, we analyzed cells cultured for 5 days in high K medium, which doubled their [H]TBZOH binding site content, and characterized their secretory granules. We determined the subcellular distribution of [H]TBZOH binding sites in these cells, and we investigated the functional meaning of this increase.


MATERIALS AND METHODS

Electron Microscopy and Morphometry

For ultrastructural analysis, cell cultures grown on collagen-coated glass coverslips were rinsed in phosphate-buffered saline (PBS) and fixed for 1 h at room temperature in 2% glutaraldehyde in phosphate buffer (0.1 M, pH 7.2). Cells were post-fixed for 1 h in 1% osmium tetroxide, dehydrated in ascending ethanol solutions, and flat-embedded in Spurr epoxy resin (17). For morphometric analysis, sections of randomly chosen areas were cut at uniform thickness (silver interference color) from control and KCl-treated chromaffin cell cultures. To eliminate variations due to differences in distributions between the various cell compartments, entire sections were photographed at a constant microscope magnification ( 3900), and negatives were subsequently enlarged to give a final magnification of 8900. The number of secretory granules was counted and recorded for each cell profile in each micrograph. Granules were classified as either noradrenergic or adrenergic on the basis of their ultrastructural appearance. Adrenergic granules were taken as those with a larger diameter and lower uniform density of granule matrix, whereas granules with very dense contents separated from the granule membrane by an electron-lucent space were classified as noradrenergic(18) . Each cell profile was then cut out on a photocopy the same size as the micrograph and weighed, to provide a relative estimate of the area. The actual surface area was determined by comparison with the weight of a known area of photocopy paper, which enabled granule density/100 µm to be calculated.

Immunofluorescence

Chromaffin cells plated on collagen-coated 35-mm coverslips at about 10 cells/coverslip were fixed for 30 min in phosphate-buffered 4% formaldehyde (freshly prepared from p-formaldehyde), washed in PBS, and incubated for 30 min in ``blocking solution'' (5% BSA in PBS). Double immunofluorescence was performed by incubating cultures simultaneously for 2 h at room temperature with the two primary antisera: mouse monoclonal anti-tyrosine hydroxylase (Euromedex, Strasbourg, France) and a previously characterized (19) rabbit polyclonal antibody prepared against a synthetic peptide named CAP14, residues 316-329 in the sequence of bovine chromogranin A. These were diluted 1:500 and 1:1000, respectively, in PBS containing 1% BSA and 0.1% Triton X-100. After PBS washes, bound antibody was detected with affinity-purified rhodamine-conjugated goat anti-mouse and fluorescein-conjugated goat anti-rabbit immunoglobulins (Euromedex, Strasbourg, France), diluted 1:200 in PBS/BSA. Coverslips were examined with a Zeiss Microscope II equipped with Neofluor objectives and the appropriate fluorescent barrier filters, and selected fields were photographed on Ilford HP5 film.

Culture of Chromaffin Cells

Bovine chromaffin cells were isolated and purified as described (16). They were suspended at 10 cells/ml in Dulbecco's modified Eagle's medium (Life Technologies, Inc.), supplemented with 10% fetal calf serum (Life Technologies, Inc.) containing 10 µM cytosine arabinoside, 10 µM 5-fluoro-2`-deoxyuridine, 50 µg/ml of streptomycin, and 50 units/ml of penicillin and plated at a density of 5 10 cells in 2-cm diameter wells or 5 10 cells in 5-cm diameter Petri dishes or 20-30 10 cells in 75-cm flasks. They were incubated for 2 days at 37 °C in a 5% CO-saturated atmosphere. The culture medium was then changed, and salts or drugs were added. Cells were harvested by scraping in Locke medium or in 0.2% Triton X-100.

Biochemical Assays

The vesicular monoamine transporter was assayed by measuring [H]TBZOH binding(20) . Samples from cell homogenates or fractions of centrifugation gradients were incubated for 1 h with 3 nM [H]TBZOH in 1 ml of Locke medium containing 25 mM Hepes buffer (pH 8) at 25 °C, and filtered on HAWP filters (Millipore). Nonspecific binding was obtained by adding 3 µM TBZ to the incubation. Previous analyses (16) indicated a homogenous class of binding sites in control and K-depolarized cells with a K of 16 nM and a B value of 1 pmol/mg of protein for control cells homogenates.

Catecholamines were measured by the trihydroxyindole technique(21) . To estimate the cellular catecholamine content, cells were scraped with 200 µl of 0.2% Triton X-100, the extract was precipitated by addition of trichloroacetic acid (6% final concentration), and the supernatant was assayed for catecholamines. Adrenaline and noradrenaline were estimated by fluorometry after differential oxidation(22) .

Cytochrome b in cellular or subcellular fractions was estimated by semiquantitative immunoblotting(16) , using an anti-cytochrome b antiserum (generous gift from Dr. D. Apps), at 1:1500 dilution, and radioiodinated protein A. The same approach was used to assay intracellular or released chromogranin A, with an anti-chromogranin A antiserum at 1:500 dilution detected by radioiodinated protein A or by anti-rabbit immunoglobulin alkaline phosphatase conjugate. Alternatively, subcellular fractions from the centrifugation gradients were assayed for chromogranin A by enzyme-linked immunosorbent assay(23) . This assay was linear up to 20 ng of chromogranin A.

Uptake and Release Experiments

Uptake and Release of [H]Noradrenaline

Chromaffin cells were incubated for 30 min at 37 °C in Locke medium containing 2.5 mM CaCl, 1.25 mM MgSO, 0.5 mM ascorbic acid, and 1 mM Hepes, pH 7.4, and then for 45 min in 500 µl of the same medium to which 10 µM [H]noradrenaline (0.3 µCi/ml) was added. When the effects of TBZ and desipramine were tested, these drugs were added during the uptake period at 3 and 4 µM concentration, respectively. Cells were washed four times for 10 min with Locke solution containing 2.5 mM CaCl and twice for 5 min with calcium-free Locke solution. Under the conditions used, K-depolarized and control cells contained the same radioactivity. Cells were stimulated by suspension in 250 µl of Locke solution, with or without 2.5 mM CaCl, containing 55 mM KCl or 20 µM nicotine. The radioactivity in the cells and in the centrifuged extracellular fluids was measured by liquid scintillation. Release of [H]noradrenaline was expressed as the percentage of total radioactivity present in the cells prior to stimulation in calcium containing media. Secretion after permeabilization by streptolysin O was tested as described previously (24).

Release of Endogenous Catecholamines

After 5 days in control or in K-containing medium, cells were rapidly washed with 1 ml of Locke solution containing 1 mM MgCl, 0.5 mM ascorbic acid, and 10 mM Hepes buffer, pH 7.4. They were either immediately stimulated or preincubated for 45 min at 37 °C in 500 µl of calcium containing Locke solution, before stimulation. Cells were stimulated by resuspension in the wash medium containing either 55 mM KCl or 20 µM nicotine, in the presence or absence of 2.5 mM CaCl. Catecholamines were assayed fluorometrically, in the external medium and in the cell pellet. Release of catecholamines was expressed as the percentage of the total catecholamine cell content.

In Situ Vesicular Uptake of [H]Noradrenaline

Chromaffin cells were washed with 1 ml of Locke solution containing 1 mM MgCl, 0.5 mM ascorbic acid, 10 mM Hepes buffer, pH 7.4. They were permeabilized by incubation with streptolysin O (4 units/well) in 200 µl of 150 mM potassium glutamate, 0.2% BSA, 4 mM ATP, 3 mM MgSO, and 5 mM nitrilotriacetic acid, pH 7.2, for 3 min and washed twice with 500 µl of medium without streptolysin O. [H]Noradrenaline uptake was measured in 500 µl of the same medium containing 0.5 mM ascorbic acid, 1 mM pargyline, 10 µM [H]noradrenaline (0.15-0.6 µCi/ml), at 25 °C. To avoid interference from endogenous monoamines, the [H]noradrenaline concentration was adjusted with unlabeled noradrenaline to 10 µM, a concentration which is saturating for the vesicular monoamine transporter. Moreover, the concentration of endogenous catecholamines in the external medium after permeabilization was the same in K-treated and control cells. Nonspecific uptake was measured by addition of 3 µM TBZ to the incubation medium. The uptake was stopped by washing three times with 1 ml of the permeabilization medium. Cells were scraped in 200 µl of 0.2% Triton X-100 and the radioactivity of aliquots measured by liquid scintillation.

Subcellular Fractionation of Chromaffin Cells

Cells (20-30 10 cells) were cultured in 75-cm flasks or 5-cm Petri dishes. They were harvested by scraping in 5 ml of 5 mM EDTA, 20 mM Hepes buffer, pH 7.4, containing 10.5% sucrose and 2 units/ml leupeptin (buffer A). This homogenate was centrifuged for 10 min at 800 g, and the resulting pellet was resuspended in 0.5 ml of the same buffer, homogenized manually in a glass-Teflon homogenizer, and again centrifuged under the same conditions. The pooled supernatants (postnuclear supernatant) were centrifuged at 200,000 g for 80 min. The pellet was resuspended in 0.6 ml of buffer A, and 0.5 ml of the suspension was layered onto 4.5 ml of a linear sucrose gradient (23-55%) in the same EDTA/leupeptin/Hepes buffer. The tubes were centrifuged for 3 h at 4 °C at 240,000 g in a SW-65 Beckman rotor. Fractions (400 µl) were collected from the bottom of the tubes.


RESULTS

Morphological Changes Induced by KDepolarization

As described by Unsicker et al.(25) , long term depolarization of bovine chromaffin cells induces flattening of the cells and the extension of neurite-like processes. The difference between control and depolarized cells was clearly seen after 5 days of culture on both plastic dishes or collagen-coated coverslips. At the ultrastructural level (Fig. 1), dense secretory vesicles (chromaffin granules) were observed in both depolarized and control cells. Two types of granules were present in both cultures, small dense noradrenaline-containing granules in noradrenergic cells and larger lighter adrenaline-containing granules(18) . In the K-depolarized cultures, 37% of the cells were estimated to be of the noradrenergic type, whereas control cultures contained only 16% of noradrenergic cells (). Because the number of cells was the same in the two types of culture, this change was attributed to phenotype plasticity and not to differential survival(25) . K-cultured cells were more vacuolar and clearly contained less secretory granules than control ones. The granules of the K-cultured cells often appeared to have an electron-dense core within a larger vesicle. The number of granules per unit surface area was determined under both culture conditions. K depolarization resulted in a decrease in the number of granules to about 30% of the controls, in both cell types. Long term stimulation by K depolarization therefore decreases the number of chromaffin granules, presumably as a result of continuous exocytosis, but without completely depleting the cell of its secretory granules.


Figure 1: Electron micrographs of control (a) and KCl-treated (b) bovine chromaffin cell cultures. Both show cells with secretory granules more typical of adrenergic chromaffin cells, although some granules with apparently condensed matrices are present in the treated cells. Note the dramatic reduction in granule density in treated cells. Bar represents 1 µm.



An immunofluorescence study using antibodies against chromogranin A, a component of chromaffin granule matrix, confirmed the decrease in the number of mature secretory granules (Fig. 2). Almost all cells in treated cultures showed a dramatic reduction in labeling for chromogranin A (compare Fig. 2, b and d), although a faint punctate labeling could still be detected. Less than 5% of the cells, which also had undergone less obvious morphological changes, appeared to be much less affected. The two cell cultures were also labeled for cytosolic tyrosine hydroxylase (Fig. 2, a and c), used as a marker of trans-synaptic induction(2, 8) . Compared with control cultures (Fig. 2a), the intensity of tyrosine hydroxylase labeling in treated cultures (Fig. 2c) was increased in all cells, except those which were still labeled fairly strongly for chromogranin A, and this diffuse cytoplasmic labeling filled both perikarya and cell extensions. Thus, the intensity of tyrosine hydroxylase labeling in treated cultures was negatively correlated with that for chromogranin A.


Figure 2: Double immunofluorescent labeling of control (a, b) and KCl-treated cultures (c, d) for tyrosine hydroxylase (a, c) and chromogranin A (b, d). Note that cells have an apparently rounder morphology in control compared with treated cultures. Tyrosine hydroxylase fluorescence appears to be augmented in treated cultures, whereas chromogranin A fluorescence is dramatically reduced in the vast majority of cells in treated cultures. A small number, less than 5% of all chromaffin cells, retain relatively high level of labeling with this antibody (d) in treated cultures. Bar represents 25 µm.



Biochemical Analysis of K-depolarized Cells

In parallel to the morphometric study, a biochemical analysis of the catecholamine content of the two types of culture was performed. The designation of the two cell types as adrenergic and noradrenergic cells was confirmed by the ratio of the two catecholamines (). K depolarization decreased the catecholamine content to 35% of the controls. In addition, immunological assays were used to estimate the cellular contents of chromogranin A and cytochrome b, which are major components of the matrix and of the membrane of chromaffin granules, respectively(26) . K depolarization induced a marked decrease of the content not only of chromogranin A, but also of cytochrome b ().

In contrast, the number of [H]TBZOH binding sites specifically associated with the vesicular monoamine transporter in the same cultures was shown to increase to 180% of controls after long term depolarization ().

Secretory Capacity of K-depolarized Chromaffin Cells

To characterize the functional aspects of the granules, the secretory capacity of these cells was analyzed in various ways. First, K-depolarized and control cells were charged with [H]noradrenaline in a nondepolarizing Na-containing medium and challenged with 20 µM nicotine in the presence of Ca ions. [H]Noradrenaline uptake was desipramine-sensitive, indicating that it occurred through the cell membrane transporters. It was also tetrabenazine-sensitive, since 3 µM TBZ decreased the cellular [H]noradrenaline content by 30-50% in control cells and by 60% in K-depolarized ones, thus showing that the tritiated catecholamine accumulated in the vesicular compartment, through the vesicular transporter. The conditions of uptake (substrate concentration, uptake incubation time) were adjusted so that the same radioactivity accumulated in both cultures. Under these conditions, the secretory capacity of K-cultured cells, defined as the percentage of tritiated noradrenaline released, was similar to that of controls (Fig. 3A). Secretion in these cells was Ca-dependent and it occurred through the vesicular compartment, since it was inhibited when [H]noradrenaline was taken up in the presence of 3 µM TBZ. The release of [H]noradrenaline was also tested after permeabilization of the charged cells by the bacterial toxin streptolysin O(27) . When such cells were challenged with 20 µM Ca, [H]noradrenaline release was identical in K-cultured and in control cells (data not shown), confirming that release occurred from the vesicular pool (and not the cytoplasm), by an unaffected process.


Figure 3: Secretion of catecholamine by chromaffin cells cultured for 5 days in control (C) or high potassium (K) medium. A, [H]noradrenaline release. Control (C) or K-depolarized (K) cells were incubated for 45 min in the presence of [H]noradrenaline and then challenged for 15 min at 37 °C by 20 µM nicotine in the presence (black boxes) or absence (white boxes) of 2.5 mM CaCl. Released [H]noradrenaline is expressed as percentage of total radioactivity present in the cell prior to stimulation. Results are means (± S.E.) of six independent experiments. B, release of endogenous catecholamine by control (C) or K-depolarized (K) cells. After resuspension in Locke solution, cells were immediately stimulated with 20 µM nicotine for 10 min at 37 °C, in the presence (black boxes) or absence (white boxes) of 2.5 mM CaCl. Results are means of five independent experiments. C, release of endogenous catecholamine by control (C) or K-depolarized (K) cells, preincubated in nondepolarizing medium. After resuspension in Locke medium, cells were incubated for 45 min at 37 °C before stimulation. Results are means (±S.E.) of four independent experiments.



In the above approach, [H]noradrenaline uptake required repolarization of cells, since the plasma membrane transporter utilizes the Na electrochemical gradient as a driving force. The secretory capacity of long term K-treated cells was also analyzed by following the release of endogenous catecholamines by spectrofluorometry. After rapid transfer to Ca-containing Locke medium and stimulation by 20 µM nicotine, K-cultured and control cells were found to release 2.6 ± 0.2 and 14 ± 1.4 of nmol catecholamine/well, respectively, in a Ca-dependent nicotine-stimulated manner. Expressed as percentages of cellular catecholamines, the corresponding figures are 9 ± 0.2 and 17 ± 1.0%, taking into account the lower amine content of the depolarized cells (Fig. 3B). This result suggests some desensitization of the secretory pathway, which could account for the presence of a stock of secretory granules in continuously stimulated cells.

The effect of a repolarizing period was also tested. Cells were incubated in normal Locke medium for 45 min at 37 °C before being challenged as above. This repolarization period is equivalent to that used for the uptake of [H]noradrenaline. Under these conditions, 3 ± 0.1 and 9 ± 1.5 nmol of catecholamine/well were specifically released by K-cultured and control cells; however, the percentages were similar, corresponding to 14.5 ± 0.8 and 16 ± 1.4%, respectively (Fig. 3C). This might be because desensitization is rapidly reversible, and all secretory granules are functional, secretion being proportional to the number of granules.

Vesicular [H]Noradrenaline Uptake by K-depolarized Chromaffin Cells

[H]TBZOH binding to the vesicular monoamine transporter occurs even when the transporter has been uncoupled from the H electrochemical gradient(15) . Binding of this ligand has been observed using detergent-solubilized transporter(28) . The increase of binding in K-depolarized cells might have been due to proteins present on non vesicular structures, such as the endoplasmic reticulum, which are unable to generate a H-electrochemical gradient, or to inactive proteins present in the endosomal network. To test the functionality of the transporter, cells were permeabilized with streptolysin O(27) , and vesicular ATP-dependent [H]noradrenaline uptake was measured in situ (Fig. 4). The accumulation of [H]noradrenaline was insensitive to 1 µM desipramine, showing that the plasma membrane transporter was by-passed, but was blocked by 3 µM TBZ, indicating a vesicular uptake. The experiment was performed at a saturating noradrenaline concentration, 10 µM, to measure the transporter concentration and to avoid any interference with residual endogenous monoamines. In three experiments similar to that of Fig. 4, the initial rate of uptake was increased in the K-depolarized cells by a factor of 1.7 ± 0.3, which was similar to the increase of [H]TBZOH binding sites. It should be noted that in K-treated cells the uptake leveled more rapidly than in control ones, indicating a lower plateau value consistent with a lower number of granules in the treated cells.


Figure 4: Vesicular uptake of [H]noradrenaline in permeabilized cells. Chromaffin cells were permeabilized by incubation with the bacterial toxin streptolysin O. Vesicular uptake was then measured in situ by addition of [H]noradrenaline and ATP. Nonspecific uptake was estimated in the presence of 3 µM TBZ and was subtracted. Data are means (±S.E.) of two independent experiments in which measurements were performed in quadruplicate.



Subcellular Fractionation of K-depolarized Cells

To determine the localization of [H]TBZOH binding sites, the cellular extracts were fractionated by isopycnic centrifugation on linear sucrose gradients (23-55%), and the profiles of [H]TBZOH binding sites were determined for K-depolarized and control cells. Three representative experiments are shown in Fig. 5. In both types of culture, a main peak of [H]TBZOH binding sites was observed. In control cells, the peak was centered in the dense part of the gradient at 45% (1.6 M) sucrose. In more than 15 experiments, K-depolarized cells were characterized by a larger peak, the ratio of peak areas reflecting the cellular increase in [H]TBZOH binding. The peak from depolarized cells also had a lighter equilibration density, and it was often asymmetrical, being broader on the lighter side. In nine different experiments, the fraction of sites equilibrating at a density lighter than 43% sucrose in control and in depolarized cells was 33 ± 10 and 62 ± 13%, respectively. The asymmetry of the broad peak of K-cultured cells was not due to size heterogeneity, since this peak was not resolved when the force and the duration of the centrifugation were decreased.


Figure 5: Analysis of [H]TBZOH binding sites by isopycnic centrifugation on linear sucrose gradients. Resuspended high speed pellets from control () or 5-day depolarized () cells were layered onto a 23-55% (0.7-2 M) sucrose gradient and centrifuged for 3 h at 240,000 g. The fractions were analyzed for specific [H]TBZOH binding. The results of three independent experiments are shown.



Synaptic-like microvesicles are present in endocrine cells(29, 30) , and in bovine chromaffin cells, they have been reported to contain catecholamines(31) , though this view is not consistent with results obtained in rat pheochromocytoma cells(32) . To test for [H]TBZOH binding sites on these vesicles, high speed pellets were analyzed by centrifugation on a 18% sucrose buffer layer on the top of a 22-35% linear sucrose gradient. Variable amounts of [H]TBZOH binding sites were found in the 27-30% sucrose fractions, containing synaptophysin, the marker of synaptic-like microvesicles (data not shown). This density is consistent with that given by O'Grady et al. (30); however, cosedimentation of synaptophysin and [H]TBZOH binding sites is difficult to interpret, since membranes from osmotically shocked chromaffin granules also sedimented in this region. Therefore, although the presence of [H]TBZOH binding sites on synaptic-like microvesicles cannot be entirely ruled out, in control and in K-depolarized cells the major fraction of these sites (80%) is associated with structures equilibrating at heavier densities (>35% sucrose). It is of interest to note that synaptophysin immunoreactivity was clearly increased in the sucrose density gradient fractions from K-cultured cells (data not shown).

To identify the structures associated with [H]TBZOH binding sites, the distributions of catecholamines, chromogranin A, and cytochrome b were analyzed on the 23-55% linear sucrose gradients (Fig. 6). In control (A) and in K-cultured (B) cells, [H]TBZOH comigrated not only with cytochrome b, a marker of the chromaffin granule membrane, but with the secretion products, catecholamine and chromogranin A, thus indicating that [H]TBZOH binding sites were present on secretory granules.


Figure 6: Subcellular fractionation of control and K-depolarized cells. The resuspended high speed pellet from control (A) or 5-day depolarized (B) cells was analyzed by centrifugation on a 23-55% (0.7-2 M) sucrose gradient for 3 h at 240,000 g. The fractions were analyzed for [H]TBZOH binding sites (), catecholamine (), chromogranin A () and cytochrome b (). The experiment was repeated more than 12 times with similar results. For [H]TBZOH binding, one unit is 100 fmol and for catecholamine, one unit is 10 nmol.



Comparison of the distributions of the markers catecholamine, chromogranin A, and cytochrome b indicated that K depolarization induced two reproducible changes. First, the distribution was slightly shifted toward lighter densities. In four different experiments, the fraction of the three markers equilibrating at a density lighter than 45% sucrose was 42 ± 15% (catecholamine), 31 ± 14% (chromogranin A) and 30 ± 10% (cytochrome b) in control cells, and in depolarized cells, this fraction increased to 67 ± 18% (catecholamine), 46 ± 16% (chromogranin A), and 65 ± 20% (cytochrome b). Second, the intensity of the peaks, equilibrating between 35 and 55% sucrose, was markedly decreased. For catecholamine and for chromogranin A, the sums of the peak fractions in the treated cells were, respectively, 33 ± 6% and 29 ± 2% (n = 4) of those in control cells. For cytochrome b, a decrease was also observed, but it was less marked; cytochrome b values were 43 ± 6% of controls. In the same cultures, potassium depolarization increased [H]TBZOH binding to 146 ± 8% of control cells. Therefore, treated cells were characterized by secretory granules with higher transporter to marker ratios. The ratios for transporter to catecholamine, chromogranin A, and cytochrome b were increased by factors of 4.4, 5, and 3.4, respectively.


DISCUSSION

After 5 days in a medium containing depolarizing concentrations of K ions, chromaffin cells still contain secretory granules. This contention is supported by three different types of data: (i) morphologically, the depolarized cells contained subcellular structures identifiable as chromaffin granules. As in control cells, noradrenergic granules could be distinguished from adrenergic ones and the morphological differences were supported by biochemical data (); (ii) functionally, the K-cultured cells released catecholamines (Fig. 3) and chromogranin A (data not shown) from a vesicular pool in a Ca-dependent manner; (iii) subcellular fractionation experiments also indicated the presence of chromaffin granules in these cells, characterized by the colocalization of catecholamines, chromogranin A, cytochrome b, and [H]TBZOH binding sites (Fig. 6). The finding that the rate of vesicular [H]noradrenaline uptake in permeabilized cells is increased by a factor similar to that observed for [H]TBZOH binding sites is consistent with the view that the binding sites are primarily associated with chromaffin granules.

In K-depolarized cells, the four markers, catecholamines, chromogranin A, cytochrome b, and [H]TBZOH binding sites, equilibrated at a density slightly lighter than in control cells, and the major difference between the two profiles was observed between 35 and 40% sucrose. The vesicles containing these markers differ certainly from rough endoplasmic reticulum and Golgi vesicles which equilibrate below 35% sucrose (33) and from synaptic-like microvesicles which equilibrate in 28% sucrose fractions(30) . If [H]TBZOH binding sites are present on synaptic like microvesicles, these sites are certainly a minor population, representing less than 20% of the total. The lighter density of the granules from K-depolarized cells suggests the presence of newly synthesized, immature granules in the stimulated cells. Such immature granules have been characterized in PC12 cells (34). Their membrane was described as more often irregularly shaped and loosely surrounding the core, in contrast to mature secretory granules, the membrane of which surrounds the core more uniformly. Such images were frequently present in K-depolarized cells (see Fig. 1). It is thus possible that maturation of the chromaffin granule involves a membrane retrieval step occurring after catecholamine uptake, which would concentrate the matrix material and act as a late sorting step(34, 35, 36, 37) . K-depolarized cells might therefore be an interesting model to study the biogenesis of secretory granules.

K-depolarized cells contain less secretory granules. Quantitative analysis of chromaffin granules isolated from K-depolarized cells indicated that catecholamine, chromogranin A, and cytochrome b were decreased to 33, 29, and 43% of control values, respectively, whereas [H]TBZOH binding sites were increased to 146%. The decrease of the granule matrix material, catecholamine and chromogranin A, associated with an increase of [H]TBZOH binding sites might suggest the presence of incompletely filled newly formed granules. However, this hypothesis is unlikely for two reasons: (i) cytochrome b, which like the vesicular transporter is a membrane protein, is clearly decreased rather than increased. Although the regulation of this protein has not been studied at the molecular level, physiological experiments suggested that the synthesis of this protein is not regulated under our experimental conditions(5) ; this result suggests also that proteins from the chromaffin granule membrane were not quantitatively recycled after exocytosis; (ii) catecholamine and chromogranin A are decreased to similar levels, though their transport into secretory vesicles is an independent process, occurring at different steps in the maturation of the granule; chromogranin A originates from the Golgi apparatus and is an early component, whereas catecholamine is a late component, which is taken up from the cytoplasm. Finally, the morphometric data, indicating that in depolarized cells the number of granules was decreased to 30% of controls, strongly support the view that the granules from these cells contain their normal quantity of matrix components and that their membrane is enriched in vesicular monoamine transporter. The enrichment of the membrane in vesicular monoamine transporter can be calculated by normalizing the [H]TBZOH binding site increase to the markers content. Depending upon the marker chosen, the enrichment ranges from 3.4 (normalized to cytochrome b) to 5 (normalized to chromogranin A). Therefore, it can be concluded that chromaffin cells adapt to prolonged secretion by changing the composition of their secretory vesicles membrane.

It has already been suggested that the protein composition of the secretory granule membrane may change under certain conditions. After repeated reserpine injections, the specific activity of membrane bound dopamine -hydroxylase was increased to 67% above control level, whereas in the same animals, the pool of chromaffin granule membrane stayed constant, as shown by morphometric analysis(38) . In treated animals, the size of the granules was reduced and this effect was compensated by a slight increase in the number of granules. However, this study is difficult to interpret, because dopamine -hydroxylase exists in both a membrane bound and a soluble form, and the relationship between these two forms is not clearly elucidated. More recently, a different concept has been proposed by Mahata et al.(39, 40) , who reported a differential mRNA regulation for the vesicular monoamine transporter and the matrix peptide NPY. Short term neurogenic stimulation induced a 4-fold increase of the NPY mRNA level in rat adrenal medulla without changing that of the vesicular monoamine transporter VMAT, suggesting that the synthesis of proteins of the matrix, but not of the membrane, was regulated. In bovine chromaffin cells, K depolarization for 2 days induced an increase in the expression of VMAT RNA(12) , which was reproducible. The difference between the results of Mahata et al.(39) and the present data might be explained by the fact that the former ones were obtained on rat adrenal medulla, where VMAT is the only expressed gene(10) , whereas bovine chromaffin cells used in the latter study expressed predominantly VMAT. In bovine K-depolarized cells, VMAT is not induced in contrast to VMAT.()Alternatively, the difference might result from the use of more vigorous stimulation conditions in the present work (5 days of continuous depolarization in culture versus insulin shock in vivo). After an insulin shock, there is an increase in the number of [H]TBZOH binding sites, visible 96 h after the shock, but which is not significant after 24 h(5) , at the time chosen by Mahata et al.(39) .

What is the physiological meaning of this regulation? It is classically proposed that the rate-limiting step in catecholamine biogenesis is tyrosine hydroxylation and that regulation of this pathway, by phosphorylation of the enzyme or by increase of its rate of synthesis, occurs mainly at this level. However, it has also been suggested that vesicular uptake might be rate-limiting(41) . This hypothesis was formulated by comparing in vitro measurements of the time required to fill up rat chromaffin granules and in vivo estimates of the maturation time obtained either in pulse experiments followed by density gradient analysis or from the recovery of the normal granule catecholamine content and granule density after insulin stimulation. Under prolonged K stimulation, catecholamine synthesis is increased as a result of the induction of tyrosine hydroxylase(8) , and the number of chromaffin granule is decreased (see Fig. 2). Since the cytosolic catecholamine concentration of control cells is estimated to be in the 10-20 µM range (41), the vesicular monoamine transporter is already operating at its maximal rate, and its regulation would be physiologically relevant. Accordingly, the relative enrichment of the granule membrane in monoamine transporter might indicate an adaptative process, allowing a faster filling of the granules.

A more quantitative estimate of the regulation of catecholamine biogenesis might possibly be achieved by applying the metabolic network theory. In this theory, regulation is not assumed to result from a unique step, but to involve the whole pathway, each step being characterized by a partial control coefficient(42) . The regulatory properties of the vesicular monoamine transporter might be important in the early steps of neurogenerative diseases, such as Parkinson's disease, where the loss of the first neurons would be compensated by overstimulation of those remaining and the induction of adaptative processes, such as that described for the vesicular monoamine transporter. This early phase would account for the threshold effect reported in the development of the disease(43) .

Continuous stimulation clearly decreased the number of granules and changed their composition. Three different interpretations are possible, which might not be mutually exclusive. First, the decrease might easily be attributed to a dynamic state, since exocytosis was not blocked under these conditions (model I). The newly synthesized vesicles would have different properties. Alternatively, the conditions used might affect granule biogenesis, decreasing the size of the vesicular pool and thereby changing their composition (model II). Finally, it might also be proposed that the decrease in the number of secretory granules results from the phenotypic change associated with the culture in elevated potassium (model III). Unsicker et al.(25) already reported the growth of neurite-like processes and an increase of the noradrenaline to adrenaline ratio. In addition, we have also noted a marked increase of synaptophysin and a decrease in chromogranin A. These changes indicate a shift from a neuroendocrine to a neuronal phenotype, which might be associated with a decrease of the number of secretory granules.

  
Table: Morphological and biochemical characterization of K-depolarized cells



FOOTNOTES

*
This work was supported by the Centre National de la Recherche Scientifique (Unité de Recherche Associée 1112, Jean-Pierre Henry) and the Institut National de la Santé et de la Recherche Médicale (Unité 338, Dominique Aunis). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Biochemistry and Biophysics, Hormone Research Institute, University of California, San Francisco, CA 94142-0448.

To whom correspondence should be addressed; Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France. Tel.: 33-1-46-34-10-57; Fax: 33-1-40-46-83-31.

The abbreviations used are: VMAT, vesicular monoamine transporter cytochrome; TBZ, tetrabenazine; TBZOH, dihydrotetrabenazine (2-hydroxy-3-isobutyl-9,10-dimethoxy-1,2,3,4,6,7-hexahydro-11bH-benzo-[]quinolizine); [H]TBZOH, [2-H]dihydrotetrabenazine; PBS, phosphate buffered saline; BSA, bovine serum albumin.

D. Botton, B. Gasnier, and J.-P. Henry, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. D. K. Apps (University of Edinburgh) for the gift of the antibody against cytochrome b. We are indebted to Dr. C. Tougard (Collège de France, Paris) for preliminary morphological studies and to Dr. F. Darchen for fruitful discussions.


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